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Rabbit hemorrhagic disease (RHD): characterization ofthe causative calicivirus

Vf Ohlinger, B Haas, Hj Thiel

To cite this version:Vf Ohlinger, B Haas, Hj Thiel. Rabbit hemorrhagic disease (RHD): characterization of the causativecalicivirus. Veterinary Research, BioMed Central, 1993, 24 (2), pp.103-116. �hal-00902110�

Review article

Rabbit hemorrhagic disease (RHD):characterization of the causative calicivirus

VF Ohlinger B Haas HJ Thiel

Federal Research Centre for Virus Diseases of Animals Tiibingen, PO Box 1149,D-7400 Tubingen, Germany

(Received 6 July 1992; accepted 23 July 1992)

Summary ― Rabbit hemorrhagic disease (RHD) which was first recognized in China in 1984 spreadvia Eastern Europe to many countries of Western Europe and other parts of the world. The analysisof the virus outlined in this review comprises: 1) physico-chemical properties, 2) electron microscopyincluding immunoelectron microscopy, 3) demonstration of capsid protein, 4) in vivo neutralizationwith monoclonal antibodies (mabs), 5) infectivity of purified RNA, and 6) characterization of the viralgenome. Also included are clinical, pathological and epidemiological findings, different diagnosticmethods as well as disease control measures. Finally, similarities between RHD and the Europeanbrown hare syndrome (EBHS) are pointed out. The latter disease is caused by a calicivirus differentfrom RHDV.

rabbit hemorrhagic disease I etiology I epidemiology I diagnosis I calicivirus

Résumé ― Maladie hémorragique du lapin : caractérisation du calicivirus responsable. Lamaladie hémorragique du lapin, qui a été signalée pour la première fois en Chine en 1984, s’estétendue par l’Europe de l’Est à de nombreuses régions d’Europe Occidentale et à d’autres partiesdu monde. L’analyse du virus exposée dans cette revue comprend (1) les propriétés physicochi-miques, (2) l’étude en microscopie électronique incluant l’immunoélectromicroscopie, (3) la mise enévidence de la protéine de capside, (4) la neutralisation par des anticorps monoclonaux, (5)l’infectivité de l’ARN purifié, et (6) la caractérisation du génôme viral. Les résultats concernant lesétudes cliniques, la pathologie et l’épidémiologie ainsi que les différentes méthodes de diagnostic etles mesures de contrôle de la maladie sont également présentées. Enfin sont signalées les simili-tudes entre la maladie hémorragique et le syndrome du lièvre brun européen. Celui-ci est causé parun calicivirus différent du virus de la maladie hémorragique du lapin.

maladie hémorragique du lapin / étiologie / épidémiologie / diagnostic / calicivirus

*

Correspondence and reprints

INTRODUCTION

Within the last decade 2 new viral dis-eases causing acute liver necrosis with

high mortality in leporides have been re-

ported from many countries around the

world. In 1984 rabbit hemorrhagic disease(RHD), which by OIE is named &dquo;viral he-

morrhagic disease&dquo; (VHD), was first recog-nized in the People’s Republic of China(Liu et al, 1984), whereas another viral epi-zootic disease, called European brown

hare syndrome (EBHS) has caused se-vere losses in European brown hares

since the beginning of 1980 (Gavier-Widenand Morner, 1991; L61iger and Eskens,1991). In this review findings concerningthe etiological agent, clinical symptoms, di-agnosis, pathology and epidemiology of

RHD are outlined and some interestingsimilarities with EBHS are discussed.

CLINICAL SYMPTOMS

Infection usually leads to peracute or

acute RHD. Whereas no or indistinct clini-cal signs are found before death for pera-cute RHD, anorexy, apathy and tachy-pnoea can be detected in acute cases

(Boujon et al, 1989; Ohlinger et al, 1989;Xu and Chen, 1989; Du, 1990; Schirrmeieret al, 1990; Marcato et al, 1991 Further-more, the rabbits show clonal convulsions,lateral torsion of the head, paresis andsometimes bloody or bloody-foamy nasalexcretions. The animals show fever but

shortly before death subnormal body tem-perature (Xu and Chen, 1989). Experimen-tal intranasal and intramuscular infections

usually result in death after 48-72 h, butsometimes animals die after 8 d. Duringan epidemic subacute cases with limited

anorexy, apathy and convalescence havebeen reported (Marcato et al, 1991 Someof these rabbits show icteric mucosa and

die several weeks after infection. Interest-

ingly, rabbits up to 4 wk of age do not ex-hibit clinical signs and usually survive in-

fection.

