virus-host adaptation and co-evolution of … · de mixoma vírus (virulento) nas últimas cinco...

VIRUS-HOST ADAPTATION AND CO-EVOLUTION OF

MYXOMA VIRUS (MV) AND RABBIT HAEMORRHAGIC

DISEASE VIRUS (RHDV) IN THEIR NATURAL HOST, THE

WILD RABBIT (ORYCTOLAGUS CUNICULUS)

ALEXANDRA MÜLLER

Tese de doutoramento em Ciências Veterinárias

2010

3

ALEXANDRA MÜLLER

VIRUS-HOST ADAPTATION AND CO-EVOLUTION OF MYXOMA

VIRUS (MV) AND RABBIT HAEMORRHAGIC DISEASE VIRUS

(RHDV) IN THEIR NATURAL HOST, THE WILD RABBIT

(ORYCTOLAGUS CUNICULUS)

Tese de Candidatura ao grau de Doutor em Ciências Veterinárias submetida ao Instituto de Ciências Biomédicas de Abel Salazar da Universidade do Porto.

Orientador – Doutora Gertrude Averil Baker Thompson

Categoria – Professor Associado

Afiliação – Instituto de Ciências Biomédicas Abel Salazar da Universidade do Porto.

Co-orientador – Doutora Paula Cristina Gomes Ferreira Proença



Co-orientador – Doutor Júlio Gil Vale Carvalheira



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Os resultados dos trabalhos experimentais incluídos na presente Tese fazem parte

dos seguintes artigos científicos e publicações:

Müller, A., J. Freitas, E. Silva, G. Le Gall-Reculé, F. Zwingelstein, J. Abrantes, P. J.

Esteves, P. C. Alves, W. van der Loo, Y. Kolodziejek, N. Nowotny & G. Thompson (2009).

Evolution of Rabbit haemorrhagic disease virus (RHDV) in wild rabbits (Oryctolagus

cuniculus) in the Iberian Peninsula. Veterinary Microbiology 135, 368-373

Müller, A. , E. Silva, J. Abrantes, P.J. Esteves, P.G. Ferreira, J.C. Carvalheira, N.

Nowotny & G. Thompson (2010). Partial sequencing of recent Portuguese myxoma virus

field isolates exhibits a high degree of genetic stability. Veterinary Microbiology, 140, 161-

166

Müller, A. & G. Thompson (2010). Evolution of RHDV in the Iberian Peninsula: A brief

review of recent findings. II Seminario Internacional sobre el Conejo Silvestre. Córdoba

28-30 Abril 2010 (in press)

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ACKOWLEDGEMENTS

My sincere thanks go to all colleagues that accompanied me in these years for their contribution to

the present thesis, in particular to:

Professor Gertrude Thompson, for the encouragement to enrol in this postgraduate study and for

her excellent supervision and guidance through the overall progress of work, for stimulating

discussions, for the disposal of the Infectious Diseases Laboratory knowhow and facilities, for all

the numerous opportunities given within and without this project, which contributed to the

attainment of scientific maturity, and, importantly, also for all her continuous support, kind

understanding and friendship at all times.

Professor Paula Ferreira and Professor Júlio Carvalheira for their co-supervision and collaboration

during the development of this work, helpful discussions and for their continuous encouragement

and support. Professor Artur Águas for his co-supervision and constructive suggestions, especially

in the initial phases of the study.

All members of the Laboratory of Infectious Diseases, in particular: Eliane Silva and Sara Marques

for sharing their expertise, technical support, aid in troubleshooting, and for their overall friendship;

Jaime Freitas for his contribution to the work on RHD; Sónia Paupério, Isabel Santos, Teresa

Pena, Joana Correia, Maria João Vieira, Luís Pinho, Dr. Raquel Souto for their contribution to the

enriching laboratory environment. To all for the constructive lab meetings, discussions and the

good moments spent together.

The Institute for Biomedical Studies (ICBAS) and the Multidisciplinary Unit for Biomedical Research

(UMIB) of Porto University for infrastructural and financial support.

CIBIO for infrastructural support as well as for the permission to use valuable wild rabbit samples.

Professor Pedro Esteves for the opportunity to participate in the Project on RHD and Myxomatosis

(POCTI/BIA-BDE/61553/2004), him and Dr. Joana Abrantes for the interesting discussions and

collaboration throughout. A special thanks to Joana for providing “hot off the press” and “hard to

get” bibliography! Professor Paulo Célio Alves for interesting discussions and his persistent positive

reinforcement to take up a “wild rabbit subject”.

The Zoonoses and Emerging Infections Group and all members of the Clinical Virology of the

University of Veterinary Medicine, Vienna, in particular Professor Norbert Nowotny for the

acceptance and supervision of the work as well as for the kind hospitality and friendship. Dr.

Jolanta Kolodziejek, Helga Lussy and Hans Homola for sharing their experience and making my

stay at the Clinical Virology in Vienna productive, and above all, for their friendly welcome in the

group, making my stay highly enjoyable and enriching.

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Professor Támas Bakonyi, guest researcher at the Clinical Virology, Vienna, for sharing his

experience on real time PCR.

Dr. Ghislaine le Gall-Recoulé, AFSSA Ploufragan, for the collaboration and constructive

discussions of the joint work on RHD.

Bioportugal, Lda., in particular Dr. Joaquim Teixeira, Dr. Sónia Martins and Dr. Carla Simões for

technical support in the use of the StepOne Real-time PCR.

The Laboratório de Investigação Veterinária (LNIV), Vairão, for infrastructural support, and in

particular Dr. Fátima Mota for her friendship.

The Foundation for Science and Technology (FCT) for the doctoral grant (SFRH/BD/31048/2006).

And finally, my parents, for their unconditional love and support, in particular for taking care of

Natália during my participation in scientific meetings and for their presence during the three month

research period in Vienna.

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SUMÁRIO

A mixomatose e a doença hemorrágica viral (RHD) são doenças infecciosas que

emergiram em populações de coelho-bravo (Oryctolagus cuniculus) na década de 1950 e

1980, respectivamente. Nos primeiros anos após o seu aparecimento, foram observadas

elevadas taxas de mortalidade, mas em anos subsequentes, o impacto destas infecções

parecia ter diminuído. A hipótese postulada foi de que estes vírus foram co-evoluindo

com o seu hospedeiro, resultando na selecção de estirpes virais menos virulentas e de

hospedeiros mais resistentes. É neste contexto, que os presentes estudos foram

desenhados, visando contribuir para o conhecimento actual sobre a adaptação vírus-

hospedeiro e a co-evolução do vírus da mixomatose (MV) e do vírus da doença

hemorrágica viral (RHDV) ao seu hospedeiro natural, através da análise da variabilidade

genética (parcial) dos vírus. Para tal, um total de 4863bp (approximadamente 3% do

genoma) englobando 12 genes de nove estirpes de campo recentes de MV virulentos e

de uma estirpe vacinal viva atenuada (“MAV”, Alemanha) foram sequenciadas e

comparadas à estirpe virulenta originalmente introduzida “Lausanne” e ao seu derivado

de campo atenuado “6918”. As nossas estirpes de campo apresentaram um máximo de

três (estirpes C43, C95) e um mínimo de uma (estirpes CD01, CD05) substituições

nucleotídicas em comparação com “Lausanne”. Estas estavam distribuídas ao longo de

todas as regiões codificantes analisadas, excepto no gene M022L (maior proteína do

envelope), onde todas as estirpes eram idênticas a “Lausanne” e “6918”. Duas novas

inserções nucleotídicas simples foram observadas em algumas das estirpes de campo:

na região intergénica M014L/M015L e no gene M009L, onde levou a um frameshift. Estas

inserções foram localizadas após regiões homopoliméricas. A estirpe vacinal exibiu 37

substituições nucleotídicas localizadas predominantemente (95%) nos genes M022L e

M036L. As regiões M009L e M014L/M015L da vacina não foram amplificadas com

sucesso, sugerindo alterações genómicas maiores, que poderiam explicar o seu fenótipo

atenuado. Os nossos resultados demonstraram um elevado grau de estabilidade genética

de mixoma vírus (virulento) nas últimas cinco décadas. No âmbito do objectivo

supracitado, também analisámos o genoma de RHDVs obtidos entre 1994 e 2007 em

Portugal (40 amostras), Espanha (3 amostras) e França (4 amostras) de coelhos

selvagens que sucumbiram à doença. As análises filogenéticas baseadas em sequências

parciais do gene que codifica a VP60 (maior proteína estrutural do virus) permitiram um

agrupamento destes RHDVs em três grupos, denominados "Grupos Ibéricos".

Curiosamente, estas agruparam separadamente, embora não muito longe de RHDVs

mais antigas do genogrupo 1 (contendo, por exemplo, "AST89"), mas claramente

separadas de outras estirpes globais de RHDV. Este resultado deu origem à hipótese de

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que o vírus evoluiu independentemente desde a sua introdução nas populações de

coelho-bravo na Península Ibérica, com os Pirenéus agindo como uma barreira natural ao

movimento de coelhos e, portanto, à dispersão do vírus. Não foram observadas

diferenças entre RHDV obtidas a partir de regiões geográficas onde a subespécie coelho

Oryctolagus cuniculus algirus prevalece comparadas com as obtidas a partir de

Oryctolagus cuniculus cuniculus. Os resultados deste trabalho foram recentemente

citados por publicação internacional, na qual foi revista a origem e a filodinâmica de

RHDV. A hipótese frequentemente citada sobre a coevolução vírus-hospedeiro de ambas

as doenças, mixomatose e RHD, foi revista à luz dos conhecimentos actuais. Para

ambas, parece ser necessário adquirir evidência adicional, que continue a apoiar esta

hipótese. Finalmente, no âmbito dos trabalhos desta tese, foram desenvolvidos testes de

PCR em tempo real para a detecção do RHDV e do vírus da syndrome da lebre parda

(EBHSV) e apresentados os resultados preliminares do seu desempenho. Ambos os

testes parecem identificar correctamente as amostras negativas, sugerindo uma alta

especificidade. No entanto, algumas amostras positivas não foram correctamente

identificadas, requerendo investigações adicionais e a optimização dos testes. Os

trabalhos apresentados nesta tese foram desenvolvidos no âmbito do projecto

“Investigação dos mecanismos que estão na base da resistência genética do coelho à

mixomatose e à doença hemorrágica viral”, financiado pela Fundação para a Ciência e

Tecnologia (FCT; POCTI/BIA-BDE/61553/2004) e do trabalho realizado no contexto de

uma bolsa de doutoramento (SFRH/BD/31048/2006) e assim como da Unidade

Multidisciplinar de Investigação Biomédica (UMIB).

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SUMMARY

Myxomatosis and rabbit haemorrhagic disease (RHD) are highly infectious diseases that

emerged in wild European rabbit populations (Oryctolagus cuniculus) in the 1950s and

1980s, respectively. In the first years after their appearance, high mortality rates were

observed, but in subsequent years, the impact of these infections seemed to have

decreased. The hypothesis had been postulated that these viruses were co-evolving with

their hosts, leading to the selection of less virulent strains and more resistant hosts. It is

within this context, that the present studies were designed, aiming to contribute to the

current knowledge on virus-host adaptation and co-evolution of myxoma virus (MV) and

rabbit haemorrhagic disease virus (RHDV) in their natural host, by analysing the (partial)

genetic variability of field viruses. A total of 4863bp (approximately 3% of the genome)

spanning 12 genes of nine recent virulent myxoma field strains and a live attenuated

vaccine strain (“MAV”, Germany) were sequenced and compared to the originally

introduced virulent strain “Lausanne” and its attenuated field derivative strain “6918”. Our

field strains displayed a maximum of three (strains C43, C95) and a minimum of one

(strains CD01, CD05) nucleotide substitutions when compared to “Lausanne”. These were

distributed through all analysed coding regions, except gene M022L (major envelope

protein), where all strains were identical to “Lausanne” and “6918”. Two new single

nucleotide insertions were observed in some of the field strains: within the intergenic

region M014L/M015L and within gene M009L, where it lead to a frameshift. These

insertions were located after homopolymeric regions. The vaccine strain displayed 37

nucleotide substitutions, predominantly (95%) located in genes M022L and M036L.

Regions M009L and M014L/M015L of the vaccine were not amplified successfully,

suggesting major genomic changes that could account for its attenuated phenotype. Our

results support a high degree of genetic stability of (virulent) myxoma virus over the past

five decades. Within the above mentioned objective, we also analysed the genome of

RHDVs obtained between 1994 and 2007 in Portugal (40 samples), Spain (3 samples)

and France (4 samples) from wild rabbits that succumbed to the disease. Phylogenetic

analyses based on the partial gene sequences codifying the major structural protein VP60

allowed a grouping of these RHDVs into three groups, termed “Iberian Groups”.

Interestingly, these clustered separately, though not far from earlier RHDVs of Genogroup

1 (containing e.g. strain “AST89”), but clearly distinct from globally described RHDV

strains. This result gave rise to the hypothesis that the virus evolved independently since

its introduction to wild rabbit populations on the Iberian Peninsula, with the Pyrenees

acting as a natural barrier to rabbit and hence to virus dispersal. No differences were

observed in RHDV sequences obtained from geographic regions where the rabbit

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subspecies Oryctolagus cuniculus algirus prevails compared with those obtained from

Oryctolagus cuniculus cuniculus. The results of this work was recently cited by

international publication, in which the origin and phylodynamics of RHDV analised. The

frequently cited hypothesis on virus-host coevolution for both diseases, myxomatosis and

RHD, was re-assessed in the light of current knowledge. For both, further evidence seems

necessary further support this hypothesis. Finally, within work carried out for this thesis,

Real-time PCR assays were developed for the detection of RHDV and European brown

hare syndrome virus (EBHSV), and the preliminary findings on the assays performance

are presented. Both assays seem to correctly identify negative samples, suggesting high

specificity. However, some positive samples were not correctly identified, warranting

further investigations and optimization of these assays. The studies presented in this

thesis were developed within the project "Investigation of the mechanisms that underlie

the genetic resistance to myxomatosis and rabbit hemorrhagic disease virus", funded by

the Foundation for Science and Technology (FCT; POCTI/BIA-BDE/61553/2004),

supported by a doctoral grant (SFRH/BD/31048/2006) and the Multidisciplinary Unit for

Biomedical Research (UMIB).

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RESUMÉ

La myxomatose et la maladie hémorragique virale du lapin (RHD) sont des maladies

hautement infectieuses qui ont émergé dans les populations sauvages de lapin Européen

(Oryctolagus cuniculus) dans les années 1950 et 1980, respectivement. Dans les

premières années après son apparition, des taux de mortalité élevés ont été observés,

mais dans les années suivantes, l'incidence de ces infections semble avoir diminué.

L'hypothèse a été postulée que ces virus ont co-évolué avec son hôte, résultant en la

sélection de souches virales moins virulentes et d’hôtes plus résistants. C'est dans ce

contexte que les études actuelles ont été conçues en vue de contribuer aux

connaissances actuelles sur l'adaptation du virus-hôte et sur la co-évolution du virus de la

myxomatose (MV) et du virus de la maladie hémorragique (RHDV) chez son hôte naturel,

par l´analyse de la variabilité génétique (partielle) du virus. Pour ça, un total de 4863bp

(environ 3% du génome) englobant 12 gènes de neuf dernières souches virulentes de MV

et une souche de vaccin vivant atténué ("MAV", Allemagne) ont été séquencés et

comparés à la souche virulente "Lausanne" introduite à l'origine et son dérivé atténué du

champ "6918". Nos souches de terrain ont montré un maximum de trois (souches C43 et

C95) et un minimum de une (souche CD01et CD05) substitutions nucléotidiques par

rapport à "Lausanne". Elles ont été distribuées dans toutes les régions de codage

analysées, sauf dans le gène M022L (protéine majeure d'enveloppe), où toutes les

souches étaient identiques à "Lausanne" et "6918." Deux nouvelles insertions de

nucléotides simples ont été observées dans certaines des souches de terrain: au sein de

la région intergénique M014L/M015L et à l'intérieur du gène M009L, où elle conduit à un

décalage. Ces insertions sont situées après les régions homopolymériques. La souche

vaccinale affiche 37 substitutions nucléotidiques, situées principalement (95%) dans les

gènes M022L et M036L. Fait intéressant, les régions M009L et M014L/M015L du vaccin

n'ont pas été amplifiées avec succès, ce qui suggère des modifications majeures de la

génomique qui pourraient expliquer son phénotype atténué. Nos résultats démontrent un

degré élevé de stabilité génétique du virus de la myxomatose (virulent) au cours des cinq

dernières décennies. Dans le objective supra-citeé, nous avons aussi analysé le génome

de RHDVs obtenus, entre 1994 et 2007, au Portugal (40 échantillons), en Espagne (3

échantillons) et en France (4 échantillons), de lapins sauvages qui y avaient succombé de

la maladie. Les analyses phylogénétiques basées sur des séquences partielles du gène

que codifique la proteine estructurale VP60 a permis un regroupement de ces RHDVs en

trois groupes, appelés " Groupes Ibériques". Fait intéressant, ces derniers ont été

groupés séparément, bien que pas très loin de RHDVs du génogroupe 1 (contenant, par

exemple, "AST89), mais nettement séparés des autres souches de RHDV globale. Ce

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résultat conduit à l'hypothèse que les virus ont évolué indépendamment depuis leur

introduction dans les populations de lapins sauvages sur la péninsule ibérique, avec les

Pyrénées agissant comme une barrière naturelle à la circulation des lapins et donc á la

propagation du virus. Aucune différence n'a été observée entre le RHDV obtenu à partir

de régions géographiques où la sous-espèce de lapin Oryctolagus cuniculus algirus

prévaut par rapport à ceux obtenus à partir de Oryctolagus cuniculus cuniculus. Les

résultats de notre travail ont été recement cités par publication internacionale sur l'origine

et la phylodymamique de RHDV. L'hypothèse fréquemment citée sur la coévolution virus-

hôte à la fois pour la myxomatose et RHD a été réévaluée à la lumière des connaissances

actuelles. Nous avons constaté qu´il est necessaire de continuer à acquérir des éléments

de preuve pour maintenir et continuer à soutenir cette hypothèse dans le cas des deux

maladies infectieuses. Finalement, dans les travails de cette thèse, ont été développés

tests de PCR en temps réel pour la détection de RHDV et du virus du syndrome du lièvre

brun européen (EBHSV) et les résultats préliminaires de la performance des tests sont

présentés. Les deux tests semblent identifier correctement les échantillons négatifs, ce

qui suggère une spécificité élevée. D'autre part, certains échantillons positifs n'ont pas été

correctement identifiés, justifiant de nouvelles investigations et l'optimisation de ces tests.

Le travail a été élaboré dans le cadre du projet "Étude des mécanismes qui sous-tendent

la résistance génétique à la myxomatose et la maladie hémorragique virale du lapin",

financé par la Fondation pour la Science et la Technologie (FCT; POCTI/BIA-

BDE/61553/2004) et financé par FCT grâce à une subvention de doctorat

(SFRH/BD/31048/2006) et grâce au travail de l'Unité Multidisciplinaire pour la Recherche

Biomédicale (UMIB).

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CONTENTS

1. Introduction ...................................................................................................................... 21

1.1 The European wild rabbit (Oryctolagus cuniculus) ............................................................. 24

1.2 Virus-host interactions ............................................................................................................. 26

1.3 Aim and objectives ................................................................................................................... 28

2. Myxomatosis .................................................................................................................... 29

2.1 Literature review ....................................................................................................................... 31

2.1.1 History of the introduction ................................................................................................ 33

2.1.2 Clinical signs...................................................................................................................... 35

2.1.3 Aetiology and virus evolution .......................................................................................... 39

2.1.4 Pathogenesis and immunology ...................................................................................... 41

2.1.5 Immunomodulation ........................................................................................................... 43

2.1.6 Laboratory Diagnosis ....................................................................................................... 44

2.1.7 Epidemiology and control ................................................................................................ 45

2.1.8 Other areas of myxoma virus research ......................................................................... 48

2.2 Partial sequencing of recent Portuguese myxoma virus field isolates exhibits a high degree of genetic stability. ............................................................................................................ 49

3. Rabbit haemorrhagic disease (RHD)................................................................................ 63

3.1 Literature review ..................................................................................................................... 65

3.1.1 Introduction and brief history .......................................................................................... 67

3.1.2 Aetiology ............................................................................................................................ 68

3.1.3 Epidemiology ..................................................................................................................... 74

3.1.4 Clinico-pathological features ........................................................................................... 77

3.1.5 Laboratory diagnosis ........................................................................................................ 80

3.1.6 Control ................................................................................................................................ 82

3.2 Evolution of Rabbit haemorrhagic disease virus (RHDV) in wild rabbits (Oryctolagus cuniculus) in the Iberian Peninsula. ............................................................................................. 85

3.3 Evolution of RHDV in the Iberian Peninsula: A brief review of recent findings. ............. 97

3.4 Real-time PCR for the detection of rabbit haemorrhagic disease virus (RHDV) - Preliminary results ........................................................................................................................ 107

3.5 Real-time PCR for the detection of European brown hare syndrome virus (EBHSV) - Preliminary results ........................................................................................................................ 115

4. Discussion ..................................................................................................................... 125

4.1 Virus-host adaptation and co-evolution of myxoma virus in the European rabbit ........ 128

4.2 Virus-host adaptation and co-evolution of RHDV in the European rabbit ..................... 132

4.3 Development of real-time PCR assays for RHDV and EBHSV ...................................... 134

5. Conclusions and perspectives ....................................................................................... 137

6. References .................................................................................................................... 141

7. Appendices .................................................................................................................... 159

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TABLES Table 1 Virulence grading of myxoma virus according to Fenner and Marshall (1957) .... 36

Table 2 Selected genes and primers used for the genetic characterisation of myxoma virus field strains. The nucleotide positions refer to myxoma virus strain “Lausanne” (GenBank accession no. AF170726) ......................................................................................... 54

Table 3 Observed nucleotide polymorphisms and deduced amino acid variations in recent myxoma virus field strains. The nucleotide and amino acid positions refer to myxoma virus strain “Lausanne” (GenBank accession no. AF170726) ......................................................... 56

Table 4 Genbank accession numbers of RHDV sequences included in the phylogenetic analysis ........................................................................................................................................... 90

Table 5 Cycle threshold (Ct) values obtained by the application of two different primer-probe pairs in a real-time PCR assay of positive samples as determined by conventional nested RT-PCR (Moss et al., 2002) ......................................................................................... 112

Table 6 Comparison of diagnostic tests for the detection of European brown hare syndrome virus (EBHSV) ........................................................................................................... 121

Table 7 Comparison of simple and nested PCR for the detection of European brown hare syndrome virus (EBHSV) in 10-fold dilutions of samples 684/04 and 685/04 ................... 122

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FIGURES Figure 1 Monopartite, linear, single-stranded, positive-sense RNA genome of 7.3 to 8.3 kb. At 5’-terminus a virus protein (VPg)is covalently linked to genome, whereas 3’-terminus is polyadenylated (Source: ViralZone www.expasy.ch/viralzone, Swiss Institute of Bioinformatics). .......................................................................................................................... 69

Figure 2 Map of the Iberian Peninsula and South of France displaying the geographic origin of the RHDV samples analysed in this study and the time period they were collected. The distribution areas of the wild rabbit subspecies Oryctolagus cuniculus algirus and Oryctolagus cuniculus cuniculus as well as the contact zone across the Iberian Peninsula are indicated. ............................................................................................................... 88

Figure 3 RHDV strains from Portugal cluster separately from known genogroups based on phylogenetic analysis of partial VP60 gene sequences. The neighbour joining tree was rooted with RCV. Bootstrap probability values above 75% for 1000 replicate runs are indicated at the nodes. .................................................................................................................. 92

Figure 4 Alignment of EBHSV partial capsid gene sequences and primer-probe pairs selected for real-time PCR. The shown nucleotide positions correspond to positions 1332-1421 of the VP60 capsid gene and to positions 6563-6652 of the complete EBHSV genome (examples strain “GD”, Genbank accession numbers Z32526 and Z69629, respectively) ................................................................................................................................. 120

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1. Introduction

23

“Novel infectious diseases can emerge either by a species-jump into a new host or by mutation of an existing

microorganism to a more virulent form. An example of each type of emerging disease has occurred in the

European rabbit (Oryctolagus cuniculus): myxomatosis, where the poxvirus myxoma virus jumped to O. cuniculus

from the tapeti (a lagomorph, Sylvilagus brasiliensis), in which it caused an innocuous cutaneous fibroma, and

rabbit haemorrhagic disease (RHD) where a pre-existing avirulent virus of European rabbits appears to have

mutated to the lethal rabbit haemorrhagic disease virus (RHDV) that has spread around the world since 1984.” In

(Kerr et al., 2009).

Myxomatosis and rabbit haemorrhagic disease (RHD) are highly infectious diseases

which have emerged in wild European rabbit populations (Oryctolagus cuniculus) within

the past six or seven decades. In the initial months and years after their appearance in

wild and also in domestic rabbits, high mortality rates were observed. In subsequent

years, the impact of these infections seemed to decrease, and the hypothesis was

postulated that these viruses were co-evolving with their hosts, leading to adaptation by

the selection of less virulent strains and more resistant hosts (Anderson and May, 1982;

Fenner and Ross, 1994; Kerr and Best, 1998; Villafuerte et al., 1995). Much research has

been carried out on this subject. In this thesis it will briefly be reviewed in the respective

chapters on each disease. Some evidence has been gathered that this may be true for

myxomatosis and to some extent for RHD, but knowledge on the genetic mechanisms

related to host and virus is still scarce, especially for “real-life” scenario, i.e. wild rabbit

populations (Best et al., 2000; Best and Kerr, 2000; Fouchet et al., 2009). This may be

related, in part, to the difficulty in obtaining samples and controlling population

parameters. Outbreaks in nature are typically suspected by the sudden disappearance of

wild rabbits. They commonly die in their warrens. Only in areas of high rabbit density,

rabbits may be found dead and eventually be sampled.

Similar to other European countries, myxomatosis and RHD have been introduced into the

Iberian Peninsula in the 1950s and early 1990s, respectively (Anonymous, 1989;

Monteiro, 1999; Muñoz, 1960; Villafuerte et al., 1995). Within a few years of their

introduction in wild rabbit populations, both diseases caused a severe decline in rabbit

abundance in the Iberian Peninsula to the extent that in Portugal the wild rabbit is

currently considered a “vulnerable” species, i.e. of high risk of being extinguished (ICNB,

2005) and even as “near threatened” by the World Conservation Union in 2008 (Smith and

Boyer, 2008). RHD is now considered endemic in Spain and Portugal, and despite many

efforts, rabbit numbers have not fully recovered (Delibes-Mateos et al., 2008b, 2009; Dias-

Pereira et al., 2004; Moreno et al., 2007; Muller et al., 2004; Santos et al., 2006;

Villafuerte et al., 1995; Ward, 2005). The impacts of decreasing wild rabbit populations in

Spain and Portugal are mainly twofold. On one hand, wild rabbit populations are

considered a keystone species in the Iberian Mediterranean ecosystems as they

represent the major food source of currently endangered specialist predators, such as the

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Iberian lynx (Lynx paradina) and the Spanish imperial eagle (Aquila adalberti) (Delibes-

Mateos et al., 2008a; Moreno et al., 2004) and on the other hand, the decline of wild rabbit

numbers has a severe negative economic impact on the hunting industry (Angulo and

Villafuerte, 2004).

It is within this context, that a project denominated RIPAC, was developed in 2002-2004

by the Algarve´s Hunters Federation and the Regional Agricultural Directorate (RIPAC,

2004). The objectives were to determine the sanitary status and the main causes of death

of small game animals, especially the wild rabbit, in the Algarve Province, Portugal. A total

of 200 specimens were analysed by different elements of Porto University, including the

Laboratory of Infectious Diseases, ICBAS. The presence of myxoma virus and RHDV was

demonstrated in the wild rabbit subspecies Oryctolagus cuniculus algirus (Dias-Pereira et

al., 2004; Muller, 2004; Muller et al., 2004; RIPAC, 2004). However the genetic

characterisation of these and other virus strains from Portugal were subject of the present

thesis. The overall aim of the present study is to contribute to the current knowledge on

virus-host adaptation and co-evolution of MV and RHDV to their natural host, by studying

the heterogeneity of selected viral genes obtained from samples taken from wild rabbits at

different geographical locations in Portugal. Our studies formed part of a larger project

(POCTII/BIA-BDE/61553/2004), whose goal it was to study the role of natural selection on

the hypothetical increased genetic resistance of wild rabbit populations to myxomatosis

and RHD.

1.1 The European wild rabbit ( Oryctolagus cuniculus )

The European rabbit (Oryctolagus cuniculus) is a mammal that, together with the hare,

belongs to the family Leporidae of the Order Lagomorpha. The first fossil records of

lagomorphs have been attribited to the Early Paleogene, around 45 Ma (Lopez-Martinez,

2008). The first fossils of the Oryctolagus genus were dated to the Middle Pleiocene,

about 3.5 Ma, from Spain and probably southern France, and those attributed to modern

European rabbit species were dated to the Mid Pleistocene i.e. around 0.5 Ma (Lopez-

Martinez, 2008). Based on analyses of fossil records, the Iberian Peninsula is considered

the probable ancestral area of the European rabbit (Lopez-Martinez, 2008). The

evolutionary history has also extensively been studied using different molecular markers

such as mitochondrial DNA (Biju-Duval et al., 1991; Branco et al., 2000; Branco et al.,

2002), protein polymorphism and genetic diversity on the X and Y chromossomes

(Geraldes and Ferrand, 2006; Geraldes et al., 2006; Geraldes et al., 2005). Despite some

25

incongruences between these different techniques, results agree that two groups of the

European rabbit have been evolving in allopatry (i.e. in entirely separate ranges) during

the Pleistocene (Ferrand, 2008). These correspond to the subspecies Oryctolagus

cuniculus cuniculus and Oryctolagus cuniculus algirus (Ferrand, 2008). Geographically,

Oryctolagus cuniculus algirus are located in the southwest and Oryctolagus cuniculus

cuniculus in the northeast of the Iberian Peninsula. Both populations contact forming a

line of hybridization in the central region of the Iberian Peninsula (Branco et al., 2000;

Branco et al., 2002; Ferrand, 2008). From the Iberian Peninsula, and probably during the

Middle Ages, O .c. cuniculus spread or was taken by humans to many other parts of

continental Europe and domesticated, giving origin to different rabbit breeds (Ferrand and

Branco, 2007). The geographical distribution of Oryctolagus cuniculus algirus, however,

remains confined to the southwest of Spain and Portugal. Rabbits of either subspecies are

not readily distinguished phenotypically, whereby molecular testing techniques currently

also play an role as important conservation management tool (Esteves et al., 2006).

The European rabbit is small grey-brown mammal. It differs from the hare by smaller ears,

a shorter tail and the fact that newborns are blind and furless nestlings, fully dependent on

the doe. The body weight of adults ranges between 800 and 1300g (Paupério et al.,

2006). The habitat consists of a mixture of pasture and scrublands as important sources

of feed and shelter (Delibes-Mateos et al., 2008b). Also important is the consistence and

structure of the soil, as rabbits are burrowing animals, and burrows are an essential

element for social structure and reproduction (Delibes-Mateos et al., 2008b; Paupério et

al., 2006). Social structure is complex and groups are generally formed by a dominant

male and various female, juvenile and subordinate male animals. Social structure is

strongly influenced by the habitat. Abundant feed and shelter leads to less evident

hierarchy and higher reproductive indices as well as higher survival rates of juveniles. On

the contrary, fragmentated or less suitable habitats lead to higher competition between

individuals, more stringent social structures and in some cases to a discontinuous

distribution of rabbits. Rabbits living in smaller colonies that are isolated from other

colonies are also considered much more vulnerable to local extinction (Paupério et al.,

2006). Each group occupies a territory of generally less than 1 hectar and rabbits normally

graze within 500m of their burrows (Paupério et al., 2006). Only juveniles may disperse at

longer distances. The reproductive cycle of rabbits is strongly influenced by its habitat. In

mediterranean ecosystems, reproductive activities coincides with the availability of feed,

i.e. Autumn, Winter and Spring (Goncalves et al., 2002; Paupério et al., 2006). The mean

litter size is 4 kits per female, and each female may have 3 to 4 litters per year

(Goncalves et al., 2002; Paupério et al., 2006). Juveniles leave their warren at three

26

weeks of age and reach sexual maturity by 4 to 5 months. The population dynamics of

wild rabbits are influenced by various factors and display seasonal fluctuations. Mortality

rates are higher in juveniles that in adults. It has been estimated that up to 80% of rabbits

die before reaching adulthood. Rabbit densities also vary accordingly. Higher densities

are observed in Spring and early Summer, reflecting births. In Autumn, rabbit numbers

decline, related to scarcity in feed, but also other factors as hunting pressure and

infectious diseases such as myxomatosis and RHD (Paupério et al., 2006).

1.2 Virus-host interactions

Here, a more general approach is taken to elucidate virus-host interactions and to define

related terms. More specific findings related to myxomatosis and RHD will also be

reviewed in the respective disease chapters below. Viruses are small infectious agents

that require living cells for replications. An infection results if a virus is able to invade and

to replicate within a host. The outcome of infection frequently may vary, and thus not

always results in clinical disease. Disease may result when invasion and replication of the

agent and/or the host’s immune responses result in tissue damage and impair

physiological functioning. The mechanisms involved vary among different host-pathogen

scenarios (Mims et al., 1995). The term virulence is generally used to describe the ability

of any agent to cause damage and disease and may be measured, for example, by case

fatality rates or clinical scoring systems (Mims et al., 1995). Several virulence factors have

been described for infectious agents, such as, e.g. Mt-7 protein for myxomatosis

(Mossman et al., 1996). Host resistance is a term frequently used in the context of

infectious disease, particularly in the context of RHD and myxomatosis. As for other

infections, the following two situations need to be differentiated (Mims et al., 1995). On

one hand, host resistance can be defined as resistance to infection, meaning that, despite

exposure of a host to a particular virus, the virus is not able to infect the host. Typically,

these infection-resistant animals remain seronegative despite exposure. On the other

hand, it could be meaning resistance to disease, i.e. infection of the host does take place,

but does not result in clinical disease. The disease-resistant host remains healthy but

seroconversion occurs upon exposure. If specific antibodies are protective, this host may

then be considered resistant to re-infection. The term host resistance reflects the opposite

of host susceptibility. In analogy, susceptibility may mean susceptibility to either infection

or disease.

27

The factors determining host resistance to infection and/or clinical outcome are complex.

For example, the presence of specific viral receptors on host cells is considered essential

for viral attachment and entry, and thus a prerequisite for infection. In other words,

resistant individuals of a susceptible species may display altered receptor configuration

that do not allow virus attachment and thus infection does not occur. On the other hand,

the innate immune system may play a role in preventing infection. The innate immune

system includes anatomical barriers, secretory molecules as well as cellular components.

As it is non-specific for a particular agent, it is also frequently termed innate resistance. In

cases where successful infection occurs of a susceptible host, clinical outcome may vary

considerably. In many diseases, a proportion of susceptible hosts may remain healthy

(asymptomatic infection), whereas others may display mild or severe signs, and in a

proportion outcome may be fatal. Different factors may have been associated to outcome

of infection, such as body condition, concurrent disease, immunosupression, age, breed

etc. Most of these factors do affect the immune system and as such the ability to control

infection and modulate the development of disease. Both, innate and acquired immunity,

are genetically programmed, as is the expression of putative viral host cell receptors.

There is growing interest in genetically characterizing resistance to infection and disease.

Different approaches are being taken such as the genetic characterisation of individual

candidate genes up to the analysis of complete host genomes in an attempt to identify

genes related with susceptibility to infectious disease (Boon et al., 2009; Brotherstone et

al., 2010; Schnappinger and Ehrt, 2006; Tuite and Gros, 2006; Vidal et al., 2008).

Co-evolution of host and parasite (including viruses) does occur, when these complex

interactions take place over time, resulting in selective pressures over each. Generally

speaking, hosts may be under selection pressure to escape parasitism, whereas parasites

may be under selection pressure to evade host defences (Anderson and May, 1982).

