Retrospective studies on rabbit haemorrhagic disease outbreaks caused by RHDV GI.2 virus on farms in France from 2013 to 2018

[EN] Rabbit haemorrhagic disease (RHD) is a critical health threat to the rabbit industry in Europe. In 2018, the French rabbit industry adopted a voluntary control plan against this disease. In this context, two epidemiological studies were conducted on RHD outbreaks that occurred between 2013 and 2018 in France. The objectives were to describe the spread of RHD due to the new genotype RHDV GI.2 (rabbit haemorrhagic disease virus GI.2) and to identify rearing factors influencing the occurrence of the disease in order to guide the prevention measures recommended in the control plan. An analysis of cases on 295 farms between 2013 and 2017 showed that 32% of farms were affected at least once; the incidence of the disease increased in 2016-2017 compared to 2013-2015. Farms already affected in 2013-2015 had a higher risk of being infected in 2016-2017 than those that remained unaffected until 2015 (Relative Risk and 95% Confident Interval 1.7 [1.1-2.7]). A case-control study carried out between 2016 and 2018 on 37 outbreaks and 32 control farms revealed variability in biosecurity and decontamination practices between farms. The risk of being infected tends to be linked to these practices, but certain structural factors (e.g. the manure disposal system, transfer of rabbits at weaning) could also influence the risk of virus introduction into farms. In the context of a limited vaccination coverage of the farms (only females are vaccinated), these hypotheses will be studied further, using information from the RHD outbreak monitoring system implemented at the same time as the control plan in 2018.This study was founded by the French Ministry of Agriculture (2017-430 / 170274).Huneau-Salaün, A.; Boucher, S.; Fontaine, J.; Le Normand, B.; Lopez, S.; Maurice, T.; Nouvel, L.... (2021). Retrospective studies on rabbit haemorrhagic disease outbreaks caused by RHDV GI.2 virus on farms in France from 2013 to 2018. World Rabbit Science. 29(2):87-98. https://doi.org/10.4995/wrs.2021.12800OJS8798292Abrantes J., Van der Loo W., Le Pendu J., Esteves P.J. 2012. Rabbit haemorrhagic disease (RHD) and rabbit haemorrhagic disease virus (RHDV): a review. Vet. Res., 43: 12.https://doi.org/10.1186/1297-9716-43-12Capucci L., Cavadini P., Schiavitto M., Lombardi G., Lavazza A. 2017. Increased pathogenicity in rabbit haemorrhagic disease virus type 2 (RHDV2). Vet. Record., 180: 426. https://doi.org/10.1136/vr.104132Carvalho C.L., Leclerc Duarte E., Monteiro J.M., Afonso C., Pacheco J., Carvalho P., Mendonça P., Botelho A., Albuquerque T., Themudo P., Fevereiro M., Henriques A.M., Santos Barros S., Dias Duarte M. 2017. Progression of rabbit haemorrhagic disease virus 2 upon vaccination in anindustrial rabbitry: a laboratorial approach. World Rabbit Sci., 25: 73-85. https://doi.org/10.4995/wrs.2017.5708Cooke B.D., Fenner F. 2002. Rabbit haemorrhagic disease and the biological control of wild rabbits, Oryctolagus Cuniculus, in Australia and New Zealand. Wildlife Res., 29: 689-706. https://doi.org/10.1071/WR02010Dalton K.P., Balseiro A., Juste R.A., Podadera A., Nicieza I., del Llano D., González R., Martin Alonso J.M., Prieto J.M., Parra F., Casais R. 2018. Clinical course and pathogenicity of variant rabbit haemorrhagic disease virus in experimentally infected adult and kit rabbits: Significance towards control and spread. Vet. Microbiol., 220: 24-32. https://doi.org/10.1016/j.vetmic.2018.04.033Dohoo I., Martin W., Stryhn H. 2003. Measures of disease frequency. In: Veterinary Epidemiologic Research, First Edition, AVC Inc., Charlottetown, Canada, 65-84.Hall R.N., Huang N., Roberts J., Strive T. 2019. Carrion flies as sentinels for monitoring lagovirus activity in Australia. Transboundary Emerg. Dis., 66: 2025-2032. https://doi.org/10.1111/tbed.13250Henning J., Meers J., Davies R., Morris R.S. 2005. Survival of rabbit haemorrhagic disease virus (RHDV) in the environment. Epidemiol. Infect., 133: 719-730. https://doi.org/10.1017/S0950268805003766Hurand J. 2016. L'élevage de lapins de chair en France, résultats technico-économiques 2015. Tema, 40.ITAVI. 