PATHOLOGY

Pathological alterations varied from inap-parent to highly significant (Ohlinger et al,1989). Enlarged vessels were observed

especially in the abdominal cavity. As arule the coagulation of blood was inhibitedsignificantly (Xu and Chen, 1989). Some-times a hydrothorax and in few cases anicterus were found. The lungs, liver, kid-

neys and spleen were usually affected

(Marcato ef al, 1988, 1989, 1991; Boujonet al, 1989; Ohlinger et al, 1989; Xu andChen, 1989; Albert and Wenzel, 1990;Schirrmeier et al, 1990; Nowotny et al,1990). The lungs showed hemorrhagic le-sions to varying degrees. Bloody foam wasgenerally detected in the trachea and in

the bronchi. Hemorrhages were also foundin the tracheal mucosa. The liver was paleand fragile with accentuation of the lobularmarkings, or the organ was enlarged anddark reddish discolored. The kidneys wereenlarged and speckled reddish, but some-times only few petechiae were observed.Usually the spleen was enlarged and alsodark reddish discoloured. Histopathologi-cally, in the lungs alveolar oedema, hemor-rhages and infiltrates of granulocytes weredetected. The submucosal capillaries ofthe trachea were congested, and some-times infiltrates of rounded cells were

found. Diffuse or focal infiltrates of granu-locytes and single hemorrhages were alsodetected in the liver early after infection.

Futhermore, degenerative perilobular alter-ations in hepatocytes with strongly homog-enous or pale vacuolized cytoplasm, nucle-ar pyknosis, karyorrhexis or karyolysiswere observed. The kidneys showed focalhemorrhages, hyperaemia, sometimes glo-

merular hyalinic thrombosis, dilated tu-

bules and lymphocytic infiltrates. The

spleen was highly hyperaemic with singu-lar follicular karyorrhexis. By electron mi-croscopy strongly degenerative and ne-

crotic alterations were detected in

hepatocytes (Marcato et al, 1991). Virus-like electron dense particles were detectedintranuclearly at the beginning of infection,later on within the cytoplasm. Similar parti-cles were also found in endothelial cells.

RHD was characterized by the rapidcourse of the disease. Fibrinous thrombi

within the capillaries of most organs, a

thrombocytopenia, as well as prolongedprothrombin- and thrombin-times indicateda consumption coagulopathy (Xu and

Chen, 1989). This disseminated intravas-cular coagulation (DIC) could be causedeither by endothelial destruction or by ne-crosis of the liver. Thus, the hemorrhagicsyndrome would be due to a primary orsecondary disorder of coagulation factors(Xu and Chen, 1989; Marcato et al, 1991 ).

ETIOLOGY

Early after outbreak of the RHD, epidemicexperimental infections were performedwith filtrated and chloroform treated organhomogenates; it was concluded that a non-

enveloped virus was the causative agentof RHD (Ohlinger et al, 1989; Xu andChen, 1989). Homogenates from variousorgans, mainly from liver of infected rab-

bits, agglutinate human erythrocytes up tohigh titres, indicating the presence of highamounts of viral antigens. Accordingly theliver of dead rabbits was used to isolatethe causative virus usually by a methodsimilar to that described by Ohlinger et al(1990a).

Briefly, liver homogenate from infectedanimals was centrifuged at low speed andthe supernatant layered on top of a su-

crose cushion. After ultracentrifugation, thepellet was resuspended and extracted withFreon -113. Density gradient centrifuga-tion using cesium chloride resulted in a vis-ible band at a density of 1.31 to 1.36 g/ml(Deng et al, 1987; Granzow et al, 1989;Smid et al, 1989; Xu and Chen, 1989; Oh-linger et al, 1990a; Park et al, 1991 Threebands were found after sucrose densitygradient centrifugation representing parti-cles with sedimentation coefficients of 175

S, 136 S and 100 S. Similar values be-tween 150-200 S were reported by others(Deng et al, 1987; Granzow et al, 1989;Capucci et al, 1990; Liebermann et al,1991 The density gradient fractions corre-lating with visible bands showed the high-est activity with convalescent sera in ELI-SA as well as in hemagglutination tests

(Ohlinger et al, 1990a). Bands of similardensity and sedimentation coefficients

were also found for caliciviruses (Schaffer,1979). Different bands of calicivirus parti-cles have been shown to be due to loss ofRNA (Schaffer and Soergel, 1976), or dif-ferent pH values (Rowlands etal, 1971). ).