Depending on each host-parasite scenario, the outcomes of host-parasite co-evolution

may be different. Some pathogens may evolve to be harmless to their hosts. Others, such

as myxoma virus, whose selection depends on transmissibility, may be expected to evolve

to intermediate or even higher values of virulence alongside increasing proportions of

resistant rabbits (Anderson and May, 1982; Ross and Sanders, 1977, 1984). In most

cases, infectious diseases may be important drivers in the survival and adaptation of

animal populations, and in the particular context of wildlife, they may also have a

considerable impact on population size and host genetic diversity (Altizer et al., 2003;

Daszak et al., 2000; O'Brien and Evermann, 1988; Smith et al., 2009).

28

The impact and the effects of myxomatosis and RHD in European wild rabbit populations

have been subject to many investigations (Boots et al., 2004; Delibes-Mateos et al.,

2008b; Forrester et al., 2003; Fouchet et al., 2009; Queney et al., 2000). There is field

evidence of viral attenuation as well as increasing genetic resistance in some rabbits, but

underlying genetic features are far from being fully understood. Some studies aiming to

identify host factors related to disease resistance have been published. These found

unique changes in the chemokine receptors CXCR4 and CCR5 of Oryctolagus compared

to other lagomorph members, suggesting that these may be major candidates related to

resistance to myxomatosis (Abrantes et al., 2010; Abrantes et al., 2008a; Carmo et al.,

2006). On the other hand, resistance to RHD has been linked to the presence of ABH

blood group antigens and the presence of non-functional alleles of fucosyltransferase

genes such as Fut2, determining a so-called “nonsecretor phenotype” possibly resistant to

RHD (Guillon et al., 2009; Ruvoen-Clouet et al., 2000). For both diseases, these

candidate host resistance factors require further studies using infectious virus under

controlled conditions.

1.3 Aim and objectives

The overall aim of the present study is to contribute to the current knowledge on virus-host

adaptation and co-evolution of MV and RHDV to their natural host, the wild rabbits.

Specific objectives were:

1- To genetically characterize viral strains of myxoma virus and rabbit haemorrhagic

disease virus obtained from wild rabbits from different geographical locations of Portugal.

2- To assess genetic variability of selected viral genes and to compare our findings with

those obtained for other European and international strains.

3- To correlate our findings with those obtained of the genetic variation of host cell

receptors in order to approach the question related to viral and/or host adaptation and co-

evolution.

4- To develop real-time PCR assays for the detection of RHDV and EBHSV.

This manuscript is outlined into the following sections. The next two chapters are

dedicated to each disease: myxomatosis and RHD. Within each, a general literature

review is presented, followed by the original research carried out. These are followed by a

general discussion, in which the role of recent findings for virus-host co-evolution of both

diseases will be addressed. Finally, conclusions and perspectives for future research are

presented.

29

2. Myxomatosis

31

2.1 Literature review

33

Before the introduction of MV into the European wild rabbit in Europe and in Australia,

interest in myxomatosis was mainly limited to some members of the scientific community

(E.g. Hobbs, 1928; Hurst, 1937). But ever since the outbreaks of myxomatosis among

European wild rabbits (Oryctolagus cuniculus) in Australia in 1950 and in Europe in 1952,

interest exploded, not only of the larger scientific community but also of the general public.

There are two major issues related with myxomatosis. First, it is the only example of the

use of an infectious agent as a biological control weapon to eradicate a vertebral animal

species considered a “pest”, especially in Australia where rabbits compete with

autochtonous flora and fauna and also cause major agricultural losses. And second, this

infection with a very lethal virus in a large population of highly susceptible mammals

provided opportunities to observe the course of virus-host interaction, i.e. provided a

model system to study the evolution of an infectious disease agent, and the effects of this

infectious disease on the evolution of a mammal (Anderson and May, 1982; Kerr and

Best, 1998).

Numerous studies were published in scientific journals (E.g. Fenner and Chapple, 1965;

Fenner and Marshall, 1957; Fenner et al., 1953; Ross and Sanders, 1977, 1984, 1987),

book chapters on Myxomatosis (E.g. Fenner, 1994) and even whole books on

Myxomatosis were written (E.g. Fenner and Ratcliffe, 1965). In view of these excellent

scientific publications and especially reviews, which are impossible to surpass, we here

aim to succinctly review key aspects of the disease and to complete these with recent

findings.

2.1.1 History of the introduction

Myxomatosis was recognized as a new disease in European rabbits in 1896 in Uruguay

(Sanarelli, 1898). Subsequently it caused sporadic lethal infections in domestic and

laboratory rabbits in Brasil and its etiological agent was shown to be a poxvirus in 1927 by

Dr. H. B. Aragão at the Oswaldo Cruz Institute in Brasil. In 1918, Dr. Aragão suggested

the use of myxomatosis as a means to control wild rabbit populations in Australia. The

strain that was eventually introduced in Australia was termed “Standard Laboratory Strain”

(SLS) or “Moses strain”, as it was recovered from a naturally infected laboratory rabbit in

Rio de Janeiro (Moses, 1911 cit. by Fenner and Ross, 1994). It had been maintained by

passage in laboratory rabbits for nearly 40 years before its use in field trials in the Murray

Valley, Australia, in 1950, which eventually lead to the spread of the infection over this

continent (Ratcliffe et al., 1952). The mortality in rabbit populations was enormous,

exceeding 99% case fatality-rate. Ever since and up to now, myxomatosis is endemic in

34

Australia, and epizootics occur periodically in association with local and seasonal vector

activity.

In 1952, Myxomatosis was introduced in France by Dr. P. F. Armand Delille, a

paediatrician who was concerned with the excessive numbers of wild rabbits at his private

estate at Maillebois. He obtained a strain of myxoma virus from a friend at the Laboratoire

de Bacteriologie, Lausanne, Switzerland. This introduced virus was termed “Lausanne

strain”, although it originated in Campinas, Brasil, in 1949 (Bouvier 1954, cit. by Fenner

and Ross, 1994). Dr Delille released two inoculated rabbits on June 14th on his land. By

the end of August 1952, virtually all rabbits on his estate were dead and further outbreaks

of myxomatosis were occurring in surrounding villages. By 1954 about 90% of wild rabbits

had been killed, and subsequently control measures, such as immunisations, were

implemented in an attempt to limit the spread of myxomatosis (Fenner and Ross, 1994).

In the following years, rabbit numbers recovered, with slight geographic variations,

probably also due to environmental factors, agricultural habits and hunting pressures

(Arthur et al., 1988 cit. by Fenner and Ross, 1994). The disease rapidly spread to other

European countries. Myxomatosis was deliberately introduced in 1953 in Kent by a

resident who had brought an infected rabbit from France (History reviewed by Bartrip,

2008). By 1955 the disease had spread over most of Britain, killing an estimated 99% of

rabbits (Hudson et al., 1955 and Brown et al., 1956 cit. by Fenner and Ross, 1994).

Despite large local fluctuations, rabbit numbers started to increase during the 1960s,

reaching 20% of the pre-myxomatosis population in 1979 (Lloyd, 1970 and Lloyd 1981 cit.

by Fenner and Ross, 1994) and about one third of the pre-myxomatosis population in the

1990s (Flowerdew et al., 1992). Case-mortality rates observed in the 1970s were between

47 and 69%, and as such much lower than during the 1950s and 1960s (Ross et al.,

1989). Still, myxomatosis is nowadays considered to be an important mortality factor,

contributing to the control of rabbit numbers, with autumn/winter peaks of disease

reducing the numbers of rabbits present at the start of the breeding season (Ross et al.,

1989). The first case of myxomatosis was reported in northern Spain in 1953 (Muñoz,

1960 cit. by Alda et al., 2009), probably appearing concomitantly in Portugal. Following

the initial outbreak, wild rabbit populations in the Iberian Peninsula were reduced by over

90% (Cabezas-Díaz et al., 2005). In Spain, rabbit population density appeared to increase

in the 1980s, but declined again due to the introduction of rabbit haemorrhagic disease

(Calvete et al., 1997; Villafuerte et al., 1995). As in other countries, myxomatosis is now

endemic in Spain and Portugal (Calvete et al., 2002a; Muller et al., 2004).

35

2.1.2 Clinical signs

The clinical signs of myxomatosis may vary considerably due to a variety of factors, such

as host species, virus-host adaptation and attenuation of viral virulence, vector-borne

transmission, ambient temperature, genetic resistant rabbits and immune status (Fenner

and Marshall, 1957).

In its natural hosts, the South American tapeti (Sylvilagus brasiliensis) and the North

American brush rabbit (Sylvilagus bachmani) MV only induces a benign cutaneous

fibroma at the site of inoculation, reflecting the long evolutionary association between the

virus and its host. On the contrary, in its evolutionary new host, the European rabbit

(Oryctolagus cuniculus), myxoma virus predominantly causes a highly lethal disease,

termed myxomatosis. To date, actually, two forms of disease are recognized in the

European rabbit: the more frequent systemic or nodular form (E.g. Silvers et al., 2006)

and the less frequent amyxomatous, atypical or respiratory form (Marlier et al., 1999;

Marlier et al., 2000b). Clinical signs of the nodular (classic) form include protuberant skin

lesions, blepharoconjuntivitis and oedematous swellings of the head and the genital

organs. The clinical signs and high mortality rates are believed to result from multiorgan

dysfunction coupled with uncontrolled secondary gram-negative bacterial infections due to

a progressive failure of the host’s cellular immune response. The clinical signs of the

amyxomatous or atypical myxomatosis are predominantly respiratory and mortality is not

a feature. Skin nodules may appear but usually are small and in reduced numbers. As this

milder clinical manifestation has mostly been reported in France and Belgium (Marlier et

al., 1999; Marlier et al., 2000b), a possible link between the use of the SG33 vaccine

strain and the occurrence of amyxomatous myxomatosis has been postulated (Brun et al.,

1981 cit. by Marlier et al., 1999). Atypical myxomatosis has been reported in the context of

vaccination the Czech Republik, but genetic analyses have shown differences between

the vaccine and the field strain (Psikal et al., 2003).

In between these two extreme clinical forms (classical and atypical), a whole plethora of

possible clinical outcomes has been described based on field observations and on

experimental inoculations (Fenner, 1994; Fenner and Marshall, 1957; Fenner and Ross,

1994; Kerr and Best, 1998). These have been linked to the process of virus-host

adaptation that occurred after the introduction of MV into European wild rabbits

populations. So within a few years of the release of MV in Australia and Europe, a

reduction in case-fatality rates and the occurrence of attenuated MV strains has been

recorded (Fenner and Chapple, 1965; Fenner and Marshall, 1957; Kerr and Best, 1998).

36

For example, in Australia, a highly attenuated field strain of myxoma virus, denominated

Uriarra-2-53/1 (Ur), was isolated in 1953, only 2 years after the release of SLS. It also

became apparent, that there could be a selection of genetically resistant rabbits in the

field, and as a result, studies were set up for monitoring the development of genetic

resistance and the virulence of MV field strains (Edmonds et al., 1975; Fenner and

Chapple, 1965; Fenner et al., 1953; Ross and Sanders, 1977, 1984, 1987; Sobey, 1969).

The numerous experimental inoculations carried out in this context were complex,

involving virus strains obtained at different geographical and temporal points and the

inoculation of so-called genetically unselected laboratory rabbits, and of so-called selected

rabbits, which were directly obtained from the field or bred from survivors in the field

(Reviewed in Fenner, 1994; Fenner and Ross, 1994; Kerr and Best, 1998). Based on the

observation of average survival time (AST) and mortality (%) of groups of inoculated

laboratory rabbits, field strains were grouped into five (I to V) virulence grades (Fenner

and Marshall, 1957), as shown in Table 1.

Virus (virulence) grade Average survival time (AST) Mortality (%) I < 13 days 100 II 13-16 days 95-99 III 17-28 days 70-95 IV 29-50 days 50-70 V Not relevant < 50

Table 1 Virulence grading of myxoma virus accordin g to Fenner and Marshall (1957)

This classification is considered the basis for detecting and monitoring the emergence of

attenuated viruses in the field. Some discrepancies were found in more recent studies.

For example, work in Australia has shown that viral virulence (lethality) did not always

correlate with mean survival times in rabbits taken from the different localities, suggesting

that the virulence of field strains may actually be higher than previously estimated (Parer,

1995; Parer et al., 1994). These results undermine previous studies that advocated an

increase in the proportion of attenuated virus strains in Australia, and as such questions

the concept of virus-host co-adaptation. Further interesting observations related to the

virulence grading of myxoma virus strains were made recently in a pathogenicity

assessment of 20 myxoma virus field strains obtained in the 1990s in Spain (Bárcena et

al., 2000). The horizontal transmission of these viruses to in-contact rabbits was as also

evaluated. This study found, that the average length of disease was significantly longer in

the group of rabbits infected by contact in comparison to those inoculated and suggested

that the stringent experimental condition could be responsible for the observed enhanced

37

severity of the disease in the subcutaneously inoculated animals. In this study, a high

inoculation dose of 104 plaque forming units was used instead of 5 rabbit-infectious doses

of the virus (rabbit ID50), and further, very young rabbits were used: 30 days-old instead of

at least 4 month-old. The authors concluded that the results of the contact-infected rabbits

would reflect more closely the real situation in the field than those of the inoculated

rabbits. Although mortality rates between both groups of animals were identical (except for

one strain), the AST of the contact-infected animals were higher than in the inoculated

animals. As such, most of the virus strains that were classed as virulence grade I and II

according to mean survival time in the inoculated animals, would actually be classified as

grade III viruses in the contact-infected animals. In another recent study involving the

virulence grading of two Californian MV, the observation was made for grade I and III

viruses, that survival of rabbits was not altered over a dose range of 5 to 105 rabbit ID50

although the AST was reduced by around 2 days at the highest dose (Silvers et al., 2006).

These recent experiments highlight the difficulty in standardizing experimental settings for

evaluating myxoma virus virulence.

The selection and emergence of attenuated virus strains has also been strongly linked to

the vector-borne mode of transmission of myxomatosis. Although the virus can also

spread via direct contact by the respiratory route, the most important mode of

transmission is by arthropod vectors. In Australia, mosquitoes such as Culex annulirostris

and Anopheles annulipes are considered important, whereas in Europe, fleas such as

Spilopsyllus cuniculi seem to be the principal vectors (Bull and Mules, 1944 and Lockley,

1954 cit. by Fenner and Ross, 1994). Both, mosquitoes and rabbit fleas act as mechanical

vectors. The virus adheres to their mouthparts, as they probe through infected skin.

Moderately attenuated viruses such as grade III or IV are more likely to be transmitted in

the field as they are present in the skin for longer periods of time. On the contrary, highly

virulent viruses are only present shortly before the rabbits death, and very attenuated

viruses, such as grade V strains, are only infectious during a very short period as virus

replication is rapidly controlled by the hosts immune response (Edmonds et al., 1975;

Fenner and Marshall, 1957; Fenner et al., 1956 cit by Kerr and Best, 1998).

Interestingly, ambient temperature also has a considerable effect on the severity of

disease. High temperatures favour milder clinical signs and cold climates favour severe

clinical manifestation, higher case-fatality rate and higher levels of viraemia (Marshall,

1959). This may be important in that, initially, rabbits in Australia may have survived

infection with moderately attenuated viruses during early stages of evolution of resistance,

and may have favoured the selection of resistant rabbits in hotter climates (Kerr and Best,

38

1998). The exact mechanisms underlying this phenomenon are not fully understood.

Further complicating is the fact that the assessment of virulence grades by survival rates

in colder indoor laboratory conditions may have led to an underestimation of host

resistance in the field. As such, viruses may favour higher recovery rates, i.e. have lower

mortality, in field sites with higher ambient temperatures than in colder laboratory

environment (Marshall and Douglas, 1961).

Last but not least, the severity of clinical signs induced by a given myxoma virus strain is

influenced by factors linked to the individual host, which in the literature has frequently

been termed “resistance” or “genetic resistance”. The emergence of resistant rabbits has

been studied in parallel with the monitoring of viral virulence in numerous field

observations and experimental inoculations (Fenner and Chapple, 1965; Marshall and

Douglas, 1961; Ross and Sanders, 1984; Williams et al., 1990). It may be exemplified by

a longitudinal study performed at Lake Urana in New South Wales, Australia (Kerr and

Best, 1998; Marshall and Douglas, 1961; Marshall and Fenner, 1958). Briefly, rabbit

kittens taken at different field sites were taken to a central laboratory and seronegative

animals were challenged at the age of 4 months or older with myxoma viruses of known

virulence. Interestingly, a decrease in mortality rates and in severity of clinical signs was

observed in rabbits trapped after 2 to 3 epidemics in comparison to those taken after

seven epidemics. It has been postulated that the interplay between virus and host would

eventually lead to the replacement of moderately virulent strains by more virulent strains

as the proportion of resistant rabbits increased, and this was postulated to have occurred

in the field (Anderson and May, 1982; Bárcena et al., 2000; Ross and Sanders, 1977).

Attention, though, must be paid to the limitation of the diagnostic test commonly used in

the 1950s and 1960s. Seronegativity was commonly assessed by the immunodiffusion

test. The use of this test in longitudinal studies revealed that specific antibodies against

the soluble antigen were inconsistently detected after infection, contrasting a persistent

humoral response as measured by neutralisation test or ELISA (Kerr, 1997; Williams et

al., 1973). In other words, the milder disease observed in challenge-inoculated “resistant”

rabbits was probably, at least partly, due to the presence of antibodies not detected by the

assay. The selection of resistant rabbits by mortality due to myxomatosis is expected to

cause a genetic population bottleneck, leading to a reduction in genetic variation, i.e. an

increase in genetic homogeneity of rabbits. A study on the genetic structure of European

wild rabbits has found high degree of genetic differentiation several sites in Great Britain

(Surridge et al., 1999). The authors conclude that the existence of such a myxomatosis

bottleneck is possible, and that the heterozygosity observed in present populations was

caused by the rapid population growth rate of rabbits. Importantly, the interpretation of this

39

study is difficult due to the inexistence of data from before the introduction of

myxomatosis.

2.1.3 Aetiology and virus evolution

Myxoma virus is a member of the genus Leporipoxvirus, subfamily Chordopoxvirinae,

family Poxviridae (ICTVdB, 2006). Other members of the Leporipoxvirus genus include

Shope fibroma virus (SFV), hare fibroma virus and squirrel fibroma virus. There are two

geographic types of myxoma virus, the South American or Brazilian myxoma virus that

circulates in the jungle rabbit or tapeti (Sylvilagus brasiliensis), and that is now endemic in

Europe and Australasia, and the so-called Californian myxoma virus (E.g. MSW and

MSD) that circulate in the brush rabbit (Sylvilagus bachmani) in the west coast of the

United States of America and the Baja peninsula of Mexico. The leporipoxvirus Shope

fibroma virus is genetically and antigenically closely related to myxoma virus (Cameron et

al., 1999; Willer et al., 1999). Its natural host is the eastern cottontail rabbit (Sylvilagus

floridanus) in North America. As this virus does not induce disseminated disease in the

European rabbit, it is widely used as immunizing agent (OIE, 2009a).

Like all poxviruses, MV has a classic brick shape replicates exclusively in the cytoplasma

of infected cells. Poxvirus particles consist of an envelope acquired by budding through

the host cell membrane, a surface membrane, a biconcave core that contains the genome

and two lateral bodies. During their life cycle, extracellular enveloped virions (EEV) and

intracellular mature virions (IMV) are produced, which contain different envelopes and are

infectious, but the infection is initiated by extracellular virions (ICTVdB, 2006). The

genome is not segmented and contains a single molecule of linear double-stranded DNA.

The central portion of approximately 120 kb of the genome encodes approximately 100

genes that are highly conserved genes among poxviruses and which encode mostly

structural and housekeeping proteins. On the terminally inverted repeats (TIR) and on the

near-terminal unique regions many immunomodulatory genes are located, which are

presumed to have evolved closely with the natural host. As they are involved in subverting

the hosts’ immune system, they may be related with inadequate host responses in the

new host, the European rabbit, eventually leading to disseminated fatal disease (Barrett et

al., 2001; Cameron et al., 1999; Kerr and McFadden, 2002; Stanford et al., 2007b;

Stanford et al., 2007c; Zuniga, 2003).

Genetic data on poxviral evolution and thus on myxoma virus evolution is scarce, mostly

due to the very large size of the viral genome. Currently the complete genome sequences

of only two strains, the virulent strain ‘‘Lausanne’’, introduced in 1952 in Europe and its

40

naturally attenuated field derivative ‘‘6918’’, obtained in 1995 in Spain are available

(Bárcena et al., 2000; Cameron et al., 1999; Morales et al., 2009). Partial sequence

information on other strains such as the Californian myxoma MSD and MSW (Jackson et

al., 1999; Labudovic et al., 2004) and two Greek isolates (Kritas et al., 2008) are also

available. Very recently, i.e. concomitantly with our work, partial sequence analysis of 97

field strains from 12 localities in Spain have shown an extremely low genetic variability of

myxoma virus (Alda et al., 2009). Altogether, current information is still scarce to allow

more accurate phylogenetic analyses and inferences about myxoma virus evolution in its

new host, the European rabbit. Additionally, horizontal gene transfer (HGT) occurs in

poxviruses including myxoma virus, potentially confounding phylogenetic inferences

based on one or few genes (Bratke and McLysaght, 2008; Gubser et al., 2004; Hughes

and Friedman, 2005; Kerr et al., 2010; Xing et al., 2006). Therefore whole-genome based

phylogenetic analyses may be considered more appropriate. Whole genome comparisons

between both myxoma virus strains as well as with shope fibroma virus have yielded

important findings. The complete genome sequencing of Lausanne has shown that it is

161773 nucleotides long and contains a total of 171 open reading frames (ORF) encoding

structural and non-structural proteins (Cameron et al., 1999). Twelve of the ORFs exist in

two copies, one at each end of the TIR of 11.5kb (Cameron et al., 1999). The genome

comparison of the virulent MV strain “Lausanne” and its attenuated field derivative “6918”

has identified a total of 73 differences consisting of 67 base substitutions, 4 deletions and

2 insertions (Morales et al., 2009). Importantly, four disrupted genes were identified as

putative determinants for the attenuation of 6918, by order of decreasing likelihood:

M135R, M148R, M009R and M036L. The comparison of virulent MV Lausanne and the

related apathogenic Shope fibroma virus has also shown significant differences. Eleven

genes are predominantly truncated or fragmented in Shope fibroma virus suggesting that

they may have possible roles in myxoma virus virulence (Cameron et al., 1999; Willer et

al., 1999). Variation at other loci is also present. The role of these genes for virulence is

difficult to assess (Cameron et al., 1999). Other techniques, based on the determination of

restriction fragment length polymorphisms (RFLPs) have been used for the

characterisation of myxoma virus field strains (Dalton et al., 2009; Kerr et al., 2010;

Russell and Robbins, 1989; Saint et al., 2001). This technique is sensible enough to

identify restriction patterns that could be linked to specific polymorphisms between strains.

Multiple genetic types of myxoma virus were found during epidemics, which apparently

were easily be replaced by others over time (Kerr et al., 2010). However, additional

studies are required to evaluate the suitability of mutations determined by this technique

for phylogenetic studies. No distinct correlation was found between RFLP typing and viral

virulence (Kerr et al., 2010). Antigenically, most strains seem to share epitopes. High

41

antigenic similarity between myxoma virus field strains have been shown by virus

neutralisation tests on the chorioallantoic membrane of developing chick embryos and by

immunodiffusion (Fenner and Marshall, 1957). But differences seem to exist, especially

between Australian field strains and the virulent strain Lausanne, as shown by challenge

experiments (Williams et al., 1973).

2.1.4 Pathogenesis and immunology

The pathogenesis of myxomatosis has been characterised in the past (Fenner and

Woodroofe, 1953 cit. by Best and Kerr, 2000) and also more recently by experimental

inoculations and comparative immunopathological studies (Best et al., 2000; Best and

Kerr, 2000). In the latter, the virulent strain SLS and its attenuated derivative Ur were

studied in genetically susceptible (laboratory) and in genetically resistant (wild) rabbits

(Best et al., 2000; Best and Kerr, 2000). Briefly, rabbits were inoculated intradermally with

100PFUs in the metatarseal region of the left hind foot. Both, laboratory and wild rabbits

inoculated with SLS developed clinical myxomatosis, however mortality was lower in the

latter. Ur infection was characterized by moderate to severe clinical signs and occasional

death in laboratory rabbits and few or no clinical signs in wild rabbits (Best and Kerr,

2000). At autopsy, several tissue samples were taken for virus titrations including the skin

of the inoculation site, skin of the equivalent site of the right hind foot (distal skin), the left

(draining lymph node) and right (contralateral) popliteal lymph nodes, blood, spleen and

lungs. There was little difference in titres of both viruses, SLS and Ur, in the skin at the

inoculation site of laboratory and wild rabbits. In the distal skin, however, virulent SLS was

present in laboratory and wild rabbits by day 4 post-inoculation (p.i.), whereas as Ur was

detected a few days later in laboratory rabbits and only in one of three inoculated wild

rabbits. In the draining lymph nodes, either SLS or Ur viruses were present by day 2 p.i. in

laboratory rabbits and by day 4 p.i. in wild rabbits. As a measure of dissemination, virus

presence was also determined in the contralateral lymph node, where it was found

approximately 2 days later than in the draining lymph node. Generally, SLS was slower to

reach this node in wild rabbits and titres remained 10-100 times lower than in laboratory

rabbits. Ur was detectable in low titres in wild rabbits only after day 15 p.i.. Neutralizing

antibody responses were detectable after day 6 p.i.. Antibody titres against virulent SLS

were higher and appeared earlier than those against Ur in both, laboratory and wild

rabbits. This work showed, that neither attenuation nor resistance were initially strongly

manifested in the skin at the inoculation site, but were more evident at the distal skin site,

and that the draining lymph node was a critical organ for amplification and dissemination

of the virus and thus for disease outcome.

42

Tissue samples of these experiments were further analysed for virus localization and

apoptosis by immunofluorescence and TUNEL reaction, respectively (Best et al., 2000).

Initial virus replication of both, SLS or Ur, in the skin was similar in resistant and

susceptible rabbits. Virus replication initiated in MHC-II positive dendritic-like cells in the

dermis, and subsequently spread to epidermal cells and lymphocytes of T cell zone of the

draining lymph node and from there to other lymphoid tissues, lungs, testis and secondary

skin sites. However, at the inoculation site an important difference in the inflammatory

response was observed. In susceptible laboratory rabbits infected with virulent SLS limited

inflammation was observed and polymorphonuclear cells, especially neutrophils,

predominated. In infections with attenuated Ur or in resistant wild rabbits an intense

cellular inflammatory response was observed with the predomination of mononuclear

cells. From the inoculation site, virus reached the draining lymph nodes within 24h in all

infections. In the lymph nodes, infection with either virus led to lymphocyte apoptosis, but

only infection with virulent SLS led to lymphocyte depletion and influx of

polymorphonuclears. In the attenuated Ur infections an influx and local proliferation of the

cells as part of an active immune response were observed, which compensated for this

loss. Dissemination of the virus from the skin and draining lymph node to distal sites

occurred in lower titres in infections with attenuated Ur or in resistant wild rabbits than in

virulent SLS infections, again, correlating with the presence of mononuclear as opposed

to polymorphonuclear cells. The authors suggested that effective constraint of virus

replication would be mediated by a type 1 cytokine response (IFN-γ, Il-12 etc) leading to

enhanced cellular immune responses, whereas a type 2 cytokine response (IL-4, IL-10

etc.), which favours humoral immune responses, predominated in disseminated

myxomatosis.

The immune responses to myxoma virus have been described in an excellent review of

Kerr and McFadden (2002). In animals that succumb to fatal nodular myxomatosis, a

severe immune dysfunction, accompanied by supervening Gram-negative bacterial

infections of the respiratory tract, is characteristic (OIE, 2009a). In animals that recover

from infection, progressive regression of signs and lesions are observed, and these

animals are generally considered resistant to subsequent disease, although

recrudescence and viral persistence in the testis has been described (Marlier et al.,

2000b). As described above, during the first encounter of a susceptible host with myxoma

virus, to date unknown factors will determine the type of innate and subsequent specific

immune responses and thus clinical outcome of infection. An effective cellular immune

response has been linked with recovery and protection. Although, to our knowledge, no

specific work has been carried out in measuring cell-mediated immunity to myxoma virus,

43

there is substantial evidence supporting this hypothesis (Best et al., 2000; Kerr et al.,

2004). The role of the humoral immune response is more difficult to evaluate. Infected

animals develop both, IgM and IgG antibodies. Rabbits that recover from myxomatosis

have antibodies basically for the rest of their lifes (Kerr, 1997). These antibodies do

neutralise the virus, but on their own may be insufficient to protect from death in virulent

infections, as similar titres were observed in survivors and animals that died from infection

(Best et al., 2000). In analogy, similar antibody titres are induced by attenuated and

inactivated vaccines, but only the former are able to provide protection. Maternally derived

antibodies play a role in providing protection to kittens, reducing clinical signs upon

infection and allowing active immunisation to take place, which will protect against

subsequent infections, especially in endemic situations with high host density and high

transmission rates (Fenner and Marshall, 1954; Fouchet et al., 2008). Crossprotection

between myxoma virus strains and as well as with the related Shope fibroma virus is

common (Fenner and Woodroofe, 1954; Gorski et al., 1994; Williams et al., 1973).

2.1.5 Immunomodulation

Poxviruses, and as such myxoma viruses, encode a large plethora of proteins which

interact with the hosts immune system. These immunomodulatory proteins may subvert

the immune responses, and thus affect pathogenesis and outcome of infection (E.g.

Cameron et al., 1999; Graham et al., 1992; Macen et al., 1996; Messud-Petit et al., 1998;

Upton et al., 1991; Upton et al., 1992). Most of these genes are located at the terminal

region of the genome (Cameron et al., 1999). They are less conserved among poxviruses

than the central region, in which most genes with housekeeping functions are located. The

sequence similarity between some of these immunomodulatory genes and the host cell

counterparts suggests that they have been acquainted from the vertebrate host reflecting

co-evolution (Johnston and McFadden, 2003). This would explain the relative apathogenic

expression of myxoma virus infection in its natural host, Sylvilagus spp.. On the contrary,

in its evolutionary recent host, the European rabbit, the expression of these gene products

could lead to a dysregulation of the elicited immune response. Thus, the acute systemic

and fatal disease observed in the European rabbit is partly mediated by

immunopathogenic and immunosuppressive mechanisms induced by the virus (Kerr and

McFadden, 2002; Kerr et al., 2010; Stanford et al., 2007b). The disease and ultimately

death is characterised by supervening bacterial infections (Kerr and McFadden, 2002).

Supporting the role of immunomodulation and -suppression in the pathogenesis of lethal

myxomatosis, is the observation that selective deletion of viral genes (“knock-out viruses”)

encoding immunomodulatory molecules produces attenuated disease in European

44

rabbits, although it should be remembered that knock-out viruses may not reflect the full

extent to which a gene product contributes to pathogenesis (Cameron et al., 1999;

Johnston and McFadden, 2004).

According to their function, the immunomodulatory proteins of poxviruses can be divided

into the following classes: a) Virostealth, in which visible signs of virus infection are

masked, for example by downregulation of MHC-I receptors by myxoma virus M135R

gene product; b) Virotransduction, by which innate antiviral mechanisms, such as

apoptosis, are inhibited, for example myxoma virus SERP-2; and c) Viromimicry, which

are viral proteins that mimic host cytokines or their receptors, virokines and viroceptors,

respectively, for example, myxoma virus M007L is a IFN-gamma receptor homologue

(Lalani et al., 1997). Many myxoma virus gene products have been studied and assigned

to these functional groups (Johnston and McFadden, 2003; Kerr and McFadden, 2002;

Stanford et al., 2007b; Stanford et al., 2007c; Zuniga, 2002, 2003).

2.1.6 Laboratory Diagnosis

Diagnosis of typical myxomatosis is generally based on clinical signs. However, in the

case of atypical or amyxomatous forms of the disease these are much less pronounced,

requiring the application of laboratory testing such as the isolation of the virus by

inoculation of sensitive cells and identification of the virus by immunological methods to

confirm infection (OIE, 2009a). In any cases, the agent can also be identified by

demonstration of nucleic acid by polymerase chain reaction. The demonstration of MV-

specific antibodies by serological tests allows the retrospective diagnosis and may be

used to determine the prevalence of infection in a population.

Virological methods allow the isolation and identification of the virus, the detection of viral

antigens or viral nucleic acid from skin or other organ material of clinically affected rabbits.

Virus can be readily isolated by cell culture using chick embryos, primary cultures of rabbit

kidney (RK) cells, or, more commonly using established cell lines, such as the RK-13 cell

line (ATCC CCL37). A cytopathic effect (CPE) usually develops after 24–48 hours, and

consists of the formation of syncytia, followed by rounding of the cells, pyknosis and

posterior detachment from the plastic support (OIE, 2009a, own observations).

Inoculation tests of rabbits also offer a means of identifying the virus and characterizing its

pathogenicity and tissue tropism, distinguishing nodular and oculo-respiratory tropisms

characteristic of the typical nodular and the atypical amyxomatous forms of the disease.

Virulence may be assessed by the type of inflammation in lesions (local or systemic

45

infection), the extent of lesions and survival time. Viral antigen can be detected by several

immunological methods such as the agar gel immunodiffusion test (OIE, 2009a) or indirect

fluorescent antibody tests applied to cell cultures (Gilbert et al., 1989). Negative-staining

electron microscopy can be applied to a sample of skin lesion (Catroxo et al., 2009). This

method, however, does not distinguish between myxoma virus and the related shope

fibroma virus (OIE, 2009a). The detection of MV-specific nucleic acids by molecular

techniques such as PCR has been described (Farsang et al., 2003), but has not been

validated for use as a diagnostic tool (OIE, 2009a).

Various serological tests have been described for the detection of a specific antibody

response (OIE, 2009a). In order of decreasing sensitivity the following may be used:

enzyme-linked immunosorbent assay (ELISA) (Gelfi et al., 1999; Kerr, 1997),

immunofluorescent antibody test based on infected fixed cell cultures, complement

fixation and agar-gel immunodiffusion. The latter can be used for the detection of both

antigen or antibody (OIE, 2009a). For epidemiological surveys, the IFA test and the

indirect ELISA can also be carried out using blood dried on blotting or filter paper: two

discs cut by paper punch are placed in each well of a 96-well plate and 100 µl PBS is

added to extract the serum. The serum dilution corresponds to about 1/30 and can be

used for testing (Gilbert et al., 1989). Antibodies develop within 8–13 days of infection,

may be detected for 6-8 months by CF and for at least 20 months by virus neutralization

and ELISA tests (Kerr, 1997; Marlier et al., 1999; OIE, 2009a).

2.1.7 Epidemiology and control

Myxoma virus spreads within rabbit populations by blood-feeding arthropod vectors, such

as fleas and mosquitoes, although limited transmission by close direct contact is possible.

Myxomatosis is currently endemic in most of Europe as well as in Australasia. Seasonal

epidemics do occur with some geographical variation, mostly depending on the availability

of vectors and a sufficient density of susceptible rabbits (Fenner and Ross, 1994; Kerr and

Best, 1998). Thus, in France and Britain, as well as on the Iberian Peninsula, epidemics

occur predominantly in late summer and autumn, but local variations may alter

epidemiological patterns, with peaks occuring in spring (Calvete et al., 2002a; Fenner and

Ross, 1994; Ferreira et al., 2009).

Control of myxomatosis is based on two main pillars: vector control and immunisation.

Vectors may be controlled physically by hygiene and nets or medically by using

ectoparasiticides such as ivermectin or selamectin, though withdrawal periods of the latter

46

need to be taken into account in meat-production. Currently, two types of vaccines, both

live modified, are available. Heterologous vaccines consist of the related Shope fibroma

virus, which is relatively apathogenic for the European rabbit, but induces good cross-

protection to myxomatosis (Fenner and Woodroofe, 1954), and homologous vaccines,

which contain attenuated strains of myxoma virus. The composition of homologous

vaccines may vary among countries and vaccine companies (Arguëllo, 1986; McKercher

and Saito, 1964). An example is the live modified strain SG33 (Saurat et al., 1978). SG33

has been attenuated by successive cell-culture passages and displays a deletion of

approximately 13kb in the Serp2 gene (Petit et al., 1996). Recently a M063-gene deleted

mutant was constructed from the attenuated Uriarra strain and tested for use as a non-

transmissible vaccine against myxomatosis (Adams et al., 2008). This vaccine would be of

interest in Australia for protecting captive and production rabbits against myxomatosis, as

other commercially available vaccine are attenuated and as such bear some risk of

spreading and inadvertently immunising wild rabbits.