2019. Situation de la filière cunicole. Novembre 2019. 6 p. Available athttps://www.itavi.asso.fr/content/note-deconjoncture-lapins-7Accessed December 2019.Le Gall-Reculé G., Zwingelstein F., Boucher S., Le Normand B., Plassiart G., Portejoie Y., Decors A., Bertagnoli S., Guérin J.L., Marchandeau S. 2011. Detection of a new variant of rabbit haemorrhagic disease virus in France. Vet. Rec., 168: 137- 138. https://doi.org/10.1136/vr.d697Le Gall-Reculé G., Lavazza A., Marchandeau S., Bertagnoli S., Zwingelstein F., Cavadini P., Martinelli N., Lombardi G., Guérin J.L., Lemaitre E., Decors A., Boucher S., Le Normand B., Capucci L. 2013. Emergence of a new lagovirus related to Rabbit haemorrhagic disease virus. Vet. Res., 44:81. https://doi.org/10.1186/1297-9716-44-81Le Gall-Reculé G., Boucher S. 2017. Connaissances et actualités sur la maladie hémorragique du lapin. In Proc.: 17èmes Journées de la Recherche Cunicole, 21-22 November, 2017. Le Mans, France. 97-109.Le Minor O., Joudou L., Le Moullec T., Beilvert F. 2017. Innocuité et efficacité de la vaccination à 2 et 3 semaines d'âge contre le virus RHDV2 de la maladie hémorragique virale du lapin (VHD). In Proc.:17èmes Journées de la Recherche Cunicole, 21-22 November, 2017. Le Mans, France. 127-130.Le Minor O., Boucher S., Joudou L., Mellet R., Sourice M., Le Moullec T., Nicoler A., Beilvert F., Sigognault-Flochlay A. 2019. Rabbit haemorrhagic disease: experimental study of a recent highly pathogenic GI.2/RJDV2/b strain and evaluation of vaccine efficacy. World Rabbit Sci., 27: 143-156.https://doi.org/10.4995/wrs.2019.11082Le Pendu J., Abrantes J., Bertagnoli S., Guitton J.S., Le Gall-Reculé G., Lopes A.M., Marchandeau S., Alda F., Almeida T., Célio A. C., Barcena J., Burmakina G., Blanco E., Calvete C., Cavadini P., Cooke B., Dalton K., Mateos M.D., Deptula W., Eden J.S., Wang F., Ferreira C.C., Ferreira P., Foronda P., Gonçalves D., Gavier-Widén D., Hall R., Hukowska-Szematowicz B., Kerr P., Kovaliski J., Lavazza A., Mahar J., Malogolovkin A., Marques R.M., Marques S., Martin-Alonso A., Monterroso P., MorenoS., Mutze G., Naimanis A., Niedzwiedzka-Rystwej P., Peacock D., Parra F., Rocchi M., Rouco C., Ruvoën-Clouet N., Silva E., Silvério D., Strive T., Thompson G., Tokarz-Deptula B., Esteves P. 2017. Proposal for a unified classification system and nomenclature of lagoviruses. J. Gen. Virol., 98: 1658-1666. https://doi.org/10.1099/jgv.0.000840Matthaei M., Kerr P.J., Read A.J., Hick P., Haboury S., Wright J.D., Strive T. 2014. Comparative quantitative monitoring of rabbit haemorrhagic disease viruses in rabbit kittens. Virol. J., 11: 109. https://doi.org/10.1186/1743-422X-11-109Mc Coll K.A., Merchant J.C., Hardy J., Cooke B.D., Robinson A., Westbury H.A. 2002. Evidence for insect transmission of rabbit haemorrhagic disease virus. Epidemiol. Infect., 129: 655-663. https://doi.org/10.1017/S0950268802007756Neimanis A.S., Larsson Pettersson U., Huang N., Gavier-Widen D., Strive T. 2018. Elucidation of the pathology and tissue distribution of Lagovirus europaeus GI.2/RHDV2 (rabbit haemorrhagic disease virus 2) in young and adult rabbits (Oryctolagus cuniculus). Vet. Res., 49:46. https://doi.org/10.1186/s13567-018-0540-zRosell J.M., de la Fuente L.F., Parra F., Dalton K.P., Badiola Sáiz J.I., Pérez de Rozas A., Badiola Díez J.J., Fernández de Luco D., Casal J., Majó N., Casas J., Garriga R., Fernández Magariños X.M. 2019. Myxomatosis and Rabbit Haemorrhagic Disease: A 30-Year Study of the Occurrence on Commercial Farms in Spain. Animals, 9: 780. https://doi.org/10.3390/ani9100780Rouco C., Aguayo-Adán J.A., Santoro S., Abrantes J., Delibes-Mateos M. 2019. Worldwide rapid spread of the novel rabbit haemorrhagic disease virus (GI.2/RHDV2/b). Transboundary Emerg. Dis., 66: 1762-1764.https://doi.org/10.1111/tbed.1318

