Characterisation of Lagovirus europaeus GI-RHDVs (Rabbit Haemorrhagic Disease Viruses) in Terms of Their Pathogenicity and Immunogenicity.

Rabbit haemorrhagic disease viruses (RHDV) belong to the family Caliciviridae, genus Lagovirus europaeus, genogroup GI, comprising four genotypes GI.1-GI.4, of which the genotypes GI.1 and GI.2 are pathogenic RHD viruses, while the genotypes GI.3 and GI.4 are non-pathogenic RCV (Rabbit calicivirus) viruses. Among the pathogenic genotypes GI.1 and GI.2 of RHD viruses, an antigenic variant of RHDV, named RHDVa-now GI.1a-RHDVa, was distinguished in 1996; and in 2010, a variant of RHDV-named RHDVb, later RHDV2 and now GI.2-RHDV2/b-was described; and recombinants of these viruses were registered. Pathogenic viruses of the genotype GI.1 were the cause of a disease described in 1984 in China in domestic (Oryctolagus (O.) cuniculus domesticus) and wild (O. cuniculus) rabbits, characterised by a very rapid course and a mortality rate of 90-100%, which spread in countries all over the world and which has been defined since 1989 as rabbit haemorrhagic disease. It is now accepted that GI.1-RHDV, including GI.1a-RHDVa, cause the predetermined primary haemorrhagic disease in domestic and wild rabbits, while GI.2-RHDV2/b cause it not only in rabbits, including domestic rabbits' young up to 4 weeks and rabbits immunised with rabbit haemorrhagic disease vaccine, but also in five various species of wild rabbits and seven different species of hares, as well as wild ruminants: mountain muskoxen and European badger. Among these viruses, haemagglutination-positive, doubtful and harmful viruses have been recorded and described and have been shown to form phylogenogroups, immunotypes, haematotypes and pathotypes, which, together with traits that alter and expand their infectious spectrum (rabbit, hare, wild ruminant, badger and various rabbit and hare species), are the determinants of their pathogenicity (infectivity) and immunogenicity and thus shape their virulence. These relationships are the aim of our consideration in this article.

MicroRNAs Regulate the Expression of Genes Related to the Innate Immune and Inflammatory Response in Rabbits Infected with Lagovirus europaeus GI.1 and GI.2 Genotypes.

MicroRNAs (miR) are a group of small, non-coding RNAs of 17-25 nucleotides that regulate gene expression at the post-transcriptional level. Dysregulation of miRNA expression or function may contribute to abnormal gene expression and signaling pathways, leading to disease pathology. Lagovirus europaeus (L. europaeus) causes severe disease in rabbits called rabbit hemorrhagic disease (RHD). The symptoms of liver, lung, kidney, and spleen degeneration observed during RHD are similar to those of acute liver failure (ALF) and multi-organ failure (MOF) in humans. In this study, we assessed the expression of miRs and their target genes involved in the innate immune and inflammatory response. Also, we assessed their potential impact on pathways in L. europaeus infection-two genotypes (GI.1 and GI.2)-in the liver, lungs, kidneys, and spleen. The expression of miRs and target genes was determined using quantitative real-time PCR (qPCR). We assessed the expression of miR-155 (MyD88, TAB2, p65, NLRP3), miR-146a (IRAK1, TRAF6), miR-223 (TLR4, IKKα, NLRP3), and miR-125b (MyD88). We also examined biomarkers of inflammation: IL-1ß, IL-6, TNF-α, and IL-18 in four tissues at the mRNA level. Our study shows that the main regulators of the innate immune and inflammatory response in L. europaeus/GI.1 and GI.2 infection, as well as RHD, are miR-155, miR-223, and miR-146a. During infection with L. europaeus/RHD, miR-155 has both pro- and anti-inflammatory effects in the liver and anti-inflammatory effects in the kidneys and spleen; miR-146a has anti-inflammatory effects in the liver, lungs and kidneys; miR-223 has anti-inflammatory effects in all tissues; however, miR-125b has anti-inflammatory effects only in the liver. In each case, such an effect may be a determinant of the pathogenesis of RHD. Our research shows that miRs may regulate three innate immune and inflammatory response pathways in L. europaeus infection. However, the result of this regulation may be influenced by the tissue microenvironment. Our research shows that infection of rabbits with L. europaeus/GI.1 and GI.2 genotypes causes an overexpression of two critical acute phase cytokines: IL-6 in all examined tissues and TNF-α (in the liver, lungs, and spleen). IL-1ß was highly expressed only in the lungs after L. europaeus infection. These facts indicate a strong and rapid involvement of the local innate immune and inflammatory response in L. europaeus infection-two genotypes (GI.1 and GI.2)-and in the pathogenesis of RHD. Profile of biomarkers of inflammation in rabbits infected with L. europaeus/GI.1 and GI.2 genotypes are similar regarding the nature of changes but are different for individual tissues. Therefore, we propose three inflammation profiles for L. europaeus infection for both GI.1 and GI.2 genotypes (pulmonary, renal, liver, and spleen).