Vaccines may be applied subcutaneously or intradermally, the latter commonly with a

device that applies the pressurised vaccine into the skin (E.g. Dermojet®). Recommended

vaccination schemes in commercial rabbitries include vaccinating breeding rabbits at 2.5

months of age, re-vaccinating every 6 months and to vaccinate fattening rabbits once at

the age of 30 days thus reducing interference with maternal antibodies. Generally, these

vaccines are well tolerated, but some side effects due to residual virulence may occur

especially in immunocompromised animals. The signs may include transitory palpebral

oedema and respiratory signs. These post-vaccinal reactions may be avoided if the

animals are first vaccinated with the heterologous vaccine and boosted with a

homologous vaccine 6 to 8 weeks later. To increase efficacy of the vaccines, animals

should preferentially be vaccinated in spring and autumn, when susceptibility to the

vaccine virus is better than in the hotter summer months. Generally, efficacy of these

vaccines is considered good in preventing major losses in the rabbit industry, due to

classical (virulent) myxomatosis as well as to the atypical (respiratory) form of the disease

(Marlier et al., 2000a), although vaccine failures may sporadically occur (Kritas et al.,

2008; Psikal et al., 2003).

Control of myxomatosis in free-ranging wild rabbit populations is more difficult, as the

application of medication or commercially available vaccines requires the individual

handling of animals. Nevertheless, vaccination is currently considered an important

additional tool in the management of wild rabbit populations (Ferreira et al., 2009; Garcia-

Bocanegra et al., 2010; Guitton et al., 2008), despite some evidence of a negative impact

47

caused by handling and vaccination, especially in young and subadult animals with poor

body condition (Calvete et al., 2004b). To achieve higher cost-benefit efficacy and to

decrease potential adverse effects related to the handling of individual animals, as well as

to increase vaccine coverage in the population, horizontal spread by contact and/or oral

administration by baits would be desirable. The success of oral vaccination of wildlife

species has already been shown for other diseases, such as the rabies vaccination of wild

foxes in Europe (Brochier et al., 1996), but may be difficult to achieve for myxomatosis. A

live attenuated myxoma virus vaccine capaple of horizontal spread has been developed

(see below).

Poxviruses, such as vaccinia viruses are already used as viral vectors for the construction

of recombinant vaccines (Giavedoni et al., 1991; Paoletti, 1996). The use of myxoma virus

as vector is interesting, in that it would also confer protection against the vector-induced

disease, myxomatosis. To date, three recombinant myxoma-based vaccines have been

described, in which immunogenic gene sequences from different viruses have been

inserted: one with influenza virus genes (Kerr and Jackson, 1995) and two with RHDV

(Barcena et al., 2000a; Bárcena et al., 2000; Bertagnoli et al., 1996a). Both recombinant

myxomatosis-RHD vaccines use the same gene encoding the major envelope protein

VP60 of RHDV to protect against this disease, but they differ in their myxoma virus

backbone strain. One of the vaccines uses the attenuated strain SG33 (Bertagnoli et al.,

1996a), whereas the other uses the avirulent MV field strain “6918” (Barcena et al.,

2000a; Bárcena et al., 2000). The latter strain has been tested and found suitable for

immunisation of rabbits against virulent MV strains by the subcutaneous and also the oral

routes of administration, and it retained its horizontal transmission potential even after

being passaged in cell culture. The resultant vaccine was found suitable for use in free-

ranging wild rabbits because it was found safe, i.e. did not retain residual virulence, and

efficacious, i.e. conferred protection against both diseases, and extremely practical as it

could be administered orally and could immunize in contact animals (Torres et al., 2000b;

Torres et al., 2001).

Although there is growing interest, in particular by conservationists and hunting

associations of Southern Europe, to apply this vaccine in the field, the use of it has not yet

been authorised by the European Union. Concern has been expressed on the use of

genetically modified live rabbit viruses in the field, especially as research for the

management of wild rabbits in Europe and Australia has opposing goals (Angulo and

Cooke, 2002). In other words, in Europe, a transmissible recombinant myxoma virus has

been produced to protect against myxomatosis and RHD (Bárcena et al., 2000) and in

48

Australia a transmissible recombinant myxoma virus has been produced to reduce rabbit

fertility (Gu et al., 2004; Hardy et al., 2006; van Leeuwen and Kerr, 2007). As easily

illustrated by history, myxomatosis and RHD viruses do not respect boundaries and

transboundary spread may occur. The inadvertent introduction of an immunizing myxoma

virus in Australia and a virus with immunocontraceptive properties in Europe would be

disastrous (Angulo and Cooke, 2002). Additionally, the potential interaction of field viruses

and genetically modified myxoma virus has not been assessed, as the field trial of the

transmissible recombinant myxoma vaccine, has been realised in a naïve wild rabbit

population (Torres et al., 2001).

2.1.8 Other areas of myxoma virus research

There are research lines exploring the use of myxoma virus for other purposes than those

related with the biology of myxomatosis in its host, for example as viral vector for

vaccines, or as therapeutic agents to treat cancer or inflammatory diseases. Due to their

large genome size, poxviruses lend themselves as attractive candidates for the

development of recombinant vaccines. For example, recombinant vaccines based on

canarypox viruses have been developed for diseases of veterinary importance and are

being commercially available, such as feline leukaemia vaccines. Myxoma virus has

proven safe and adequate in terms of immunogenicity, and thus represents a promising

new candidate as vector for a variety of agents. The potential of myxoma virus as vaccine

vector in non-leporid species has been explored recently for cats and small ruminants

(McCabe et al., 2002; Pignolet et al., 2008). Myxoma virus exhibits tropism for many

human tumour cells, making it an interesting candidate for oncolytic virotherapy (Lun et

al., 2005; Sypula et al., 2004; Wu et al., 2008). The tropism of myxoma virus for cancer

cells has been linked to the virus ability to activate the enzyme “Akt” by the viral protein M-

T5 (Sypula et al., 2004; Wang et al., 2006). More recent studies also focus on the

synergistic effect of immunosupressant drugs, such as rapamycin, and myxoma virus on

tumour regression (Lun et al., 2010; Stanford et al., 2007a). Similar to other viral proteins,

myxoma virus proteins are interesting candidates as novel anti-inflammatory reagents

(Reviewed in (Lucas and McFadden, 2004)). As discussed above, upon infection, some of

the myxoma virus proteins display a modulatory effect on the host’s immune system,

potentially subverting the immune response. This characteristic can be – and is starting to

be – explored for the development of new anti-inflammatory therapeutics. Some promising

results have already be obtained by testing the effect of viral proteins using animal models

(Lucas and McFadden, 2004).

49

2.2 Partial sequencing of recent Portuguese myxoma virus field isolates exhibits a high degree of genetic stabilit y.

Adapted from: Veterinary Microbiology (2010) 140, 161-166

A. Mullera,b*, E. Silvaa,b, J. Abrantesc,d,h , P. J. Estevesc,e , P. G. Ferreiraf , J. C. Carvalheiraa,c , N. Nowotnyg and G. Thompsona,b

a) Department of Veterinary Clinics, Institute of Biomedical Science Abel Salazar (ICBAS), University of Porto, P-4099-003 Porto, Portugal

b) Multidisciplinary Unit of Biomedical Investigation (UMIB), University of Porto, P-4099-003 Porto, Portugal

c) Centre of Investigation for Biodiversity and Genetic Resources (CIBIO), University of Porto, P-4485-661 Vairão, Portugal

d) Department of Zoology and Anthropology, Faculty of Sciences, University of Porto, P-4150-150 Porto, Portugal

e) Centro de Investigação em Tecnologias da Saúde (CITS), CESPU, Portugal f) Department of Anatomy, Institute of Biomedical Science Abel Salazar (ICBAS), University

of Porto, P-4099-003 Porto, Portugal g) Zoonoses and Emerging Infections Group, Clinical Virology, Department of Pathobiology,

University of Veterinary Medicine, Vienna, A-1210 Vienna, Austria h) INSERM, U892, Nantes, France; Université de Nantes, France

*Corresponding author: Phone: +351-252-660410, Fax: +351-252-661780. E-mail address: [email protected]. Postal address: ICAV–UP, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal Keywords : Myxoma virus, field strains, sequence analysis, co-evolution, European rabbit

51

Summary

To study genetic changes underlying myxoma virus evolution in its new host, the

European rabbit (Oryctolagus cuniculus), we sequenced selected genomic regions of nine

recent virulent field strains and a live attenuated vaccine strain (“MAV”, Germany). DNA

was extracted from cell culture passaged myxoma virus. A total of 4863bp (approximately

3% of the genome) of ten regions spanning 12 genes of the myxoma viruses was

sequenced and compared to the original virulent strain “Lausanne” and its attenuated field

derivative strain “6918”. The field strains displayed a maximum of three (strains C43, C95)

and a minimum of one (strains CD01, CD05) nucleotide substitutions. These were

distributed through all analysed coding regions, except gene M022L (major envelope

protein), where all strains were identical to “Lausanne” and “6918”. Two new single

nucleotide insertions were observed in some of the field strains: within the intergenic

region M014L/M015L and within gene M009L, where it leads to a frameshift. These

insertions were located after homopolymeric regions. The vaccine strain displayed 37

nucleotide substitution, predominantly (95%) located in genes M022L and M036L.

Interestingly, regions M009L and M014L/M015L of the vaccine were not amplified

successfully, suggesting major genomic changes that could account for its attenuated

phenotype. Our results support a high degree of genetic stability of myxoma virus over the

past five decades. None of the analysed genome regions by its own seems sufficient for

the genetic characterisation of field strains.

52

Introduction

Myxoma virus (MV) is a large double stranded-DNA virus of the genus Leporipoxvirus. In

its natural hosts (Sylvilagus brasiliensis and Sylvilagus bachmani) it causes benign

cutaneous fibromas. However, its introduction in the 1950s into a new host, the European

rabbit (Oryctolagus cuniculus), resulted in devastating epizootics with mortality rates

above 99%. Interestingly, within a few years of the introduction of the virus into naïve

European wild rabbit populations in Australia and Europe, a decrease from the initial

nearly 100% mortality rates were observed. This process has been related to virus-host

adaptation consisting of the evolution of attenuated viral strains and the natural selection

of resistant rabbits (reviewed by Fenner & Ross, 1994). Extensive knowledge has been

gained on subjects related to this co-evolution, such as the determination of virulence

grades of circulating myxoma viruses (e.g.: Bárcena et al., 2000; Fenner & Ross, 1994;

Marlier et al., 1999), the pathogenesis of myxoma virus infection of different clinical

outcomes (Best & Kerr, 2000; Best et al., 2000), the rabbits’ immune response (reviewed

by Fenner & Ross, 1994), and the characterisation of virally encoded proteins able to

modulate the host’s immune response and thus clinical outcome of infection (e.g.

Johnston & McFadden, 1994; Stanford et al., 2007; Willer et al., 1999; Zúñiga, 2002).

Contrasting little is known about the genetic changes related to the evolution of myxoma

virus in its new host (Kritas et al., 2008; Morales et al., 2008; Saint et al., 2001). The

evolution of poxviruses has been studied based on whole-genome phylogenetic analyses

rather than single gene phylogeny due to the occurrence of phenomena such as

horizontal gene transfer (Bratke & McLysaght, 2008; Gubser et al., 2004; Hughes &

Friedmann, 2004; Xing et al., 2006). To date, the complete genomes of only two myxoma

viruses are available: the virulent strain “Lausanne”, introduced in 1952 in Europe, and its

attenuated field derivative “6918”, obtained in 1995 in Spain (Bárcena et al., 2000;

Cameron et al., 1999; Morales et al., 2008). The comparative analysis of both genomes

has identified 73 differences consisting of 67 base substitutions, 4 deletions and 2

insertions (Morales et al., 2008). Interestingly, events such as whole gene loss or gain

were not observed.

The understanding of genetic changes driving myxoma virus evolution in the European

rabbit requires the genetic analysis of larger numbers of field strains. The large size of the

viral genome has hampered the availability of this kind of information, and partial gene

sequences have shown little if any differences compared to the virulent strain Lausanne

(Kritas et al., 2008; Saint et al., 2001). As gene order and content remained unchanged in

the attenuated myxoma virus field strain “6918” (Morales et al., 2008), it may be sufficient

to study virus evolution by analysing specific genomic regions. Our objectives were to

53

study selected genomic regions of recent myxoma virus field isolates and to evaluate the

suitability of these as molecular marker for virus evolution within the European rabbit.

Material and Methods

1. Selection of viral genes

The genome comparison between the virulent myxoma virus strain “Lausanne” (GenBank

accession no. AF170726) and the attenuated field strain “9618” (GenBank accession no.

AF552530), suggested that the observed disruptions in ORFs M135R, M148R, M009L,

M036L may be related to loss of viral virulence (Morales et al., 2008). Thus, we included

these genome regions in our analysis (Table 2). We also partially amplified gene M020L

to confirm the insertion of the CTC codon for leucine at position 52 (Morales et al., 2008).

Further five viral genome regions were selected, which include the following partial genes:

M002L/R and M007L/R for being located in the terminal inverted repeats and encoding

immunomodulatory proteins similar to TNF receptor and to rIFN gamma receptor,

respectively (Johnston & McFadden, 1994; Stanford et al., 2007; Willer et al., 1999),

M014L and M015L, which encode a kelch-like protein and the small subunit of

ribonucleotide reductase, respectively; M022L encoding the major envelope protein, as

this protein is exposed to the immune system of the host, and may reflect the effect of

immune pressures; M137R and M138L because the corresponding sequence of the

California myxoma virus strain “MSD” (GenBank accession no. AF030894) was available

for comparison and displayed major differences when compared with “Lausanne”

(Jackson et al., 1999). The “MSD” strain naturally infects the brush rabbit (Sylvilagus

bachmani) but causes fulminant disease in the European rabbit. A further locus, M061R,

coding for the thymidine kinase gene, was selected for diagnostic purposes only (see

below). The term “genomic region” was used to describe the amplified sequences, which

correspond to either part of a single gene, or, in the case of M014L/M015L and

M137R/M138R, to two adjacent genes and their respective intergenic noncoding region.

2. Primers and sequencing

The targeted genes and used primers are shown in Table 2. A hot start Taq polymerase

(Hotstar Taq Polymerase, Qiagen) was used. The annealing conditions of the PCR

reactions varied between 50 and 65ºC. The PCR products were directly sequenced in

both directions.

54

Table 2 Selected genes and primers used for the ge netic characterisation of myxoma virus field strain s. The nucleotide positions refer to myxoma virus strain “Lausanne” (GenBank ac cession no. AF170726)

Gene

(position)

Gene description Forward primer

(position)

Reverse primer

(position)

Annealing

temperature

Size PCR

amplicon

Genomic Region:

Positions of obtained

sequence

Length of

viral protein

Positions of deduced

amino acids

M002L/R

(1665-2645;

160109-159129)

Similar to TNF receptor MT2F

(1682-1702)

MT2R

(2614-2634)

55ºC 932bp 1706-2613;

160068-159161

326 12-313

M007L/R

(7985-8776; 153789-

152998)

Similar to rIFNgamma

receptor

MT7F

(7990-8009)

MT7R

(8748-8767)

55ºC 758bp 8006-8741;

153768-153033

263 13-257

M009L

(11601-13130)

Contains kelch motifs M009F

(11859-11878)

M009R

(12383-12364)

55ºC 525bp 11859-12336 509 263-424

M014L

(15012-16565)

Kelch-like protein

NN1F= 16496F

(16496-16515)

NN1R= 16931R

(16912-16931)

65ºC

436bp

16501-16931

517 1-21

M015L

(16618-17586)

Ribonucleotide reductase,

small subunit

322 220-322

M020L

(19194-20531)

Serine/threonine protein

kinase

M020F

(20145-20164)

M020R

(20458-20439)

55ºC 314bp 20146-20413 445 38-125

M022L

(22558-23673)

Major envelope protein NN2F= 22913F

(22913-22932)

NN2F= 23298R

(23279-23298)

60ºC 386bp 22914-23298 371 126-253

M036L

(37209-39251)

Leucine zipper motif M036F

(37558-37577)

M036R

(37845-37864)

55ºC 307bp 37551-37858 680 464-564

M061R

(57797-58333)

Thymidine kinase TKU

(57822-5783)

TK3

(58375-58393)

50ºC 571bp Diagnostic PCR

M135R

(131699-132235)

IL-1/IL-6 receptor-like M135F

(131647-131668)

M135R

(131856-131876)

55ºC 230bp 131642-131871 178 1-59

M137R

(132908-133840)

Similar to vacA51R

A1F

(133767-133787)

A1R

(134405-134424)

60ºC

658bp

133788-134746

310 295-310

M138L

(133874-134746)

alpha 2,3-sialyltransferase A2F

(134340-134360)

1160R

(134938-134957)

60ºC 618bp 290 1-290

M148R

(141626-143653)

Ankyrin motif; host range M148F

(142701-142720)

M148R

(143119-143138)

55ºC 438bp 142696-143132 675 360-504

55

The obtained sequences were edited, aligned and analysed using the software Bioedit (Hall,

1999). The obtained sequences were compared to sequences available in GenBank by

performing BLAST searches. The deduced amino acid sequences were compared to the

proteins of myxoma virus strain “Lausanne” and “6918”.

3. Myxoma virus isolates

Nine field strains were obtained from European rabbits displaying signs and/or lesions

compatible with myxomatosis. They were collected between 2004 and 2007 in different

locations in Portugal: four were obtained from wild rabbits in the southern province Algarve

(C43, C95, C116, C152), three from wild rabbits in the north-eastern province Trás-os-

Montes e Alto Douro (CB31, CB32, CB191), and two from an outbreak in domestic rabbits in

the north-western province Douro Litoral (CD01, CD05). The vaccine strain “MAV” was

originally derived from a cell culture attenuated Californian myxoma MSD virus (McKercher

and Saito, 1964). It is currently used as seed for commercially available vaccines in Europe

(Gorski et al., 1994). This vaccine strain was kindly made available by the company IDT

Biologika GmbH, Dessau, Germany, through the Friedrich-Loeffler-Institut, Germany, and

was used here as reference strain.

The diagnosis of myxomatosis was made by PCR targeting the thymidine kinase gene

(M061R) using DNA extracts from eyelids (Table 2). In order to increment viral yield for the

genetic characterisation, the viruses were passaged twice in RK-13 cells. Virus was

harvested after submitting the infected cell culture twice to freeze-thaw cycles at -80ºC. Cell-

free supernatant was used for nucleic acid extraction using the QIAamp Viral RNA Mini Kit

(Qiagen, Hilden, Germany) according to the manufacturer’s instructions.

4. Nucleotide sequence accession numbers

The GenBank accession numbers of 16 sequences representing the observed nucleotide

changes are FJ970492-FJ970507.

Results

Ten genomic regions spanning 12 genes were selected for the partial genetic analysis of

nine virulent myxoma virus field isolates obtained from seven wild and two domestic rabbits

between 2004 and 2007 in Portugal, as well as from the German live attenuated vaccine

strain “MAV”. The sequence information on 4863bp (corresponding to 3% of the viral

genome) was compared to that of strains “Lausanne” and “6918”.

56

Table 3 Observed nucleotide polymorphisms and dedu ced amino acid variations in recent myxoma virus fi eld strains. The nucleotide and amino acid positions refer to myxoma virus stra in “Lausanne” (GenBank accession no. AF170726)

The GenBank accession numbers of selected sequences (FJ970492-FJ970507) are indicated in bold italics below the respective polymorphic nucleotide positions.

Terminal region Central region Terminal region

Gene M002L/R

907bp

M007L/R

735bp

M009L

477bp

M014L

M015L

430bp

M022L

384bp

M036L

307bp

M135R

229bp

M137R/M138L

958bp

M148R

436bp

NT AA NT AA NT AA NT NT AA NT AA NT AA NT AA NT AA

Strain 6918

(EU552530)

T1981

A2497

T2594

D222

F104

S18

A8064 F238 Deletion

11942-

11951

F/S after

aa395,

start Met 398

ID ID ID Insertio

n

C37687

F/S after

aa523,

start Met 542

Insertion

G131750

F/S after

aa19,

start Met

44

ID ID Deletion

C142959

F/S

after

aa446,

start

Met

505

C43 T1981 FJ970494

D222

A8261 FJ970496

ID ID ID ID*)

FJ970498 ID ID ID ID ID ID ID ID G142867

FJ970506 S416

C95 T1981 D222 ID ID Insertion

C12297 FJ970497

F/S after

aa280,

start Met 303

ID*)

ID ID T37588 FJ970500

M496 ID ID ID ID G142867 S416

C116 T1981 D222 ID ID Insertion

C12297

F/S after

aa280,

start Met 303

ID*)

ID ID ID ID ID ID ID ID G142867 S416

C152 T1981 D222 ID ID ID ID ID ID ID T37759 FJ970501

M553 ID ID ID ID ID ID

CB31 T1981 D222 ID ID Insertion

C12297

F/S after

aa280,

start Met 303

ID*)

ID ID ID ID T131772 FJ970503

C27 ID§)

FJ970504 ID ID ID

CB32 T1981 D222 ID ID Insertion

C12297

F/S after

aa280,

start Met 303

ID*)

ID ID ID ID T131772 C27 ID§)

ID ID ID

CB191 T1971 FJ970492

ID ID ID Insertion

C12297

F/S after

aa280,

start Met 303

ID*)

ID ID ID ID ID ID A133966 FJ970505

C261 ID ID

CD01 T1971 ID

ID ID ID ID ID ID ID ID ID ID ID ID ID ID ID

CD05 C1975 FJ970495

G224

ID ID ID ID ID ID ID ID ID ID ID ID ID ID ID

Vaccine

strain “MAV”

ID ID G8492 FJ970507

ID No PCR

amplicon

(2 weak

bands 100

+ 400kb

approx.)

- No PCR

amplicon

G23118

C23296

T23212

C23218

A23235

C23236

All ID 27 nt

subst. FJ970502

L458

S491

S530

S532

S549

All other 22

ID ID ID ID T142804 FJ970493

V395

57

G23254

T23257 FJ970499

ID

ID Identical to “Lausanne”

*) Insertion (A) after nucleotide 16616 of a poly-A in the non-coding region.

§) Point mutation (C -> T) at position 133846 in the non-coding region.

F/S

58

We also confirmed the insertion of the CTC codon in gene M020L of all field strains as

well as the vaccine strain (data not shown). The results are shown in Table 3. Within gene

M002L/R (907bp) all field isolates displayed one single nucleotide substitution, albeit at

three different locations. Six of the nine field strains displayed a nonsynonymous

substitution at position 1981, which was also observed in strain “6918”. The field strains

CB191 and CD01 showed a synonymous substitution at position 1971. In field strain

CD05, which originated from the same outbreak as CD01, a nonsynonymous nucleotide

change at a different position (1975) was observed. The vaccine strain was identical to

“Lausanne”. Within gene M007L/R (735bp), two nonsynonymous nucleotide substitutions

were observed in field strain C43 and in the vaccine strain (positions 8261 and 8494,

respectively). Within gene M009L (477bp), five field strains (C95, C116, CB31, CB32,

CB191) displayed a single nucleotide insertion (C) at position 12297 after a

homopolymeric region of 5 cytosins, causing a frameshift after amino acid 280. The PCR

amplification of the vaccine strain yielded two very weak bands of approximately 100 and

400bp, inadequate for successful sequencing. Within the region spanning M014L/M015L

(430bp) a single nucleotide insertion (A) was observed at position 16616 of the intergenic

region of six field strains (C43, C95, C116, CB31, CB32, CB191) after a homopolymeric

region of 10 adenosines. PCR amplification of this region of the vaccine strain was

unsuccessful. At gene M022L (384bp) all field strains were identical to “Lausanne” and the

vaccine strain displayed eight synonymous nucleotide substitutions. Within gene M036R

(229bp), nonsynonymous nucleotide substitutions were observed at positions 37588 and

37759, of the two field strains C95 and C152, respectively. The vaccine strain displayed

27 nucleotide substitution, of which 5 were nonsynonymous. In gene M135R (229bp), the

same nonsynonymous nucleotide substitution was observed at position 131772 in the field

strains CB31 and CB32. The remaining, as well as the vaccine strain, were identical to

“Lausanne”. Within the region spanning M137R/138L (958bp), a nonsynonymous

nucleotide substitution was observed at position 133966 of field strain CB191, and a

single nucleotide substitution at position 133846 in the intergenic non-coding region of

strains CB31 and CB32. Within gene M148R (436bp), nonsynonymous nucleotide

substitutions were observed at position 142867 of isolates C43, C95, C116, and at

position 142804 of the vaccine strain.

59

Discussion

Since the introduction of Myxoma virus into its new host population, the European rabbit,

a decrease in disease mortality has been observed and myxomatosis has turned into a

frequently cited example of virus-host adaptation, but genetic information on field strains is

scarce (Kritas et al., 2008; Morales et al., 2008; Saint et al., 2001). In the present study

only virulent virus strains were included, because field evidence of attenuated or atypical

forms of disease is difficult to obtain, as these probably go undetected, e.g. we were

unable to find evidence of myxoma virus infection in eyelid samples from 30 healthy wild

rabbits hunted in southern Portugal (Pancas) and tested by PCR analysis of the TK gene

as well as by three consecutive passages on RK-13 cells (data not shown). The analysed

nucleotide sequences presented here corresponded to only approximately 3% of the

complete viral genome, but they were selected based on the above-mentioned criteria and

thus considered good candidates for detecting changes reflecting virus - host co-

evolution. Interestingly, a maximum of only three (strains C43, C95) and a minimum of

one nucleotide substitutions (strains CD01, CD05) were observed in each field strain. The

attenuated strain “6918” only displayed four nucleotide substitutions within the analysed

genome regions, one of which (position 1981 of gene M002L/R) was also found in six of

the field strains, suggesting that this mutation is being fixed in some European wild rabbit

populations. The major envelope gene (M022L) of myxoma is exposed to the rabbit’s

immune system, and expected to be a good target for identifying viral variability.

Surprisingly, the analysed gene sequences of the field isolates were identical to strain

“Lausanne”, similar to that of a pathogenic myxoma virus isolated from an outbreak in

vaccinated and non vaccinated commercial rabbits in Greece (Kritas et al., 2008). Within

M022L of the vaccine strain, eight synonymous nucleotide substitutions were found. No

equivalent sequence information on the MSD strain was found in GenBank to understand

whether these mutations are characteristic for this strain, or whether they may reflect

pressures related to its attenuation process. Although our findings support the limited

degree of genetic alteration found in recent myxoma virus field strains (Morales et al.,

2008; Saint et al., 2001), the mutations observed in the selected genome regions allowed

the individual identification and some grouping of the strains according to their geographic

origin. For example, CB31 and CB32, which were collected from wild rabbits during the

same occasion, are identical to each other but differ from CB191, which was sampled at a

different geographic location, but also in the north of Portugal. Interestingly the strains

obtained from domestic rabbits during an outbreak in a family farm (CD01, CD05)

displayed mutations at different locations of gene M002L/R, but were otherwise identical

to “Lausanne”.

60

Within myxoma viruses, whole gene gain or loss does not seem to occur, but single or

multiple base pair insertions and deletions which resulted in frameshift mutations have

been described (Morales et al., 2008). We have found two homopolymeric sites, where a

single nucleotide has been inserted in some of the virulent field strains: within gene

M009L and in the noncoding region between M014L and M015L. Thus these single

nucleotide insertions do not seem to affect virulence. However, major disruptions, at least

in M009L, may indeed play an important role in attenuation, e.g. in strain “6918” (Morales

et al., 2008). Although it has been argued that integrity of gene M009L may not be a

critical virulence factor in myxoma virus, because of its disruption in the virulent

Californian strain MSW (Labudovic et al., 2004) and its partial duplication in the

attenuated Shope fibroma virus (Willer et al., 1999), it should be remembered, however,

that these leporipoxviruses have evolved in different hosts, Sylvilagus bachmani and S.

floridanus, respectively, whereas the natural host of myxoma virus is S. brasiliensis

(Fenner & Ross, 1994). They present a considerable degree of genetic heterogeneity (84-

89%) and may not entirely be suitable for comparison with “Lausanne” and “6918”. Major

changes are likely to be present in gene M009L of the vaccine strain, hampering PCR

amplification and yielding two products of smaller sizes. The use of two other primer pairs

overlapping the site of the deletion in “6918” (positions 11937-11947) was unsuccessful

(not shown). The region spanning genes M014L/M015L of the vaccine strain could also

not be amplified successfully, suggesting the occurrence of genomic changes requiring

further investigations. Single indels in genes M135R and M148R where considered of

putative relevance for the attenuation of “6918” (Morales et al., 2008). Our results support

this hypothesis as these changes were not observed in the virulent field strains. However,

they were neither found in the vaccine strain, indicating that they are not essential for

attenuation. The disruption of another gene (M135R) was considered a potentially

important determinant for the attenuation of the field strain “6918” (Morales et al., 2008).

Again, this disruption does not seem essential for attenuation, as it did not occur in the

vaccine strain. The sequence spanning genes m137R/m138L of the vaccine strain

displayed 100% nucleotide indentity with “Lausanne”. This is rather unexpected, as the

sequence comparison with the “MSD” strain (Genbank accession no. AF030894), from

which the vaccine strain was derived, displayed only 84% nucleotide identity to strain

“Lausanne”. The vaccine strain has a long history of cell culture passaging, and it is thus

very difficult to rule out possible cross contaminations with “Lausanne”-derived isolates or

strains at any point.

61

Altogether, these results support those obtained with specific gene knockout viruses, in

that various disruptions, on their own or in synergy, may affect virulence of myxoma

viruses (Johnston & McFadden, 1994; Stanford et al., 2007; Willer et al., 1999). Further

field strains should be analysed to assess the frequency of single base substitutions and

indels as well as their importance in reflecting viral evolutionary processes, also

considering that misincorporations due to polymerase infidelity (in vivo and in vitro) may

account for some of the observed differences between strains. Although focussing on only

small parts of the viral genome, this study supports the relatively high degree of genetic

stability of myxoma field strains over the past five decades (Saint et al., 2001, Morales et

al., 2008). Based on our findings it is difficult to identify unique single gene markers of

virus attenuation or evolution, indicating that analyses of larger portions of the genome are

required.

Acknowledgements:

This study was supported by the Foundation for Science and Technology Portugal:

Project POCTI/BIA-BDE/61553/2004 and grants SFRH/BD/31093/2006,

SFRH/BD/31048/2006, SFRH/BPD/27021/2006 to A. M., J. A. and P. J. E., respectively.

Our thanks go to IDT Biologika GmbH, Dessau, Germany, in particular to Dr. Neubert for

the permission to use the vaccine strain “MAV” and for supplying information on its origin,

and to the Friedrich-Loeffler-Institut, Bundesforschungsinstitut für Tiergesundheit, Insel

Riems, Germany, in particular to Dr. Dauber and Dr. Riebe, for supplying the RK-13 cells

and the above mentioned myxoma vaccine strain. Our thanks also go to Dr. S. Bertagnoli,

École Nationale Veterinaire, Toulouse, France, for the primers and protocol for amplifying

the thymidine kinase gene of myxoma virus. We also thank the National Laboratory for

Veterinary Investigation (LNIV, Delegação do Norte) for infrastructural support.

63

3. Rabbit haemorrhagic disease (RHD)

65

3.1 Literature review

67

3.1.1 Introduction and brief history

Rabbit haemorrhagic disease was first reported in China in 1984. The disease spread

throughout Europe between 1987 and 1989 and is considered endemic since. In the

1990s, the etiological agent of RHD was characterised as a calicivirus (Ohlinger et al.,

1990), and the virus was introduced into Australia and subsequently New Zealand as bio-

control agent against wild European rabbit populations (Forrester et al., 2003).

In an attempt to understand the molecular epidemiology and ultimately the origins of RHD,

phylogenetic analyses based on partial sequences of the viral capsid protein VP60 of

many viral isolates from different countries were conducted (Asgari et al., 1999; Forrester

et al., 2006; Le Gall-Recule et al., 2003; Matiz et al., 2006; McIntosh et al., 2007; Moss et

al., 2002; Nowotny et al., 1997). These showed that there were up to six or more viral

groups or subgroups and that virus strains may or may not cluster according to their

geographical origin and/or the year of isolation, but that they did not always do so. Also,

evidence was gathered on the current and previous existence of RHDV-like viruses,

which may have been circulating more or less harmlessly in Europe for many years before

its first epidemic appearance in China in 1984 (Forrester et al., 2006; Moss et al., 2002;

Rodak et al., 1990). RHDV-specific signals were detected retrospectively by RT-PCR in

rabbit sera taken in the 1950s, concomitantly with the detection of RHD antibodies.

Infectiousness of viral RNA could not be proven, and recent work has shown that theses

sequences appear to be modern laboratory contaminants (Kerr et al., 2009; Moss et al.,

2002). Furthermore, rabbits with cross-reacting “pre-existing” antibodies against any

putative RHD-like viruses were not always fully protected against RHD, indicating that

antigenic differences may exist between viruses (Marchandeau et al., 2005).

New light was shed on these issues by recent extensive phylogenetic analyses (Kerr et

al., 2009; Kinnear and Linde, 2010). In these studies, four phylogenetic groups or lineages

were defined for RHDV: a) antigenic variants (RHDVa), b) strains from the Iberian

Peninsula, based on our work presented here (Muller et al., 2009), c) strains representing

the documented beginning of the epidemic, including the isolate obtained from China in

1984, and d) isolates mostly from Central Europe collected between 1989 and 2004. The

rabbit caliciviruses (RCV) displayed considerable divergence between each other and

also from all RHDV lineages, and a common ancestor has been estimated for over 200

years ago. Thus, virulent RHDV most likely did evolve from an avirulent RCV, but this

would not be a recent event and therefore would not explain the first observed epidemic in

1984 (Kerr et al., 2009). It is postulated that virulence of RHDV emerged in all RHDV

lineages on multiple occasions in the beginning of the 20th century (Kerr et al., 2009;

68

Kinnear and Linde, 2010). Various possible explanations as to why it was only in the

1980s that disease has been described were discussed (Kerr et al., 2009). The most likely

being that virulent RHDV emerged in farmed rabbits in Asia, in particular in China, aided

by a rapid expansion and intensification in that industry particularly in the second half of

the 20th century. Crossprotection conferred by co-circulating avirulent RHDV together with

concurrent socio-economical conditions would have hampered large scale disease

outbreaks earlier. Thus, the disease was not reported until 1984, when it was observed in

probably serologically naïve rabbits imported from Germany to China (Kerr et al., 2009).

3.1.2 Aetiology

Taxonomy

RHDV is a member of the Caliciviridae family (Meyers et al., 1991b; Ohlinger et al., 1990).

Based on sequence comparisons of the capsid gene, caliciviruses have been subdivided

into four genera (Green et al., 2000; ICTV, 2002). A fifth genus, Beco- or Nabovirus,

affecting cattle has been proposed (Oliver et al., 2006). The two genera Noro- and

Sapovirus include important human enteric pathogens. The genus Vesivirus comprises

San Miguel sea lion virus, vesicular exanthema virus, feline calicivirus and a recently

characterized putative new member, rabbit vesivirus (Martin-Alonso et al., 2005). The

genus Lagovirus contains RHDV and European brown hare syndrome virus (EBHSV)

(Green et al., 2000). RHDV and EBHSV are species-specific for rabbits and hares,

respectively. Despite an overall amino acid similarity of 76% of the major capsid protein,

they are serologically distinct, thus representing distinct virus species (Capucci et al.,

1991; Chasey et al., 1992; Lavazza et al., 1996; Wirblich et al., 1994). The most important

property of the genome of lagoviruses is the presence of only two ORFs as opposed to

the three ORFs observed in the other caliciviruses. Similar to other caliciviruses, RHDV is

nonenveloped with a capsid of icosaedrical symmetry. The virus is small, with a diameter

of 35-39nm. The depressions between the capsomers are cup-shaped, giving the family

its name “Calici”. The capsid is formed by the major structural protein, VP60.

Genome organization and replication The complete genome of RHDV consists of a single positive-stranded RNA of 7.437 kb

with a virus-encoded protein (VPg) covalently attached at its 5´end and a polyadenylated

3´end (Figure 1).