Characterisation of a non-pathogenic and non-protective infectious rabbit lagovirus related to RHDV

The existence of non-pathogenic RHDV strains was established when a non-lethal virus named rabbit calicivirus (RCV) was characterised in 1996 in Italy. Since then, different RNA sequences related to RHDV have been detected in apparently healthy domestic and wild rabbits, and recently a new lagovirus was identified in Australia. We have characterised from seropositive healthy domestic rabbits a non-lethal lagovirus that differs from RHDV in terms of pathogenicity, tissue tropism and capsid protein sequence. Phylogenetic analyses have revealed that it is close to the Ashington strain and to the RCV, but distinct. We proved experimentally that it is infectious but non-pathogenic and demonstrated that, contrary to the other described non-pathogenic lagoviruses, it induces antibodies that do not protect against RHDV. Our results indicate the existence of a gradient of cross-protection between circulating strains, from non-protective, partially protective to protective strains, and highlight the extent of diversity within the genus Lagovirus

Histo-Blood Group Antigens Act as Attachment Factors of Rabbit Hemorrhagic Disease Virus Infection in a Virus Strain-Dependent Manner

Rabbit Hemorrhagic disease virus (RHDV), a calicivirus of the Lagovirus genus, and responsible for rabbit hemorrhagic disease (RHD), kills rabbits between 48 to 72 hours post infection with mortality rates as high as 50–90%. Caliciviruses, including noroviruses and RHDV, have been shown to bind histo-blood group antigens (HBGA) and human non-secretor individuals lacking ABH antigens in epithelia have been found to be resistant to norovirus infection. RHDV virus-like particles have previously been shown to bind the H type 2 and A antigens. In this study we present a comprehensive assessment of the strain-specific binding patterns of different RHDV isolates to HBGAs. We characterized the HBGA expression in the duodenum of wild and domestic rabbits by mass spectrometry and relative quantification of A, B and H type 2 expression. A detailed binding analysis of a range of RHDV strains, to synthetic sugars and human red blood cells, as well as to rabbit duodenum, a likely gastrointestinal site for viral entrance was performed. Enzymatic cleavage of HBGA epitopes confirmed binding specificity. Binding was observed to blood group B, A and H type 2 epitopes in a strain-dependent manner with slight differences in specificity for A, B or H epitopes allowing RHDV strains to preferentially recognize different subgroups of animals. Strains related to the earliest described RHDV outbreak were not able to bind A, whereas all other genotypes have acquired A binding. In an experimental infection study, rabbits lacking the correct HBGA ligands were resistant to lethal RHDV infection at low challenge doses. Similarly, survivors of outbreaks in wild populations showed increased frequency of weak binding phenotypes, indicating selection for host resistance depending on the strain circulating in the population. HBGAs thus act as attachment factors facilitating infection, while their polymorphism of expression could contribute to generate genetic resistance to RHDV at the population level

Serological evidence for the presence of non-pathogenic rabbit haemorrhagic disease virus-like strains in rabbits <i>Oryctolagus cuniculus</i> of the Kerguelen archipelago

Antibodies raised against a Lagovirus were found in healthy rabbits Oryctolagus cuniculus sampled in 2003 and 2004 in the Kerguelen archipelago. The serological test we used enabled the detection of antibodies due to both pathogenic and non-pathogenic viruses related to the rabbit haemorrhagic disease virus (RHDV). The overall proportion of seropositive rabbits was 35% and differed between sites. Since previous studies have failed to detect antibodies raised against pathogenic RHDV strains, the antibodies detected in the present study were likely due to non-pathogenic strains. The interest of these strains circulating in the Kerguelen archipelago is that they have evolved independently of those detected in the other parts of the world and should derive from an ancestral RHDV precursor. Their characterization may help understanding the evolution of the virus and the emergence of the disease

Large-scale lagovirus disease outbreaks in European brown hares (Lepus europaeus) in France caused by RHDV2 strains spatially shared with rabbits (Oryctolagus cuniculus)

