Cairo University Faculty Of Veterinary Medicine Department Of Virology

MOLECULAR CHARACTERIZATION OF FIELD AND VACCINAL STRAINS OF NEWCASTLE

DISEASE VIRUS

A thesis presented By

Mohamed Mounir Abd El-Fattah Radwan [B. V. Sc., Cairo University, 2006]

For the degree of

Master in Veterinary Medical Sciences [Virology]

Under supervision of

Prof. Dr. Mohamed Abd El-Hameed Shalaby Professor of Virology

Faculty of Veterinary Medicine Cairo University

Prof. Dr. Dr. Ahmed Abd El-Ghani El-Sanousi Ibrahim M. A. El-Sabagh

Professor of Virology Associate Professor of Virology Faculty of Veterinary Medicine Faculty of Veterinary Medicine

Cairo University Cairo University

2012

Name: Mohamed Mounir Abd El-Fattah Radwan

Nationality: Egyptian

Specialty: Virology

Degree: Master in Veterinary Medical Sciences.

Title: Molecular characterization of field and vaccinal strains of Newcastle disease virus

Supervisors:

- Prof. Dr. Mohamed Abd El-Hameed Shalaby Professor of virology, Faculty of Veterinary Medicine, Cairo University.

- Prof. Dr. Ahmed Abd El-Ghani El-Sanousi Professor of virology, Faculty of Veterinary Medicine, Cairo University.

- Dr. Ibrahim Mohamed Ahmed El-Sabagh Associate professor of virology, Faculty of Veterinary Medicine, Cairo University.

Abstract

Using the NDV RRT-PCR M-gene assay followed by the two step RT-PCR to amplify the hyper variable region of the NDV F-gene coupled with direct sequencing, followed by bioinformatical analysis to the sequence product including; multiple-sequence alignment, phylogenetic analysis, pairwise alignment and RDP, enables us to diagnose both; the old NDV genotype VI which used to be circulating in Egypt since 1970s, and the new NDV genotype VIId, which is currently circulating in Egypt, causing very high mortalities in birds. It enables us also to differentiate between the NDV vaccine and field strains, beside detection of mutations that could have been occurred in the neutralizing epitope A4, and the possible recombination events.

Keywords: Newcastle disease virus, genotype VIId, goose paramyxovirus, hyper variable region, recombination

ACKNOWLEDGEMENTS

"Praise to Allah, who has guided us to this; and we would never have been

guided if Allah had not guided us."

I would like to express my gratitude and appreciation to the following:

Prof. Dr. Mohamed Abdel-Hameed Shalaby and Prof. Dr. Ahmed Abdel-Ghani El-Sanousi (Professors of Virology, Faculty of Veterinary Medicine, Cairo University) and Dr. Ibrahim M. A. El-Sabagh (Associate Professor of Virology, Faculty of Veterinary Medicine, Cairo University) for their supervision, patience, unlimited trust, believe that I can do it, guidance and support throughout the study.

Dr. Samah Fekry Mohamed Ali (Senior Researcher, Biotechnology Research Unite, Animal Reproduction Research Institute, Agriculture Research Center) for her continuous advice, efforts, technical assistance that facilitate the completion of the study, critically reading the manuscript and assistances that are too numerous to mention.

Dr. Khaled M. Mahgoub (Researcher, Poultry Disease Department, Veterinary Research Division, National Research Center) for his continuous advice, supports, and providing me the old Newcastle disease virus isolates that were used in this study.

Staff members of the Virology Department, Faculty of Veterinary medicine, Cairo University for their support.

Staff members of the Biotechnology Research Unite, Animal Reproduction Research Institute, Agriculture Research Center for their assistances.

Members of the Arab Poultry Breeders Co. "OMMAT" for their assistances and support.

Finally, my Mother and Father for their duáa, patience, sacrifice, moral and financial supports.

I

CONTENTS

CONTENTS...........................................................................................................................I

LIST OF TABLES...................................................................................................................III

LIST OF FIGURES...............................................................................................................IV

LIST OF ABBREVIATIONS..................................................................................................V

I. INTRODUCTION.................................................................................................................1

II. REVIEW OF LITERATURE II. 1. Nomenclature..............................................................................................................................4

II. 2. Taxonomy and Classification...........................................................................................4

II. 3. History............................................................................................................................................6

II. 4. Properties of the Virion........................................................................................................7

II. 5. Properties of the Genome....................................................................................................9

II. 6. Viral Proteins...........................................................................................................................11

II. 7. Replication Cycle..................................................................................................................22