69

Figure 1 Monopartite, linear, single-stranded, pos itive-sense RNA genome of 7.3 to 8.3 kb. At 5’-terminus a virus protein (VPg)is cova lently linked to genome, whereas 3’-terminus is polyadenylated (Source: ViralZone ww w.expasy.ch/viralzone, Swiss Institute of Bioinformatics).

In virions and infected hepatocytes, both, genomic and a subgenomic RNA are found,

covalently linked to VPg and packaged into non-enveloped icosahedral capsids, (Meyers

et al., 1991a; Ohlinger et al., 1990; Parra and Prieto, 1990).

The genomic RNA of RHDV is composed by one long open reading frame (ORF1) that

encodes a large polyprotein of 257kDa which is proteolytically cleaved into non-structurals

proteins and a capsid protein (Gould et al., 1997; Meyers et al., 1991a; Meyers et al.,

2000; Wirblich et al., 1996). Most of the cleavage reactions are executed by the virus-

encoded trypsin-like cysteine protease (“3-C-like cysteine protease”), which is similar to

3C-proteases of picornaviruses. After cleavage, the seven non-structural proteins and the

major capsid protein are produced in the order NH2-p16-p23-p37-p29-p13-p15-p58-VP60-

COOH. Some of these proteins have larger precursor proteins, such as p60 (p23-p37),

p41 (p29+p13) and p72 (p15+p58), which are further cleaved or post-translationally

modified within the hepatocyte (see (Konig et al., 1998; Meyers et al., 2000) for review).

In RHD-infected cells a subgenomic RNA of 2.2 kb is transcribed, which is collinear with

the genomic RNA, and identical at the 3´end (Meyers et al., 1991a). The 5´end

corresponds to position 5296 of the genomic RNA. This subgenomic RNA also encodes

the viral capsid protein VP60 (Sibilia et al., 1995; Wirblich et al., 1996). It is thought that

VP60 produced by the subgenomic RNA is the type predominantly assembled into mature

virions and that it is two amino acids larger at the N-terminal than the genomic VP60.

However, both capsid proteins seem to be antigenically very similar, as shown by

monoclonal antibody reactivity and immunisation experiments (Sibilia et al., 1995). For the

generation of viral subgenomic RNA from genomic RNA template, two basic mechanisms

have been proposed, internal initiation or premature termination (Miller and Koev, 2000).

70

Recent studies suggest that internal initiation seems to be the strategy used by RHDV

(Morales et al., 2004). In any case, viral replication must occur because the RNA

dependent RNA polymerase is required for subgenomic RNA production (Miller and Koev,

2000). A second ORF (ORF2) is located at the 3´end of the genomic and subgenomic

RNA and encodes the structural protein VP10. This protein is thought to be important for

encapsidation of the viral genome with VP60, as VP60 alone is able to assemble into

virus-like but empty particles indistinguishable from those found in livers of RHDV-infected

animals (Sibilia et al., 1995; Wirblich et al., 1996).

The genomic RNA is infectious and serves as both genome and viral messenger RNA.

For replication, complementary negative sense ssRNA is synthesized from the genomic

RNA by the virally encoded RNA dependent RNA polymerase which is translated initially

from the RNA genome entering the cell. This minus strand then acts as a template for the

synthesis of new genomic RNA as well as of subgenomic RNA encoding for ORF2 protein

(Clarke and Lambden, 1997). The encapsidated nucleic acid is mainly of genomic origin,

but virions may also contain subgenomic RNA.

As RHDV cannot easily be propagated in tissue culture, many of the studies of genome

organization and viral replications have been made using different in vitro systems as RT-

PCR, ELISA and sequence analyses. However, uncertainties remain as the life cycle may

be influenced by cellular co-factors (Joubert et al., 2000; Wirblich et al., 1996). Using

cultured hepatocytes, additional proteins were detected, which were apparently not

observed in vitro, and which probably were produced by cellular post-translational

modifications (Konig et al., 1998). The results strongly suggested that virus replication had

taken place in the cultured hepatocytes, but whether infectious virus was produced

remained unanswered.

Structural proteins and antigenicity

The viral genome encodes two structural proteins, VP10, a small protein that probably

interacts with the genome during encapsidation and the major capsid protein VP60

(Wirblich et al., 1996). The major capsid protein VP60 is composed of 579 aminoacids.

This single structural protein determines functions related with capsid assembly, receptor

recognition, host specificity, strain diversity, and immunogenicity (Capucci et al., 1991;

Chen et al., 2004; Martinez-Torrecuadrada et al., 1998; Wirblich et al., 1996). The three-

dimensional structure of different caliciviruses has been studied by electron

cryomicroscopy and other methods (Chen et al., 2004; Prasad et al., 1994). Specific

information on lagoviruses are scarce, but conserved structural features across different

71

caliciviruses allow extrapolations. The RHDV capsid is composed of 90 dimers that form a

shell domain from which arch-like capsomers protrude (Prasad et al., 1994). The first 227

N-terminal amino acids form the shell domain (S), and the remaining C-terminal residues

(codons 287-579) make up the protruding domain (P) (Chen et al., 2004; Laurent et al.,

2002; Prasad et al., 1994). The S domain is the most internally, lying next to the RNA

genome. It is highly conserved among caliciviruses. This domain corresponds to the well-

conserved region B (Neill, 1992). The P domain is more variable, corresponding to the

region E (Neill, 1992). It can be further divided into the subdomains P1 and P2. The P1

domain lies more internally and is moderately conserved among caliciviruses. The P2

domain (codons 287-449) represents the most external part of the capsid protein and is

implicated with receptor and antibody interactions. This subdomain is hypervariable

between caliciviruses, also in the number of residues thus accounting for the structural

variation observed between caliciviruses (Chen et al., 2004). Antigenic determinants seem

to be located on both domains, S and P, as shown by binding studies of monoclonal

antibodies (mAb) (Capucci et al., 1995; Capucci et al., 1991; Martinez-Torrecuadrada et

al., 1998). Interestingly, mAb that reacted to internal epitopes cross-reacted with the

antigenically related but distinct EBHSV and also with so-called “smooth” RHDV particles

(s-RHDV), but were unable to inhibit haemagglutination. The other group of mAbs seemed

to react with the RHDV projections. They were able to inhibit haemagglutination, and to

confer in vivo protection. Thus, it is thought, that in the course of natural infection, the

main antigenic determinants of RHDV inducing a humoral immune response are located

on the C-terminal End of VP60 (Capucci et al., 1995; Capucci et al., 1991; Laurent et al.,

2002; Martinez-Torrecuadrada et al., 1998; Schirrmeier et al., 1999).

Only one single serotype of pathogenic RHDV is known, which contains two major

subtypes, denominated RHDV and the antigenic variants denominated RHDVa. The

antigenic variants were first detected in 1996 simultaneously in Italy and Germany

(Capucci et al., 1998a; Schirrmeier et al., 1999), but soon were described worldwide

(Farnos et al., 2007; Kerr et al., 2009; Le Gall-Recule et al., 2003; Matiz et al., 2006;

McIntosh et al., 2007). The RHDVa-specific antigenic epitope has been located to

residues 344 to 370 in the hypervariable N-terminal region E of VP60. A RHDVa strain-

specific monoclonal antibody (3B12) has been developed, which allows the detection of

these antigenic variants (Capucci et al., 1998a).

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Types of RHDV or related caliciviruses

In the context of RHD, several types of viruses or particles have been described, such as

the virus-like particles, core-like or smooth particles, and rabbit calicivirus (RCV). Some

have been reviewed recently (Kerr et al., 2009; Lavazza and Capucci, 2008). Virus-like

particles (VLPs) result from the expression of recombinant VP60. These capsid proteins

assemble spontaneously (Laurent et al., 1994; Sibilia et al., 1995). VLPs are

morphologically and structurally similar to native RHDV particles, but are empty, i.e.

devoid of viral genome. They are highly immunogenic and able to confer protection, and

as such useful for RHD structural analyses or vaccine development (Laurent et al., 1994;

Sibilia et al., 1995). Core-like particles (CLPs), also denominated smooth particles or s-

RHDV, are detected in the liver of rabbits that die with protracted forms of the disease, or

in non-lethal intestinal infections (Capucci et al., 1991; Granzow et al., 1996). The

structural protein of CLPs has a molecular weight of 28-30kDa and particles have a

diameter of 25-27nm. Thus, they are smaller than RHDV whose structural protein is of

60kDa and that have a diameter of 32-40nm (Granzow et al., 1996). CLPs are also

described as smooth particles because the spike projections - or cup-shaped depressions

- are absent (Capucci et al., 1991). Their smaller capsid protein represents the N-terminal

fragment and thus the conserved S domain of RHDV. For that reason they cross-react

with convalescent rabbit sera as well as mAbs directed towards the inner shell. They also

lack the biological activities of the C-terminal half of VP60 such as haemagglutination

(Capucci et al., 1991; Granzow et al., 1996). Liver homogenates with these particles do

not show hemagglutinating activity (Granzow et al., 1996). Not all of the CLPs contain

nucleic acid, some may remain empty. It is not clear if they are infectious, as infection

experiments are complicated by the fact that CLP preparations do retain some complete

RHDV particles (Granzow et al., 1996). The presence of these CLPs is associated with

sub-acute cases and lower mortality, and it has been discussed that the higher

immunogenicity of these particles could enhance the specific humoral immune response

(Martinez-Torrecuadrada et al., 1998). The origin of these CLPs is unclear. They could

represent a soluble protein that either derived from the proteolytic digestion of the capsid

protein, or from a truncated genome or defective gene expression (Granzow et al., 1996;

Lavazza and Capucci, 2008). It has also been postulated by Barbieri et al. (1997), that

these particles result from the immunological clearance by its host, especially by the

interaction with RHDV specific IgM (Lavazza and Capucci, 2008). That would explain the

occurrence of these particles in the livers of rabbits with subacute or chronic forms of the

disease.

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Rabbit caliciviruses are a group of viruses that display some similarities, but are

phylogenetically distant to RHDV (Bergin et al., 2009; Capucci et al., 1996; Forrester et

al., 2009; Forrester et al., 2007; Kerr et al., 2009; Moss et al., 2002; Strive et al., 2010;

Strive et al., 2009). The presence of a nonpathogenic virus closely related to RHDV was

already suspected at the time of the first RHD seroepidemiological surveys, when specific

RHDV antibodies were found in sera of farm and laboratory rabbits where no disease was

reported, and also in rabbit sera collected before the appearance of RHD (Moss et al.,

2002; Rodak et al., 1990). The first apathogenic calicivirus, denominated RCV, was

identified in Italy in the 1990s in rabbits from commercial farms, in which no history of

RHD had been recorded (Capucci et al., 1996). The virus was characterised antigenically

and genetically, and its infectious nature was proven by experimental inoculations.

Antigenically, this virus displayed an average amino acid identity of 91.5% to virulent

RHDV. Most of the differences were located in the C-terminal half of the capsid protein,

which constitutes the external surface of the virion. The main distinctive feature of the

RCV capsid protein was found to be a three amino acid deletion, corresponding to N-308,

A-309 and T-310 of RHDV. Importantly, despite these differences, RCV infection induced

antibodies which were cross-reactive with RHDV and able to confer protection from

challenge with pathogenic RHDV (Capucci et al., 1996). Western blot analysis using a

monoclonal antibody (mAb 5G3), that recognizes a conserved epitope on VP60 of all

lagoviruses, showed that similar to RHDV, a 60kDa protein and sometimes also a 30kDa

protein were recognized in intestinal samples of RCV infected rabbits. The latter smaller

protein is generally associated with a smooth capsid surface of the virions (Capucci et al.,

1996; Capucci et al., 1991). RCV genome organization and polyprotein processing were

found similar to RHDV (Capucci et al., 1995; Wirblich et al., 1995). A difference was found

for the putative ORF2 initiation codon, which in RCV is located 9 nucleotides downstream

as compared to RHDV(Capucci et al., 1996). In biological terms, RCV infections differed

from RHDV in that they were asymptomatic, tissue tropism was predominantly intestinal

and not hepatic, and much lower viral titres were found in internal organs. Whether the

differences on the capsid protein of RCV are directly responsible for the different virus

tropism and the lack of pathogenicity requires further clarification.

Several other RCV-like viruses have since been described. Partial or complete sequence

data is available for all, but pathogenesis studies have only been conducted for some. The

Ashington virus was obtained in the UK from a wild rabbit within a few hours of death

(Moss et al., 2002). It is assumed that the rabbit died from RHD, but certainty on the

virulence of the strain would require experimental infection. A few years later, an avirulent

RCV-like strain, denominated Lambay virus, was obtained from healthy wild rabbits on an

Irish island (Forrester et al., 2007). A high RHD seroprevalence was also observed in this

74

population, probably contributing to the absence of overt disease; however it was not

possible to isolate infectious virus. Recently an avirulent rabbit calicivirus, denominated

RCV-A1, has been characterised in Australia (Strive et al., 2010; Strive et al., 2009).

Detailed pathogenesis data is available. Viral loads have been determined by quantitative

real-time PCR for several organs. Genetically, RCV-A1 forms a branch of its own,

separating before the branches containing the Italian RCV, Ashington and Lambay

viruses, and thus possibly representing a more ancient lineage. Interestingly, strong

similarities were found between infections with RCV-A1 and those with the Italian RCV,

such as predominantly intestinal tropism, absence of clinical signs and seroconversion

capable of conferring protection against virulent RHDV (Strive et al., 2010; Strive et al.,

2009). Other novel divergent strains have been described recently from a longitudinal

study in Pitroddie, Scotland (Forrester et al., 2009). Here, healthy wild rabbits were

sampled for serology and RT-PCR on a monthly basis, during 12 months. Interestingly,

two types of viruses were obtained from liver samples, and thus were discussed to be co-

circulating. The divergent strains, RCV-like, were collectively termed Pit-WD, and strains

which clustered with other virulent RHDV strains were termed Pit-EP. Pit-WD strains were

obtained from healthy adult rabbits during months in which no RHD was observed. On the

contrary, Pit-EP strains were obtained from young seropositive rabbits, possibly survivors,

during an outbreak of RHD (Forrester et al., 2009). Noteworthy, it was not possible to

obtain viral RNA from wild rabbits that died with signs of RHD, so molecular diagnosis of

RHD remains to be confirmed. Further, pathogenesis and virulence of either Pitroddie

strains were not characterised by experimental inoculations. Whether intestinal tropism

plays a role in these infections remains unknown. Another RCV-like virus, denominated

MRCV, was reported from a clinical RHD outbreak in a commercial rabbitry in Michigan,

USA (Bergin et al., 2009). A case-fatality of 32.5% was observed in young and adult

rabbits. The virus was detected in livers, but no other organs were analysed. Interestingly,

inoculation experiments failed to reproduce disease, but viral RNA could be demonstrated

in several tissues, including the intestinal tract of inoculated rabbits. Sequence analysis

revealed similarities with the Italian RCV, especially the presence of one initiation codon

of ORF2, opposed to two observed in RHDV (Bergin et al., 2009).

3.1.3 Epidemiology

RHDV is an extremely contagious disease. The virus is very resistant to ambiental factors

(Smíd et al., 1991) and is readily transmitted by direct contact as well as by insect vectors.

The European rabbit (O. cuniculus) is the only susceptible host (OIE, 2009b).

Environmental temperature and humidity play an important role in the epidemiology of

RHD in immunologically naïve populations (Cooke and Fenner, 2002). Viral spread in wild

75

rabbit populations is slower in the hotter summer months. Viral particles are more rapidly

inactivated by temperatures above 37ºC, but they may survive up to weeks in rabbit

carcasses. Insect vectors such as flies and fleas have been shown to play a role in the

mechanical transmission of RHDV (Asgari et al., 1998). Studies in Australia have shown

that although RHD caused high mortalities in arid areas, it was less efficient in reducing

rabbit numbers in cooler areas of higher rainfall (Cooke and Fenner, 2002). The most

obvious declines in rabbit abundance were seen in Spain and Portugal and to some

extent in France, and much less in Northern Europe or Britain. On the Iberian Peninsula

(Cooke and Fenner, 2002), for example an early outbreak was observed in June 1988 in

Almeria, the arid south-east of Spain. Sequential sampling and analyses showed that

many of the surviving rabbits, both adult and subadult, had antibodies. During the

following year, the proportion of seropositive rabbits declined, as more young joined the

adult population. In May 1990, many adults were again seropositive, indicating that a

second outbreak had occurred. It took more than 5 years for RHD to reach all rabbits in

Spain. Even after 5 years of virus introduction, rabbit populations across Spain were being

held at approximately half of their former levels, but, in areas generally more favourable

for rabbits, populations recovered better than in others (Cooke and Fenner, 2002; Delibes-

Mateos et al., 2009).

Once RHD becomes established, its epidemiology changes by a variety of factors.

Rabbits that survive RHD have high levels of antibodies and are immune. Antibody titres

may decrease with time, but if animals are re-exposed to the virus, their titres may be

boosted (Cooke et al., 2000). It is unlikely that re-infection kills them, although this has

been described (Calvete et al., 2002b). So, further outbreaks depend on the presence of

susceptible individuals within given population, which most commonly arise through the

appearance of young animals. Breeding of rabbits is seasonal and associated with

pasture growth. In Southern Europe, typically, rabbits are born in the spring. Now, these

young rabbits, apart from the natural resistance during the first 4-6 weeks, may be further

protected until 13 weeks of age by passively transferred antibodies from immune mothers.

Maternal antibodies may not necessarily protect against infection, but may protect against

the lethal effect of the virus, assisting the young to become immune (Calvete et al., 2002b;

Cooke et al., 2000). This means, that there will be a whole range of susceptible to partially

or fully immune “new population” a few months after the breeding season. So, for

example, in the semi-arid Ebro Valley in northern Spain RHD outbreaks tended to occur in

the winter (Calvete et al., 2002b). Based on these observations, it has been proposed that

the pattern of RHD outbreaks was determined by the resistance of the young, and that the

impact of disease was determined by population density and hence level of contact

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between rabbits (Calvete et al., 2002b; Cooke and Fenner, 2002). This means that, as the

contact between rabbits increases, there will be initially higher mortality, because the virus

affects a high number of animals. The higher rate of virus spread then leads to a reduction

in the median age of infection, towards an age where the young rabbits are still resistant.

This means that many will survive, so the impact on RHD on the population is reduced, as

sufficient numbers of rabbits survive to maintain the basic breeding population.

These considerations agree with mathematical concepts of infectious disease

transmission and the R0 value or basic reproductive number. R0 is defined as the

expected number of secondary cases which one case would produce in a completely

susceptible population. R0 is based on the duration of the infectious period, transmissibility

(i.e. probability of infecting a susceptible individual during one contact) and contact rates

(number of new susceptible individuals contacted per unit of time) (Anderson and May,

1982; Dietz, 1993). Therefore R0 varies not only for different infectious diseases, but also

for the same disease in different populations (Dietz, 1993). The density of susceptible

hosts is important. The threshold density for transmission is the density of susceptible

hosts at which R0 = 1. At a lower density, each infectious individual will produce less than

1 new infection and so the disease will eventually die out in the population. Outbreaks of

epidemics and the persistence of endemic levels are thus associated with R0 greater than

one. This model has been applied to RHD (Boots et al., 2004). However, in this work, the

hypothesis is laid out in a slightly different way. The authors discuss that avirulent strains

will be favoured in dense populations with high population immunity, and that more

virulent strains will tend to circulate in sparse populations (Boots et al., 2004). Laid out in a

more classical way, in high density wild rabbit populations the virus may persist creating a

high proportion of seropositive and protected animals, thus reducing RHDV impact. In low

density wild rabbit populations, the virus is unable to persist, and eventually new fully

susceptible animals will replace older ones. The introduction of RHDV into this population

is likely to have an enormous impact in terms of mortality, i.e. will be noticed as an

epidemic. Field evidence exists that supports this concept (Delibes-Mateos et al., 2009;

Santos et al., 2006). For example, RHDV epidemiology was described in a low density

population of wild rabbits in northwestern Portugal, based on serology and virology data

obtained from hunted, apparently healthy animals (Santos et al., 2006). No RHDV was

detected in 66 liver samples, whereas antibodies were detected in 2/72 sera. The authors

discussed that RHDV could become epidemic in this population. Some mathematical

modelling has been carried out for RHD (Fouchet et al., 2009; White et al., 2002; White et

al., 2001; White et al., 2004). These models are based on field observations and

assumptions such as the existence of chronic infection that is lifelong and accompanied

77

with low level viral excretion (Fouchet et al., 2009; White et al., 2002), or the existence of

persistent infection and propagation of RHDV by pathogenic and apathogenic modes

(White et al., 2004). To our knowledge however, neither has been proven, and although

the persistence of viral RNA inspite the presence of antibodies has been demonstrated,

this viral RNA does not seem to be highly infectious (Gall et al., 2007; Moss et al., 2002).

Further studies are required to elucidate and clarify the role of RHDV persistence in

recovered hosts. Regarding the discussion on non-pathogenic rabbit caliciviruses, there is

field and experimental evidence of their existence (Capucci et al., 1998b; Forrester et al.,

2009; Monteiro and Alves, 1999; Strive et al., 2009). However many open questions

pertain and require further investigations.

3.1.4 Clinico-pathological features

RHD is clinically characterised by a peracute or acute clinical disease course with high

morbidity and mortality. Infected animals typically die within 24-72 hours after infection. In

the peracute form, clinical sign may absent, and animals that die are in good body

condition. In acute forms, various signs may be present, such as fever, anorexia, apathy,

prostration, respiratory signs, nervous signs and cries (Ferreira et al., 2006c; Lavazza and

Capucci, 2008; Shien et al., 2000). Severe leukopaenia and marked increase in liver

enzymes characterise the late stages of acute RHD (Ferreira et al., 2006c). Very few

animals display subacute or chronic infection (Fuchs and Weissenbock, 1992; Gall et al.,

2007; Teifke et al., 2002). Frequently these survive the acute disease, but may exhibit

jaundice and die 1 or 2 weeks later, probably due to liver dysfunction (Lavazza and

Capucci, 2008). The most consistent pathological lesions are hepato- and splenomegaly.

The liver typically appears with friable consistency, yellow discolouration and marked

lobular pattern. The tracheal mucosa may appear hyperaemic and the lungs congested. In

subacute and chronic cases the mucosae may be icteric. Hepatocytes are the most

important cell type for viral replication in early phases of infection and characteristic

findings are severe necrotising hepatitis and disseminated intravascular coagulation

(Ferreira et al., 2006c; Fuchs and Weissenbock, 1992; Gelmetti et al., 1998; Prieto et al.,

2000). Death is considered to result from massive liver damage and associated

disseminated intravascular coagulation which causes the haemorrhages observed in

various organs.

Upon infection, viral antigen or RNA can be detected in macrophages, circulating

monocytes as well as in reticuloendothelial cells in liver, lung, spleen and lymph nodes

(Gall et al., 2007; Gelmetti et al., 1998; Gould et al., 1997; Guittre et al., 1996; Kimura et

al., 2001; Prieto et al., 2000; Shien et al., 2000; Yang et al., 2008). In situ hybridization

78

using antisense and sense probes allows the identification of sites where active virus

replication is taking place. Antisense probes detect positive sense genomic RNA

representing replicated virions, and sense probes detect negative sense template RNA,

representing intermediates of active viral replication. The application of this technique in

acute RHDV infections in adults, has shown that viral replication in the liver begins as

early as 8 hours post-infection, and that viral replication takes place in hepatocytes and

also in Kupffer cells, alveolar and splenic macrophages (Gelmetti et al., 1998; Kimura et

al., 2001). Although intestinal lesions have been described in acute RHD, viral RNA has

not been detected in intestinal epithelium (Kimura et al., 2001). By PCR, positive signals

were found in liver, bile, spleen at 18 hours post-infection, in lung, kidney, mesenteric

lymph nodes, buffy coat at 26 hours, in thymus at 30 hours and finally in urine and faeces

at 36 hours post-infection, until death of the animal (Shien et al., 2000). Subacute

infections or chronic infections in adults are characterised by a longer clinical course, of

around 10 days, with death as consequence of infection. These forms seem to be rather

uncommon under natural conditions, but may be achieved experimentally by lowering the

infectious dose of the inoculum (Teifke et al., 2002). The subacute form of RHD is

characterised by progressive icterus due to early stages of liver cirrhosis (Teifke et al.,

2002). RHDV positive sense RNA was detected by in situ hybridization mainly in

macrophages of periportal areas of the liver, those lining the sinuses and red pulp of the

spleen and also in a few alveolar macrophages, which was interpreted as possible

attempts of clearance by phagocytosis rather than as active viral replication (Teifke et al.,

2002). Another less frequent outcome of infection with virulent RHDV of adult rabbits is

survival and seroconversion. Recently, the viral loads of survivors after experimental

infection have been determined (Gall et al., 2007). These animals showed fever between

2 and 4 days p.i., but no further clinical signs or RHDV-specific lesions were observed at

5, 7 or 15 weeks post-infection. Surprisingly however, viral RNA was detected by

quantitative real-time PCR in several organs and excretions throughout the experiment,

but with levels of viral loads decreasing with time (Gall et al., 2007). The highest viral load

was found in bile. No viral antigen could be detected in bile or the different tissues,

possibly due to masking by the high antibody titres present in these animals (Gall et al.,

2007). Importantly, infectiousness of the detected viral RNA could not be proven.

Seronegative in contact rabbits did not seroconvert, neither did the rabbit that was

inoculated with concentrated RT-PCR positive liver material of a survivor (Gall et al.,

2007). The authors discussed the possible inhibitory effect of antibodies present in the

inoculum, but it is also possible, that ongoing immune clearance mechanisms in

recovering rabbits actually inactivated any virus and that the detected viral RNA does not

represent infectious particles, and that these rabbits eventually turn into RNA-negative

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seropositive animals. The hypotheses related to RHDV persistence in these rabbits and

their epidemiological relevance through potential reactivation and viral excretion would

require further studies.

Susceptibility to disease upon RHDV exposure is strongly influenced by age. Even in the

absence of maternal antibodies, domestic rabbits less than four weeks do not develop

clinical signs or lesions upon infection (Prieto et al., 2000). Susceptibility to disease

increases from that age onwards, and by 40-50 days of age most rabbits are fully

susceptible (OIE, 2009b). Several studies have been conducted to understand possible

factors related to this innate resistance of young rabbits to RHD (Ferreira et al., 2006a;

Ferreira et al., 2004; Ferreira et al., 2005; Prieto et al., 2000; Shien et al., 2000). Clinically,

4-5 week old rabbits may show fever on the first days post-infection with virulent RHDV,

but remain otherwise asymptomatic (Shien et al., 2000). Antibodies appear from day 4 to

6 and remain for many weeks and are protective against disease in adulthood (Ferreira et

al., 2008; Lavazza and Capucci, 2008; Shien et al., 2000). Viral RNA could be detected by

RT-PCR in liver, bile and spleen from day 1 and in other organs such as lung, kidney,

mesenteric lymph node, buffy coat and faeces from day 2 p.i. (Shien et al., 2000). The

number of RNA positive organs then started to decrease after day 4 and at day 42 p.i.

viral RNA was only detected in bile and spleen, which has been related to virus clearance

by the immune system. Attempts to reactivate the infection by immunosuppressive doses

of dexamethasone were unsuccessful (Shien et al., 2000). Viral antigen detection using

immunohistochemistry, failed to observed VP60 antigen in 3 week-old infected rabbits. In

6 week-old infected rabbits a very small number of positive cells were observed in the liver

between 18 and 72 hours post-infection, but no labelling was found in lungs, spleen,

heart, intestine, thymus or brain (Prieto et al., 2000). The authors discussed that in young

rabbits only a small number of hepatocytes supported RHDV replication, possibly

indicating that changes in liver structure and function could be a determinant for RHDV

infectivity (Prieto et al., 2000). Recent studies showed that RHDV infection of young

resistant rabbits resulted in a transient decrease of blood heterophils and transient

hepatitis, characterised by an increase in liver transaminases between 18 and 72 hours

p.i. (Ferreira et al., 2004). The authors proposed that rabbits infected at a young age may

be long-term carriers for RHDV as they age. Differences in young versus adult RHDV-

infected rabbits were also found in the type of liver inflammatory reaction, which may

account for the different clinical outcomes. In the liver of young resistant rabbits, early

inflammatory cells were mostly heterophils, which were subsequently substituted by

mononuclear cells. In adult rabbits heterophils were the most predominant inflammatory

cell type, probably representing the response to the extensive hepatic cell death caused

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by the virus (Ferreira et al., 2004; Ferreira et al., 2005). To our knowledge however, the

infectivity of the observed RHDV components in the liver of young infected but

asymptomatic rabbits were not yet assessed.

Susceptibility of rabbits to RHD has also been linked to differential receptor expression

and binding. It has been argued that young rabbits do not readily become infected with

RHDV because receptors that enable the virus to attach to the intestinal mucosa are not

yet fully developed. RHDV binds to antigens of the ABH histo-blood group family, which

occur on human erythrocytes and also in rabbits on the mucosa of the upper respiratory

tract and the intestine, where they become fully functional at about 6 weeks of age

(Ruvoen-Clouet et al., 2000). However, they seem not to be the only receptors or factors

explaining infection, as intra-muscular RHDV inoculation bypassing the mucosae did

result in infection and in liver pathology similar to that observed with intra-nasal and oral

inoculation (Ferreira et al., 2006a; Ferreira et al., 2004). Further, ABH tissue antigen

receptors do not appear to be expressed on liver cells, which are considered the main

host cell for RHDV replication (Ruvoen-Clouet et al., 2000), thus the role of other putative

receptors needs to be determined. Recent studies have shown genetic polymorphism on

Fut2 and Sec1 genes of on rabbits, which are related to the expression of the H histo-

blood group antigen binding to RHDV (Guillon et al., 2009). Different allele profiles have

been related to sensitivity to RHDV but this association was based on estimates of local

mortality rates of wild rabbits. The true status of these rabbits as to RHDV infection or

recovery was not assessed. Sophisticated technology such as nuclear magnetic

resonance has been applied to further understand RHDV binding to histo-blood group

antigens, and has shown that L-fusose is a minimal structural requirement (Rademacher

et al., 2008). Age-related changes in liver structure and function as well as the continuing

maturation of the immune system may thus contribute to pathogenicity of RHD, but further

studies are required to enhance our understanding on RHDV-receptor binding, viral entry

and, of course, resistance to disease.

3.1.5 Laboratory diagnosis

RHD can be suspected by history and clinical signs, pathological lesions and confirmed by

a variety of specific laboratory tests (Capucci et al., 1991; Lavazza and Capucci, 2008;

OIE, 2009b). Direct diagnostic methods allow the detection of viral particles, viral antigen

or viral nucleic acids. As no cell culture systems are available, the demonstration of

infectious virus requires the experimental inoculations of rabbits. The liver is considered

the target sample, as in acute infections it contains large amounts of virus. Other suitable

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samples are spleen and serum (OIE, 2009b). Electron microscopy has been used for the

visualisation and morphological studies of RHDV particles as well as induced hepatic

lesions (Capucci et al., 1991; Ferreira et al., 2006b; Ferreira et al., 2006c; Granzow et al.,

1996; Ohlinger et al., 1990; Prasad et al., 1994). Despite being a valuable research tool, it

is time-consuming and not very sensitive and thus of limited use as diagnostic test.

RHDV antigen can be detected by several test systems. The haemagglutination (HA) test

on 10% liver or spleen homogenates was used as the first diagnostic test, and is still

widely used (Capucci et al., 1991; Nowotny et al., 1997; Shien et al., 2000). Human

erythrocytes type “O” are required, as it as the virus binds to antigens of the ABH histo-

blood group family (Ruvoen-Clouet et al., 2000), which is probably absent on erythrocytes

of other species. A small percentage of false negative HA results can be observed in

cases of protracted forms or chronic RHD, in which core-like (smooth) particles exist

(Capucci et al., 1991). Various ELISA systems have been developed to detect RHDV

antigen (Capucci et al., 1991; OIE, 2009b). Most are based on a sandwich system, using

varying types of monoclonal or polyclonal RHDV antibodies as catcher and tracer

reagents (Capucci et al., 1995; Ohlinger et al., 1990). An ELISA based on monoclonal

antibodies for distinguishing RHDV subtypes is also available (Capucci et al., 1991; OIE,

2009b). RHDV antigen can be detected in cryosections or in formalin fixed tissues by

immunofluorescence, immunohistochemistry and in situ hybridization, but these assays

are mainly used for research purposes rather than diagnosis (Gelmetti et al., 1998;

Kimura et al., 2001; OIE, 2009b; Prieto et al., 2000; Teifke et al., 2002). Since the mid

1990s, several PCR-based techniques have been described for the detection of RHDV

RNA, including single and nested PCR (Bascunana et al., 1997; Forrester et al., 2003;

Gould et al., 1997; Moss et al., 2002; Shien et al., 2000; Yang et al., 2008),

immunocapture RT-PCR (Le Gall-Recule et al., 2001) and quantitative real-time RT-PCR

(Gall et al., 2007). All these methods vary slightly in their analytical sensitivity, but are

considered more sensitive than antigen ELISA, and thus currently represent the

diagnostic test of choice (Gall et al., 2007; Guittre et al., 1995; OIE, 2009b). For example,

real-time RT-PCR allowed the detection of 10 copies of viral RNA per well, whereas

positive ELISA results corresponded to a minimum of 107 copies of viral RNA (Gall et al.,

2007). However, it should be kept in mind, that the different types of direct tests detect

different components of the virus, and thus may yield different results during the course of

infection. In particular, in the course of recovery from acute infection or in non-lethal

infections of young animals, as well as in vaccinated but infected rabbits, viral nucleic acid

has been detected (Gall et al., 2007; Gall and Schirrmeier, 2006; Shien et al., 2000). Viral

antigen or infectious virus has not always been demonstrated in these PCR positive

samples, and so PCR results may require careful interpretation (Forrester et al., 2003;

82

Gall et al., 2007; OIE, 2009b; Shien et al., 2000). There are at least two possible

explanations for this incongruence. In animals where specific RHD-antibodies are also

detectable, these may interfere with the antigen detection system. In other words, viral

structural proteins are produced but not detected by the assays. Another possible

explanation is that virus clearance mediated by specific immune responses is ongoing and

that virus replication is aborted and antigen is produced below detection threshold,

whereas remnants of viral RNA are being amplified by PCR.

Indirect or serological methods for the detection of RHD-specific antibodies include the

haemagglutination inhibition (HI) test and several ELISAs. Animals that recover from

infection develop detectable specific antibodies 4-6 days p.i., and this humoral immune

response has been correlated with protection. The HI was the first test to be developed. It

is convenient, as it uses Human erythrocytes type “O” and 4-8 haemagglutination units of

RHDV antigen, usually prepared from infected liver homogenates. Nevertheless, this

method is quite time consuming as it requires heat inactivation and/or kaolin pre-treatment

of sera to increase sensitivity (Capucci et al., 1991; OIE, 2009b). Many laboratories have

now replaced HI by ELISAs, which are quicker and much easier to perform. For routine

diagnosis, the competition ELISA (cELISA) using monoclonal antibodies detecting

antigenic determinants on the external surface of RHDV is considered highly specific and

is currently the reference test for RHD (Lavazza and Capucci, 2008). This test correlates

strongly with protection from disease. An indirect ELISA (iELISA) has been developed, in

which purified RHDV is adsorbed to the solid phase, and in which internal antigenic

determinants are exposed. This ELISA is less specific than the cELISA, as it allows to

detect cross-reactive antibodies, induced by different lagoviruses such as RCV or EBHSV

(Capucci et al., 1991; OIE, 2009b). These do not necessarily correlate with protection.

Isotype-specific ELISAs have been described which allow the detection of IgM, IgA and

IgG, and which have been used for epidemiological and pathogenesis studies (Lavazza

and Capucci, 2008; OIE, 2009b).

3.1.6 Control

There is no treatment for RHD infected rabbits, thus prophylactic measures are essential

to reduce the impact of RHD in a given population. In that context, it is useful to make

separate considerations for rabbits held in captivity, such as domestic pet or commercially

reared rabbits, and those free-living in nature, i.e. wild rabbits.

Domestic rabbits are typically regularly handled by man. This makes the application of

control measures relatively easy. Sanitary measures include vector control, appropriate

83

hygiene, good management practices and bio security which prevent the contact with wild

rabbits or other wildlife that could serve as mechanic vectors of the virus.