Material obtained from the bands after

density gradient centrifugation was nega-tively stained in suspension and analyzedby electron microscopy (EM). Virions ofuniform size were detected in all samples,but preparations of 175 S particles showedthe highest degree of homogeneity andpurity. The virions were 40 nm in diameter(Ohlinger et al, 1990a) and displayed aclearly structured surface consisting of reg-ularly arranged cup-shaped depressions(Granzow et al, 1989; Ohlinger et al, 1989,1990a; Smid et al, 1989; Soike et al, 1989;Capucci et al, 1990; Du, 1990; Tesouro-

Vallejo et al, 1990; Valicek et al, 1990;Marcato et al, 1991; Park et al, 1991; Ro-dak et al, 1991). Following a 5-fold axis ofsymmetry, 10 short projections most parti-cles exhibit. Few particles showed 3- or 2-fold axes of symmetry (Capucci et al,1991; Rodak et al, 1991) and smaller parti-

cles with a smooth surface were found in

proteolytically degraded preparations (Ca-pucci et al, 1991). Whereas the electronmicrographs from several authors showedcaliciviral particles, the reported size of thevirions varied from 29-40 nm in diameter.These variations can either be explainedto be dependent on different stainingmethods, or on non-uniform presentationsof the projections. Rodak et al (1991 Mar-cato et al (1991) and Park et al (1991)demonstrated specific immune complexesby EM, which had been prepared after in-cubation of such virions with convalescentsera.

To detect virus specific proteins in livertissue from infected rabbits, materials froma sucrose gradient were first separated bySDS-polyacrylamide gel electrophoresis(SDS-PAGE). Fractions with highest hem-agglutinating activity resulted in a singlevisible band after Coomassie blue staining(Ohlinger et al, 1990a): Western blot anal-

yses of such fractions with convalescentsera again resulted in a single proteinband with an identical apparent molecularweight of 60-61 kDa; such a protein couldnot be observed with RHD-antibody nega-tive sera as well as with material from non-

infected rabbits (Ohlinger et al, 1990a).Others performed equivalent experimentsand reported similar molecular weights(Capucci et al, 1990; Rodak et al, 1990a).Virions of the Caliciviridae family consistprimarily of RNA and 180 copies of a sin-gle capsid protein with a molecular weightof 60-71 kDa (Schaffer and Soergel,1976; Francki et al, 1991 The results out-lined so far for RHD virus (RHDV) are con-sistent with its preliminary status as a ca-licivirus.

In less pure preparations of virions addi-tional proteins with apparent molecular

weights of 24-25 kDa and 35-44 kDa

were found (Ohlinger et al, 1989; Capucciet al, 1990; Rodak et al, 1990a, 1990b).Proteins with apparent molecular weights

of 120, 85-87, 52-56, 29, and < 20 kDa

were detected only in homogenates fromlivers of infected rabbits and may repre-sent nonstructural proteins (Ohlinger et al,1989; Parra and Prieto, 1990). Proteins ofsimilar molecular weights were previouslydescribed for other caliciviruses (Black andBrown, 1975/1976; Fretz and Schaffer,1978). So far, nonstructural proteins en-coded by calciviruses have not been identi-fied.

Capucci et al (1991) produced about 50monoclonal antibodies (mabs) directed

against RHDV. Eight of these mabs werefurther characterized and reacted with or-

gan homogenates from infected animals ina double sandwich-ELISA, using polyclo-nal trapping sera. Two of these mabs re-acted exclusively with RHDV, whereas theother 6 mabs showed crossreactivity withEBHS virus (EBHSV). When purified intactvirions were employed, only the 2 non-

crossreactive mabs exhibited strong bind-ing (Capucci et al, 1991 ).