Hepatobiliary organoids derived from leporids support the replication of hepatotropic lagoviruses.

The genus Lagovirus of the family Caliciviridae contains some of the most virulent vertebrate viruses known. Lagoviruses infect leporids, such as rabbits, hares and cottontails. Highly pathogenic viruses such as Rabbit haemorrhagic disease virus 1 (RHDV1) cause a fulminant hepatitis that typically leads to disseminated intravascular coagulation within 24-72 h of infection, killing over 95 % of susceptible animals. Research into the pathophysiological mechanisms that are responsible for this extreme phenotype has been hampered by the lack of a reliable culture system. Here, we report on a new ex vivo model for the cultivation of lagoviruses in cells derived from the European rabbit (Oryctolagus cuniculus) and European brown hare (Lepus europaeus). We show that three different lagoviruses, RHDV1, RHDV2 and RHDVa-K5, replicate in monolayer cultures derived from rabbit hepatobiliary organoids, but not in monolayer cultures derived from cat (Felis catus) or mouse (Mus musculus) organoids. Virus multiplication was demonstrated by (i) an increase in viral RNA levels, (ii) the accumulation of dsRNA viral replication intermediates and (iii) the expression of viral structural and non-structural proteins. The establishment of an organoid culture system for lagoviruses will facilitate studies with considerable implications for the conservation of endangered leporid species in Europe and North America, and the biocontrol of overabundant rabbit populations in Australia and New Zealand.

Genetics of the rabbit haemorrhagic disease virus strains responsible for rabbit haemorrhagic disease outbreaks in Japan between 2000 and 2020.

Rabbit haemorrhagic disease (RHD) is a highly contagious and fatal disease in rabbits caused by the rabbit haemorrhagic disease virus (RHDV), which includes two genotypes, RHDV-GI.1 and RHDV2-GI.2. RHDVs tend to recombine among different strains, resulting in significant genetic evolution. This study evaluated the genetics of Japanese RHDV strains associated with six outbreaks between 2000 and 2020 using whole-genome sequencing, genomic recombination and phylogenetic analyses. Genomic recombination analysis using near-complete genomic sequences revealed that two Japanese strains detected in 2000 and 2002 were non-recombinant GI.1 (variant RHDVa-GI.1a) strains of different origins, most closely related to strains identified in PR China in 1997 and the USA in 2001, respectively. In contrast, four recent Japanese GI.2 strains detected between 2019 and 2020 were recombinant viruses harbouring structural protein (SP) genes from GI.2 strains and non-SP (NSP) genes from a benign rabbit calicivirus (RCV) strain of genotype RCV-E1-GI.3 (GI.3P-GI.2) or an RHDV G1-GI.1b variant (GI.1bP-GI.2). Phylogenetic analysis based on SP and NSP regions revealed that the GI.1bP-GI.2 recombinant virus detected in Ehime prefecture and the GI.3P-GI.2 recombinant viruses detected in Ibaraki, Tochigi and Chiba prefectures were most closely related to recombinant viruses identified in Australia in 2017 and Germany in 2017, respectively. These results suggested that past RHD outbreaks in Japan did not result from the evolution of domestic RHDVs but rather represented incursions of foreign RHDV strains, implying that Japan is constantly at risk of RHDV incursion from other countries.

The potential role of scavenging flies as mechanical vectors of Lagovirus europaeus/GI.2.