II. 8. Phylogenitic analysis and Molecular Epidemiology of NDV....................25

II. 9. Molecular Basis of Viral Virulence............................................................................30

II. 10. Structural difference between virulent and avirulent/vaccinal NDV

strains..................................................................................................................................................37

II. 11. Newcastle Disease..............................................................................................................38

II. 12. Diagnosis.................................................................................................................................39

II. 13. Immune Response..............................................................................................................47

II. 14. Vaccination.............................................................................................................................48

II. 15. NDV in Egypt.......................................................................................................................53

III. MATERIAL AND METHODS

III. 1. Material......................................................................................................................................57

III. 2. Methods.....................................................................................................................................65

II

IV. RESULTS

IV. 1. Isolation of NDV circulating in Egypt..................................................................76

IV. 2. Molecular detection of NDV using quantitative RT-PCR........................76

IV. 3. Sequencing of the highly variable region of the F-gene..............................79

IV. 4. Epidemilogical assessment of the origin and spread of NDV.................81

IV. 5. Molecular differentiation between vaccinal and field NDV.....................85

IV. 6. Determination of possible mutation that could have been occurred in

NDV……………………………………………………………………………………………….....85

V. DISCUSSION..............................................................................................................................92

VI. Summary...................................................................................................................................103

VII. REFERENCE......................................................................................................................105

Arabic Summary

III

LIST OF TABLES

Table No. Description Page No.

1 Place and year of isolation of the laboratory isolate

samples 57

2 Place and year of isolation of the field collected

samples 57

3 components of 2X QuantiTect RT-PCR master mix 58

4 RRT-PCR primer and probe details 59

5 Details of the PCR primers 60

6 Components of Dream Taq green PCR master mix

(2X) 61

7 Sequences retrieved from the GenBank 62

8 Quantitative RT-PCR reaction mix 69

9 cDNA reaction mix 70

10 PCR reaction mix 71

11 Abbreviation assigned to the samples 76

12 Cycle threshold result of the M-gene assay 77

13 Molecular characterization of the sequenced isolates 81

14 Pairewise alignment 84

15 Pairewise alignment (percent identity) based on

Wilber-Lipman method with currently available live

vaccines 85

16 The average P-value results of the methods used in

the RDP4 86

17 Analysis of the deduced amino acid sequences for

F-genes 87

IV

LIST OF FIGURES

Figure No. Description Page No.

1 Electron micrograph of NDV particles purified from allantoic fluid 8

2 NDV viron 8

3 NDV genome 11

4 An electron micrograph of the NDV nucleocapsid 12

5 Topology Diagram of the Head, Neck, and Stalk Regions of NDV-F, Including Schematic Representations of the Individual Domains within Each Monomer 19

6 Proposed Arrangement of the Fusion Peptide, HR-A, HR-B of NDV-F in its Metastable Form and Stable Form 20

7 Replication cycle of family Paramyxoviridae 23

8 Phylogenetic tree of 161 NDV isolates containing an amino acid analysis of the full fusion protein using the neighbour-joining method/JTT matrix-based. Class I with 7 of 10 genotypes and class II with 7 of 10 genotypes are represented 28