After simultaneous injection of RHDV

and single mabs into rabbits, only one ofthe RHDV specific mabs (1 H8) mediatedstrong in vivo-neutralizing activity. In vivo-

neutralizing activity was not detected withthe crossreactive mabs (Capucci et al,1991 ).

These results led to the hypotheses thatRHDV specific mabs are directed againstexternal epitopes and that the crossreac-tive mabs recognize internal epitopes (Ca-pucci et al, 1991 Whereas Capucci et al(1991) found only 3 crossreactive mabs tobe positive by Western blot, Ohlinger et al(1990b) could show by Western blottingthe binding of the in vivo-neutralizing mab1 H8 to the 60 kDa protein. The mabs,which did not react by Western blot, alsorecognize the capsid protein as shown byimmunoprecipitation and subsequent dem-onstration by Western blot (Ohlinger andThiel, 1991). Rodak et al (1990a, 1990b)

also produced mabs against RHDV, whichreacted in ELISA and recognized a 60 kDaand 38 kDa protein by Western blotting.

MOLECULAR BIOLOGY

The findings outlined so far are consistentwith the idea that a member of the familyCaliciviridae represents the causative

agent of RHD. However, up to this pointdata about the genetic material of the virusare missing. Caliciviruses represent plusstrand RNA viruses; the genomic RNA hasa size of = 8 kb. Virus infected cells contain1 additional RNA with a size of = 2.4 kb

(Ehresmann and Schaffer, 1977; Black etal, 1978; Neill and Mengeling, 1988; Cart-er, 1990).

It was first shown that the RNA from

purified RHD virions has a size of = 8 kb.Liver tissue from infected animals con-

tained in addition to the genomic RNA asubgenomic RNA with a size of 2.2 kb.The demonstration of genomic and subge-nomic RNA with expected sizes suggestedagain a calicivirus as the causative agentof RHD (Ohlinger et al, 1990a).

After reverse transcription of the RHDVgenome, the cDNA was cloned in a bacte-

riophage vector. To detect virus specificclones a radioactive cDNA probe was em-ployed, which was obtained after incuba-tion of the reverse transcriptase with RNAfrom purified virions. Following cloning ofthe whole RHDV genome as cDNA the

complete nucleotide sequence was deter-mined; this represents the first completesequence of a calicivirus (Meyers et al,1991 a). The RHDV genome comprises7 437 nucleotides (without polyA tail) andcontains one long open reading frame. Thenonstructural proteins are encoded in the

5’ region of the genome, whereas the 60kDa capsid protein is encoded in the 3’ re-gion. The 2.2 kb subgenomic RNA men-

tioned above is colinear with the 3’ regionof the genome and encodes the capsidprotein. Sequence comparisons with felinecalicivirus (FCV) and RHDV indicated re-

gions of significant homology between

nonstructural proteins (Meyers et al,1991a). In the meantime additional se-

quences of the capsid protein genes fromFCV and RHDV have become available,indicating a higher degree of homogeneityamong RHDV isolates than among FCVisolates (Neill et al, 1991; Tohya et al,1991; Milton et al, 1992).

Further studies showed that genomic aswell as subgenomic RNA from RHDV areprotein-linked (Meyers et al, 1991 b). Equiv-alent findings have been reported for thegenomic RNAs of other caliciviruses. In

analogy to picornaviruses, which also rep-resent a virus family with plus-strand RNAgenome and a protein covalently linked tothe 5’ end of the genome, the respectivepolypeptide was termed VPg. In contrast,almost all eukaryotic mRNAs possess acap at the 5’ end.

CONCLUSIONS CONCERNINGTHE CAUSATIVE AGENT OF RHD

The above outlined analysis of the virus

comprises so far: 1) physico-chemicalproperties, 2) electron microscopy includ-ing immunoelectron microscopy, 3) dem-onstration of one capsid protein, 4) in vivoneutralization with a mab which recognizesthe capsid protein, 5) infectivity of purifiedviral RNA after intrahepatic injection, and6) characterization of viral nucleic acids.All of the results show that RHD is caused

by a calicivirus. One of our RHDV cDNAclones has been made available to a re-

search group in the People’s Republic ofChina to test the conflicting findings of sci-entists from China (Xu et al, 1988) andalso from the USA (Gregg and House,1989).