Immunoprophylactic measures represent the centrepiece of RHD control. Currently

several companies offer commercially available vaccines. All are of parenteral

administration. In Portugal all vaccines are based on whole inactivated RHDV, obtained

from liver homogenates of experimentally infected rabbits. All but one vaccines are

monovalent. The bivalent vaccine confers additional protection against myxomatosis.

Recommended vaccination schemes vary slightly, but centre on the following guidelines:

all animals shall be vaccinated between 8 and 10 weeks of age, fattening animals only

once and breeders shall be revaccinated yearly thereafter. In situations of high

epidemiological pressure, early vaccination may be practiced, starting at 4 weeks of age,

revaccinating 4 to 6 weeks later and thereafter annually breeders only. Several subunit

vaccines based on recombinant VP60 have been developed since the mid 1990s, but to

our knowledge none is available commercially. Different expression systems have been

used such as vaccinia virus (Bertagnoli et al., 1996b), canarypox virus (Fischer et al.,

1997), baculovirus (Laurent et al., 1994; Nagesha et al., 1995; Soledad Marín et al.,

1995), insect larvae (Pérez-Filgueira et al., 2007), myxoma virus (Barcena et al., 2000b;

Bertagnoli et al., 1996a; Torres et al., 2000a) and others. Most of these recombinant VP60

proteins or viral vectors encoding VP60 are immunogenic and successfully protect rabbits

against RHD.

The application of current RHD control measures in wild rabbit populations is, similar to

other wildlife species, hampered by several factors, such as difficult access to individual

animals, unknown populations sizes, poorly understood morbidity and mortality rates

because ill animals may die hidden and undetected, undefined responsibility as wildlife

seldom is under any ownership, etc. (Artois et al., 2001). Thus, the application of sanitary

measures and of commercially available vaccine which require parenteral application is

considered impractical and cost-inefficient for long-term and large scale control of RHD in

wild rabbits. In many areas of the Iberian Peninsula RHDV has had a major long lasting

impact on the reduction of wild rabbit densities, but other factors acting in synergy have to

be considered. Most cited examples include hunting pressures and habitat loss and

fragmentation mainly due to intensification of agriculture as well as urbanisations (Ward,

2005). Thus, RHDV control measures and in particular vaccination, form part of other

important actions aiming to increase wild rabbit populations. Habitat management

measures that can be applied to increase wild rabbit populations include improvement of

the availability of foodstuff, water and shelter, decrease of natural predators. These may

be coupled with active restocking initiatives (Delibes-Mateos et al., 2009; Letty et al.,

84

2008; Rouco et al., 2006a). As such, RHD vaccination of wild rabbits do have a

predominantly sporadic application in these local translocation and restocking events

(Cabezas et al., 2006; Calvete et al., 2004a; Moreno et al., 2004; Piorno, 2006; Rouco et

al., 2006b). Although vaccination is a reasonably common practice, depending on local

RHD infection pressures and population immunity, it may not be necessary to vaccinate

rabbits to successfully increase wild rabbit numbers (Delibes-Mateos et al., 2009; Rouco

et al., 2006a; Rouco et al., 2006b), and in one study, vaccination has even been found to

have a negative impact on the survival of wild rabbits due to the stress caused by

handling (Calvete et al., 2004b). Thus, most drawbacks of the use of RHD vaccines in wild

rabbits have been related to the need of individual delivery of the vaccine. As a

consequence, novel approaches are being explored to specifically immunise wild rabbit

populations by alternative delivery systems, such as oral uptake or horizontal spread. For

example, the immunodominant protein VP60 expressed in vaccinia virus (Bertagnoli et al.,

1996b) or transgenic potato plants (Martin-Alonso et al., 2003) has been shown to confer

protection upon oral administration. It is likely that VP60 obtained by other expression

systems are also capable to do so, but this has often not specifically been assessed

(Fischer et al., 1997; Laurent et al., 1994). The formulation of oral RHD vaccine baits for

wild rabbits in analogy to those applied for rabies and classical swine fever control in

European wild life reservoirs (Artois et al., 2001) is thus theoretically conceivable.

Horizontal spread of vaccines among wild rabbits is another attractive option, as only a

small proportion of animals would need to get actively immunized. One of the above

mentioned recombinant vaccines based on myxomavirus expressing VP60 meets this

criterion (Barcena et al., 2000b; Torres et al., 2000a). Rabbits immunised by either routes,

oral or subcutaneous, were protected against RHD and myxomatosis. This life modified

vaccine virus was capable of immunising rabbits by direct contact. Importantly other

inconvenient or risks are associated with this vaccine in particular, as has been discussed

above, in the context of myxomatosis control.

85

3.2 Evolution of Rabbit haemorrhagic disease virus (RHDV) in wild rabbits ( Oryctolagus cuniculus ) in the Iberian Peninsula.

Adapted from: Veterinary Microbiology (2009), 135, 368-373

A. Mullera,b*, J. Freitasa,b, E. Silvaa,b, G. Le Gall-Reculéc, F. Zwingelsteinc, J. Abrantesd,e, P. J. Estevesd, P. C. Alvesd,e, W. van der Lood,e, J. Kolodziejekf,g, N. Nowotnyf and G. Thompsona,b

i) Department of Veterinary Clinics, Institute of Biomedical Science Abel Salazar (ICBAS), University of Porto, P-4099-003 Porto, Portugal

j) Multidisciplinary Unit of Biomedical Investigation (UMIB), University of Porto, P-4099-003 Porto, Portugal

k) French Agency for Food Safety (AFSSA); Laboratory for studies and research on poultry, pig and fish farming; Virology, immunology, and parasitology in poultry and rabbits Unit; B.P. 53, F-22440 Ploufragan, France

l) Centre of Investigation for Biodiversity and Genetic Resources (CIBIO), University of Porto, P-4485-661 Vairão, Portugal

m) Department of Zoology and Antropology, Faculty of Sciences, University of Porto, P-4150-150 Porto, Portugal

n) Zoonoses and Emerging Infections Group, Clinical Virology, Department of Pathobiology, University of Veterinary Medicine, A-1210 Vienna, Austria

o) Animal Production Unit, FAO/IAEA Agriculture & Biotechnology Laboratory, Agency's Laboratories, Seibersdorf, International Atomic Energy Agency, Austria *Corresponding author: Phone: +351-252-660410, Fax: +351-252-661780. E-mail adress: [email protected]. Postal address: ICAV–UP, Rua Padre Armando Quintas, 4485-661 Vairão, Portugal

87

Abstract

To date information on rabbit haemorrhagic disease virus (RHDV) in Spain and Portugal

has been scarce, although the disease is endemic and continues to have a considerable

impact on species conservation and hunting industry. We analysed RHDVs obtained

between 1994 and 2007 at different geographic locations in Portugal (40 samples), Spain

(3 samples) and France (4 samples) from wild European rabbits (Oryctolagus cuniculus)

that succumbed to the disease. Phylogenetic analyses based on partial VP60 gene

sequences allowed a grouping of these RHDVs into three groups, termed “Iberian”

Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups clustered separately,

though not far from earlier RHDVs of Genogroup 1 (containing e.g. strain “AST89”), but

clearly distinct from globally described RHDV strains of Genogroups 2-6. This result,

supported by a bootstrap value of 76%, gives rise to the hypothesis that the virus evolved

independently since its introduction to wild rabbit populations on the Iberian Peninsula,

with the Pyrenees acting as a natural barrier to rabbit and hence to virus dispersal. No

differences were observed in RHDV sequences obtained from geographic regions where

the rabbit subspecies Oryctolagus cuniculus algirus prevails compared with those

obtained from Oryctolagus cuniculus cuniculus.

Key words: RHDV; Phylogeny; Iberian Peninsula; oryctolagus cuniculus; wild rabbit

88

1. Introduction

Rabbit haemorrhagic disease (RHD) outbreaks on the Iberian Peninsula were first

described in 1988 and the disease is endemic since (Anonymous, 1989; Arguello Villares

et al., 1988). Wild rabbit abundance has declined over 30% since, raising conservational

concerns and having a negative economic impact on the hunting industry (Alves and

Ferreira, 2004; Moreno et al., 2007; Villafuerte et al., 1995). The European rabbit that

nowadays is distributed in many continents originated from the Iberian Peninsula, where

two well separated subspecies exist, O. c. cuniculus in the northeast and O. c. algirus in

the southwest, which form a contact zone in the central region (Branco et al., 2000;

Geraldes et al., 2006; Monnerot et al., 1994) (Figure 2).

Toledo1 sample 1994

Pyrénées-Orientales3 samples 2000-2

O. c. cuniculus

Northern Portugal:3 samples 1996-715 samples 2006-7

Central Portugal:33 samples 1994-7

Southern Portugal:14 samples 2004-5

O. c. algirus

O. c. cuniculus

PORTUGAL

S P A I N

Alicante1 sample 2004Albacete1 sample 2004

Contact zoneO. c. cuniculus +

O.c.algirus

F R A N C E

Manche1 sample 2005

Figure 2 Map of the Iberian Peninsula and South of France displaying the geographic origin of the RHDV samples analysed in t his study and the time period they were collected. The distribution areas of the wild rabbit subspecies Oryctolagus cuniculus algirus and Oryctolagus cuniculus cuniculus as well as the contact zone across the Iberian Peninsula are indic ated.

The subspecies O. c. algirus is endangered due to various factors, but, most importantly,

due to high mortality rates of RHD epizootics (Alves and Ferreira, 2004; Moreno et al.,

2007).

89

Surprisingly, the available genetic information on rabbit haemorrhagic disease virus

(RHDV) in Spain and Portugal is scarce (Boga et al., 1994; Nowotny et al., 1997; Parra

and Prieto, 1990), although many studies have been conducted in other countries

(Forrester et al., 2007; Le Gall-Recule et al., 2003; Le Gall et al., 1998; Matiz et al., 2006;

Moss et al., 2002; Nowotny et al., 1997). These phylogenetic analyses focus

predominantly on sequences located on the viral capsid protein VP60 gene, and although

inferences could be made in some studies, linking RHDV genetic groups to variables such

as the years of sampling, geographic origin, or virulence of strains, these relations were

not always observed and therefore cannot be generalized.

Our objectives were to characterise RHDV strains obtained from wild rabbits presumed to

have died from RHD in different years and geographic locations in the Iberian Peninsula

as well as to characterise RHDV strains obtained from rabbits in distribution areas of O. c.

algirus.

2. Materials and Methods

2.1. Samples

In Portugal, a total of 65 wild rabbits that were found dead during known epidemics of

RHD were collected and immediately stored at -20ºC. The animals were collected from

different geographic areas in different years (Figure 2). Thirty-six samples were collected

between 1994 and 1997 for a study aiming to determine the presence of RHDV as cause

of death in wild rabbits. These had previously tested positive by antigen capture ELISA

(Capucci et al., 1991). These and the remaining liver samples (collected 2004 - 2007)

were processed in the Laboratory of the University of Porto.

Seven RHDV sequences were obtained from wild rabbits from Spain and France (Table 4;

Figure 2). The sequences of the two Spanish RHDVs collected in Alicante and Albacete in

2004 were kindly provided by Dr. Ramon Soriguer (CSIC). The sequence from the Toledo

specimen was obtained in the Laboratory of the University of Porto. It was the only nested

RT-PCR positive sample out of 39 hunted wild rabbits collected in 1994. The four recent

RDHV sequences from France corresponded to three strains (“2000-08”, “2001-23” and

“2002-20”) that had been collected in 2000, 2001 and 2002, respectively, in the

Department “Pyrénées-Orientales”, and to strain “2005-01” that was obtained in 2005 in

the Department “Manche”. These sequences were obtained in the Laboratory of AFSSA,

Ploufragan.

90

Table 4 Genbank accession numbers of RHDV sequence s included in the phylogenetic analysis

RHDV Group Strain GenBank Reference

Group IB3 2004-03 EU192134

France_2001-23 AM746980

Spain_Alicante_2004 AM884394

Spain_Albacete_2004 AM884395

2006-01 EF571322

2006-04 EF571325

2006-09 EF571330

2005-01 EU192140

France_2000-08 AJ319594

France_2002-20 AM746981

2007-01 EU192135

Group IB2 1995-01 EU192132

1994-02 EU192136

1997-03 EU192139

Spain_Toledo_1994 EU192137

1996-08 EU192138

Group IB1 1997-02 EU192133

1994-07 EU192131

Genogroup 1 Eisenhuettenstadt Y15440

AST89 Z49271

France_SD Z29514

Spain_MC-89 L48547

Genogroup 2 China_WX/1984 AF402614

Mexico89_ AF295785

Germany_FRG M67473

Czech_V351 U54983

Genogroups 3-5 France_00-13 AJ495856

France_2005-01 AM085133

Wriezen Y15427

Hagenow Y15441

Meiningen Y15426

Frankfurt Y15424

France_95-10 AJ535094

Genogroup 6 CUB5-04 DQ841708

France_03-24 AJ969628

France_99-05 AJ302016

Root RCV X96868

2.2. RT-PCR and sequencing

RNA was extracted from liver homogenates and cDNA was synthesized using random

priming and M-MLV reverse transcriptase (Invitrogen). Nested PCR was used to amplify

partially the RHDV VP60 capsid protein gene (Moss et al. 2002), corresponding to

positions 6157 to 6703 of strain AST89 (GenBank accession number Z49271). The

91

amplicons of 547bp were directly sequenced in both directions employing the nested PCR

forward and reverse primers.

2.3. Sequence analysis

From Portugal only the 40 non-identical sequences were further analysed. For

phylogenetic analysis, different methods (minimum evolution, maximum parsimony,

maximum likelihood and neighbour-joining) were applied using the software package

MEGA3.1 (Kumar et al., 2004). The obtained trees showed similar clustering of the

sequences. The neighbor-joining tree was presented as considered adequate for

comparing relatively short sequences (Takahashi and Nei, 2000). The nucleotide

substitution model of Kimura-2-parameter was used, and the reliability of the tree was

tested by bootstrap analysis of 1000 replicates. The obtained sequences were compared

with published homologues from GenBank database (NCBI). Eighteen sequences

representing previously described RHDV genogroups were selected and included (Table

1). These sequences were grouped into Genogroup1 to 6, adapted to the classification

used by Le Gall-Reculé et al. (2003). The Italian non-pathogenic rabbit calicivirus “RCV”

(GenBank accession number X96868) was used to root the tree. The amino acid

sequences and the consensus sequences of each group were deduced and aligned.

Results

The phylogenetic tree estimated by the neighbor-joining method (Figure 3) shows the

clusters formed by RHDV strains from the Iberian Peninsula and the south of France,

which have been termed “Iberian” Groups IB1 to IB3, together with the clusters containing

“known” RHDV strains representing Genogroup 1, Genogroup 2, Genogroups 3-5 and

Genogroup 6 antigenic variants or “RHDVa” strains (Capucci et al., 1998). The Groups

IB1 and IB2 contain sequences collected between 1994 and 1997, whereas Group IB3

includes more ones collected between 2000 and 2007. Each group contains sequences

from different geographic locations, except Group IB1, that contains five sequences

obtained in 1994 and 1997 from Santarém district of central Portugal. Group IB2 contains

strains collected between 1994 and 1997 from northern and central Portugal as well as

the virus obtained in 1994 from Toledo, Spain. Group IB3 includes the more recent (2004-

2007) Portuguese strains collected in the southern province Algarve (2004-2005) as well

as in the North (2006-2007). Also included in Group IB3 are the sequences from three

French RHDVs that had been identified between 2000 and 2002 in the Department

“Pyrénée-Orientales”, and the two Spanish strains “Albacete” and “Alicante” collected in

2004.

92

Figure 3 RHDV strains from Portugal cluster separa tely from known genogroups based on phylogenetic analysis of partial VP60 gene sequences. The neighbour joining tree was rooted with RCV. Bootstrap probabi lity values above 75% for 1000 replicate runs are indicated at the nodes.

Group IB 32000-2007

Group IB 21994-1997

Genogroup 1

Group IB 11994-1997

Genogroup 2

Genogroups 3-5

Genogroup 6

2004-02 2004-10

2004-07 2004-03 2004-01 2004-09 2004-05

France 2001-23 Spain Alicante 2004

Spain Albacete 2004 2006-01 2006-04 2006-09

2004-08 2005-03

2005-01 2005-04

France 2000-08 France 2002-20 2007-01 2007-02

1995-01 1994-10 1995-09

1994-02 1995-05

1996-01 1997-03 Spain Toledo 1994

1996-04 1997-01

1995-04 1996-05 1997-05 1994-12

1995-02 1995-12

1996-07 1996-08

1995-10 1995-11

Eisenhuettenstadt AST89

France SD Spain MC-89

1997-02 1994-01

1994-03 1994-04

1994-07 France 88

China WX/1984 Mexico89

Germany FRG Czech V351

France 00-13 France 2005-01

Wriezen Hagenow

Meiningen Frankfurt

France 95-10 Saudi Arabia

CUB5-04 France 99-05 France 03-24

RCV

100

100

100

99

99

9199

9699

78

99

99

98

8698

94

93

97

85

77

76

82

94

87

84

82

90

0.02

93

All described RHDV sequences from Portugal, Spain and the South of France

represented in Groups IB1, IB2 and IB3 formed, together with Genogroup 1 sequences, a

distinct cluster separated from all previously described RHDV genogroups, supported by a

bootstrap value of 76%. Group IB2 and IB3 viruses share a common ancestor with

Genogroup 1 viruses (bootstrap 82%). Group IB1, which contains five sequences from

central Portugal (bootstrap 97%), separated before the known RHDV Genogroup 1. The

recent French strain “2005-01” obtained in the Department “Manche” clustered within

Genogroups 3-5, close to another French strain collected in 2000 (“00-13”).

The grouping of the RHDVs was supported by the bootstrap values as well as a

comparison of nucleotide proximities. Nucleotide similarities of 97%, 98% (excluding strain

“1996-08”) and 99-94% were observed within groups IB1, IB2 and IB3, respectively.

Lower values were observed between groups and genogroups. Group IB2 strain “1996-

08” displayed a higher genetic distance proportionally to all other strains, but was still

closer to RHDVs of its own group IB2 (96-95%) than to Genogroup 1 strains (94%) or

others (less than 94%).

The comparison of the group consensus sequences of the deduced RHDV VP60

polymorphic amino acid composition showed that, in comparison to Genogroup 1, Group

IB1 presents three replacements: A2176N, N2178S and S2194N. The strains contained in

Groups IB2 and 3 are characterised by five amino acid replacements when the respective

group consensus sequences are compared to those of Genogroups 1 to 6: R2062Q, L2152P,

T2163I, A2176T and V2230I. The more recent Iberian and French RHDV strains of Group IB3

are further characterised by the residues P2069N, N2071G and I2092V, which is not a feature

of the other described strains.

4. Discussion

In Spain and Portugal, rabbit mass mortality due to RHD was observed in the late 1980s

and early 1990s, coinciding with the observation of the epidemic in other European

countries, suggesting that the origin of the virus was the same. Accordingly, Genogroup 1

sequences were obtained from RHD outbreaks in 1989 not only in Spain (Boga et al.,

1994; Parra and Prieto, 1990), but also in France (“France SD”) and Germany

(“Eisenhuettenstadt”). No genetic information has ever been published on more recent

RHDVs circulating in wild rabbits in Spain, and none ever from Portugal. Here, forty-seven

RHDV strains from wild rabbits (Oryctolagus cuniculus) obtained between 1994 and 2007

at different geographic locations in Portugal, Spain and France (Fig. 2) were characterised

based on their partial VP60 gene sequences and grouped into three groups, termed

“Iberian” Groups IB1, IB2 and IB3. All these “Iberian” Groups sequences formed, together

94

with those of Genogroup 1, a distinct cluster separated from all other described RHDV

genogroups. Group IB2 and IB3 viruses share a common ancestor with Genogroup 1

viruses, whereas Group IB1 strains separated before Genogroup 1 RHDV.

In geographically neighbouring France, virulent RHDVs of two distinct Genotypes (1 and

2) were initially present between 1987 and 1990, but were subsequently replaced by other

RHDV strains (Le Gall-Reculé et al., 2003). Despite the lack of data between 1989 and

1994, none of the RHDV sequences from Spain and Portugal clustered within Genogroup

2, suggesting that only Genogroup 1-like virulent RHDV strains predominated initially and

that these were subsequently replaced by strains here grouped as IB2 and IB3, indicating

that RHDV could have evolved separately in the Iberian Peninsula since. Alongside the

nucleotide sequence analysis, the observed amino acid polymorphisms suggest that the

substitutions observed in Groups IB2 and IB3 seem to have become fixed around 1994

and are still present in RHDVs circulating in wild rabbits in the Iberian Peninsula, but not

elsewhere. It is therefore tempting to speculate that the Pyrenees may act as a natural

barrier, constraining wild rabbit and hence virus dispersal and evolution, similar to what

was observed on Lambay island or in New Zealand (Forrester et al., 2007; Forrester et al.,

2003).

Phylogenetic, serological and epidemiological studies related to RHD have led to the

hypothesis that attenuated or avirulent forms of the virus have been circulating in Europe

before the 1980s (Forrester et al., 2003; Moss et al., 2002; Nowotny et al., 1992; Rodak et

al., 1990). The drastic reduction in wild rabbit numbers observed on the Iberian Peninsula

has been historically unprecedented, suggesting that, if any RHDV-like viruses were

circulating in wild rabbit populations at that time, they must have been highly host-adapted

but not cross-protective. Among the 39 wild rabbits obtained from hunters in 1994 from

the Toledo region, only one tested positive by nested RT-PCR (2-3% of the total). Also,

more recently in 2006, liver samples obtained from 30 healthy wild rabbits hunted in 2006

in Pancas (Lisbon district) tested negative by nested RT-PCR (data not shown). This data

is different from the high percentage (40-60%) of PCR positive healthy wild rabbits found

in New Zealand (Forrester et al., 2003), suggesting that in the Iberian Peninsula RHDV-

like viruses are either not present or are present at a very low incidence. Climatic

differences together with potentially circulating avirulent “RHDV-like” strains in cooler and

more humid countries (Cooke, 2002) may explain the observed differences.

The categorisation of the samples obtained in Portugal between 1994 and 1997 in two

distinct groups (IB1 and IB2) was quite surprising, as all were collected during the same

time period, and because the five RHDVs that formed Group IB1 originated from the same

district (Santarém) as others that grouped within IB2 (RHDV strains “1994-10”, “1995-01”,

“1995-10”, “1995-11”), suggesting that two different RHDVs were circulating concomitantly

95

in the presence of disease and mortality. The alignment of the amino acid sequence group

consensus sequences further strengthens this hypothesis, as Group IB1 differed in eight

positions when compared to Group IB2, but in only three when compared to Genogroup 1.

Bearing in mind, that RHDV infection can perpetuate in rabbit holdings due to virus

persistence (Gall et al., 2007; Moss et al., 2002), i.e. without repeated introduction of the

virus from the environment, the question arises whether the detected subgroups are

typical for wild rabbits of this region. Our data seem to support this hypothesis, as e.g.

shown by the proximity of all recent wild rabbit RHDVs collected on the Iberian Peninsula

and South of France forming Group IB3. This does not necessarily mean that the same

RHDV circulate in wild and farmed or pet rabbits. We characterised a RHDV from an

outbreak that occurred in January 2007 in a commercial rabbit farm in the North of

Portugal, which was classified as an antigenic variant RHDVa, based on partial VP60

gene sequence analysis (own observations) and on antigenic characterisation using a

panel of monoclonal antibodies performed by the OIE Reference Laboratory (Dr. L.

Capucci).

Phylogenetic analyses of RHDV strains are commonly based on sequences representing

only a fragment of the capsid VP60 protein gene, however, discrepancies between

authors in relation to the number of RHDV groups and subgroups and also in tree

topology, warrant harmonisation of RHDV typing. Due to recombination events

contributing to RHDV variability, it may be more appropriate to investigate the complete

sequence encoding VP60, or ideally, the full length genome, rather than partial capsid

gene sequences (Abrantes et al., 2008b; Forrester et al., 2008).

In this study we report the genetic characterisation of RHDV strains which have been

obtained in Portugal, where the rabbit subspecies O. c. algirus is prevalent, from locations

in Spain within the contact zone, and from France where only O. c. cuniculus has been

described. No significant epidemiological, clinical or pathological differences have been

observed between the rabbit subspecies, suggesting that O. c. algirus is as susceptible to

RHDV as O. c. cuniculus, and that the virus is equally virulent for both rabbit subspecies.

No significant differences have been observed in RHDV partial VP60 gene sequences

obtained from wild rabbit specimens in either region. To our knowledge this is the first

genetic characterisation and molecular epidemiology of RHDV sequences obtained from

O. c. algirus.

Acknowledgements

This study was supported by the Foundation for Science and Technology Portugal:

Project POCTI/BIA-BDE/61553/2004 and grants SFRH/BD/31093/2006,

SFRH/BD/31048/2006, SFRH/BPD/27021/2006 to A.M., J. A. and P. J. E., respectively.

96

We are grateful to Dr. R. Soriguer (CSIC, Spain) for providing the RHDV sequences from

Albacete and Alicante, and to Oporto City Council, in particular to Dr. A. Pereira, for

providing the samples of the RHD outbreak of 2006. We thank the OIE Reference

Laboratory in Brescia and in particular Dr. L. Capucci for the antigenic characterisation of

the RHDV strain “Viseu”. We also thank the National Laboratory for Veterinary

Investigation (LNIV, Delegação do Norte) for infrastructural support.

Addendum

Detailed information on the samples and respective sequences referred to in this chapter

can be found in Appendices 1 and 2.

97

3.3 Evolution of RHDV in the Iberian Peninsula: A b rief review of recent findings .

Adapted from: II Seminario Internacional sobre el Conejo Silvest re. Córdoba 28-30

Abril 2010 (in press)

Muller, A. and Thompson, G. Departamento de Clínicas Veterinárias, Instituto de Ciências Biomédicas Abel Salazar (ICBAS), Universidade do Porto Adress: R. Padre Armando Quintas, P- 4485-661, Vairão-Portugal Telef.: ++351-252-660400, Email: [email protected] and [email protected]

99

Abstract

Since the early 1990s, rabbit haemorrhagic disease (RHD) has caused high mortality

rates in wild rabbit populations in the Iberian Pensinsula. It is now considered endemic

and continues to have considerable impact on species conservation and hunting industry.

Here we review current knowledge on the evolution of rabbit haemorrhagic disease virus

(RHDV) in the Iberian Peninsula. Our previous work (Muller et al. 2009, Vet. Microbiol.

135: 368-373) as well as recent work on the origin and phylodynamics of RHD (Kerr et al.,

2009, J. Virol. 83, 12129-12138; Kinnear and Linde, 2010, J. Gen. Virol. 91, 174-181) is

presented and discussed. Currently available sequence data of RHDV of the Iberian

Peninsula derive from the beginning of RHD outbreaks (AST89, MC89) and from different

geographic locations in Portugal (40 samples) and Spain (3 samples), as well as Southern

France (3 samples) obtained between 1994 and 2007. Phylogenetic analyses based on

partial VP60 gene sequences allowed a grouping of these RHDVs into three groups,

termed “Iberian” Groups IB1, IB2 and IB3. Interestingly, these three Iberian groups

clustered separately, though not far from earlier RHDVs of Genogroup 1 (containing e.g.

strain “AST89”), but clearly distinct from globally described RHDV strains. This result gave

rise to the hypothesis that the virus evolved independently on the Iberian Peninsula, with

the Pyrenees acting as a natural barrier to rabbit and virus dispersal (Muller et al 2009).

No differences were observed in RHDV sequences obtained from geographic regions

where the rabbit subspecies O. c. algirus prevails compared with those obtained from O.

c. cuniculus. The distinct clustering of these same Iberian RHDV sequences was

confirmed by more sophisticated phylogenetic analyses (Kerr et al., 2009). Times to most

common recent ancestor (TMRCA) for the different RHDV branches were estimated.

These gave a mean date of 1948 for the origin of “Iberian” strains. Both studies also

estimated that virulent RHDV probably emerged in the early 1900s, and that multiple virus

lineages were already circulating before the first reports of disease in 1984 (Kerr et al.,

2009; Kinnear and Linde, 2010). Both studies relate the emergence of virulent RHDV to

the intensification of rabbit production. We discuss these findings and conclude that if

virulent RHDV already existed in the Iberian Peninsula in the 1950s, their impact could

have been masked by mortalities due to the concomitant recent introduction of

myxomatosis. Despite evidence of genetic differences between RHDV field strains

currently circulating in the Iberian Peninsula and those circulating elsewhere; there is no

evidence that these affect protection induced by current commercially available vaccines.

Antigenic variants (RHDVa) have been found in a commercial rabbit farm in Portugal, but

not yet in wild rabbits. To our knowledge, no avirulent forms of RHDV or RCV-like viruses

have yet been found in Iberian wild rabbit populations, but further investigations are

100

warranted to address these issues. Also, more strains, especially from different locations

in Spain, should be analysed to enhance current understanding of RHDV evolution on the

Iberian Peninsula. Work was funded by FCT: SFRH/BD/31093/2006.

101

Introduction

Similar to other European countries, myxomatosis and rabbit haemorrhagic disease

(RHD) have been introduced into the Iberian Peninsula in the 1950s and early 1990s,

respectively (Anonymous, 1989; Monteiro, 1999; Muñoz, 1960; Villafuerte et al., 1995).

Within a few years of their introduction in wild rabbit populations, a severe decline in rabbit

abundance was recorded in the Iberian Peninsula to the extent that the wild rabbit is

currently considered “near threatened” by the World Conservation Union in 2008 (Smith

and Boyer, 2008). RHD is now considered endemic in Spain and Portugal, and despite

many efforts, rabbit numbers have not fully recovered (Delibes-Mateos et al., 2008b,

2009; Dias-Pereira et al., 2004; Moreno et al., 2007; Muller et al., 2004; Santos et al.,

2006; Villafuerte et al., 1995; Ward, 2005). The objective of the present manuscript is to

review current knowledge on the evolution of rabbit haemorrhagic disease virus (RHDV) in

the Iberian Peninsula. We will briefly present our previous work (Muller et al., 2009) as

well as recent work on the origin and phylodynamics of RHD (Kerr et al., 2009; Kinnear

and Linde, 2010) and discuss the practical implications of these findings.

1. Brief history

RHD was first reported in China in 1984. The disease spread throughout Europe, also

reaching the Iberian Peninsula, between 1987 and 1989. In the 1990s, the etiological

agent of RHD was characterised as a calicivirus (Ohlinger et al., 1990), and the virus was

introduced into Australia and subsequently New Zealand as bio-control agent against the

wild European rabbit population (Forrester et al., 2003). In an attempt to understand the

molecular epidemiology and ultimately the origins of RHD, phylogenetic analyses based

on partial sequences of the viral capsid protein VP60 of many viral isolates from different

countries were conducted (Asgari et al., 1999; Forrester et al., 2006; Le Gall-Recule et al.,

2003; Matiz et al., 2006; McIntosh et al., 2007; Moss et al., 2002; Nowotny et al., 1997).

These showed that there were up to six or more viral groups or subgroups and that virus

strains may or may not cluster according to their geographical origin and/or the year of

isolation, but that they did not always do so. Also, evidence was gathered on the current

and previous existence of RHDV-like viruses, collectively denominated rabbit caliciviruses

(RCV), which may have been circulating more or less harmlessly in Europe for many

years before the first epidemic appearance in China in 1984 (Forrester et al., 2006; Moss

et al., 2002; Rodak et al., 1990). RHDV-specific signals were detected by RT-PCR in

rabbit sera taken in the 1950s, concomitantly with the detection of RHD antibodies, but

infectiousness of viral RNA could not be proven (Moss et al., 2002). So, until recently, a

102

lot of information on the molecular epidemiology of RHDV in Europe and other countries

was gathered. However, detailed information on RHDV in the Iberian Peninsula was still

lacking, and further questions on the origin of RHD remained.

2. Evolution of rabbit haemorrhagic disease virus ( RHDV) in the European rabbit

(Oryctolagus cuniculus ) from the Iberian Peninsula

RHDV strains obtained from wild rabbits in different years and geographic locations in the

Iberian Peninsula were recently characterised (Muller et al., 2009). Briefly, 47 partial

RHDV sequences were analysed, that were obtained from wild rabbits that were found

dead during known epidemics of RHD in Portugal, Spain and France between 1994 and

2007. The majority, 40 viral sequences, were obtained from different geographic locations

of Portugal. Three RHDV sequences were obtained from wild rabbits from Spain: two from

Alicante and Albacete collected in 2004 (Dr. Ramon Soriguer, CSIC), and one from

Toledo collected in 1994 (CIBIO). Three of the four recent RDHV sequences from France

were collected between 2000 and 2002 in the Department “Pyrénées-Orientales”, and the

fourth in 2005 in the Department “Manche” (Dr. Le Gall-Recoulé, AFSSA). Nested PCR

was used to amplify partially the RHDV VP60 capsid protein gene (Moss et al. 2002). The

amplicons of 547bp were directly sequenced in both directions. The phylogenetic tree was

estimated by the neighbor-joining method as considered adequate for comparing relatively

short sequences (Takahashi and Nei, 2000). The obtained sequences were compared

with published homologues from GenBank database (NCBI). Eighteen sequences

representing previously described RHDV genogroups were selected and included. These

sequences were grouped into Genogroup 1 to 6, adapted to the classification used by Le

Gall-Reculé et al. (2003). The results of this phylogenetic analysis allowed a grouping of

these RHDV strains into three groups, termed “Iberian” Groups IB1, IB2 and IB3.

Interestingly, these three Iberian groups clustered separately, though not far from earlier

RHDVs of Genogroup 1 (containing e.g. strain “AST89”), but clearly distinct from globally

described RHDV strains. The Groups IB1 and IB2 contained sequences collected

between 1994 and 1997, whereas Group IB3 included those collected between 2000 and

2007. Each group contained sequences from different geographic locations, except Group

IB1, that contained five sequences obtained in Santarém district of central Portugal.

Interestingly, Group IB2 also included the virus obtained in 1994 from Toledo, Spain, and

Group IB3 also included the three sequences from the French Department “Pyrénée-

Orientales”, as well as the Spanish strains “Albacete” and “Alicante”. On the contrary, the

sequence obtained in 2005 from the Departement Manche in the North of France,

clustered amidst other European RHDV sequences of Genogroups 3-5. These findings

103

led to the hypothesis that virus evolution occurred independently in wild rabbit populations

on the Iberian Peninsula, with the Pyrenees acting as a natural barrier to rabbit and hence

to virus dispersal

3. The origin and phylodynamics of RHDV

Recently, two detailed and sophisticated phylogenetic analyses on RHDV were published

(Kerr et al., 2009; Kinnear and Linde, 2010). Importantly, Bayesian Markov Chain Monte

Carlo analyses were applied, allowing the estimation of viral nucleotide substitution rates,

as well as times to most common recent ancestors (MCRA), adding a temporal dimension

to the evolutionary reconstruction of RHDV. Despite some differences in methodology and

results, both studies agree on the identification of four distinct monophyletic groups or

clades of RHDV. Although contrasting previous studies (Forrester et al., 2003; Le Gall-

Recule et al., 2003; Moss et al., 2002), these findings are contributing substantially to the

warranted harmonisation of RHDV classification. Group 1 (Clade D) contained the

antigenic variants. Group 2 (Clade C) contained predominantly strains from the Iberian

Peninsula and South of France. Group 3 (Clade A) contains the “original strain” dating to

China 1984 as well as viruses from central Europe between 1987 and 1993, Korea and

Mexico. Group 4 (Clade B) contained strains from central Europe between 1989 and 2004

and Bahrein. The estimated dates for MRCA of the different groups of RHDV indicated

that virulent RHDV probably emerged in the early 1900s, and that multiple lineages were

likely circulating long before the first reports of disease in 1984. Both studies related the

evolution of RHDV with the intensification of rabbit production. Importantly, one of the

studies included some of our recent sequence data on Iberian RHDV, and confirmed our

findings of distinct clustering of Iberian strains (Kerr et al., 2009). Times to MRCA

indicated a mean date of 1948 for the origin of “Iberian” strains. Within this predominantly

“Iberian” group, two subgroups were described: viruses isolated between 1989 and 1997

and those isolated between 2000 to 2007, which have a mean TMRCA of around 1962.