9 Amplification plots of the NDV M-gene assay 78

10 Agrose gel electrophoreses result 79

11 Nucleotide sequence of samples under study 80

12 Phylogenetic relationship between the isolates used in this study and other sequences retrieved from the GenBank 82

V

LIST OF ABBREVIATIONS

AIV : Avian Influenza Virus

AV : Australia-Victoria strain

BAP : Bioactive Amplification with probing

bp : base pair

cDNA : Complementary Deoxyribonucleic acid

Chimaera : Maximum mismatch Chi-square

Ct : Cycle threshold

DNA : Deoxyribonucleic acid

ELISA : Enzyme linked immune-sorbent assay

F-protein : Fusion glycoprotein

GenConv : Gene conversion

GPMV : Goose paramyxovirus

HA : Haemagglutinin

HB : Helix bundle

HI : Haemagglutinin inhibition

HN : Haemagglutinin neuraminidase glycoprotein

HPAI : Highly pathogenic avian influenza

HR : Heptads repeat

ICPI : Intracerebral pathogenicity index

IFN : Interferon

IGs : Intergenic sequence

IVPI : Intravenous pathogenicity index

L-protein : Large polymerase protein

LATE : Linear after the exponential

LBM : Live bird market

loNDV : Low virulence Newcastle disease virus

VI

M-protein : Matrix protein

MaxChi : Maximum Chi-square test

MDT : Mean death time

MEGA : Modern Evolutionary Genetic Analysis

NASBA : Nucleic acid sequence based amplification

ND : Newcastle disease

NDV : Newcastle disease virus

NP-protein : Nucleocapsid protein

nt : neucleotide

NTC : No template control

OIE : Office international des epizooties

ORF : Open reading frame

P-protein : Phosphoprotein

PCR : Polymerase chain reaction

PPMV : Pigeon paramyxovirus

RAPID : Reliable assay protocol for identification of disease

RdRp : RNA dependant RNA polymerase

RDP : Recombinant detection program

RNA : Ribonucleic acid

RNP : Ribonucleoprotein

rPCR : random polymerase chain reaction

RRT-PCR : Real-time reverse-transcriptase polymerase chain

reaction

RT-PCR : Reverse transcriptase polymerase chain reaction

SiScan : Sister-scanning

SISPA : Sequnce independent single primer amplification

SNP : Single nucleotide polymorphism

VII

SSCP : Single-stranded conformation polymorphism analysis

USDA : United States department of agriculture

vNDV : Virulent Newcastle disease virus

VVND : Velogenic viscerotropic Newcastle diseasae virus

Amino acids abbreviations

A : Ala : Alanine

R : Arg : Arginine

N : Asn : Asparagine

D : Asp : Aspartic acid

C : Cys : Cysteine

E : Glu : Glutamic acid

Q : Gln : Glutamine

G : Gly : Glycine

H : His : Histidine

I : Ile : Isoleucine

L : Leu : Leucine

K : Lys : Lysine

M : Met : Methionine

F : Phe : Phenylalanine

P : Pro : Proline

S : Ser : Serine

T : Thr : Threonine

W : Trp : Tryptophan

Y : Tyr : Tyrosine

V : Val : Valine

Introduction

1

I. Introduction

Newcastle disease (ND) is regarded throughout the world together

with highly pathogenic avian influenza as the two most important diseases of

poultry and other birds, because of the severe nature of the disease and the

associated consequences, ND is included as an Office International des

Epizooties (OIE) list A disease.

The importance of ND for poultry is not only due to devastating

NDV infections, with flock mortality rates up to 100%, but also the

economic impact that may ensue due to trading restrictions and embargoes

placed on areas and countries where outbreaks have occurred. (Aldous and

Alexander, 2001) Economic losses also resulted from decreased egg

production rates (egg drops); even on well-vaccinated farms have raised

questions about the antigenic variation of NDV and the efficacy of

conventional vaccines. Conventional vaccines can protect against mortality

caused by field NDV strains, but protection against egg drop resulting from

contemporary field NDV strains has never been demonstrated. (Cho et al.,

2008)

Because of the highly contagious nature of NDV and its clinical

similarity to highly pathogenic avian influenza, accurate monitoring and

rapid diagnosis of bird infections are crucial to any control and eradication

program. Many challenges still remain on the detection of virulent NDV

(vNDV), because vaccine and endemic viruses are often serologically

indistinguishable. Rapid differentiation of low pathogenic NDV (loNDV)

strains from vaccines and identification of new forms of vNDV needs to be

investigated further. (Miller et al., 2010b)

Introduction

2

Being a member of the Paramyxoviridae family, which include

viruses such as human parainfluenza viruses, which are the leading cause of

respiratory disease in children, studying the structure of NDV proteins, and

there role in recognition of the receptors and replication, and the spread of

virus makes it an attractive target for designing and the development of

drugs against important childhood respiratory viruses. (Zaitsev et al., 2004)

A case history involving a poultry farmer whose metastatic gastric

carcinoma underwent regression coinciding with an outbreak of ND in his

chickens led to the application of an attenuated NDV vaccine for treatment

of a few terminal cancer patients with favorable results. Various NDV

strains have been used in cancer treatment. (Swayne and King, 2003)

In the past 2 decades, due to strict vaccination program, occurrences

of ND were mild and sporadic and caused few deaths in chicken flocks in

Egypt. The sporadic cases, showing no typical clinical and pathological

manifestation of the disease. Since the past year (2011), however, outbreaks

of ND have continuously occurred in the well vaccinated chickens flocks of

Egypt. This raises the question about either the presence of new genotype

that entered Egypt that is very virulent, or the presence of mutation in

currently present genotype.

Introduction

3

So this study aims to:

1. Isolation of NDV currently circulating in Egypt.

2. Rapid, accurate and sensitive molecular detection and diagnosis of

NDV.

3. Genotyping of NDV.

4. Epidemiological assessment of the potential origin and method spread

of NDV.

5. Molecular differentiation between vaccine and field strain of NDV.

6. Determination of possible mutation that could have been occurred in

the NDV.