DIAGNOSIS

Together with the description of the firstRHD outbreaks, Liu et al (1984) reportedhemagglutination of human erythrocytesafter incubation with diluted organ homog-enates from infected rabbits. Later the

hemagglutination test was frequently rec-ommended by several authors as a diag-nostic test for the detection of RHDV anti-

gen (Pu et al, 1985; Ohlinger et al, 1989;Schirrmeier et al, 1990; Schliater et al,1990) and it is still being used as a simpletest. Whereas the test appears to be inde-

pendent of the blood group of human

erythrocytes, no or only weak reactionsare found with erythrocytes from other

species (Xu, 1991 The test is performedas a microtest, at pH values of 6-7.2 andwith 0.5 to 1 % erythrocytes in phosphate-buffered saline. Between incubation tem-

peratures of 4-37 °C no significant varia-tions in the titres were found (Capucci etal, 1991 The hemagglutinating activity is

stable against ether, chloroform, and for

60 min against temperatures up to 50 °C.No significant loss of activity was foundwith organ material, which was stored at 4°C for several months (Capucci et al,1991). Xu (1991) reported hemagglutina-tion titres of = 10 x 2!$ in liver homogen-ates, spleen and peripheral blood. Signifi-cantly lower titres were found in other

organ homogenates.At the Federal Research Centre for Vi-

rus Diseases of Animals in Tubingen, thehemagglutination test (HAT) was per-formed with human erythrocytes at 1% in

phosphate buffered saline, pH 6.4; about2% of organ homogenates from experi-mentally infected rabbits reacted negative-ly in HAT.

For the detection of RHDV antigens,sandwich-ELISAs have also been reported(Ohlinger et al, 1990a; Capucci et al,1991 ). In all these assay systems, rabbit

antisera to RHDV were bound to ELISA-

plates to trap RHDV. Thereafter, the boundantigen was detected using either guinea-pig antisera to RHDV (Ronsholt, personalcommunication) or biotinylated anti-RHDVIgG purified from RHDV-convalescent rab-bits (Haas et al, unpublished observations)followed by peroxidase-conjugate and sub-strate (Ohlinger et al, 1990a). The peroxi-dase was also directly conjugated to

RHDV specific IgG (Capucci et al, 1991 ).Normal rabbit sera were used to exclude

nonspecific reactions. An increase in spec-ificity and sensitivity was reported by usingmabs against RHDV instead of polyclonalantisera (Capucci et al, 1991). Thesemabs were either directly conjugated to al-kaline phosphatase (Capucci et al, 1991 ),or detected by peroxidase conjugated antispecies sera (Haas and Ohlinger, 1990).

After ELISAs became available for thedetection of RHDV antigen, the sensitivityof the HAT is being discussed much morecritically. Capucci et al (1991 ) performed acomparative survey of 1 000 samples,which were tested by immunoelectron mi-croscopy (IEM), antigen-ELISA and HAT;after direct comparison of the 3 tech-

niques, they reported that HAT resulted in8% of the samples being false positive and9% being false negative. Similar results

were obtained at our institute. HAT-titres

up to 1 in 500 should be considered ques-tionable; for example nonspecific HAT pos-itive results can be due to Pasteurella spp.Western blotting experiments demonstrat-ed that HAT-negative samples, which re-

acted positive in ELISA, contained a de-graded 60 kDa RHDV-specific protein(Capucci et al, 1991). The presence of

hemagglutination inhibiting antibodies in vi-rus positive samples has also been dis-

cussed to interfere with the detection of

hemagglutinating activity (Biermann et al,1992). Especially with regard to sampleswhich have undergone degradation the EL-ISA is thus certainly preferable to HAT.

Direct immunofluorescence (Nowotny etal, 1990; Rodak et al, 1991) or peroxidaseprocedures are also used to detect viral

antigen in organ material. However, strongnonspecific fluorescence is commonly ob-served when cryosections of infected,highly necrotic livers and various fluores-

cence-conjugates are used. Therefore, thefluorescence has to be evaluated verycarefully and it is not used for routine diag-nosis in our institute. Peroxidase staininghas also been reported for the detection ofviral antigens (Marcato et al, 1988; Man-delli et al, 1990; Stoerckle-Berger et al,1990; Park and Itakura, 1992).