The European rabbit (Oryctolagus cuniculus) populations of the Iberian Peninsula have been severely affected by the emergence of the rabbit haemorrhagic disease virus (RHDV) Lagovirus europaeus/GI.2 (RHDV2/b). Bushflies and blowflies (Muscidae and Calliphoridae families, respectively) are important RHDV vectors in Oceania, but their epidemiological role is unknown in the native range of the European rabbit. In this study, scavenging flies were collected between June 2018 and February 2019 in baited traps at one site in southern Portugal, alongside a longitudinal capture-mark-recapture study of a wild European rabbit population, aiming to provide evidence of mechanical transmission of GI.2 by flies. Fly abundance, particularly from Calliphoridae and Muscidae families, peaked in October 2018 and in February 2019. By employing molecular tools, we were able to detect the presence of GI.2 in flies belonging to the families Calliphoridae, Muscidae, Fanniidae and Drosophilidae. The positive samples were detected during an RHD outbreak and absent in samples collected when no evidence of viral circulation in the local rabbit population was found. We were able to sequence a short viral genomic fragment, confirming its identity as RHDV GI.2. The results suggest that scavenging flies may act as mechanical vectors of GI.2 in the native range of the southwestern Iberian subspecies O. cuniculus algirus. Future studies should better assess their potential in the epidemiology of RHD and as a tool for monitoring viral circulation in the field.

Multiple mechanisms of reinitiation on bicistronic calicivirus mRNAs.

Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long open reading frames (ORFs) on cellular mRNAs, reinitiation occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG, designated "termination upstream ribosomal binding site" (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2(∗). To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes and diminishes dependence on eukaryotic initiation factor 3 (eIF3) of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes.

Seromolecular surveillance of rabbit haemorrhagic disease virus in Nigeria.

Following the first 2020 rabbit haemorrhagic disease virus (RHDV) outbreak in Nigeria which caused massive mortalities in several rabbitries, there was a need to know the spread and strains circulating in the affected states. Over 100 rabbitries still existing post-RHDV outbreak in Ogun and Kwara States were investigated. A commercial enzyme-linked immunosorbent assay kit was used to screen for RHDV immunoglobulin G in 192 rabbit sera, while RHDV VP60 gene was amplified in RNA extracted from these sera and tissues (liver and/or spleen harvested from 37 carcasses necrotized) by reverse transcription-polymerase chain reaction (RT-PCR). Sequences obtained from the amplicons were subjected to phylogenetic analysis. The results revealed a seroprevalence of 82.3% (158/192). RHDV VP60 gene was detected in 15/17 (88.2%) and 2/20 (10.0%) carcasses from Ogun and Kwara States, respectively, while none of the sera was positive. Sequences of the two positive amplicons selected (one from each states) shared 98.95% nucleotide identity and belonged to RHDV 2/GI.2 strain. Also, nBLAST of these sequences revealed 98.43-99.55% homology with the prototype Nigerian RHDV strain RHDV/NGR/ILN/001 (MT996357.1). Furthermore, these strains clustered with this prototype and a German RHDV strain (LR899166.1). Pathologic lesions affecting the respiratory, cardiovascular, renal, lymphatic, and digestive systems were observed in necropsied carcasses. This study indicated that RHDV 2/GI.2 strain was the cause of 2020 RHD outbreak in Nigeria. Thus, while continuous public sensitization about RHD especially among rabbit farmers in Nigeria is important, efforts aimed at design and implementation of RHD vaccination policy, preferably using indigenous seed, should be expedited.

Early pathogenesis in rabbit hemorrhagic disease virus 2.

To detail early tissue distribution and innate immune response to rabbit hemorrhagic disease virus 2 (RHDV2), 13 rabbits were orally (Oryctolagus cuniculus) inoculated with liver homogenate made from a feral rabbit that succumbed to RHDV2 during the 2020 outbreak in Oregon, USA. Rabbits were monitored regularly, with euthanasia and collection of tissues and swabs, at 12, 24, 36, 48, 96, and 144 h post inoculation. Livers from these rabbits were positive by RT-rtPCR for presence of the virus. Using RNAscope for viral and replicative intermediates, rabbits had detectable viral genomic RNA at each time point, initially within the gastrointestinal tract, then in the liver by 36 h post inoculation. Also using RNAscope, there were increasing amounts of mRNA coding for TNF-α, IL-6, and IL-1ß within the liver and spleen through 48 h post inoculation. The results of this study aided our understanding of the local innate immune response to RHDV2, as well as aspects of pathogenesis.