The rabbit caliciviruses (RCV) displayed considerable divergence between each other and

also from all RHDV lineages, and a common ancestor was estimated to fall in 19th or early

20th century (Kerr et al., 2009; Kinnear and Linde, 2010). Thus, virulent RHDV most likely

did evolve from an avirulent RCV, but this would have occurred approximately 300 years

ago, and therefore would not explain the first observed epidemic in 1984 (Kerr et al.,

2009).

104

4. Discussion and Conclusions

Genetic differences have been found between current RHDV field strains of the Iberian

Peninsula and those elsewhere (Kerr et al., 2009; Muller et al., 2009). We hypothesized

that virus evolution occurred independently in wild rabbit populations on the Iberian

Peninsula (Muller et al., 2009). A mean date of 1948 for the origin of “Iberian” strains has

been estimated recently (Kerr et al., 2009). This suggests that virulent RHDV must have

been circulating already before the first recorded rabbit mass mortality in the late 1980s

and early 1990s in Spain and Portugal. The following question arises: is it possible that

RHD outbreaks already occurred in wild rabbit populations well before the 1980s, e.g. in

the 1950s and 1960s? We think it is possible and that they would likely have gone

unnoticed. Rabbit mortalities from the 1950s onwards coincided with the known

introduction of myxomatosis. RHD is an acute infection that induces discrete if any visible

signs and lesions, requiring necropsy for diagnosis. Other co-factors may have played a

role in incrementing the impact of RHD in wild rabbit populations between the 1950s and

1980s. Maybe the most notorious being major changes in agricultural practices, leading to

an increase in rabbit habitat defragmentation. Finally, only with the first description of RHD

in 1984, investigations on this new disease exploded and the disease was subsequently

actively searched for and thus reported from many countries, including Spain and Portugal

(Anonymous, 1989; Monteiro, 1999; Villafuerte et al., 1995). Current sequence data

consists mostly of strains obtained in Portugal, and it is unclear whether these are

representative for the whole of the Iberian Peninsula. Analyses of more RHDV field strains

especially from Spain and South of France are required to strengthen current findings and

to gain further insight into viral evolution and hopefully origins of RHD in the Iberian

Peninsula.

Only one single serotype of pathogenic RHDV is known, which contains two major

subtypes, denominated RHDV and the antigenic variants denominated RHDVa (Capucci

et al., 1998a; Schirrmeier et al., 1999), which have by now been described worldwide

(Farnos et al., 2007; Kerr et al., 2009; Le Gall-Recule et al., 2003; Matiz et al., 2006;

McIntosh et al., 2007). Although antigenic characterisations of current Iberian RHDV has

not been carried out, to our knowledge there is no evidence of antigenic variation. Current

vaccines are thus expected to be fully protective. Interestingly, although antigenic variants

(RHDVa) have been found in a commercial rabbit farm in Portugal (Muller et al., 2009),

these have not yet been found in wild rabbits in the Iberian Peninsula. Rabbit caliciviruses

(RCV) are a group of predominantly apathogenic viruses that display some antigenic

similarities, but are phylogenetically distant to RHDV (Bergin et al., 2009; Capucci et al.,

105

1996; Forrester et al., 2009; Forrester et al., 2007; Kerr et al., 2009; Moss et al., 2002;

Strive et al., 2010; Strive et al., 2009). The presence of a nonpathogenic virus closely

related to RHDV was already suspected at the time of the first RHD seroepidemiological

surveys, when specific RHDV antibodies were found in sera of farm and laboratory rabbits

where no disease was reported, and also in rabbit sera collected before the appearance

of RHD (Moss et al., 2002; Rodak et al., 1990). The drastic reduction in wild rabbit

numbers observed on the Iberian Peninsula has been historically unprecedented,

suggesting that RHDV-like apathogenic viruses were either not circulating, or, if they did,

they were probably not fully cross-protective. Active investigations on the presence of

avirulent RCV-like viruses on the Iberian Peninsula seem limited. We have analysed 68

samples from 2 healthy wild rabbit populations by nested RT-PCR. All samples were

negative, however serology was not performed (Muller et al., 2009). Thus to our

knowledge, no avirulent forms of RHDV nor RCV-like viruses have been found in Iberian

wild rabbit populations, but further investigations are warranted to address these issues as

they are expected to significantly add to current understanding of RHDV epidemiological

history.

Acknowledgements

This study was supported by the FCT grant SFRH/BD/31093/2006 to A.M.

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3.4 Real-time PCR for the detection of rabbit haemo rrhagic disease virus (RHDV) - Preliminary results

Manuscript in preparation

109

Introduction

Rabbit haemorrhagic disease (RHD) is a contagious disease associated with high

mortality rates due to acute liver necrosis caused by a calicivirus, termed rabbit

haemorrhagic disease virus (RHDV) (Meyers et al., 1991b; Ohlinger et al., 1990). Since its

first description in the 1980s, RHD has established endemicity in wild rabbits in many

European countries (Anonymous, 1989; Calvete et al., 2002b; Le Gall-Recule et al., 2003;

Nowotny et al., 1997). Recent studies have shown that RHDV strains obtained from wild

rabbits in the Iberian Peninsula are genetically different from most others, forming a group

of its own (Kerr et al., 2009; Muller et al., 2009). Despite genetic variability, Iberian strains

are antigenically similar to other virulent strains, and distinct from the antigenic variants,

RHDVa. Current diagnostic methods include conventional PCR (Bascunana et al., 1997;

Moss et al., 2002) and antigen ELISA (OIE, 2009b). Recently, a real-time PCR was

developed for the detection and quantification of viral RNA of rabbits infected with RHDV

(Gall et al., 2007). This assay allowed the demonstration of viral RNA loads in different

organs at different time points after infection, as well as RNA persistence during at least

15 weeks in recovered and also in vaccinated and subsequently challenged rabbits (Gall

et al., 2007; Gall and Schirrmeier, 2006). Whether infectious virus is present in these

animals requires further investigations (Gall et al., 2007). Here, we describe a new real-

time PCR for the detection of RHDV strains, aiming a more sensitive detection of RHDV

circulating in the Iberian Peninsula as well as to quantify viral loads in experimental

infections using the Spanish strain AST89 (Genbank accession number Z49271).

Materials and methods

A total of 45 liver samples from 43 wild and 2 domestic European rabbits (Oryctolagus

cuniculus) from the Iberian Peninsula were tested. Twenty-one samples have previously

tested positive by convention nested PCR (Moss et al., 2002). Of these, nineteen were

obtained from wild rabbits and have recently been characterized genetically based on

sequence analysis of the partial major capsid VP60 gene (Muller et al. 2008) and two

(CD09 and CD10) derived from an outbreak in a commercial rabbit farm near Viseu, North

Portugal. The latter were characterized as antigenic variants or RHDVa strains by genetic

(HM450410) and antigenic analysis (L. Capucci, personal communication). Twenty four

samples were obtained from clinically healthy wild rabbit populations: five samples were

obtained 1994 near Toledo, Spain, ten samples were obtained 2006 in a hunting estate in

Pancas, Portugal and nine samples were obtained in 1992 near Santarém, Portugal. The

latter were obtained before the observation of RHD related mortality in that region (P.C.

110

Alves, personal communication). As positive control, liver homogenate was used from a

domestic rabbit that succumbed to RHD following experimental infection with the Spanish

strain AST89 (kindly made available by P. G. Ferreira, ICBAS-UP). The negative control

consisted of the mastermix with double distilled DEPC-treated water instead of RNA

template.

RNA was extracted from 10% w/v liver homogenates in DEPC-treated double distilled

water using QIAamp viral RNA extraction kit (Qiagen) according to the instructions of the

manufacturer. For the real-time PCR, a dual labeled probe, using FAM as fluorophor and

TAMRA as quencher was designed using the software Primer Express version 2 (Applied

Biosystems) based on the sequence encoding the partial VP60 gene sequence of the

spanish RHDV strain AST89 (Genbank accession number Z49271). After analysis of the

primer-probe pairs in light of the alignment of 60 international partial RDHV capsid

sequences available on GenBank (Appendix 3), the following degenerate primer probe

pair was designed to accommodate single nucleotide substitutions observed among

strains: probe rhdv_fam: 5´- FAM-TGGCATGCAGTTYCGCTTCATAGTTGC-TAMRA-3´

covering nucleotide positions 5649-5675 on AST89, forward primer rhdv_for: 5´-

GCCGTGCTGAGCCAGAT-3´ covering nucleotide positions 5613-5630 on AST89 and

reverse primer rhdv_rev: 5´-CGATGCCYGGTGGTATCA-3´ covering nucleotide positions

5731-5714 on AST89.

The qPCR mastermix was prepared using the SuperscriptTM Platinum One Step

Quantitative RT-PCR System (Invitrogen) in a final volume of 25µl, consisting of 20µl

mastermix and 5µl template RNA. Primers and probes at a concentration of 10µM were

added to each reaction at a volume of 0.5µl of each primer and of 0.25µl of the probe. The

thermocycling reaction was carried out in a Applied Biosystems Step One Thermocycler

with the following protocol: Reverse transcription: 50ºC for 15 minutes, activation of the

Taq enzyme: 95ºC for 2 minutes and 40 cycles at 95ºC for 15 seconds and 60ºC for 30

seconds. The performance of this real-time PCR was compared with an established fully

validated assay (Gall et al., 2007).

111

Results

A total of 45 samples were tested by a real-time PCR using the newly designed primer-

probe pair spanning nucleotide positions 5613 to 5731 of the RHDV AST89 capsid. These

samples were also tested by an established assay whose primer-probe pair spans

nucleotide positions 6941-7044 (Gall et al., 2007). The samples were classified as positive

or negative according to previous results obtained by a nested RT-PCR (Moss et al.,

2002; Muller et al., 2009) and partly by antigen ELISA (Monteiro, 1999).

The cycle threshold (Ct) values obtained by real-time PCR of the 21 positive samples are

shown in Table 5. Sequence information spanning nucleotide positions 6157 to 6703 of

the RHDV AST89 capsid were available for all samples and Genbank references are

indicated of those submitted. All except the two samples containing the antigenic variants

(CD09 and CD10) were included in a previous phylogenetic analysis, and are

representative of Iberian groups 2 and 3 (Muller et al., 2009). Sixteen of the samples were

detected by both real time assays. Six samples yielded a higher Ct value in the assay

described by Gall et al. 2007, six yielded higher values in the assay described here, and

four samples had very similar values. The positive control (strain AST89) had a slightly

lower Ct value in the assay described here. Five of the samples were not amplified in

either or both of the real-time PCRs. These include the four samples from a virulent RHD

outbreak observed in 2006 in Porto City Park and one sample obtained in Loulé, Algarve,

in 2005.

The twenty four negative samples as well as the no template control tested negative in the

assay described by Gall et al. 2007, and one sample (CB08 Pancas) gave a Ct value of

33.93 in the new real-time PCR assay described here.

112

Table 5 Cycle threshold (Ct) values obtained by th e application of two different primer-probe pairs in a real-time PCR assay of posi tive samples as determined by conventional nested RT-PCR (Moss et al., 2002)

Sample/Origin Comment Genbank

reference Geno-group Muller et al. 2009

Ct value Probe “VP60” Gall et al. 2007

Ct value Probe “RHD” this paper

CD09 Antigenic variant

HM450410 Geno-group 6

7.39 11.85

CD10 Antigenic variant

As HM450410 (Sample CD09)

Geno-group 6

7.68 11.32

2007-03 Virulent RHD

Sequence not submitted

IB 3 20.66 14.72

2007-04 Virulent RHD


IB 3 19.94 13.31

CB98 Porto Virulent RHD

As EF571322 (Sample 2006_01)

IB 3 7.76 Below threshold


As EF571322 (Sample 2006_01)

IB 3 Below threshold Below threshold


As EF571322 (Sample 2006_01)

IB 3 31.03 Below threshold

2006-09 Porto Virulent RHD

EF571330 IB 3 Below threshold Below threshold

2005-01 Loulé Virulent RHD

EU192140 IB 3 Below threshold 17.78

2004-10 Loulé Virulent RHD


IB 3 17.31 28.36

1995-01 Santarém

Virulent RHD

EU192132 IB 2 12.29 12.21

1994-02 Coruche

Virulent RHD

EU192136 IB 2 9.7 10.45

1997-03 Fornos

Virulent RHD

EU192139 IB 2 11.0 10.38

1997-01 Bragança

Virulent RHD


IB 2 10.96 23.98

CB168 1996-08

Virulent RHD

EU192138 IB 2 10.67 10.61

1997-02 Santarém

Virulent RHD

EU192133 IB 2 11.91 15.33

1995-10 Coruche

Virulent RHD


IB 2 14.52 14.34

1995-11 Coruche

Virulent RHD


IB 2 10.93 10.18

1996-05 Guarda

Virulent RHD


IB 2 34.71 27.81

1997-05 V. Conde

Virulent RHD


IB 2 34.26 30.66

CB66 TOLEDO94

Unknown EU192137 IB 2 28.47 24.89

Control C15 - AST89

Positive control

Z49271 Geno-group 1

23.51 21.98

113

Discussion and conclusions

Here we show preliminary findings on the performance of a new real-time PCR for the

dual purpose of diagnostic detection of RHDV circulating in the Iberian Peninsula as well

as quantification of viral loads in infections using the Spanish strain AST89. A primer-

probe pair was designed based on nucleotide positions 5613-5731, which was compared

to a previously described, spanning nucleotides further downstream (positions 6941-7044)

of the major capsid protein gene (Gall et al., 2007). These positions were chosen, as the

region appears slightly more conserved, justifying the use of less wobble bases in the

primer-probe. Initially, an attempt was made to design a primer-probe pair for capsid

region typically targeted for sequencing, especially spanning nucleotides 6157-6703,

which were available for the RHDV obtained in wild rabbits in Portugal. The options given

by the primer design software were inspected visually on an alignment of 60 international

partial RDHV capsid sequences available on GenBank. The high variability in this region

did not allow succeeding, and none was found suitable. Hence, a different region from the

already described by Gall et al., 2007 was targeted, and is here presented.

Twenty one positive and 24 negative samples as classified by conventional PCR were

tested by real time PCR using the herein described primer-probe pair as well as the

previously described by Gall et al., 2007. A high agreement between both assays was

observed with the negative samples. Only one sample (CB08 Pancas) gave a Ct-value of

33.93 in the new real-time PCR. Interpretation of this sample is difficult, especially taking

into account that high Ct-values could be indicative of minimal amounts of target nucleic

acid which could represent an infection state or otherwise environmental contamination.

Further investigations would be necessary to clarify the true status of this sample as this

sample was taken from a healthy wild rabbit population. For example, the real-time assay

could be repeated with a larger amount of RNA template. Unfortunately serum was not

available for the determination of RHDV antibodies. If antibodies were present, viral RNA

persistence could be a possibility (Gall et al., 2007). Otherwise, seronegativity would not

rule out an early incubation period, so other methods should be used. A lower agreement

between tests was observed with the positive samples. Sixteen samples tested positive by

both assays, despite some variability between the individual ct-values. Only one well was

tested per sample, and pipetting errors are likely to account for this difference. Hence, the

use of two or more wells per sample must be considered during the validating of this

assay. Three samples were discordant, i.e. positive by one primer-probe pair but not the

other. And two samples were not detected by either real time PCR. Interestingly, the two

of the discordant as well as the two false negative samples corresponded to samples from

114

a virulent RHD outbreak in Porto City Park. These results suggest a rather lower

sensitivity of real-time PCR in comparison to conventional PCR. Although positive

samples have been used that have been genetically characterised, the sequence

information does not cover the genetic region targeted by the real-time PCR, so is of

limited value. The sequences targeted by both primer-probe pairs should be determined at

least for the samples that were not detected.

In conclusion, further studies are required to determine the tests performance. Especially

if intended for diagnostic screening, more samples of known status as determined by

conventional PCR should be tested, and the apparent low sensitivity be clarified. For the

quantification of viral loads in RHDV “AST89”-infections, the existent validated assay may

be used (Gall et al., 2007). Alternatively, the herein described primer-probes may be used,

as sequence alignments show high complementarity and ct-values are similar between

both tests. Ideally, the herein described primer-probe pairs should also be calibrated with

an internal control.

115

3.5 Real-time PCR for the detection of European bro wn hare syndrome virus (EBHSV) - Preliminary results

Manuscript in preparation

117

Introduction

European Brown hare syndrome (EBHS) affects wild and farmed hares and has been

described since the 1980s in many European countries (Billinis et al., 2005; Frolich et al.,

2001; Frolich et al., 2003; Frolich et al., 1996; Gavier-Widen and Morner, 1993; Le Gall-

Recule et al., 2006; Nowotny et al., 1997; Syrjala et al., 2005). The syndrome is

characterized by an acute and severe contagious necrotizing hepatitis with varying levels

of associated mortality. It is caused by a calicivirus termed European Brown hare

syndrome virus (EBHSV), which is similar to but distinct from rabbit haemorrhagic disease

virus (RHDV) which naturally infects the European rabbit (Oryctolagus cuniculus) (Wirblich

et al., 1994). Mortality rates of EBHS are difficult to estimate in nature as there are other

factors that may influence the density of hare populations such as climate, agricultural and

hunting practices or concurrent diseases such as tularaemia. Therefore, despite

associated morbidity and mortality, EBHS epidemics may not necessarily have any

negative effect on free living hare populations, and could remain unnoticed if the virus´

presence was not actively searched for (Gavier-Widen and Morner, 1993). Mortality rates

of EBHS are considered to be lower than for RHDV, thus it is possible, that more

attenuated or even avirulent EBHSV circulate in hare populations, similar to what has

been postulated for RHDV (Forrester et al., 2003; Moss et al., 2002). To date, the virus

has been found to cause death in two hare species, the European hare (Lepus

Europaeus) and the Mountain hare (Lepus timidus) (Frolich et al., 2001; Gavier-Widen

and Morner, 1993; Syrjala et al., 2005). To our knowledge, however, neither EBHS-related

mortality nor seroconversion has ever been reported in the Iberian or Granada hare

(Lepus granatensis). The Iberian hare occupies a large variety of habitats in the central

and southern Iberian Peninsula (Alves et al., 2008; Melo-Ferreira et al., 2005). Just a

small fringe from Galicia and Asturias in the Northwest of Spain over to Catalonia in the

east is occupied by the European hare. In these, EBHS has been documented (P. C.

Alves, personal communication). The habitat of both hare species overlap in a contact

zone, so virus spread can be expected (Alves et al., 2008; Melo-Ferreira et al., 2005).

Further studies are required to clarify the presence of EBHSV in Iberian hare populations.

Different diagnostic methods have been validated for EBHS, such as histopathology

(Fuchs and Weissenbock, 1992), haemagglution tests and antigen ELISA (OIE, 2009b),

an immunocapture RT-PCR (Le Gall-Recule et al., 2006) and a nested RT-PCR

(Bascunana et al., 1997). For its ease of use and high sensitivity, the latter is frequently

used, either in its nested form (Syrjala et al., 2005) or using the external primer pair only

(Billinis et al., 2005). Recently, quantitative real-time assays have become available for

the detection and quantification of viral genomes, including the related RHDV (Gall et al.,

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2007). Advantages of these assays are that they are even more rapid than conventional

molecular techniques, they reduce laboratory contaminations and are potentially more

sensitive, facilitating higher sample throughput. Here we describe the development of a

real-time PCR assay for the detection of EBHSV in hares, and its applications in field

samples of Iberian hares.

Materials and Methods

A total of 22 liver samples from wild hares and 1 liver sample of a domestic rabbit

(Oryctolagus cuniculus) infected with RHDV were tested. Five samples were from

European hares from Austria that had died with clinical signs and lesions suggestive of

EBHS (kindly made available by Dr. Nowotny, Department of Clinical Virology, University

of Veterinary Medicine, Vienna). These samples have previously tested positive by the

haemagglutination test using human group O red blood cells as described (OIE, 2009b).

The remaining samples were obtained from the Iberian hare (Lepus granatensis) in

Portugal. Five specimens were obtained from a hunting bag of healthy hares in

Benavente, in the North of Portugal, whereas 12 were taken from animals that were found

dead in the southern Province Algarve. The latter were subjected to necropsy and

displayed heavy infestation with the parasite cysticercus pisiformis. Gross pathological

and histopathological lesions did not suggest EBHS infection (RIPAC, 2004).

RNA was extracted from 10% w/v liver homogenates in DEPC-treated double distilled

water using QIAamp viral RNA extraction kit (Qiagen) according to the instructions of the

manufacturer. Conventional nested PCR was carried out as described (Bascunana et al.,

1997) using a one step RT-PCR kit (Qiagen). PCR amplicons were visualized under UV

light after GelRedTM (Biotium) nucleic acid gel staining.

For the real-time PCR, a dual labeled probe, with FAM as fluorophor and TAMRA as

quencher, was designed using the software Primer Express version 2 (Applied

Biosystems) based on the sequence encoding the partial VP60 gene sequence of the

EBHSV strain Austria 94b (Genbank accession number U65359). A multiple sequence

alignment of 52 international partial EBHSV capsid sequences available at GenBank was

carried out (Figure 4) using the software Bioedit Version 7 (Hall, 1999). The following

degenerate primer probe pair was designed to accommodate single nucleotide

substitutions observed among strains: Probe ebhs_fam: 5´- FAM- TGC RAT TGT YAC

AAC ACC TGG RAC ACC-TAMRA-3´, forward primer ebhs_for: 5´- CCA CAT AYA CCC

CAC AAC CA-3´ and reverse primer ebhs_rev: 5´- RCC AAT RGG TGC RGC RA . The

119

qPCR mastermix was prepared using the SuperscriptTM Platinum One Step Quantitative

RT-PCR System (Invitrogen) in a final volume of 25µl, consisting of 20µl mastermix and

5µl template RNA. Primers and probes at a concentration of 10µM were added to each

reaction at a volume of 0.5µl of each primer and of 0.25µl of the probe. The thermocycling

reaction was carried out in a Applied Biosystems Step One Thermocycler with the

following protocol: Reverse transcription: 50ºC for 15 minutes, activation of the Taq

enzyme: 95ºC for 2 minutes and 40 cycles at 95ºC for 15 seconds and 60ºC for 30

seconds.

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Figure 4 Alignment of EBHSV partial capsid gene se quences and primer-probe pairs selected for real-ti me PCR. The shown nucleotide positions correspond to positions 1332-1 421 of the VP60 capsid gene and to positions 6563-6 652 of the complete EBHSV genome (examples strain “GD”, Genbank accession num bers Z32526 and Z69629, respectively)

111 111 111 122 222 222 223 333 333 333 444 444 444 455 555 555 556 666 666 666 777 777 777 788 888 888 889 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 U65359_Austria_94b ggc cgc cac cac ata cac ccc aca acc aag tgc aat tgt cac aac acc tgg aac acc tgt tgc tgc acc cat tgg caa gaa tac acc gat U65365_Germany_89b ... t.. ... ... ... ... .. . ... ... ... ... ... ... t.. ... ... ... ... ... . .. ... ... ... ... ... ... ... c.. ... ... U65371_Sweden_93 ... t.. ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... U65363_Finland_90 ... t.. ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... Z32526_EBHSV-GD ... t.. ... ... ... ... ... t.. ... ... ... g.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. ... ... U65362_Czech_R_91 ... t.. ... ... ... ... ... ... g.. ... ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... U65361_Belgium_90 ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... t.. U65358_Austria_94a ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... t.. U65357_Austria_93 ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... t.. U65367_Germany_90b ... t.. ... ... ... t.. ... ... ... ... ... ... ... t.. ... ... c.. ... ... ... c.. ... ... ... ... ... ... ... ... ... U65356_Austria_92 ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... t.. X98002_Italy_BS89 ... t.. ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... U65366_Germany_90a ... t.. ... ... ... t.. ... ... ... ... ... ... ... t.. ... ... c.. ... ... ... c.. ... ... ... ... ... ... ... ... ... U65364_Germany_89a ... t.. ... ... ... ... ... c.. ... ... ... ... ... t.. ... ... ... g.. ... ... ... ... ... ... ... ... ... c.. ... ?.. U09199_pEB-2_and_pEB-4 ... t.. ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... ... ... ... ... ... ... t.. ... ... ... c.. ... ... U65372_UK_91 ... t.. ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... ... ... ... ... ... ... t.. ... ... ... c.. ... ... U65360_Belgium_89 ... t.. ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... ... ... ... ... ... ... t.. ... ... ... c.. ... ... U65368_Sweden_81 ... t.. ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... g.. ... ... ... ... ... ... ... ... ... ... ... ... U65370_Sweden_82b ... t.. ... ... ... ... ... g.. ... ... ... ... ... t.. ... ... ... g.. ... ... ... ... ... ... ... ... ... ... ... ... U65369_Sweden_82a ... t.. ... ... ... ... ... g.. ... ... ... ... ... t.. ... ... ... g.. ... ... ... ... ... ... ... ... ... ... ... ... Z69620_France_GD ... t.. ... ... ... ... ... t.. ... ... ... g.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. ... ... AJ971300_France_9101 ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... c.. ... ... DQ862478_Slovakia_DV1 ... t.. ... ... ... ... t.. ... ... ... ... g.. ... t.. ... ... ... ... g.. ... ... ... ... ... ... ... ... c.. ... ... AJ971311_France_0102 ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... g.. ... ... ... c.. ... c.. ... ... ... ... ... ... ... t.. AJ971299_France_9005 ... t.. ... ..a t.. ... ... ... ... ... ... ... ... t.. ... ... ... g.. ... ... ... ... ... t.. ... ... ... c.. ... ... AJ971310_France_0022 ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... g.. ... ... ... c.. ... c.. ... ... ... ... ... ... ... t.. AJ971302_France_9930 ... t.. ... ... ... t.. t.. ... ... ... ... g.. ... t.. ... ... ... ... ... ... ... ... g.. ... ... ... ... ... ... ... AJ971308_France_9917 ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. ... t.. AJ971314_France_0257 ... t.. ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... c.. c.. c.. ... ... ... ... a.. ... ... t.. AJ971313_France_0149 ... t.. ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. c.. ... ... ... ... ... ... ... t.. AJ971309_France_0006 ... t.. ... ... ... ... ... ... ... ... ... c.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. ... t.. AJ971305_France_0101 ... t.. ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... c.. ... ... AJ971301_France_0252 a.. t.. ... ... ... ... ... ... ... ... ... ... ... t.. ... ... ... g.. ... ... ... ... ... t.. ... ... ... c.. ... ... AJ971304_France_0251 ... t.. ... ... ... ... ... ... ... ... ... g.. ... t.. t.. ... ... ... ... ... ... ... ... ... ... t.. ... c.. ... ... AJ971303_France_0004 ... t.. ... ... ... ... ... ... ... ... ... g.. ... t.. t.. ... ... ... ... ... ... ... ... ... ... t.. ... c.. ... ... AJ584643_Greece ... t.. t.. ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. c.. ... ... ... t.. ... ... ... c.. AJ971315_France_0305 ... t.. ... ... ... t.. ... ... ... ... ... ... ... t.. ... ... ... ... ... ... c.. c.. ... ... ... ... ... ... ... t.. AJ971312_France_0129 ... t.. t.. ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. c.. ... ... ... ... a.. ... ... t.. AJ971306_France_0256 ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. ... ... AJ971307_France_0303 ... t.. ... ... ... ... ... ... ... ... ... ... ... a.. ... ... ... ... ... ... ... ... ... ... ... ... ... c.. ... ... DQ862480_Slovakia_N8k ... t.. ... ... ... ... ... ... ... ... ... g.. ... ... ... g.. ... ... ... ... ... ... ... ... c.. t.. ... ... ... ... DQ862478_Slovakia_DV1(2) ... t.. ... ... ... ... t.. ... ... ... ... g.. ... t.. ... ... ... ... g.. ... ... ... ... ... ... ... ... c.. ... ... AM048854_Greece_GRE-8 ... t.. t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .a. ... ... ... ... ... ... ... U80981_Sweden_77 ... t.. ... ... ... ... ... g.. ... ... ... ... ... t.. ... ... ... g.. ... ... ... ... ... ... ... ... ... ... ... ... DQ862479_Slovakia_bc3 ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... t.. ... c.. ... t.. AM048851_Greece_GRE-5 ... t.. ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. c.. ... ... ... ... ... ... ... c.. AM048853_Greece_GRE-7 ... t.. t.. ... ... t.. ... ... ... ... ... ... ... ... ... ... ..a ... ... ... c.. ... ... ... ... ... ... c.. ... ... AM048849_Greece_GRE-3 ... t.. t.. ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. c.. ... ... ... t.. ... ... ... c.. AM048848_Greece_GRE-2 ... t.. ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... g.. ... c.. c.. ... ... ... t.. ... c.. ... c.. AM048850_Greece_GRE-4 ... t.. ... ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... g.. ... c.. c.. ... ... ... t.. ... c.. ... c.. AM048847_Greece_GRE-1 ... t.. t.. ... ... t.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... c.. c.. ... ... ... t.. ... ... ... c.. AM048852_Greece_GRE-6 ... t.. ... ... ... tt. ... ... t.. ... ... ... ... ... ... ... ... ... ... ... c.. c.. ... ... ... ... ... ... ... c.. PRIMER-PROBE ggc cgc cac cac ata yac ccc aca acc aag tgc rat tgt yac aac acc tgg rac acc tgt rgc rgc acc rat tgg raa gaa tac acc gat

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Results

Of the five samples from European hares obtained from Austria, four tested positive by

conventional PCR and yielded ct-values between 22 and 25 by real-time PCR, and one

tested negative by both, conventional and real-time PCR (Table 6). These five samples

had been found positive by the haemagglutination test. The 17 samples obtained from

Iberian hares were found negative by the conventional nested PCR as well as by real-time

PCR (Table 6). The sample of a RHDV-positive rabbit tested negative by real-time PCR.

Table 6 Comparison of diagnostic tests for the det ection of European brown hare syndrome virus (EBHSV)

Sample Host History PCR-I PCR-II qPCR

(Ct value) 594/04 Wien Lepus Europeus Died, EBHS POSITIVE not tested 22,94 684/04 Wien Lepus Europeus Died, EBHS POSITIVE not tested 22,78 273/04 Wien Lepus Europeus Died, EBHS negative negative undetermined*) 685/04 Wien Lepus Europeus Died, EBHS POSITIVE not tested 25,92 717/04 Wien Lepus Europeus Died, EBHS POSITIVE not tested 24,96 Benavente 1 Lepus granatensis Healthy, hunted negative negative undetermined Benavente 2 Lepus granatensis Healthy, hunted negative negative undetermined Benavente 3 Lepus granatensis Healthy, hunted negative negative undetermined Benavente 4 Lepus granatensis Healthy, hunted negative negative undetermined Benavente 5 Lepus granatensis Healthy, hunted negative negative undetermined Algarve L8 Lepus granatensis Found dead negative negative undetermined Algarve L10 Lepus granatensis Found dead negative negative undetermined Algarve L11 Lepus granatensis Found dead negative negative undetermined Algarve L12 Lepus granatensis Found dead negative negative undetermined Algarve L17 Lepus granatensis Found dead negative negative undetermined Algarve L18 Lepus granatensis Found dead negative negative undetermined Algarve L19 Lepus granatensis Found dead negative negative undetermined Algarve L20 Lepus granatensis Found dead negative negative undetermined Algarve L21 Lepus granatensis Found dead negative negative undetermined Algarve L22 Lepus granatensis Found dead negative negative undetermined Algarve L23 Lepus granatensis Found dead negative negative undetermined Algarve L24 Lepus granatensis Found dead negative negative undetermined RHD AST89 Oryctolagus

cuniculus Rabbit with RHD not tested not tested undetermined

*) undetermined means ct-value below threshold

Ten-fold dilution series of two positive samples were tested by simple and nested

conventional PCR assay (Table 7). The threshold of detection for the nested reaction was

the same as the observed after a single PCR. Positive reactions were obtained at dilutions

of 10-5 and 10-7 of samples 685/04 and 684/04, respectively.

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Table 7 Comparison of simple and nested PCR for th e detection of European brown hare syndrome virus (EBHSV) in 10-fold diluti ons of samples 684/04 and 685/04

Sample Dilution PCR-I PCR-II 685/04 Neat + +

10-1 + + 10-2 + + 10-3 + + 10-4 + + 10-5 + + 10-7 - - 10-8 - - 10-9 - -

684/04 Neat + + 10-1 + + 10-2 + + 10-3 + + 10-4 + + 10-5 + + 10-6 + + 10-7 + + 10-8 - - 10-9 - -

Discussion

Since the 1980s mortalities have been described in wild hares in many European

countries. Interestingly, in the Iberian hare (Lepus granatensis), which occupies most

areas of central and south of the Iberian Peninsula, to our knowledge, neither unusual

mortalities due to EBHS nor the presence of the virus have been reported. This may partly

be due to the lack of active diagnostic investigations. In order to be able to engage in

further investigations on the presence of EBHS in the Iberian Peninsula, we developed an

EBHS-specific real time PCR. Here, we present preliminary findings on its performance.

As the virus has not yet been detected in the Iberian hare, we used five samples from

European hares obtained in 2004 in Austria. These samples had previously tested

positive by the haemagglutination test, and thus were considered positive for EBHSV.

Interestingly one of these samples tested negative by both PCR assays. The reasons for

this discrepancy have not been determined. Both situations are possible: a false positive

result by the agglutination test, or alternatively, a false negative result by the molecular

methods. The former may be induced by any unspecific agglutination reaction and the

latter by viral strains not detected by the specific primers. The assessment of the

sensitivity of the real-time PCR requires the testing of larger numbers of positive samples

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by haemagglutination as well as both PCR assays. Serial dilutions of two positive samples

were tested by a single and nested PCR reaction. No differences were observed in the

detection threshold of either. This suggests that the nested reaction may be omitted for

diagnostic purposes. These dilutions should also be tested by real-time PCR to allow

estimation of the detection thresholds of both assays. Typically, the development and

validation of real-time PCR includes the testing of dilution series of a positive control

consisting of a vector containing the appropriate template (Gall et al., 2007). This was not

performed as we pretend to use the EBHS real-time PCR for diagnostic purposes and not

for the measurement of viral loads in infected animals. Thus we consider the

determination of performance of the presented real-time PCR satisfactory by comparing it

to currently established assays. Seventeen samples from Iberian hares were available.

Five samples were obtained from healthy hares during hunting, and twelve samples were

obtained from hares found dead in the field. The lesions observed in the latter were not

considered typical of EBHS. These samples were tested by conventional and real-time

PCR, and found negative by both, suggesting good agreement. However, considering the

possibility of virus variants going undetected by the specific primers, these samples

should also be tested by the more robust haemagglutination test. A sample of a RHDV

infected rabbit tested by real-time PCR, suggesting no cross-reactivity between viruses,

similar to the observed for the conventional PCR (Bascunana et al., 1997). To obtain a

more accurate estimation of the specificity of the real-time PCR, more samples should be

tested, ideally by the three assays.

In conclusion, our preliminary studies on the performance of a real-time PCR for the

detection of EBHSV in liver samples suggest that this test may be a valuable tool for the

rapid screening of large numbers of samples. More samples need to be tested to

determine the sensitivity and specificity of this real-time PCR. Further studies on the

putative presence or absence of EBHS in the Iberian hare will also require the application

of complementary diagnostic tools, such as serology to rule out asymptomatic infections.

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4. Discussion

127

Myxomatosis and rabbit haemorrhagic disease (RHD) are highly infectious diseases

which have emerged in wild European rabbit populations (Oryctolagus cuniculus) in the

1950s and 1980s, respectively. In the first years after their appearance, high mortality

rates were observed, but in subsequent years, the impact of these infections seemed to

have decreased, and the hypothesis was postulated that these viruses were co-evolving

with their hosts, leading to adaptation by the selection of less virulent strains and more

resistant hosts (Fenner and Ross, 1994; Kerr and Best, 1998; Villafuerte et al., 1995). It is

within this context, that the present studies were designed, aiming to contribute to the

current knowledge on virus-host adaptation and co-evolution of MV and RHDV in their

natural host, by analysing the (partial) genetic variability of the viruses.