Several groups failed to propagateRHDV in tissue culture cells. Interestingly,Ji et al (1991) reported the establishmentof a permissive rabbit kidney cell line

(DJRK). Data obtained by electron micros-copy and immunofluorescence, successfulprotective immunization, as well as infec-tion with material obtained from the 10-16th passage of infected DJRK cells, sug-gested replication of RHDV in cell culture.However, the titres of infectious virus areapparently extremely low. Additional exper-iments have to be performed, especially di-rect demonstration of viral nucleic acid andvirus encoded protein(s).

HAT and ELISAs established for diag-nosis of RHD were also employed for de-tection of EBHSV antigen(s) in diagnosticsamples. Using mabs against RHDV,which are crossreactive with EBHSV, theELISA can be used as a sensitive assayfor the detection of EBHSV antigens.RHDV specific mabs and mabs crossreac-tive to EBHSV allow discrimination be-tween RHDV and EBHSV antigens in diag-nostic samples (Ohlinger et al, 1990b;Capucci et al, 1991 Comparative studiesby Capucci et al (1991) resulted in much

higher sensitivity and specificity of the ELI-SA when compared to the HAT. Hemag-glutinating activities were found with semi-purified preparations of EBHSV, when thetest was performed on ice. Only 50% of

the samples, which reacted positively in

ELISA and by IEM, showed in HAT low ti-tres of hemagglutinating activity.

In order to detect antibodies to RHDV,the hemagglutination inhibition test (HIT),indirect ELISAS, as well as the competitionELISAs are used. The HIT is performedwith 0.5-1% of human erythrocytes in

phosphate buffered saline and 4 to 16

hemagglutinating units of RHDV antigen(Maess et al, 1989; Ohlinger et al, 1989;Sodan et al, 1990; Capucci et al, 1991; Xu,1991 To eliminate nonspecific hemagglu-tination inhibition activities, the sera haveto be pretreated by incubation at 56 °C for30 min, treatment with potassium tetrajo-date, kaolin or preadsorbtion to erythrocy-tes. The pretreatment of test samples inter-feres with the examination of largenumbers of diagnostic sera and the HIT isdifficult to standardize. Therefore variousELISAs for the detection of antibodies toRHDV were established. Rodak et al

(1990a) and Schirrmeier et al (1990) em-ployed an indirect ELISA. In this test puri-fied RHDV antigens are coated to ELISAplates and antibodies to RHDV are detect-ed by anti-species enzyme conjugates andsubstrate; such an ELISA is commerciallyavailable. To avoid laborious purificationsteps of the antigen and to increase thespecificity, ELISAs were established to de-tect antibodies by competition. Competitiveantisera to RHDV were raised in guineapigs and detected with anti-species en-

zyme conjugates (Ronsholt, personal com-munication). Alternatively sera derivedfrom convalescent rabbits were either di-

rectly conjugated to enzymes (Capucci etal, 1991) or biotinylated and subsequentlydetected by streptavidin-enzyme conju-gates (Ohlinger et al, 1990a). In our ELI-

SA, RHDV antibody-negative sera resultedin < 30% inhibition compared to values de-rived from phosphate buffer controls. Thus,this 30% inhibition was taken as cut-off val-ue to differentiate between RHDV anti-

body-positive and -negative samples. 304

sera were tested in a comparative surveyusing our competitive ELISA and the HIT.In both systems 77 sera reacted positiveand 219 sera gave negative results. Sevensera, which originated from infected hold-ings, but were negative in the HIT, reactedpositively in the ELISA. After comparativetitration of 52 sera in the HIT and the

ELISA, we found a coefficient of correla-tion of 0.62.

Animals which survived an infectionwith RHDV or infected juvenile rabbits de-veloped antibodies to RHDV, which weredetectable at d 5 post infection. These an-tibody titres reached a plateau one weekafter infection and persisted for at least 8months.