Comparative Analysis of RNA Virome Composition in Rabbits and Associated Ectoparasites.

Ectoparasites play an important role in virus transmission among vertebrates. Little, however, is known about the nature of those viruses that pass between invertebrates and vertebrates. In Australia, flies and fleas support the mechanical transmission of two viral biological controls against wild rabbits-rabbit hemorrhagic disease virus (RHDV) and myxoma virus. We compared virome compositions in rabbits and these ectoparasites, sequencing total RNA from multiple tissues and gut contents of wild rabbits, fleas collected from these rabbits, and flies trapped sympatrically. Meta-transcriptomic analyses identified 50 novel viruses from multiple RNA virus families. Rabbits and their ectoparasites were characterized by markedly different viromes, with virus abundance greatest in flies. Although viral contigs from six virus families/groups were found in both rabbits and ectoparasites, they clustered in distinct host-dependent lineages. A novel calicivirus and a picornavirus detected in rabbit cecal content were vertebrate specific; the newly detected calicivirus was distinct from known rabbit caliciviruses, while the picornavirus clustered with sapeloviruses. Several picobirnaviruses were also identified that fell in diverse phylogenetic positions, compatible with the idea that they are associated with bacteria. Further comparative analysis revealed that the remaining viruses found in rabbits, and all those from ectoparasites, were likely associated with invertebrates, plants, and coinfecting endosymbionts. While no full genomes of vertebrate-associated viruses were detected in ectoparasites, small numbers of reads from rabbit astrovirus, RHDV, and other lagoviruses were present in flies. This supports a role for flies in the mechanical transmission of RHDV, while their involvement in astrovirus transmission merits additional exploration.IMPORTANCE Ectoparasites play an important role in the transmission of many vertebrate-infecting viruses, including Zika and dengue viruses. Although it is becoming increasingly clear that invertebrate species harbor substantial virus diversity, it is unclear how many of the viruses carried by invertebrates have the potential to infect vertebrate species. We used the European rabbit (Oryctolagus cuniculus) as a model species to compare virome compositions in a vertebrate host and known associated ectoparasite mechanical vectors, in this case, fleas and blowflies. In particular, we aimed to infer the extent of viral transfer between these distinct types of host. Our analysis revealed that despite extensive viral diversity in both rabbits and associated ectoparasites, and the close interaction of these vertebrate and invertebrate species, biological viral transmission from ectoparasites to vertebrate species is rare. We did, however, find evidence to support the idea of a role of blowflies in transmitting viruses without active replication in the insect.

Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d strain: genome sequencing, in vivo virus replication kinetics, and viral dose effect.

BACKGROUND: Rabbit haemorrhagic disease virus Lagovirus europaeus/GI.1d variant (GI.1d/RHDV) was identified in 1990 in France, and until the emergence of the new genotype GI.2, it was the main variant circulating in the country. The early stages of RHDV infection have been described in a few studies of rabbits experimentally infected with earlier strains, but no information was given on the minimum infective dose. We report the genomic and phenotypic characterisation of a GI.1d/RHDV strain collected in 2000 in France (GI.1d/00-21). RESULTS: We performed in vivo assays in rabbits to study virus replication kinetics in several tissues at the early stage of infection, and to estimate the minimum infective dose. Four tested doses, negligible (10- 1 viral genome copies), low (104), high (107) and very high (1011) were quantified using a method combining density gradient centrifugation of the viral particles and an RT-qPCR technique developed to quantify genomic RNA (gRNA). The GI.1d/00-21 genome showed the same genomic organisation as other lagoviruses; however, a substitution in the 5' untranslated region and a change in the potential p23/2C-like helicase cleavage site were observed. We showed that the liver of one of the two rabbits inoculated via the oral route was infected at 16 h post-infection and all tissues at 39 h post-infection. GI.1d/00-21 induced classical RHD signs (depression) and lesions (haemorrhage and splenomegaly). Although infective dose estimation should be interpreted with caution, the minimum infective dose that infected an inoculated rabbit was lower or equal to 104 gRNA copies, whereas between 104 and 107 gRNA copies were required to also induce mortality. CONCLUSIONS: These results provide a better understanding of GI.1d/RHDV infection in rabbits. The genome analysis showed a newly observed mutation in the 5' untranslated region of a lagovirus, whose role remains unknown. The phenotypic analysis showed that the pathogenicity of GI.1d/00-21 and the replication kinetics in infected organs were close to those reported for the original GI.1 strains, and could not alone explain the observed selective advantage of the GI.1d strains. Determining the minimum dose of viral particles required to cause mortality in rabbits is an important input for in vivo studies.