Four specific objectives were initially outlined, but as work was being carried out, some

modifications were made. The first and second objectives, to genetically characterize viral

strains of myxoma virus and RHDV obtained from wild rabbits from Portugal and to

compare findings with those obtained for other international strains, were considered to be

completed successfully, and two research papers were published (Muller et al., 2009;

Muller et al., 2010). The third objective, to correlate our findings with those obtained of the

genetic variation of host cell receptors in order to approach the question related to viral

and/or host adaptation and co-evolution was not achieved in form of a research paper and

will form the centrepiece of this discussion. The fourth objective, to development of real

time PCR assays for RHDV and EBHS was added in the course of progressing work, as it

was increasingly being considered a priority for our research group, as collaborations with

other research groups and new research projects were being developed. Primers and

probes were developed and the preliminary findings on the performance of these real time

assays were presented. In addition, an invite was received from the “Junta de Andalucia”

to participate in the “2nd International Seminar on the Wild Rabbit” with an oral

presentation on the evolution of RHD in the Iberian Peninsula. This Seminar was held in

the context of the Project “Conservation and Reintroduction of the Iberian Lynx in

Andalucia”, LIFE 06/NAT/000209. A more detailed manuscript written for inclusion into the

Seminar´s Report is therefore also presented in this thesis.

Within each publication or chapter on original work of this thesis, discussions on the

respective subjects have been presented. Here we shall integrate all recent findings on

the current knowledge of virus-host adaptation of myxomatosis and RHD.

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4.1 Virus-host adaptation and co-evolution of myxom a virus in the European rabbit

Several studies and models have led to the following hypothesis on virus-host co-

evolution for myxomatosis (Anderson and May, 1982; Fenner, 1994; Fenner and Chapple,

1965; Kerr and McFadden, 2002; Ross and Sanders, 1987). Within the first years of the

introduction of highly virulent MV into wild rabbit populations, high case-fatality rates were

observed. The following years, viral virulence dropped and attenuated strains appeared,

that allowed the rabbits to survive for longer. Rabbits with high innate resistance to

disease survived initial epidemics, raising the proportion of resistant rabbits over time. In

these resistant rabbits, viruses of moderate or low virulence would be too attenuated to be

successfully transmitted and thus, more strains of intermediate to high virulence started

again to be selected for and to become more prevalent.

The first question requiring analysis on the virus-host adaptation and co-evolution of

myxoma virus in the European rabbit in the Iberian Peninsula is: What evidence do we

currently have, to support that there has been a change in viral virulence of the virus since

its introduction? Recent information in Europe is scarce, and mostly relates to studies in

Great Britain. For example, between 1962 and 1981, similar proportions (62-67%) of

moderately virulent grade III viruses were observed, together with a slight increase in

grade II viruses since from 1962 (17.6%) to 1981 (35.8%) (Fenner and Chapple, 1965;

Ross and Sanders, 1987). Information on the virulence of field strains in the Iberian

Peninsula relates to only one study, carried out in the 1990s (Bárcena et al., 2000). Here,

the virulence of twenty field strains from different locations in Spain was assessed in

inoculated and in-contact rabbits. Interestingly, three quarter of the viruses were classified

as highly virulent grades I (50%) and II (25%). The remaing strains were graded as

moderately virulent grade III (10%), and attenuated grades IV (5%) and V (10%).

However, very stringent experimental conditions using very high inoculation doses in very

young animals were employed, not following the established protocol (Fenner and

Marshall, 1957). This experiment included transmission to other rabbits by contact.

Interestingly, the average survival time recorded in the contact-infected rabbits allowed

the classification of over half (55%) of the field strains as moderately virulent grade III, and

none as grades I and II. The results obtained by the in-contact animals were considered

more suitable for comparison to previous studies in Great Britain (Bárcena et al., 2000). In

our study we present the partial genetic characterisation of nine myxoma virus field strains

obtained from rabbits with signs and/or lesions of myxomatosis. Based on the presence of

signs and/or lesions of myxomatosis, we classified the analysed strains as virulent and

129

discussed, that the genetic changes found on these strains were compatible with a

virulent phenotype. Field evidence of attenuated or atypical forms of disease is very

difficult to obtain, as these may go undetected. In an attempt to detect less virulent strains,

we also tested eyelid samples from 30 apparently healthy wild rabbits by three

consecutive passages on the highly susceptible cell line RK-13 as well as PCR, but

results were negative. Strictly speaking however, inoculation experiments and virulence

grading (Fenner and Marshall, 1957) would have to be performed with our field samples.

This would be of particular importance, as strains of virulence grades III and IV also

induce signs and lesions, but average survival times are longer than for strains of higher

virulence grades. Mortality rates are also higher for the former, so a varying proportion of

infected and symptomatic animals may actually recover, which is not the case for

infections with viruses of virulence grades I and II, where mortality is above 95%. So, the

possibility does exist, that the nine field strains herein analysed, actually pertained to

different virulence grades. On the other hand, the difficulty in standardizing the

experimental conditions for the assessment of virulence grades has been described

(Bárcena et al., 2000; Parer, 1995; Parer et al., 1994). Factors such as ambient

temperature, age, dose and route of challenge influence outcome of infection (Bárcena et

al., 2000; Fenner and Ross, 1994). Reliable and more simple systems of measuring viral

virulence would be extremely utile for the evaluation of current field strains aiming to

further corroborate the above described hypothesis on virus-host co-evolution for

myxomatosis. To bypass ethical concerns and reduce potencial variability related to the in

vivo virulence testing, the development of an equivalent in vitro assay, would be highly

desirable. Attempts have been made using diverse assays, including pock counts on the

allantoic membrane of chick embryos (Fenner and Marshall, 1957) or RFLP assays (Kerr

et al., 2010; Saint et al., 2001), but no consistent correlation has been found with virulence

grades. Futher studies are required to address this subject, for example, by using different

cell cultures and/or measuring the production of cytokines induced by different viruses in

cell cultures, or assessing other putative molecular markers of virulence.

What are the molecular mechanisms underlying myxoma virus virulence? A whole

genome comparison of the virulent myxoma strain “Lausanne”, and its attenuated field

derivative “6918” mapped the attenuated phenotype to potentially four disrupted genes

(Morales et al., 2009). Inoculation experiments of genetically engineered “knock-out”

viruses have shown that many unrelated gene deletions are also related with an

attenuated phenotype (Johnston and McFadden, 2003, 2004; Stanford et al., 2007c;

Willer et al., 1999). In other words, the disruption of many different genes may lead to

attenuated phenotypes, suggesting that attenuation is not a ”site-specific” genetic event,

130

but that many different disruptions may have that consequence. This raises yet another

question. Are the attenuated myxoma viruses observed in the field a consequence of

evolutionary pressures imposed by adaptation to its new host, or are they due to

stochastic events related, for example, to “errors” during virus replication? In a recent

study in Australia, up to three different genetic types were found in epidemics, suggesting

that multiple viruses may co-exist in a given area at given time-point (Kerr et al., 2010).

Further studies attempting to clarify this subject are necessary for the European context,

first, because different circulating strains derived from a different ancestor, and second,

because in Australia multiple virus introductions were realized. Whole genome or at least

RFLP analyses should be carried out on field viruses, as partial genetic approaches, e.g.

as taken in our study and elsewhere (Alda et al., 2009; Kritas et al., 2008; Muller et al.,

2010) are likely to miss important changes.

Summarizing, myxoma virus evolution over the past six decades, in particular in the

Iberian Peninsula, is difficult to assess. Data on virus strains shortly after the introduction

of the virus is lacking, but as the same strain was introduced elsewhere in Europe,

information from other countries may be extrapolated. Recent studies on the virulence and

the genetic characterisation of circulating field strains have been carried out (Alda et al.,

2009; Bárcena et al., 2000; Muller et al., 2010). The interpretation on the virulence

gradings may be somewhat ambiguous, but ultimately suggests that virus strains of

intermediate virulence are predominating in the field (Bárcena et al., 2000). Our findings

did not specifically address the virulence gradings of field strains, but we were only able to

include field strains from animals with signs and/or lesions, as no virus could be isolated

from apparently healthy animals (Muller et al., 2010). Similar to other viruses, evolution of

myxoma viruses is expected to be reflected by genetic changes in the viral genome. The

genetic characterisation of poxviruses is difficult due to the large size of the virus and

different approaches have been taken to detect genetic changes either reflecting

attenuation and/or evolution (Alda et al., 2009; Kerr et al., 2010; Muller et al., 2010; Saint

et al., 2001). Our data on the partial genetic characterisation has shown little differences

between field strains (Muller et al., 2010). A recent study on field strains from different

locations in Spain has obtained similar findings, further strengthening the idea of a

relatively high genetic stability of myxoma virus over time (Alda et al., 2009). Although this

this is quite expected for large DNA viruses such as poxviruses (Gubser et al., 2004; Xing

et al., 2006), it seems to contradict the hypothesized evolutionary pressures suffered by

myxoma virus in its new host, which are reflected by changes in virulence.

131

The second question on virus-host adaptation and co-evolution of myxoma virus in the

European rabbit in the Iberian Peninsula is related to the putative selection of

myxomatosis-resistant rabbits within the past 5-6 decades. As above, this subject is also

difficult to address, because it typically requires animal inoculation experiments. Most

older studies relied on the capture of young rabbits in the field and the selection of

seronegative individuals in challenge experiments with viruses of known virulence. The

immunodiffusion test was initially widely used to determine if rabbits were immunologically

naïve prior to experimental inoculation, but this test was later shown to be rather

inconsistent and substituted by others such as ELISA (Kerr and McFadden, 2002; Kerr,

1997; Williams et al., 1973). So, initial data may carry some bias due to the inclusion of

immunised animals. Most studies were carried out either in Australia or in Great Britain

(Edmonds et al., 1975; Ross and Sanders, 1984). We are not aware of studies addressing

this subject in the Iberian Peninsula, where two different subspecies of the wild rabbit

exist, displaying a higher genetic diversity than elsewhere (Branco et al., 2000; Ferrand,

2008; Ferrand and Branco, 2007). Theoretically it would be conceivable that selection of

resistant rabbits would be a slower process in the Iberian Peninsula and that currently a

much smaller proportion of resistant rabbits would be present than elsewhere. But specific

studies are required to detect and characterise the proportion of resistant rabbits in the

field. The genetic basis for resistance of rabbits to myxomatosis remains unknown. Some

work has focussed on the identification and characterisation of putative viral receptors. In

particular chemokine receptors such as CCR-5 and CXCR4, that present features unique

to Oryctolagus may be related with the species´ susceptibility to infection (Abrantes et al.,

2010; Abrantes et al., 2008a; Abrantes et al., 2008b; Carmo et al., 2006). Others have

based on the characterisation of microsatellites (Surridge et al., 1999). In whichever, in

vivo and/or in vitro infection assays are required to consolidate these hypotheses. Another

possible explanation for resistance are differences in tissue tropism or an enhanced

immune response (Kerr and McFadden, 2002). Differences may sometimes but not

always be found in the virus tropism for lymphocytes of different rabbits (Best and Kerr,

2000; Kerr and McFadden, 2002). This issue merits further investigations.

The investigation of a putative enhanced immune response is complex, because during

successful myxoma virus replication, many different proteins may be secreted that may

interact in different ways, modulating the hosts’ immune responses, affecting clinical

outcome (Johnston and McFadden, 2003; Kerr and McFadden, 2002; Messud-Petit et al.,

1998; Stanford et al., 2007c; Zuniga, 2002). Current knowledge on the in vivo secretion

and physiological effect of these proteins is scarce. The up or down regulation of the

secretion of these proteins may be triggered by complex, to date unknown, host factors

132

present in some rabbits but not others. The interaction between myxoma virus and the

hosts immune system is more difficult to assess than putative viral receptors, and may

require the study of complete host genomes as proposed in the context of susceptibility to

infectious disease in general (Schnappinger and Ehrt, 2006; Tuite and Gros, 2006; Vidal

et al., 2008).

4.2 Virus-host adaptation and co-evolution of RHDV in the European rabbit

In analogy to myxomatosis, the following questions on virus-host adaptation and co-

evolution of RHDV in the European rabbit require attention. What evidence do we

currently have, to support that there has been attenuation of the virus? Not much, really.

Actually, the contrary hypothesis prevails. Virulent RHDV strains appear to have resulted

from avirulent strains which probably have been circulating in wild rabbit populations for

decades before the first description of RHD (Forrester et al., 2006; Forrester et al., 2007;

Kerr et al., 2009; Kinnear and Linde, 2010; Moss et al., 2002). The molecular base related

to this switch in virulence has yet to be defined. The presence of avirulent strains similar

to RHDV, denominated rabbit caliciviruses or RCV-like viruses, has been described in

some countries, but generally seem difficult detect consistently (Forrester et al., 2009;

Forrester et al., 2007; Strive et al., 2010; Strive et al., 2009). In an attempt to detect

avirulent RHDV or RHDV-like viruses, we analysed 30 liver samples from a healthy wild

rabbit population by nested RT-PCR, but found no evidence of virus infection (Muller et

al., 2009). Further studies are required to amplify these preliminary findings, bearing in

mind that RCV-like viruses, which are genetically different from RHDV, may also be

circulating in the Iberian Peninsula, but have not yet been described there. Hence,

sampling should include intestine as well as liver, and diagnostic assays should include

serology as well as RCV and RHDV-specific (or crossreactive) methodology.

Pathogenesis studies must be carried out to determine if detected viruses are virulent or

not. This is essential, because viral RNA but apparently not infectious virus may persist for

several weeks after recovery from acute RHDV infection or even upon challenge of

vaccinated rabbits (Gall et al., 2007; Gall and Schirrmeier, 2006).

We have genetically characterised so called “virulent” RHDV, obtained from wild rabbits

that died from RHD in different locations of the Iberian Peninsula (Muller et al., 2009).

Similar work has previously been carried out for many other European RHDV field strains

(Asgari et al., 1999; Forrester et al., 2006; Le Gall-Recule et al., 2003; Le Gall-Recule et

al., 2006; Matiz et al., 2006; McIntosh et al., 2007; Moss et al., 2002; Nowotny et al.,

133

1997). Surprisingly, our results appointed a distinct grouping of field strains from the

Iberian Peninsula. We hypothesized that field strains from the Iberian Peninsula and

South of France may have evolved distinctly from most other European or worldwide

RHDV, probably due to a physical barrier to wild rabbit dispersal formed by the Pyrenees

(Muller et al., 2009). Two recent independent studies now agree on the identification of

four distinct monophyletic clades of RHDV (Kerr et al., 2009; Kinnear and Linde, 2010).

Importantly, one of these four RHDV clades is formed predominantly by the sequences of

the Iberian Peninsula and South of France (Kerr et al., 2009). Our study is mostly based

on samples from Portugal. To further corroborate the hypothesis of distinct RHDV

evolution on the Iberian Peninsula, strains from other geographical areas, especially the

central and eastern Spain, must be characterised.

Two recent publications on RHDV evolution introduced a temporal dimension to the

reconstruction of RHDV evolution (Kerr et al., 2009; Kinnear and Linde, 2010). This

prompted us to review our current knowledge on RHD evolution in the Iberian Peninsula in

the light of these studies (Chapter 3.3). Briefly, the year 1948 was estimated as mean time

to most common recent ancestor for Iberian strains (Kerr et al., 2009). This suggests that

virulent RHDVs were already emerging in the Iberian Peninsula long before 1984, maybe

already in the 1950s or 1960s. The emergence of virulent RHDV would probably be a

gradual process, by in large obscured by the then observed mass mortality caused by

myxomatosis. This hypothesis contrasts with some field reports on the drastic reduction of

rabbit numbers in some locations presumably due to RHD observed in the 1980s and

1990s (Delibes-Mateos et al., 2008b, 2009; Villafuerte et al., 1995). On one hand these

observations may reflect true incidence of RHD and variability in local epidemiological

circumstances, as they are still observed today (Villafuerte et al., 1995). On the other

hand, as they resulted after the enlarged awareness on RHD, they may to some extent be

biased, as more objective rabbit population census and surveillance schemes were

implemented a posteriori, so pre-RHD information on rabbit densities is in many cases

lacking or merely empirical. Ultimately, there is no factual evidence that RHDV-like viruses

have really been circulating before the 1980s in the Iberian Peninsula. This may partly

because wild rabbit sera taken before 1980s are not available, so retrospective analyses

are not possible. Further studies are required to test this hypothesis and to clarify the

origin of RHD in wild rabbits of the Iberian Peninsula and elsewhere. The genetic

characterisation of larger numbers of field samples of different geographic areas of the

Iberian Peninsula are required to corroborate our findings (Muller et al., 2009) and to

support statistical inferences on viral evolution and, potentially, its origins.

134

A few considerations on the putative selection of RHD-resistant rabbits shall also be

made. Recent studies have determined ABH tissue antigen as putative RHDV cellular

receptor (Ruvoen-Clouet et al., 2000). But these are not expressed on liver cells, so other

receptors need to be identified. Different allele profiles of Fut2 and Sec1 genes have been

identified in wild rabbits (Guillon et al., 2009). Functional alleles are thought to be present

in so-called “secretor phenotypes” which are thought to be present in individuals

susceptible to RHD. However, this hypothesis is based on empirical observations of the

donor populations and requires testing by infection experiments. To our knowledge,

virulence grading assays by rabbit inoculations similar to those realised for myxomatosis

have not been carried out for RHDV. We are also not aware of the identification of

resistant rabbits, excluding young animals in the phase of natural resistance (Ferreira et

al., 2006a; Ferreira et al., 2004; Ferreira et al., 2005; Prieto et al., 2000; Shien et al.,

2000). If the “virus-host co-evolution” hypothesis is to be maintained as representing a

major force driving rabbit evolution, further studies are required to determine a) if, and in

which proportions, either disease- or infection-resistant rabbits do exist in the field, and b)

what the underlying molecular mechanisms are.

4.3 Development of real-time PCR assays for RHDV an d EBHSV

The development of real-time PCR has had a major impact not only in the rapid and more

sensitive diagnosis of infectious diseases, but importantly also on current knowledge on

the respective pathogenesis. For example, the application of real-time PCR to different

tissues has allowed the quantification of viral RNA sequentially after RHDV infection (Gall

et al., 2007). Real-time PCR has the further advantage of reducing cross-contaminations

which may occur during the process of traditional and in particular nested PCR, which is

currently a widely used diagnostic assay for RHD (Bascunana et al., 1997; Moss et al.,

2002; Nowotny et al., 1997). In order to improve diagnosis of RHD and also to detect viral

RNA in experimental inoculation experiments using the Spanish RDHV strain AST89, a

new primer-probe pair was designed and compared to a previously described assay (Gall

et al., 2007). The preliminary results on the performance of this new real-time PCR were

obtained using RHDV field strains, for which partial genetic information was available

(Muller et al., 2009). However, the available sequence information does not cover the

regions targeted by the primer-probe pair annealing. This is due to the fact that variable

regions are required for the study of viral evolution and that more conserved regions are

usually targeted for diagnostic purposes. Our preliminary results showed good

agreements between tests using negative samples. However, some positive samples, in

135

particular those obtained in the recent RHD outbreak of Oporto City Park, were not

unanimously detected by both real-time assays. The underlying causes require further

analysis, such as sequencing of the regions targeted by both real-time assays. Further

work is also required to compare both primer-probes for the detection of the strain AST89

in samples from experimental infections. It may be considered necessary to contruct an

internal control to allow proper RNA quantification.

A real-time PCR assay was also developed for EBHSV and the preliminary findings on the

performance of this test are presented. To our knowledge, this is the first description of

such assay for EBHS. This assay should ideally substitute current traditional PCR assays

for diagnostic purposes (Bascunana et al., 1997; Nowotny et al., 1997), which are more

time-consuming and, as mentioned above for RHD, bear considerable risks of cross

contaminations. A primer-probe pair was designed and the performance tested on positive

and negative samples as determined by conventional PCR and haemagglutination test.

The agreement between tests on the 17 negative samples was excellent, however, more

samples should be tested to give a more accurate estimate on the tests specificity. Only

five positive samples were available from Austria (kindly made available by Dr. Nowotny,

Department of Clinical Virology, University of Veterinary Medicine, Austria). No positive

samples from the Iberian Peninsula were available, because the EBHS has not been

described in the Iberian hare (Lepus granatensis), and further investigations shall be

carried out by our group, ideally using a real time as screening tool. Of the five positive

samples as determined by the haemagglutination test, one was neither detected by

conventional nor real time PCR. The reason for this incongruence should be investigated.

Also, more positive samples from different geographical origins should be tested to give a

more accurate estimate on the tests sensitivity.

Summarizing, real-time PCR for the detection of EBHS and RHD remain attractive and

potentially sensitive tools, but further work is required to validate and optimize their

performance, and before allowing their application in diagnostic or experimental settings.

137

5. Conclusions and perspectives

139

In the present thesis original studies on the partial characterisation of myxoma virus and

RHDV strains obtained from wild rabbits in the Iberian Peninsula, in particular from

Portugal, and on the preliminary results of newly developed real-time PCR assays for the

detection of RHDV and EBHSV were presented and discussed in the light of current

knowledge, allowing the following conclusions to be made:

• The analysis of selected genomic regions corresponding to approximately 3% of the

viral genome suggests a high genetic stability of myxoma virus field strains over the

past five decades. Based on our findings it is difficult to identify unique single gene

markers of virus attenuation or evolution, indicating that analyses of larger proportions

of the genome or even the whole genome may be required.

• The phylogenetic analysis based on the partial VP60 sequences of the RHDV capsid

gene of field strains from Portugal, Spain and South of France allowed the grouping of

these into three groups, determined “Iberian Groups” IB1 to IB3. These Iberian groups

clustered separately from most globally described RHDV, giving rise to the hypothesis

of independent viral evolution on the Iberian Peninsula, with the Pyrenees acting as

natural barrier to rabbit and thus viral dispersion.

• Two real-time PCR assays for the detection of RHDV and EBHSV have been

developed and preliminary assessment of its performance suggests high specificity

but less satisfactory sensitivity.

• The frequently cited hypothesis on virus-host coevolution for both myxomatosis and

RHDV was assessed in the light of current knowledge. According to this hypothesis,

the co-evolution of viruses and their host would lead to the emergence of attenuated

viruses and more resistant hosts. We found that further field and experimental

evidence is required to maintain and further support this hypothesis in the case of both

infectious diseases. We also highlight the complexity of these subjects requiring the

integration of field and experimental studies at different levels.

140

Perspectives

The work presented in this thesis allowed the identification of several questions that may

be investigated in the near future, such as:

1. What evidence do we have that attenuated strains of myxomatosis occur in the

field and what is their importance? Is it possible to develop an in vitro method for

the assessment of virulence grades and/or host genetic resistance to the virus?

The latter question could be addressed with inoculation experiments using field

viruses and targetting primary lymphocyte cultures from different rabbits.

2. What are the origins of RHDV in the Iberian Peninsula? Do avirulent RHDV or

RCV-like strains exist in healthy wild rabbit populations? This topic shall be

approached by a joint project already approved for funding, and that also covers

assessing the role of EBHS in the Iberian Peninsula (see below).

3. To complete the development of real-time PCR assays for the detection of RHDV

and EBHSV: to fully validate and optimize their performance, and to apply these

assays in diagnostic or experimental settings. This topic shall be addressed within

two joint projects. One is already ongoing and includes the detection of RHDV-

specific RNA in AST89-infected young rabbits. Another joint project on the

screening of Iberian hares for EBHS has recently been approved for funding (see

above).

141

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7. Appendices

Appendix 1 Identification of Iberian RHDV field strains obtained from European wild rabbits between 1994-2007. Information of non-identical sequences was included in our study (Muller et al., 2009) ........................................................................................................... 161

Appendix 2 Sequence alignment of variable nucleotide positions of RHDV field strains obtained from European wild rabbits between 1994-2007 in Portugal. The represented region numbered 1-546 represent positions 853-1398 of the major capsid VP60 gene and positions 6157-6702 of RHDV whole genome strain AST89 (Genbank accession number Z49271), respectively. ................................................................................................................ 163

Appendix 3 Nucleotide sequence alignment of the major capsid gene VP60 of RHDV strains. The localisation of two primer probe pairs for the use in real-time PCR are boxed and highlighted in bold letters. The newly designed primer-probe pair “RHD” spans the positions 309 to 427 of VP60, corresponding to positions 6513 to 5731 on strain AST89 (Genbank accession number Z49271). The primer-probe pair “VP60” described by Gall et al. 2007 spans the positions 1637 to 1740 of VP60, corresponding to positions 6941 to 7044 on strain AST89. The partial VP60 for mostly targeted for sequencing and phylogenetic studies spans positions 853 to 1399 of VP60, corresponding to positions 6157 to 6703 on strain AST89, here represented as shaded background on strain AST89. ........................................................................................................................................................ 165

Appendix 4 Publications ............................................................................................................ 181

161

Appendix 1 Identification of Iberian RHDV field strains obtained from European wild rabbits between 1994-2007. Information of non-identical sequences was included in our study (Muller et al., 2009)

Identification NEW identification Collection site Acession number

1994 1994

CB107 1994-01 Coruche /Infantado

CB109 1994-02 Coruche EU192136

CB110 1994-03 Santarém/Pernes



CB114 1994-06 Vila Viçosa

CB116 1994-07 Santarém/Pernes EU192131

CB117 1994-08 Golegã


CB119 1994-10 Santarém/Casével

CB120 1994-11 Golegã

CB150 1994-12 Porto Santo

1995 1995

CB112 1995-01 Santarém/Pernes EU192132

CB121 1995-02 Penamacor/Malcata

CB123 1995-03 Idanha/Sra. Do Almortão

CB125 1995-04 Cast. Rodrigo/Devesas

CB128 1995-05 Sabugal/Lageosa

CB129 1995-06 Sabugal/Foios

CB130 1995-07 Guarda

CB131 1995-08 Belmonte

CB133 1995-09 Belmonte

CB135 1995-10 Coruche

CB136 1995-11 Coruche

CB137 1995-12 Alpiarça

1996 1996

CB141 1996-01 Sabugal /Malcata

CB145 1996-02 Santarém


CB151 1996-04 Bragança/Montesinho

CB160 1996-05 Guarda

CB161 1996-06 Vila do Conde

CB167 1996-07 Avis

CB168 1996-08 Coimbra/Pena EU192138

1997 1997

CB153 1997-01 Bragança

CB154 1997-02 Santarém/Casével EU192133

CB156 1997-03 Fornos EU192139


CB161 1997-05 Vila do Conde

2004 2004

C134 2004-01 Alcoutim


C142 2004-03 Castro Marim/Alcoutim EU192134

C143 2004-04 Castro Marim





C153 2004-09 Tavira

C154 2004-10 Loulé/S.Brás

2005 2005

Coelho1 2005-01 Loulé/Tor EU192140

Coelho2 2005-02 Loulé/Tor

Coelho3 2005-03 Loulé/Alte

Coelho4 2005-04 Loulé/Alte

2006 2006

CB97 Opo 01/06 = 2006-01 Oporto City Park EF571322

CB98 Opo 02/06 Oporto City Park



162







2007 2007

CB193 2007-01 Chaves EU192135

CB194 2007-02 Chaves

CB195 2007-03 Chaves

CB196 2007-04 Valpaços

CB197 2007-05 Valpaços

OTHERS OTHERS

CB66 Toledo 1994 Spain 1994 EU192137

France 00-08 France 2000-08 France 2000 Pyrenée AJ319594

France 01-23 France 2001-23 France 2001 AM46980



Alicante 04-05 Alicante 2004 Spain 2004 AM884394

Albacete 04-08 Albacete 2004 Spain 2004 AM884395

163

Appendix 2 Sequence alignment of variable nucleotide positions of RHDV field strains obtained from European wild rabbits between 1994-2007 in Portugal. The represented region numbered 1-546 represent positions 853-1398 of the major capsid VP60 gene and positions 6157-6702 of RHDV whole genome strain AST89 (Genbank accession number Z49271), respectively. 1111 1111111111 1111111111 1112222222 2222222222 2222223333 3333333333 3333333333 3333333344 4444444444 4444444555 55555 112233344 4455566666 6667880111 1122223334 5567788889 9990001123 3333445566 7888990001 1123344444 5566777788 8888999900 2223344455 6678888012 34444 3594706913 4812401236 7895145147 8913690254 0621403690 2581473622 3467395814 3028174590 1883623489 4723234812 3479036907 0365714709 2570369198 70346 AST89 TCCTCCCTGA GCTGACCCTG AACACGTGTA ATGCTCACCA CTTCGTCCTG CTACGGCATA ACGTCCCTGT TTATCGGGCC TCAATGACCG GCATAACCAG CCAACCCATT TTCAGCCCCG CATACACCGC CGTGC 1994-01 ...C...... .......... ....T..... ....C..... .....C.... ...T...G.. .....T.C.. C..C..AA.. ...G...... .........A AT.G.A.... ....A..... ....T..... .A... 1994-02 ..c..c..A. ......T... .G........ ......G... ......T... ......T... TT........ .......... C...C..TT. ...C.....A ....T...C. .......... .......T.. ...A. 1994-03 ...t...... g......... ....T..... ....C..... .....C.... .......G.. .....T.C.. C..C..A... ...G...... .........A AT.G.A.... ....A..... ....T..... ..... 1994-04 .....c...G ...A...... ....T..... ....C..... .....C.... .......... .....T.C.. C..C.A.... .......... .........A AT.G.A.... ....A..... ....T..... ..... 1994-07 .........G ...A...... ....T..... ....C..... .....C.... .......... .....T.C.. C..C...... .......... .........A AT.G.A.... ....A..... ....T..... ..... 1994-10 ........A. ......T... .G........ .......... .......... ......T... TT........ ..G....... C...C..T.. .........A ....T...C. .C........ .......... .A... 1994-12 ........A. ......T... .a...AC... .........G ..C..Cc... ...T..T..G CT......A. ..g....... C......T.. g....GG..A ........C. .......... .G........ ...A. 1995-01 ........A. ......T... .G........ .......... .......... ......T... TT........ ..G....... C...C..T.. .........A ....T...C. .C........ .......... ..... 1995-02 ........A. ......T... .....A.... .C.......G ..C..C.... .C.T..T... CT........ ....A..... C......T.. ......G..A ........C. .......... .......... .A... 1995-04 ........A. ......T... .G...A.... .........G ..C..C..C. ...T..T.C. T......... .......... C.....GT.. ......G..A .......GC. .......... .......... ...A. 1995-05 C.......A. ......T... ....T..... G...C..T.. .C...C.... .CG...T... .T...A.... ..G...A... C.G.C..T.. .........A ....T...C. .......... ......T... ..... 1995-09 t.c..c.CA. ......T... .G........ .......... .......... ......T... TT........ .......... C...C..T.. .........A ....T...C. .......... .......... .A... 1995-10 C.......A. ....G.T... GGT....T.. .C.......G T.C..C.... .C.T..T..G CT........ C...A..... C......T.. ......G..A ......T.C. .......... ........A. .A.A. 1995-11 cc...c..A. ....G.T... GGT....T.. .C.......G T.C..C.... .C.T..T..G CT........ C...A..... C......T.. ......G..A ......T.C. .......... ...G....A. .A.A. 1995-12 ........A. ......T... .....A.... .C.......G ..C..C.... .C.T..T... CT........ ....A..... C......T.. ......G..A ........C. .....T.... .......... .A.A. 1996-01 ........A. .....TT... ........C. .......... ..C....T.. ......T... .T...T.C.. ..G....... C...C..TT. .........A ....T...C. .......... ......T... ..... 1996-04 ........A. ......T... .....A.... .........G ..C..C...A ...T..T..G T........C .......... C......T.. ......G..A ........C. .......... .......... ...A. 1996-05 .....c..A. A.....T... .....AC... .........G ..C..C.... ...T..T..G CT......A. ..G....... C......T.. A...G.G..A ........C. .......... .G........ ...A. 1996-06 .....c..A. ......T... .....AC... .........G ..C..C.... ...T..T..G CT......A. ..G....... C......T.. A...G.G..A ........C. .......... .G........ ...A. 1996-07 c.......A. ....G.T... GG....tT.. .CA.t....G T.C..C.... .C.T..T..G CTA....... t...A..... C......T.A g.T...G..A ......T..G ........cC T..G....g. .A.A. 1996-08 c....c..A. ....G.T... GG.....T.. .C.......G T.C..C.... .C.T..T..G CT........ C...A..... C....A.T.. ......G..A ......T.C. .......... ..CG...... .A.A. 1997-01 ..A.....A. ......T... .G...A.... .........G ..C..CT..A ...T..T..G T........C .......... C......T.. ......G..A ........C. .......... .......... ...A. 1997-02 .......... ........C. ....T..... ....C..... ..C..CT... TC....T... .....T.C.. CC.C..A... .......... .........A AT...A.... ....A....A .......... T.... 1997-03 .....c..A. .T....T... .........G G......... T.C....... ......T... TT..T..... .........A C......T.. .........A ........C. .......... .......... ..... 1997-05 .....c..A. ......T... .....AC... .........G ..C..C.... ...T..T..G CT......A. ..G....... C......T.. A...G.G..A ........C. .......... .G........ ...A. 2004-01 .T....T.A. A.....AA.. GGT....... ...TCTG... ..C....... T...ATT... TT........ ..G.....T. C...C..T.. ....GT.AGA ........C. C.T..TT.T. .....G.... ...A. 2004-02 .T....T.A. A.....AA.. GGT....... ...TCTG... ..C....... T...ATT... TT....T... ..G.....T. C......T.. ....GT.AGA ........C. C.T..TT.T. .....G.... ...A. 2004-03 .T....T.A. A.....AA.. GGT....... ...TCTG... ..C....... T...ATT... TT........ ..GC....T. C......T.. ....GT.AGA ........C. C.T..TT.T. .....G.... ...A. 2004-05 .T....T.A. A.....AA.. GGA....... ...TCTG... ..C....... T...ATT... TT........ ..G.....T. C......T.. ....GT.AGA ........C. C.T..TT.T. .....G.... ...A. 2004-07 .T....T.A. A.....AA.. GGT....... ...TCTG... ..C....... T...ATT... TT........ ..G.....T. C......T.. ....GT.AGA ........C. C.T..TT.T. .....G.... ...A. 2004-08 .....T..A. .T....AAC. GG........ ....CTG... ..C.a..... T...A.TG.. TT........ ..G....... C......T.. ....GT...A .....T..C. .C...TTT.. .......Aa. ..cA. 2004-09 .T....T.A. A.....AA.. GGT....... ...TCTG... ..C....... T...ATT... TT........ ..G.....T. C......T.. ....GT.AGA .....T..C. C.T..TT.T. .....G.... ...A. 2004-10 .T.C..T.A. A.....AA.. GGT....... ...TCTG... ..C....... T...ATT... TT........ ..G.....T. C......T.. ....GT.AGA ........C. C.T..TT.T. .....G.... ...A. 2005-01 .....T..A. .T....AAC. GG........ ....C.G... ..C....... T...A.TG.. TT....T... ..G....... C......T.. ....GT.T.A ........C. .C...TTT.. .......Aa. ..cAt 2005-03 .....T..A. .T....AACA GG........ ....C.G... ..C..C.... T...A.TG.. TT....T... ..G....... C......T.. ....GT.T.A ........C. .C...TTT.. .......Aa. ..cAt 2005-04 .....T..A. .T....AAC. GG........ G...C.G... ..CT...... T...A.TG.. TT....T... ..G....... C......T.. ....GT.T.A ........C. .C...TTT.. .......Aat ..cAt 2006-01 ..A.....A. A.....AA.. GG.T...... G...C.GT.. ..C....... T...A.TG.. TT.....C.. ........T. C......T.. .T..GT..GA ..G.....C. ...T.TTTT. .....G..A. ...A. 2006-04 ..A.....A. A.....AA.. GG.T...... G...C.GT.. ..C....... T...A.TG.. TT.....C.. ........T. C......T.. .T..GT..GA ..G.....C. ...T.TTTT. .....G..A. t..A. 2006-09 ..A.....A. A.....AA.. GG.T...... G...C.GT.. ..C....... T...A.TG.. TT.....C.. ......A.T. C......T.. .T..GT..GA ..G.....C. ...T.TTTT. .....G..A. t..A. 2007-01 ....T...A. A.C...T... GGT....... ....C.G.A. .......... ....A.T.C. TT.C.T.... ..GC....T. CT.....T.. ....GT..GA ........C. .......T.. ...G...... ...A. 2007-02 ....T...A. A.C...T... GGT....... ....C.G.A. ..C....... ....A.T.C. TT.C.T.C.. ..GC....T. CT.....T.. ....GT..GA ........C. .......T.. ...G...... ...A.