Surprisingly, antibodies to RHDV werefound in sera, which were taken prior tothe first reports of viral hemorrhagic dis-eases of lagomorphs in Europe between1960-1980, and also in rabbit sera, whichcame from holdings with no history of

RHDV (Ohlinger et al, 1989; Rodak et al,1990a; Nowotny et al, 1992). Three sero-positive rabbits from such a holding sur-vived experimental challenge infectionswith RHDV, whereas the control rabbitsdied. Rodak et al (1991) challenged 121non-RHD diseased antibody positive rab-bits and correlated the anti-RHDV anti-

body titres with the rates of survival. Eightsero-negative rabbits were introduced intoa clinically unsuspicious holding, which

comprised rabbits with antibodies to

RHDV, seroconverted without any clinicalsymptoms of RHD (Capucci et al, 1991).Taken together these findings indicate theexistence of an apathogenic virus which isrelated to RHDV.

EPIDEMIOLOGY

Rabbit hemorrhagic disoase was first re-

ported in the People’s Republic of China in1984 (Liu et al, 1984) and connected to an

import of angora rabbits from Germany (Xuet al, 1988). Further outbreaks occured in

Korea (An et al, 1988), in Italy (Cancelottiet al, 1988, 1991) and in nearly all Europe-an countries (Morisse, 1988; Argbllo Vil-

lares et al, 1989; L61iger et al, 1989; Pes-chev et al, 1989; Smid et al, 1989; Soike etal, 1989; Boujon et al, 1989; K61bl et al,1990; Mocsari, 1990; SchlOter and Schirr-meier, 1991; OIE, 1992). RHD epidemicswere also described in Mexico (Gregg andHouse, 1989) and in Israel. Most investiga-tors suppose that the spread of the dis-

ease started with exported rabbit meat

from China. In contrast, L61iger and Esk-ens (1991) hold unrecognized caliciviral in-fections of wild-life lagomorphs in Europeresponsible for RHD. However RHD ap-

peared in wildlife rabbits at the same timeas in domestic rabbits. In contrast EBHS

was recognized in European brown haresprior to the first reports of RHD. Followingthe hypothesis of L61iger and Eskens,EBHS and RHD should show epidemio-logical correlations and one would expectthat the viruses can overcome species bar-riers. After infection with material from

EBHS diseased hares, Morisse et al

(1991) as well as Di Modugno and Nasti(1990) reported clinical symptoms in rab-

bits identical to RHDH, and Du (1991) aswell as Nowotny et al (1992) found clinicalsigns identical to EBHS in hares infectedwith material from rabbits with RHD. How-

ever, most of the infection experimentsacross species failed (Capucci et al, 1991;Ohlinger, unpublished results). Further-

more, EBHS was observed in Scandinavi-

an countries several years before the first

outbreaks of RHD occurred and from most

of these countries also no RHD was report-ed in wild rabbits.

Lagomorphs are the only animals whichare susceptible to experimental infectionswith RHDV and EBHSV (Xu and Chen,1989; Smid et al, 1991; Xu, 1991). Otherspecies like rats, mice, hamster, guinea

pigs, horses, donkeys, cattle, buffaloes,sheep, goats, dogs, cats and chicken weretested with negative results. Furthermorethere are no indications for RHD and

EBHS as zoonoses. Significant differencesbetween various races of rabbits (Xu,1991) or between males and females

(Pages Mante, 1989) have not been found.Using an RHDV isolate from Spain, PagesMante (1989) reported a higher susceptibil-ity of wild rabbits to experimental infectionswhen compared with rabbits from holdings.

The disease is spread by oral, nasal orparenteral transmission. The virus is ex-

creted with all secretions and excretions ofdiseased rabbits. In the field, the fecal-oralway of transmission is probably the mostimportant one (Morisse et al, 1991 ). Gregget al (1991) found that faeces from surviv-ing rabbits were infectious for susceptibleanimals up to 4 wk after infection. In exper-iments with 3 susceptible rabbits, whichwere held in contact with young RHD con-

valescent animals (4 wpi) no clinical symp-toms and no seroconversion could be de-

tected even after immunosuppression of

the convalescent rabbits (Haas, unpub-lished observations). Other ways of trans-mission could be contamination of feed,materials and persons (Erber et al, 1991 ),which was shown to be important especial-ly in small extensive, non-commercial hold-ings. The significance of wild rabbits withregard to contamination of green feed aswell as epidemiology of RHD is describedby Maess et al (1990). The importance ofinsects and rodents as passive vectors isunclear (Cancelotti and Renzi, 1991; Gehr-mann and Kretzschmar, 1991 ). RHDV re-mained infectious for 225 d at 4 °C and for

2 d at 60 °C. Infective particles could bedemonstrated in dried material for 105 d at

room temperature (Smid et al, 1991 Fur-thermore, RHDV is resistant to pH 3.0 (Xuand Chen, 1989).