Review

4

II. REVIEW OF LITERATURE

II. 1. NOMENCLATURE

The name “Newcastle disease” (ND) (after the geographical location

of the first outbreaks in Great Britain), was coined by Doyle as a temporary

measure because he wished to avoid a descriptive name that might be

confused with other diseases. The name has, however, continued to be used,

although when referring to ND virus (NDV), the synonym “avian

paramyxovirus type 1” (APMV-1) is now often employed. Sometimes

APMV-1 has been used to describe ND strains of low virulence, to avoid

terming them ND viruses, as the definitions used by the World Organisation

for Animal Health and other international agencies reserve ND for virulent

viruses. (Alexander, 2009)

II. 2. TAXONOMY AND CLASSIFICATION

Newcastle disease virus is a member of the genus Avulavirus of the

family Paramyxoviridae in the order Mononegavirales. (Lamb and Parks,

2007) The order contains four families of enveloped viruses possessing

linear, nonsegmented, negative-sense, single-stranded Ribonucleic acid

(RNA) genomes. The family Paramyxoviridae is further divided into two

subfamilies: Paramyxovirinae and Pneumovirinae. The subfamily

Paramyxovirinae comprises five genera, Rubulavirus, Avulavirus,

Respirovirus, Henipavirus, and Morbillivirus, as well as a number of

unclassified viruses that might become the basis of one or more additional

future genera, in Paramyxovirinae. The division of this subfamily into five

genera and the unclassified group is based on: (1) amino acid sequence

Review

5

relationship between the corresponding proteins; (2) the number of

transcriptional units; (3) RNA editing products of the phosphoprotein (P)

gene; and (4) the presence of neuraminidase and hemagglutinin activities in

the attachment protein. The subfamily Pneumovirinae contains two genera:

Pneumovirus and Metapneumovirus. (Samal, 2010)

The Avulavirus genus contains, until 2010, nine distinct avian

paramyxovirus (APMV) serotypes grouped or separated by serological tests.

Six of these serotypes were classified in the latter half of the 1970s, when

the most reliable assay available to classify paramyxoviruses was the

hemagglutination inhibition (HI) assay. However, there are multiple

problems associated with the use of serology, including the inability to

classify some APMVs by comparing them to the sera of the nine defined

APMVs alone. In addition, one-way antigenicity and cross-reactivity

between different serotypes have been documented for many years.

However, as more and more isolates become available and new intermediate

isolates are discovered, it is possible that a serology-based classification may

not provide enough resolution for exact classification. (Miller et al., 2010a)

Miller et al. (2010a) isolated and characterized a paramyxovirus

isolated from rock hopper penguins (Eudyptes chrysocome) in Falkland

Islands. The morphology, biological and genomic characteristics, and

antigenic relatedness have proved that the virus belongs to a new serotype

(APMV 10). Currently there are at least eight other avian paramyxoviruses

isolated from different penguin species from various geographical locations

that are not inhibited by known APMV sera and likely representing at least

Review

6

three serotypes different from each other and from APMV10. (Miller et al.,

2010a)

Newcastle disease virus (APMV-1) remains the most important

pathogen for poultry, but APMV-2, APMV-3, APMV-6, and APMV-7 are

known to cause disease in poultry. (Alexander, 2003)

Newcastle disease virus was previously placed in genus Rubulavirus,

(Emmerson, 1999) but the fully sequenced La Sota NDV genome found to

be only distantly related to other members of the genus Rubulavirus.

(Sinkovics and Horvath, 2000) So NDV was separated in separate genus

Avulavirus. (Lamb and Parks, 2007)

II. 3. HISTORY

The first outbreaks of the severe disease of poultry known as

Newcastle disease occurred in 1926, in Java, Indonesia, (Alexander, 2009)

and it is thought likely that the virus was transported to the port of

Newcastle-upon-Tyne by ship from Southeast Asia. (Emmerson, 1999)

Whether the outbreaks of 1926 marked the emergence of ND has been

the subject of some discussion, as there are earlier reports of similar disease

outbreaks in Central Europe before this date. (Alexander, 2009)

Macpherson (1956) attributes the death of all the chickens in the Western

Isles of Scotland in 1896 as being due to Newcastle disease. (Macpherson,

1956) It is possible; therefore, that ND did occur in poultry before 1926, but

its recognition as a specifically defined disease of viral aetiology dates from

the outbreaks during that year in Newcastle-upon-Tyne. (Alexander, 2009)