165

Appendix 3 Nucleotide sequence alignment of the major capsid gene VP60 of RHDV strains. The localisation of two primer probe pairs for the use in real-time PCR are boxed and highlighted in bold letters. The newly designed primer-probe pair “RHD” spans the positions 309 to 427 of VP60, corresponding to positions 6513 to 5731 on strain AST89 (Genbank accession number Z49271). The primer-probe pair “VP60” described by Gall et al. 2007 spans the positions 1637 to 1740 of VP60, corresponding to positions 6941 to 7044 on strain AST89. The partial VP60 for mostly targeted for sequencing and phylogenetic studies spans positions 853 to 1399 of VP60, corresponding to positions 6157 to 6703 on strain AST89, here represented as shaded background on strain AST89.

167

111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 122 222 222 223 333 333 333 444 444 444 455 555 555 556 666 666 666 777 777 777 788 888 888 889 999 999 999 000 000 000 011 111 111 112 222 222 222 333 333 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 Z49271_RHDV-AST89 ATG GAG GGC AAA GCC CGC ACA GCG CCG CAA GGC GAA GCA GCA GGC ACT GCC ACC ACA GCA TCA GTC CCT GGA ACC ACA ACC GAT GGC ATG GAT CCC GGC GTT GTG GCC ACT ACC AGC GTG GTC ACT GCA GAG AAT AB300693_Hokkaido/2002/JPN ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..A ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... AF231353_NZ ... ... ... ... ... ..T G.. ... ... ... ... ... ... ..G ... ... .T. ... ... ... ... ..T ..C ... ... ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... A.. ... ... ..A ... AF258618_Iowa2000 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... AF295785_Mexico89 ... ... ... ... ... ..T G.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... A.. ... ... ..A ... AF402614_WX/China/1984 ... ... ... ... ... ..T G.. ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ... ... ... ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... A.. ... ... ..A ... AF453761_China/Harbin/TP ... ... ... ... A.. ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... AJ302016_99-05FR ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... AJ302016_99-05FR(2) ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... AJ303106_00-ReuFR ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ... ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... AJ319594_00-08FR ... ... ... ... ... ..T ... ... ... ... ... ... ..T ..G ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ..T ... ... ... ... ..C ..T ..T ... ... ... ... ..A ... AJ495856_00-13FR ... ... ... ... ... ... ... ... ... ... A.. ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ..C ..T ... ..C ..T ... ..C ... ... ..A ..T ... ... ... ..C A.C ... ... AJ535092_95-05FR ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ... ... ... ..G ... ... ..T ... ... ..T ... ... ... ... ..C ... ... ... ... ... ... ... ... AJ535094_95-10FR ... ... ... ... ... ..T ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... T.. ... ..T ... ... ... ... ..C ... ... ... ... ... ... ... ... AJ969628_03-24FR ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... AM085133_05-01FR ... ... ... ... ... ... ... ... ... ... A.. ..T ... ..G ... ... ..T ... ... ... ... ... ..C ... ... ... ... ..C ..T ... ..C ..T ... ..A ... ... ..A ..T ... ... ... ..C A.T ... ..C AY269825_NJ/China/1985 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ..T ..A ... ... G.A ..T ..T ... ... ... ... ..A ... AY523410_CD/China ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..A ... ..T G.A ..T ..T ... ... ... ... ... ... AY926883_Ireland12 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... T.. ... ..T ... ... ... ... ..C ..T ..T ... ... ... ... ... ... AY928268_Ireland18 ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... T.. ... ..T ... ... ... ... ..C ..T ..T ... ... ... ..G ... ... AY928269_Ireland19 ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... T.. ... ..T ... ... ... ... ..C ..T ..T ... ... ... ..G ... ... DQ069280_whn/China/01/2005 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..A ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... DQ069281_whn/China/02/2005 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..A ... ... G.A ..T ..T ... ... ... ... ..A ... DQ069282_whn/China/03/2005 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..A ... ... G.A ..T ..T ... ... ... ... ..A ... DQ189077_Bahrain ... ... ... ... ... ..T ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... T.. ..C ... ... ... ... ... ..C ..T ..T ... ... ... ... ... ... DQ189078_SaudiArabia ... ... ... ... ... ..T ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ... ... ... ..G ..T ... ... ... ... ..T ..T ... ..A ... ... ... ..T ... A.. ... ... ..A ... DQ205345_JX/CHA/97 ... ... ... ... A.. ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... DQ280493_ChinaWHNRH ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..A ... ... G.A ..T ..T ... ... ... ... ..A ... DQ530363_China-Yangling(YL) ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ..A ..A ... ... G.G ..T ..T ... ... ... ... ..A ... DQ841708_CUB5-04 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... .TC ... ... ..G ... ..C ... ... ... ..T ... ..A ... ... G.A ..T ..T ... ... ... ... ..A ... EF363035_clonepJG-RHDV-DD06 ... ... ... ... ... ... ... ... ... ... ... ... .T. ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... T.. ... ..T ... ... ... ... ..C ..T ..T ... ... ... ... ... ... EF558572_Frankfurt12 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... T.. ... ..T ... ... ... ... ..C ... ... ... ... ... ... ... ... EF558573_Frankfurt5 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... T.. ... ..T ... ... ... ... ..C ... ... ... ... ... ... ... ... EF558574_Wika_Germany ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... T.. ... ..T ... ... ... ... ..C ... ... ... ... ... ... ... ... EF558575_Ascot_UK ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... T.. ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... EF558576_Jena_Germany ... ... ... ... ... ..T ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... T.. ... ..T ... ... ... ... ..C ..T ... ... ... ... ... ... ... EF558577_Meiningen_Germany ... ... ... ... ... ..T ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ..T ... ... ... ... ... ... ... EF558578_Eisenhuttenstadt ... ... ... ... ... ... ... ... ... ... ... C.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ..C EF558579_NZ54 ... ... ... ... ... ..T G.. ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ..C ... ... ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... A.. ... ... ..A ... EF558580_NZ61 ... ... ... ... ... ..T G.. ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ..C ... ... ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... A.. ... ... ..A ... EF558581_Erfurt ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..A ... ... G.A ..T ..T ... ..T ... ... ..A ... EF558582_Dachswald ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... EF558583_Triptis ... ... ... .G. ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... EF558584_Rossi ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... EF558585_Hagenow ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... EF558587_Ashington --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --- --. ... ... ... ... ..C ... ... ... ..T ... ..T ... ..C ..T ..T ... ... ... ..A ... ..T ... ... ..C A.T ... ..C EU003578_IN-05 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ... ... ... ..T ... ..A ... ... G.A ..T ..T ... ... ... ... ..A ..C EU003579_Italy90 ... ... ... ... ... ..T G.. ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ..C ... ... ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... AC. ... ... ..A ... EU003580_Korea90 ... ..A ... ... ... ..T G.. ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ... ... ... ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... A.. ... ... ..A ... EU003581_NY-01 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ..T ..C ... ... ..G ... ..C ..T ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... EU003582_UT-01 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ..C ... ... ..G ... ..C ... ... ... ..T ... ..G ... ... G.A ..T ..T ... ... ... ... ..A ... L48547_MC-89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... M67473_FRG ... ... ... ... ... ..T G.. ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ..C ... ... ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... A.. ... ... ..A ... NC_001543_FRG ... ... ... ... ... ..T G.. ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ..C ... ... ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... A.. ... ... ..A ... RHU49726_Haute-Saone/FR88 ... ... ... ... ... ..T G.. ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ..T ... ... ..T ..G ..T ... ... ... ... ..T ... ... ... ... ... ... ... ... A.. ... ... ..A ... 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NC_001543_FRG ... ... ..C ... ... ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ..A ... ... ... ... ... ..A C.. ..C CG. RHU49726_Haute-Saone/FR88 ... ..T ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ..A ... ... ... ... ... ..A ... ..C ... U54983_V351 ... ... ..C ... ... ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ..A ... ... ... ... ... ..A C.. ..C CG. X87607_BS89 ... ... ..C ... ... ... ... ..A ... ... ... ..T ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ..A ... ... ... ... ... ..A C.. ..C ..A X96868_RCV ..T ..T ... ... ..C ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ..C ..C ... ... ... ... ... ... ... ..C ... ... ... ... ..T ..G ..A ... ... ... ..C ..A ..A C.. ... ..A Y15424_Frankfurt ... ... ..C ... ... ... ... ..A ... ... ... ..T ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ..A ... ... ... ... ... ..A C.. ..C ..A Y15427_Wriezen ... ... ..C ... ... ... ... ..A ..T ... ..G ..T ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ..A ... ... ... ... ... ..A C.. ..C ..A Z24757_AST/89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ATC ... ... ... ... ... ... ... ... ... Z29514_SD ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... Primer-Probe “RHD” A GCC GTG CTG AGC CAG AT FAM-T GGC ATG CAG TTY CGC TTC ATA GTT GC-TAMRA

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444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 444 455 555 555 555 555 555 555 555 555 555 555 555 555 555 000 011 111 111 112 222 222 222 333 333 333 344 444 444 445 555 555 555 666 666 666 677 777 777 778 888 888 888 999 999 999 900 000 000 001 111 111 111 222 222 222 233 333 333 334 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 Z49271_RHDV-AST89 GCC GTG ATA CCA CCG GGC ATC GAG ATT GGA CCA GGG CTG GAG GTC AGG CAA TTC CCC CAT GTT GTC ATC GAC GCT CGT TCA CTT GAA CCT GTC ACC ATC ACC ATG CCA GAC TTG CGT CCC AAC ATG TAC CAT CCA AB300693_Hokkaido/2002/JPN ..T ... ... ... ... ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..G ... ... ..T ... ... ..C ... ... ..C ... ... ..T ..T ... ... ... ... ... C.. ... ... ... ... ... ..C ... AF231353_NZ ..T ... ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ..G ..T ..T ... ... ... ... ..T ..C ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... AF258618_Iowa2000 ..T ... ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ..T ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... AF295785_Mexico89 ..T ... ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ..T ..T ... ... ... ... ... ..C ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... AF402614_WX/China/1984 ..T ... ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ... ... ... ..C ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... AF453761_China/Harbin/TP ..T ... ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ..T ... ... ..C ... ... ..C ..G ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... 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AJ535092_95-05FR ..T ... ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ... ... ... ... ... ... ..C ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... AJ535094_95-10FR ... ... ..T ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ..T ... ... ..C ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... AJ969628_03-24FR ..T ..A ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ..T ..T ... ..C ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... AM085133_05-01FR ... ... ... ... ..A ... ... ... ... ... ... ... T.A ... ... ... ... ..T ..T ..C ... ... ..T ... ..C ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... AY269825_NJ/China/1985 ..T ... ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ..T ... ... ..C ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ..T AY523410_CD/China ..T ... ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ..T ... ... ... ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... AY926883_Ireland12 ..T ... ... ... ..A ... ... ... ..C ... ... ... T.. ... ... ... ... ... ..T ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... AY928268_Ireland18 ..T ... ... ... ..A ... ... ... ..C ... ... ... T.. ... ... ... ... ..T ..T ... ... ... ..T ... ..C ... ... ... ... ... ... ... ... ... ... ... ... C.. ... ... ... ... ... ..C ... AY928269_Ireland19 ..T ... ... ... ..A ... ... ... ..C ... ... ... T.. ... ... ... ... ..T ..T ... ... ... ..T ... ..C ... ... ... ... ... ... ... ... ... ... ... ... C.. ... ... ... ... ... ..C ... DQ069280_whn/China/01/2005 ..T ... ... ... ... ... ... ... ... ... ... ... T.. ... ... ... ... ... ... ... ... ..T ... ... ..C ... ... ..C ... ... ..T ..T ... ... ... ... ... ..A ... ... ... ... ... ..C ... DQ069281_whn/China/02/2005 ..T ..C ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ..T ... ... ... ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ..T ... ... ... ... DQ069282_whn/China/03/2005 ..T ..C ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ..T ... ... ... ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ..T ... ... ... ... DQ189077_Bahrain ..T ... ... ... ..A ... ... ..A ..A ... ... ... T.. ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... DQ189078_SaudiArabia ..T ..A ... ... ..A ... ... ... ... ..G ... ... 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RHU49726_Haute-Saone/FR88 ..T ... ... ... ..A ... ... ..A ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ... ... ... ..C ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... U54983_V351 ..T ... ... ... ..A ... ... ... ... ... ... ... T.. ... ..T ... ..G ..T ..T ... ... ... ... ..T ..C ... ... ..C ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... X87607_BS89 ..T ... ... ... ..A ... ... ... ... ... ... ... T.. ... ... ... ... ..T ..T ... ... ... ... ... ..C ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... X96868_RCV ..A ... ... ... ..A ... ..T ..A ..C ... ... ... T.A ... ..T ... ... ..T ... ... ... ..T ... ..T ... ... ... ..C ..G ..G ..T ..T ... ... ... ... ... ... ... ... ... ... ... ... ... Y15424_Frankfurt ... ... ... ... ..A ... ... ... ... ... ... ... T.. ..A ... ... ... ..T ..T ... ... ... ... ... ..C ... ... ... ... ... ..T ... .A. ... ... ... ... ... ... ... ... ... ... ... ... Y15427_Wriezen ..T ... ... ... ..A ... ... ... ..C ... ... ... T.. ... ... ... ... ..T ..T ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ..A ... ..A ... ... ... ... ... Z24757_AST/89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... Z29514_SD ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... Primer-Probe “RHD” TG ATA CCA CCY GGC ATC G

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172

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173

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GG. ... ... ..T ... ... ... ... ..C DQ205345_JX/CHA/97 ..T ..C ... ... ... ... ... ... ... ..A ..C ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ..T T.. ... ... .G. T.C ... ... ... ..C ... ... DQ280493_ChinaWHNRH ..T ..C ... ... ... ... ... ... ... C.. ..C ... ... ..G ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ..T T.. ... ... ... T.C ... ... ... ... ... ... DQ530363_China-Yangling(YL) ..T ..C ... ... ... ... ... ... ... C.. ..C ..T ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ..C ..T T.. ... ... ... T.C ... ... ... ... ... ... DQ841708_CUB5-04 ..T ..C ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ..T T.. ... ... ... T.C ... ... ... ..C ... ... 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174

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175

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GCT .C. ... ... ... ... ..C ... ... ... ... ... ..T ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... AF231353_NZ ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... AF258618_Iowa2000 ... ... ... ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... AF295785_Mexico89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... AF402614_WX/China/1984 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... AF453761_China/Harbin/TP ... ... ... ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ..T ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... AJ302016_99-05FR ... ... ... ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... AJ302016_99-05FR(2) ... ... ... ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... AJ303106_00-ReuFR ... ... ... ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... AJ319594_00-08FR ... .T. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... .C. ... ... ... ... ... ... ... ... ..G ... .T. ... ... ... ... ... ... AJ495856_00-13FR ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.G ..A ... ... ... ..A ... ..T ... AJ535092_95-05FR ... ... ... ... .A. ... ... ... ..T ... ... ... ... ... ..C ... ... ... ..C ... ... ... ... ... ... A.. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... AJ535094_95-10FR ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ..T ... ... ... ..T ... AJ969628_03-24FR ... ... ... ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... AM085133_05-01FR ... ... ..C ..T ... ... ..T ... ... ..C ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.G ..A ... ... ... ..A ... ..T ... AY269825_NJ/China/1985 ... ... ... ... .A. ... ... ... GCT .C. ..A ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AAT ... ... ... ... ... ... ... ... ..G ... G.. ... ... ... ..T ... ... ..T ... AY523410_CD/China ... ... ... ... .A. ... ... ..T GCT .CC ... ..C ... ... ..C ... ... ... ... ... ... ... ... ... ... AAT ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... AY926883_Ireland12 ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ..C ... ... ..G ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... G.. ..A ... ... ... ... ... ..T ... AY928268_Ireland18 ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ..T ... ... ... ... ... G.. ..A ... ... ... ... ... ..T ... AY928269_Ireland19 ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ..T ... ... ... ... ... G.. ..A ... ... ... ... ... ..T ... DQ069280_whn/China/01/2005 ... ... ..C ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... DQ069281_whn/China/02/2005 ... ... ..C ... .A. ... ..T ... GCT .C. ... ... ... ... ..C ... ... ... ..C ... ... ... ... ... ... AAT ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... DQ069282_whn/China/03/2005 ... ... ..C ... .A. ... ..T ... GCT .C. ... ... ... ... ..C ... ... ... ..C ... ... ... ... ... ... AAT ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... DQ189077_Bahrain ... ... ... ... ... ... ..T ... ... ..A ... ... ... ... ..C ... ... C.. ... ... ..T ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ..A .T. ... ... ... ... ..T ... DQ189078_SaudiArabia ... ... ... ... ..T ... ... ... ... ..C ... ... ... ... ..C ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... DQ205345_JX/CHA/97 ... ... ... ... .A. ... ... ... GCT .CC ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... DQ280493_ChinaWHNRH ... ... ..C ... .A. ... ..T ... GCT .C. ... ... ... ... ..C ... ... ... ..C ... ... ... ... ... ... AAT ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... DQ530363_China-Yangling(YL) ... ... ..C ... .A. ... ... ... GCT .CC ..A ... ... ... ..C ... ... ... ... ... ... ..C ... ... ... AAT ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... DQ841708_CUB5-04 ... ... ... ... .A. ... ... ... GCT .C. ..A ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AAT ... .C. ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... EF363035_clonepJG-RHDV-DD06 ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ..T ..T ... ... ... ... ... ... ... ... G.. ..A ... ... ... ... ... ..T ... EF558572_Frankfurt12 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ..T ... ... ... ..T ... EF558573_Frankfurt5 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ..T ... ... ... ..T ... EF558574_Wika_Germany ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ..T ... ... ... ..T ... EF558575_Ascot_UK ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ..A ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... EF558576_Jena_Germany ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ..A ... ... ... ... ... ..T ... EF558577_Meiningen_Germany ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... EF558578_Eisenhuttenstadt ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ..A ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558579_NZ54 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..C ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... EF558580_NZ61 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..C ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... EF558581_Erfurt ... ... ... ... .A. ... ... ... GCT .C. ..A ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AAT ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... EF558582_Dachswald ... ... ... ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ..A ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... EF558583_Triptis ... ... ... ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... EF558584_Rossi ... ... ... ..T .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... EF558585_Hagenow ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... A.T ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... EF558587_Ashington ... ... ..C ... ..T ..C ... ... ..T ..C ... ... ..T ... ..A ... C.A ..G ... ... ... ..C ..T ... ... A.. ..T ..T ... ... ..T ... ... ..C ... ... G.G ... GT. ... ... ... ... ..T ... EU003578_IN-05 ... ... ..C ... .A. ... ... ... GCT .CC ... ..C ... ... ..C ... ... ... ... ... ... ... ... ... ... AAT ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... EU003579_Italy90 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... EU003580_Korea90 ... ... ... ... .A. ... ... ... ... ... ..A ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... EU003581_NY-01 ... ... ..C ... ... ... ... ... GCT .C. ... ... ... ... ..C ... ... ..G ..C ... ... ... ... ... ... AA. ... ... ... ... ..T ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... EU003582_UT-01 ... ... ..C ... .A. ... ... ... GCT .C. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... AA. ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ..T ... ... ..T ... L48547_MC-89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... M67473_FRG ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... NC_001543_FRG ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... RHU49726_Haute-Saone/FR88 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... U54983_V351 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... X87607_BS89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ..T ... X96868_RCV ... ... ... ... ..T ... ... ... ... ..C ..A ..C ... ... ..C ... ... ..G ... ... ... ..C ..A ... ... AA. ... ... ... ..T G.. ... ... ... ... ..C ... ..A .T. ..T ... ..A ... ..T ... Y15424_Frankfurt ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... G.. ... ... ..T ... ... ... ..T ... Y15427_Wriezen ... ... ... ... ... ... ..T ... ... CG. ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ..A ... ... ... ... ... ..T ... Z24757_AST/89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... Z29514_SD ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ...

176

111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 222 333 333 333 333 333 333 333 333 333 333 333 333 333 333 333 333 333 111 122 222 222 223 333 333 333 444 444 444 455 555 555 556 666 666 666 777 777 777 788 888 888 889 999 999 999 000 000 000 011 111 111 112 222 222 222 333 333 333 344 444 444 445 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 Z49271_RHDV-AST89 GCC GTG GTA ACT GGC ACA GCC CAA AAC CCC GCC GGA TTG TTT GTG ATG GCC TCG GGT GTT ATC TCC ACC CCA AGT GCC AAC GCC ATC ACA TAC ACG CCC CAA CCA GAC AGA ATT GTA ACC ACA CCC GGC ACT CCT AB300693_Hokkaido/2002/JPN ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... ... ... G.. ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ... ... AF231353_NZ ... ..A ..G ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... A.. ... ... ... ... .A. ... .G. ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... AF258618_Iowa2000 ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.A ... ... ... ... ... ... ... ... ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ..T ... ... ..G ..T ... ... ... ... ... AF295785_Mexico89 ... ... ... ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... .A. ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ..T ... ... AF402614_WX/China/1984 ... ..A ... ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... .A. ... .G. ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... AF453761_China/Harbin/TP ... ... ..G ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ..T ... ... ..G ..T ... ... ... ... ... AJ302016_99-05FR ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ..T ... ... ..G ..T ... ... ... ... ... AJ302016_99-05FR(2) ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ..T ... ... ..G ..T ... ... ... ... ... AJ303106_00-ReuFR ... ... ..G ..C ... ... AA. ... ... ..A A.. ... C.A ... ... ... ... ... ... ... ... ... ... ... .AC ... ... ... G.. ... ... ... ... ... ... ..T ... ... ..G ..T ... ... ... ... ... AJ319594_00-08FR ... ... ..G T.. ... ..G A.. ... ... ... ... ... C.. ... ... ... ... ... ... ... ... ... ... ... ... ..T ..T ..T ..T ... ..T ..A ... ... ... ... ... ... ... ... ... ... ... ... ... AJ495856_00-13FR ... ... ... ... ... ... .G. ... ... ..A ... ..G ... ... ... ... ..T ... ..C A.C ... ..T ... ... .A. ... ... ... ... ... ... ..A ..T ... ..G ..T ... ..C ... ... ... ... ... ... ..A AJ535092_95-05FR ..T ... ... ... ... ... A.. ... ... ..A ... ..G ..A ... ..A ... ... ... ..C ... ... ... ... ... .A. ... .G. ... ... ... ... ..A ... ... ... ... ... ... ... ..T ... ... ... ... ..A AJ535094_95-10FR ... ... ... ... ... ... ... ... ... ..A ... ..G ... ... ... ... ... ... ... A.. ... ... ... ... .A. ... ... ... ..T ... ... ..A ..T ... ... ... ... ... ... ..T ... ... ... ... ..A AJ969628_03-24FR ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ..T ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ..A ... AM085133_05-01FR ... ... ... ... ... ... .G. ... ... ..A ... ..G ... ... ... ... ... ... ... A.C ... ..T ..T ... .A. ..T ... ... ... ... ... ..A ..T ... ... ... ... ... ... ... ... ... ... ... ..A AY269825_NJ/China/1985 ... ... ... ..C ... ... AA. ... ..T ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... ... ... G.. ... ... ... ... ..G ... ... ... ... ..G ... ... ... ... ... ... AY523410_CD/China ... ... ... ..C ... ... AA. ... ..T ..A A.. ... C.. ... ... ... ... ... ... A.. ... ... ... ... ..C ... .G. ... G.. ... ... ..A ... ... ... ... ... ... ..G ..T ... ... ... ... ... AY926883_Ireland12 ... ... ... ... ... ... .G. ... .G. ..A ... ..G ... ..C ... ... ... ... ..C A.C ... ... ... ... .A. ... ... ... ... ... ... ..A ..T ... ... ... ... ... ... ... ... ... ... ... ..A AY928268_Ireland18 ... ... ... ... ... ... .G. ... .G. ..A ... ..G ... ... ... ... ... ... ..C A.. ... ... ... ... .A. ... ... ... ... ... ... ..A ..T ... ... ... ... ... ... ... ... ... ... ... ..A AY928269_Ireland19 ... ... ... ... ... ... .G. ... .G. ..A ... ..G ... ... ... ... ... ... ..C A.. ... ... ... ... .A. ... ... ... ... ... ... ..A ..T ... ... ... ... ... ... ... ... ... ... ... ..A DQ069280_whn/China/01/2005 ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ..T ... .AC ... ... ... G.. ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ... ... DQ069281_whn/China/02/2005 ... ... ... ..C ... ... AA. ... ..T ..A A.. ..G C.. ... ... ... ... ... ... ..C ... ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ... ... DQ069282_whn/China/03/2005 ... ... ... ..C ... ... AA. ... ..T ..A A.. ..G C.. ... ... ... ... ... ... ..C ... ... ... ... .AC ... ... ... G.. ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ... ... DQ189077_Bahrain ... ... ... ... ... ... .G. ... ... ..A ... ..G ... ... ... ... ... ... ..C A.A ... ... ... ... .A. ... ... ... ... ..C ... ..A ..T ... ... ... ... ... ... ..T ... ... ... ..C ..G DQ189078_SaudiArabia ... ... ... ... ... ... A.. ... ... ..G ... ... C.. ... ... ... ..T ... ... A.. ... ... ... ... .A. ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... DQ205345_JX/CHA/97 ... ... ..G ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ..T ... ... ..G ..T ... ..T ... ... ... DQ280493_ChinaWHNRH ... ... ... ..C ... ... AA. ... ..T ..A A.. ..G C.. ... ... ... ... ... ... ..C ... ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ... ... DQ530363_China-Yangling(YL) ... ... ... ..C ... ... AA. ... ..T ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... ... ... G.. ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ... ... DQ841708_CUB5-04 ... ... ... ..C ... ... AA. ... ..T ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... .G. ... G.. ..T ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... EF363035_clonepJG-RHDV-DD06 ... ... ... ... ... ... .G. ... .G. ..A ... ..G ... ... ... ... ... ... ..C A.. ... ... ... ... ... ... ..T ... ... ... ... ..A ..T ... ... ... ... ... ... ... ... ... ... ..C ..A EF558572_Frankfurt12 ... ... ... ... ... ... A.. ... ... ..A ... ..G ... ... ... ... ... ... ... A.C ... ... ... ... .A. ... ... ... ... ... ... ..A ..T ... ... ... ... ... ... ... ... ... ... ... ..A EF558573_Frankfurt5 ... ... ... ... ... ... ... ... ... ..A ... ..G ... ... ... ... ... ... ... A.C ... ... ... ... .A. ... ... ... ... ... ... ..A ..T ... ... ... ... ... ... ... ... ... ... ... ..A EF558574_Wika_Germany ... ... ... ... ... ... ... ... ... ..A ... ..G ... ... ... ... ... ... ... A.. ... ... ... ... .A. ... ... ... ... ... ... ..A ..T ... ... ... ... ... ... ... ... ... ... ... ..A EF558575_Ascot_UK ... ... ... ... ... ... ... ... ... ..A ... ..G ... ... ... ... ... ... ..C ... ... ... ... ... .A. ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ..T ... ... ... ... ..A EF558576_Jena_Germany ... ... ... ... ... ... .G. ... ... ..A ... ..G ... ... ... ... ... ... ..C A.. ... ... ... ... .A. ... ... ... ... ... ... ..A ..T .C. ... ... ... ... ... ..T ... ... ... ..C ..A EF558577_Meiningen_Germany ... ... ... ... ... ... A.. ... ... ..A ... ..G ... ... ... ... ... ... ..C ... ... ..T ... ... .A. ... .G. ... ... ... ... ..A ... ... ... A.. ... ... ... ..T ... ... ... ... ..A EF558578_Eisenhuttenstadt ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558579_NZ54 ... ..A ..G ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... A.. ... ... ... ... .A. ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ..C EF558580_NZ61 ... ..A ..G ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... A.. ... ... ... ... .A. ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ..C EF558581_Erfurt ... ... ... ..C ... ... AA. ... ..T ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ... ... EF558582_Dachswald ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... .GT ... G.. ... ... ... ... ... ... ..T ... ... ... ..T ... ... ... ... ... EF558583_Triptis ... ... ... ..C ... ..T AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... .G. ... G.. ... ... ... ... ... ... ..T ... ... ..G ..T ... ... ... ... ... EF558584_Rossi ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... G.. ... ... ... ... ... ... ..T ... ... ..G ... ... ... ... ... ... EF558585_Hagenow ... ... ... ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... ..C ... ... ... ... .A. ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ..T ... ... ... ... ..A EF558587_Ashington ... ..T TC. ... ..T GTG ... ... ... ..G ... ..G ... ..C ... ... ... ... ... A.C ..A ... ... ... .A. ... .CT ... ... ..G ... ..A ... ... ... ... ... ... ..C .AT G.. ... ..T ... ..C EU003578_IN-05 ... ... ... ..C ... ... AA. ... ..T ..A A.. ... C.. ... ... ... ... ... ... A.. ... ... ... ... .AC ... .G. ... G.. ... ... ..A ... ... ... ... ... ... ..G ..T ... ... ... ... ..A EU003579_Italy90 ... ... ..G ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... A.. ... ... ... ... .A. ... .G. ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... EU003580_Korea90 ... ... ... ... ... ... A.. ... ... ..A ... ... ... ... ... ... ..T ... ... ... ... ... ... ... .A. ... .G. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EU003581_NY-01 ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ... ... ... ... ... ... .AC ... ... ... G.. ... ... ... ... ... ... ... ... ... ..G ..T ... ... ... ... ... EU003582_UT-01 ... ... ... ..C ... ... AA. ... ... ..A A.. ... C.. ... ... ... ... ... ..C A.. ... ... ... ... .A. ... .G. ... G.. ... ... ..A ... ... ... ... ... ... ..G ..T ... ... ... ... ... L48547_MC-89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G CG. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... M67473_FRG ... ... ..G ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... A.. ... ... ... ... .A. ... .G. ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... NC_001543_FRG ... ... ..G ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... A.. ... ... ... ... .A. ... .G. ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... RHU49726_Haute-Saone/FR88 ... ... ..G ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ..C ... ... ... ... ... .A. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... U54983_V351 ... ..A ..G ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... A.. ... ... ... ... .A. ... .G. ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... 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177

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178

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NC_001543_FRG ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ..G ... RHU49726_Haute-Saone/FR88 ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ..C ..C ... ... ... ... ..G ... U54983_V351 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ..G ... X87607_BS89 ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... X96868_RCV ... ..T ..G ... ... ... ... ... ..A ... ... ..C .CA ... ..G ... ... ... ... ... ... ... .AT ... ..C ... ..C ... ... ... ... ..G ..G .A. ..T ..T ... ... ... ... ... ..G ... ... ..T Y15424_Frankfurt ... ... ... ... ... ... ... ... ... ... T.. ... ... ... ... ... ... ... ..A ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... Y15427_Wriezen ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... Z24757_AST/89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... Z29514_SD ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ...

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111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 111 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 666 677 777 777 777 777 777 777 777 777 777 777 777 777 777 222 222 222 333 333 333 344 444 444 445 555 555 555 666 666 666 677 777 777 778 888 888 888 999 999 999 900 000 000 001 111 111 111 222 222 222 233 333 333 334 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 123 456 789 012 345 678 901 234 567 890 Z49271_RHDV-AST89 TCA ACC ACA CTC ATT GAC TTG ACT GAA CTC ATT GAC GTA CGC CCT GTG GGA CCC AGG CCA TCC AAG AGC ACA CTC GTG TTC AAC CTG GGG GGC ACA GCC AAT GGC TTT TCT TAT GTC TGA AB300693_Hokkaido/2002/JPN ... ... ..G ... ... ... C.. ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. A.T ... ... ... ... ... ... ... AF231353_NZ ... ... ... ... ... ... C.. ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... AF258618_Iowa2000 ... ... ..G ..T ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... AF295785_Mexico89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ..T ..A ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... AF402614_WX/China/1984 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... AF453761_China/Harbin/TP ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... AJ302016_99-05FR ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... AJ302016_99-05FR(2) ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... AJ303106_00-ReuFR ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... AJ319594_00-08FR ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ... ... AJ495856_00-13FR ... ... ... ..T ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... G.. ... ... ..T ... ... ... ... ... AJ535092_95-05FR ... ... ..G ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..A ... ... ... AJ535094_95-10FR .T. ..T ... ... ... ... ... ... ... ... ... ..T ..T ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... AJ969628_03-24FR ... ... ..G ... ... ... ... ..C ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ..T ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... AM085133_05-01FR ... ... ... ..T ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ..T ..A ... ... ... .C. ... ... ... ... ... G.. ... ... ... ... ... ... ... ... AY269825_NJ/China/1985 ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... AY523410_CD/China ... ..T ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... G.. A.. ... ... ... ... ... ... ... AY926883_Ireland12 ... ... ... ... ... ... ... ... ... ..G ... ... ..C ... ... ... ... ... ... ..G ... ..A ... ... ... ... ..T ... ... ... ... G.. ... ... ... ..C ... ... ... ... AY928268_Ireland18 ... ... ... ... ... ... ... ... ... ... ..C ... ..C ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... G.. ... ... ... ..C ... ... ... ... AY928269_Ireland19 ... ... ... ... ... ... ... ... ... ... ..C ... ..C ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... G.. ... ... ... ... ... ... ... ... DQ069280_whn/China/01/2005 ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ..G ... ... ... ... ... ... ... ... ... ..T ... ... ... ... G.. A.. ... ... ... ... ... ... ... DQ069281_whn/China/02/2005 ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... DQ069282_whn/China/03/2005 ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... DQ189077_Bahrain ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... DQ189078_SaudiArabia ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..A ... ... ..T ..A ... ... ... ... ... G.. ... ... ... ... ... ... ... ... DQ205345_JX/CHA/97 ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... DQ280493_ChinaWHNRH ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... C.. ... ... ... ... DQ530363_China-Yangling(YL) ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... DQ841708_CUB5-04 ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... EF363035_clonepJG-RHDV-DD06 ... ... ... ... ... ... ... ... ... ... ..C ... ..C ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... G.. ... ... ... ..C ... ... ... ... EF558572_Frankfurt12 .T. ... ..T ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558573_Frankfurt5 .T. ... ..T ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558574_Wika_Germany .T. ... ..T ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558575_Ascot_UK ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558576_Jena_Germany ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558577_Meiningen_Germany ... ... ... ... ... ... ... ... ... ..T ... ... ..T ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... .G. ... ... ... ... A.. ... ... ... ... ... ... ... EF558578_Eisenhuttenstadt ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558579_NZ54 ... ... ... ... ... ... C.. ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558580_NZ61 ... ... ... ... ... ... C.. ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558581_Erfurt ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... EF558582_Dachswald ... ... ..G ... ..C ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558583_Triptis ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558584_Rossi ... ... ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EF558585_Hagenow ... ... ... ... ... ... ... .TC ... ... ... ... ... ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... EF558587_Ashington ... ... ..C ..T ... ... ... ..C ... ... ... ... A.. ... ... ... ... ... ... ... ... .CA ... ... ... ... ..T ..- --- --- --- --- --- --- --- --- --- --- --- --- EU003578_IN-05 ... ..T ..G ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... EU003579_Italy90 ... ... ... ... ... ... C.. ... ... ... ... ... ... ... ..C ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EU003580_Korea90 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... EU003581_NY-01 ... ... ..G ... ... ... C.. ... ... ... ... ... ..G ... ..C ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... EU003582_UT-01 ... ... ..G ... ... ... ... ..A ... ... ... ... ... ... ..C ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... L48547_MC-89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..C ... ... ... ... ... ... ... ... ... ... ... M67473_FRG ... ... ... ... ... ... C.. ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... NC_001543_FRG ... ... ... ... ... ... C.. ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... RHU49726_Haute-Saone/FR88 ..T ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ..A ... ... ... ... ... ... ... ... ... U54983_V351 ... ... ... ... ... ... C.. ... ... ..T ... ... ... ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... X87607_BS89 ... ... ... ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... X96868_RCV ..G ... ..C ..T ..C ... ... ... ... ... ... ... A.. ... ... ... ... ... ... ... ... .C. ... ..T ..T ... ... ... ... ... ... G.. A.. .G. ... ... ... ... ... ... Y15424_Frankfurt .T. ... ..T ... ... ... ... ... ... ... ... ... ..T ... ... ... ... ... ... ..G ... ..A ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... Y15427_Wriezen ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ..G ... ... ... ... ... ... ... ... ... ... A.. G.. ... ... ... ... ... ... ... ... Z24757_AST/89 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... Z29514_SD ... ... ... ... ... ... ... .T. ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... PRIMER-PROBE “VP60” AC YTG ACT GAA CTY ATT GAC G FAM-CC AAR AGC ACR CTC GTG TTC AAC C T-TAMRA CC AAT GGC TTT TCT TAT GTC TGA

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Appendix 4 Publications

virus-host adaptation and co-evolution of … · de mixoma vírus (virulento) nas últimas cinco...

Documents