After introduction of RHD into free re-

gions, the virus shows rapid spreading and

leads to epidemics with high morbidity andmortality with rates up to 100% for adult

rabbits (Xu and Chen, 1989). In endemic

regions, the morbidity and mortality are

significantly lower due to higher frequen-cies of anti-RHDV- antibody positive ani-mals.

DISEASE CONTROL

Effective disease control measures can so

far only be taken for non-wildlife rabbits

and hares. Vaccination with an inactivated

vaccine is currently the most effective wayto control RHD. All the available vaccines

are derived from organ homogenates,which are prepared from infected rabbits.The virus is inactivated by different meth-ods, like !-propiolactone-, formaldehyde-,ethylenediamine- (Pages Mante, 1989;

ArgOllo Villares, 1991; Smid etal, 1991) orheat (68 °C) treatment (Haralambiev et al,1990, 1991a, 1991b). Whereas ArgOllo Vil-lars (1991) added aluminium hydroxide,Pages Mante (1989) used an oil adjuvant.Even after multiple vaccinations, no sideeffects were reported except for rare localreactions. Rabbits can be vaccinated after

weaning, and protective immunity lasts

from 6 months up to over 1 year (ArgOlloVillars, 1991 ). Huang (1991) and Peschlej-ski et al (1991 ) reported protective immuni-ty induced by hyperimmune sera. Haralam-biev et al (1991a) detected protectiveimmunity in rabbits only 2 d after vaccina-tion. Since sera from these animals con-

tained no detectable antibodies to RHDV,protection may also be explained by othermeans.

Antibodies to RHDV induced after either

vaccination or infection cannot be differen-

tiated by the various serological assays.Vaccines can thus not be used in commer-

cial holdings, which want to export serone-gative rabbits. To avoid the introduction ofRHDV into such holdings the following pro-

phylactic control measures are recom-

mended:

- rabbits, which have to be introduced intothe holding should be seronegative to

RHDV and taken into quarantine for a peri-od of = 2 wk. Sentinel rabbits should be

added;- avoid contact to wild rabbits and hares

as well as to rabbits from non commercial

holdings;- avoid green feed, use prepared feed;- take care of high standards of hygiene,like change of cloths, use of shower, etc.

In Germany and other countries RHD isa notifiable disease. During an outbreak,RHD can be eradicated by stamping out af-fected holdings as was exercised in Mexi-co. If an eradication is not possible due tointeractions with wild rabbits, the followingcontrol measures are recommended:

- killing of diseased and RHD suspiciousrabbits and innocuous disposal of the car-casses;

- cleaning and disinfection procedures us-ing 10% formaldehyde, 2% NaOH (Xu andChen, 1989) or other disinfectants, whichare recommended for use and effective

against nonenveloped viruses;- vaccination of the rabbits within the hold-

ing, in contact holdings or in affected re-

gions, which are suspicious of becominginfected;- no introduction of rabbits of unknown ori-

gin.

OUTLOOK

The virus family Calici’viridae presentlyconsists of several members, like vesicularexanthema virus of swine, feline calicivi-

rus, San Miguel sealion virus and calicivi-rus of dogs. The Calicivirus study grouprecently included RHDV into the family.

The studies on EBHSV demonstrate that

this virus also represents a calicivirus (man-uscript in preparation). An infectious agentisolated from man, the Norwalk agent, is

now also a member of the virus family.

Surprisingly little is known about calicivi-ruses with regard to genome organization,strategy of gene expression, replication aswell as synthesis of virions. Such studiesare not only of basic interest but will alsobe important for applied approaches like

development of recombinant vaccines. Ananimal virus could serve as a model for

agents which are relevant for man. Finallythe techniques of molecular biology mayallow to isolate the so far hypothetical rab-bit calicivirus. Such an approach may notonly answer the question whether RHDVinitially originated from rabbits imported toChina from Western Europe, but also pro-vide a tool to study the molecular basis ofRHDV pathogenicity.

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