Review

7

Newcastle disease was first recognized as highly pathogenic disease

with up to 100% mortality. (Emmerson, 1999) It is later become more clear

that other less sever disease were caused by viruses indistinguishable from

NDV by conventional method. (Alexander, 2003) As in California, United

States, a relatively mild respiratory disease was observed in the mid 1930s,

with mortality usually less than 15% which was first called

pnemoencephalitis, but it was later shown to be caused by a virus which was

indistinguishable immunologically from NDV. The observation that NDV

was not always highly pathological was important and was followed by

numerous reports of other strains with low virulence. Such isolates were

used later as live vaccines. (Emmerson, 1999)

II. 4. PROPERTIES OF THE VIRION

NDV is a pleomorphic (as shown in figure 1), enveloped virus of

roughly spherical shape, ranging in diameter from 150-400 nm. It contains a

helical nucleocapsid structure 1000 nm long and 17-18 nm in diameter, with

a center hole of 5 nm. (Emmerson, 1999) NDV virions are made of three

envelope and three core proteins. Envelope proteins include a large

glycoprotein with both hemagglutinin and neuraminidase activities (HN), a

smaller glycoprotein with cell-fusing activity (F), and a nonglycosylated

protein (M) localized at the inner surface of the envelope, (Long et al.,

1986) interposed between the nucleocapsid and the viral envelop. The core

of the virion, contain the nucleocapsid, which is formed by the nucleocapsid

(NP) protein, associated with the NP the phosphoprotein (P) and the large

(L) protein, as shown in figure 2. (Emmerson, 1999)

Review

8

Figure 1: (a) Electron micrograph of NDV particles purified from allantoic fluid. (b) A partly disrupted NDV particle exposing the nucleocapsid. The bars represent 100 nm. (Yusoff and Tan, 2001)

Figure 2: NDV viron

Review

9

II. 5. PROPERTIES OF THE GENOME

The genome NDV is linear, single stranded of RNA, negative sense

(Emmerson, 1999) have at least three genome lengths; 15,186, 15,192 and

15,198 nucleotides. Class I viruses have the longest of the APMV-1

genomes at 15,198 nucleotides. (Miller et al., 2010b)

Negative-strand RNA viruses are unique in their RNA genomes are

always encapsidated by a viral coded nucleoprotein to form a

ribonucleoprotein (RNP) complex. This complex serves as the template for

viral RNA synthesis and forms the structural core of the viruses when

packaged into virions. The RNP is formed concurrently with

transcription/replication by the viral RNA-dependent RNA polymerase

(RdRp). The RdRp complex is composed of the negative-sense RNA

genome and three proteins: nucleoprotein (NP), phosphoprotein (P) and the

large subunit polymerase protein (L). The RNA genome of this complex is

always found associated with the nucleoprotein as the RNP. This structure is

resistant to nucleases, even during synthesis. (Cleveland et al., 2011)

The viral genome must be transcribed into mRNA by the virion

associated RNA-dependent RNA polymerase before translation can take

place, as the genomic RNA itself is not infectious. (Emmerson, 1999)

The NDV genome is comprised of six genes: nucleoprotein (NP),

phosphoprotein (P), matrix protein (M), fusion glycoprotein (F),

hemagglutinin-neuraminidase (HN) glycoprotein, and large polymerase

protein (L), as shown in figure 2. (Oberdorfer et al., 1999) The genes are

arranged in the order: 3´-NP-P-M-F-HN-L-5´. (Emmerson, 1999) The

Review

10

genome contains neither a 5´ cap nor a 3´ end poly (A) tail. At the 3 ́and 5´

ends of the genome are short extragenic (noncoding) regions known as the

‘leader’ and ‘trailer’ region, respectively. (Samal, 2010) The leader

sequence at the 3´ end, of 55 nucleotides long, upstream of the NP gene, and

the trailer sequence at the 5 ́end, 56 or 114 nucleotides long according to the

strain, downstream of the L gene. (Emmerson, 1999)

The beginning and end of each gene are conserved transcriptional

control sequences, known as the gene start and gene end, respectively.

Between the gene boundaries are noncoding intergenic sequences (IGSs),

which vary in length from 1 to 47 nt. Each of the first three IGSs, the NP-P,

P-M, and M-F gene junctions, has only 1 nt, while the other two IGSs, the F-

HN and HN-L gene junctions, are 31 nt and 47 nt, respectively. The lengths

of IGSs are generally conserved in most NDV strains. However, the

sequences of IGSs vary among NDV strains. (Yan and Samal, 2008)

For most members of the Paramyxovirinae, including NDV, efficient

replication is dependent on the genome length being a multiple of six. This

requirement is known as the ‘rule of six’. It is assumed that each NP subunit

is in contact with exactly six nucleotides and that this arrangement is

required for efficient replication. A shift of the NP subunit other than 6n

causes a shift in the position of the promoter resulting in improper initiation

of replication. (Zhao and Peeters, 2003)