Abstract Rabbit haemorrhagic disease virus (RHDV) is a lagovirus that causes rabbit haemorrhagic disease (RHD) in European rabbits (Oryctolagus cuniculus). In 2010, a new genotype called RHDV2 emerged in France. It exhibits a larger host range than classical RHDV strains by sporadically infecting different hare species, including the European hare (Lepus europaeus). Phylogenetic analyses revealed that closely related RHDV2 strains circulate locally in both hares and rabbits, and therefore that RHDV2 strains infecting hares do not belong to a lineage that has evolved only in this species. We showed that RHDV2 is widely distributed in France and that it was responsible for more than a third of cases of lagovirus disease in European hare populations in 2015. The oldest RHDV2 positive hare was sampled in November 2013 and we reported two hares co-infected by EBHSV and RHDV2. All together, our results raise important epidemiological and evolutionary issues. In particular, along with the potential emergence of recombinant EBHSV/RHDV2 strains in hares, the enlargement of the host range changes the host population structure of RHDV2 and may alter the impact of the virus on rabbit and hare populations

Development and Evaluation of a Duplex Lateral Flow Assay for the Detection and Differentiation between Rabbit Haemorrhagic Disease Virus Lagovirus europaeus/GI.1 and /GI.2

Rabbit Haemorrhagic Disease Virus 2 (RHDV2, recently named Lagovirus europaeus/GI.2) was first reported in France in 2010 and has spread globally since then, replacing most of the circulating former RHDV (genotype GI.1) in many countries. The detection and differentiation of both genotypes is of crucial importance for the surveillance of the disease. In this article, a duplex lateral flow assay (LFA) for antigen detection is described and evaluated, providing the first description of a quick and easy-to-use test that allows for the simultaneous detection and differentiation of RHDV genotypes GI.1 and GI.2. A panel of GI.1- or GI.2-infected and non-infected rabbit liver samples and liver exudates (136 samples) was analysed, obtaining a total sensitivity of 94.4% and specificity of 100%. These data confirm that the developed duplex LFA can be used as a reliable diagnostic test for RHD surveillance, especially in farms and the field

A new HaCV-EBHSV recombinant lagovirus circulating in European brown hares (Lepus europaeus) from Catalonia, Spain

Abstract In 2020/2021, several European brown hare syndrome virus (EBHSV) outbreaks were recorded in European hares (Lepus europaeus) from Catalonia, Spain. Recombination analysis combined with phylogenetic reconstruction and estimation of genetic distances of the complete coding sequences revealed that 5 strains were recombinants. The recombination breakpoint is located within the non-structural protein 2C-like RNA helicase (nucleotide position ~ 1889). For the genomic fragment upstream of the breakpoint, a non-pathogenic EBHSV-related strain (hare calicivirus, HaCV; GII.2) was the most closely related sequence; for the rest of the genome, the most similar strains were the European brown hare syndrome virus (EBHSV) strains recovered from the same 2020/2021 outbreaks, suggesting a recent origin. While the functional impact of the atypical recombination breakpoint remains undetermined, the novel recombinant strain was detected in different European brown hare populations from Catalonia, located 20–100 km apart, and seems to have caused a fatal disease both in juvenile and adult animals, confirming its viability and ability to spread and establish infection. This is the first report of a recombination event involving HaCV and EBHSV and, despite the recombination with a non-pathogenic strain, it appears to be associated with mortality in European brown hares, which warrants close monitoring

Retrospective Analysis Shows That Most RHDV GI.1 Strains Circulating Since the Late 1990s in France and Sweden Were Recombinant GI.3P–GI.1d Strains

Recombination is one of the major sources of genetic variation in viruses. RNA viruses, such as rabbit hemorrhagic disease virus (RHDV), are among the viruses with the highest recombination rates. Several recombination events have been described for RHDV, mostly as a consequence of their genomic architecture. Here, we undertook phylogenetic and recombination analyses of French and Swedish RHDV strains from 1994 to 2016 and uncovered a new intergenotypic recombination event. This event occurred in the late 1990s/early 2000s and involved nonpathogenic GI.3 strains as donors for the nonstructural part of the genome of these recombinants, while pathogenic GI.1d strains contributed to the structural part. These GI.3P&ndash;GI.1d recombinant strains did not entirely replace GI.1d (nonrecombinant) strains, but became the dominant strains in France and Sweden, likely due to a fitness advantage associated with this genomic architecture. GI.3P&ndash;GI.1d (P stands for polymerase) strains persisted until 2013 and 2016 in Sweden and France, respectively, and cocirculated with the new genotype GI.2 in France. Since strains from the first GI.2 outbreaks were GI.3P&ndash;GI.2, we hypothesize that GI.3P&ndash;GI.1d could be the parental strain. Our results confirm the outstanding recombination ability of RHDV and its importance in the evolution of lagoviruses, which was only revealed by studying complete genomic sequences