Structure-function relationship in the 'termination upstream ribosomal binding site' of the calicivirus rabbit hemorrhagic disease virus.

Caliciviruses use a termination/reinitiation mechanism for translation of their minor capsid protein VP2. A sequence element of about 80 nucleotides denoted 'termination upstream ribosomal binding site' (TURBS) is crucial for reinitiation. RNA secondary structure probing and computer aided secondary structure prediction revealed a rather low degree of secondary structure determinants for the TURBS of the rabbit hermorrhagic disease virus. Mutation analysis showed that prevention of duplex formation had major impact on the VP2 expression levels. Restoration of complementarity of the respective sequences by reciprocal mutation at least partially restored reinitiating rates. Synthetic TURBS structures preserving only the secondary structure forming sequences and the known short motifs important for TURBS function were found to drive reinitiation when the altered sequence could be predicted to allow establishment of the crucial secondary structures of the TURBS.

Detection and molecular characterization of a first isolate of rabbit haemorrhagic disease virus in Nigeria.

Rabbit haemorrhagic disease virus (RHDV) was recovered from necropsied rabbits that died during an outbreak characterized by epistaxis, incoordination, paralysis, and multi-organ haemorrhages in Ilorin, Nigeria. The haemagglutination test (HA) and RT-PCR assay targeted against a fragment of the RHDV VP60 gene were performed on liver, spleen, and kidney homogenates; faeces; and urine obtained from the rabbits. Amplicons were purified, sequenced, and phylogenetically analysed. The liver homogenates yielded the highest HA titres while RT-PCR of liver, spleen, and kidneys yielded the expected 1252 bp band. Sequence and phylogenetic analyses revealed that the Nigerian RHDV strain (RHDV/NGR/ILN/001) was 98.57%, 97.95%, and 96.70% homologous with RHDV2 (RHDVGI.2) strains from the Netherlands, Germany, and France, respectively. RHDV/NGR/ILN/001 induced tracheal, intestinal, and mediastinal lymph node haemorrhages, pulmonary oedema and congestion, and enlarged, necrotic liver in experimentally inoculated rabbits. The implications of this study, which is the first report of RHDV in Nigeria, are discussed.

Adaptive changes in the genomes of wild rabbits after 16 years of viral epidemics.

Since its introduction to control overabundant invasive European rabbits (Oryctolagus cuniculus), the highly virulent rabbit haemorrhagic disease virus (RHDV) has caused regular annual disease outbreaks in Australian rabbit populations. Although initially reducing rabbit abundance by 60%, continent-wide, experimental evidence has since indicated increased genetic resistance in wild rabbits that have experienced RHDV-driven selection. To identify genetic adaptations, which explain the increased resistance to this biocontrol virus, we investigated genome-wide SNP (single nucleotide polymorphism) allele frequency changes in a South Australian rabbit population that was sampled in 1996 (pre-RHD genomes) and after 16 years of RHDV outbreaks. We identified several SNPs with changed allele frequencies within or close to genes potentially important for increased RHD resistance. The identified genes are known to be involved in virus infections and immune reactions or had previously been identified as being differentially expressed in healthy versus acutely RHDV-infected rabbits. Furthermore, we show in a simulation study that the allele/genotype frequency changes cannot be explained by drift alone and that several candidate genes had also been identified as being associated with surviving RHD in a different Australian rabbit population. Our unique data set allowed us to identify candidate genes for RHDV resistance that have evolved under natural conditions, and over a time span that would not have been feasible in an experimental setting. Moreover, it provides a rare example of host genetic adaptations to virus-driven selection in response to a suddenly emerging infectious disease.

Lagovirus europeus GI.2 (rabbit hemorrhagic disease virus 2) infection in captive mountain hares (Lepus timidus) in Germany.

BACKGROUND: Rabbit hemorrhagic disease virus (RHDV, Lagovirus europeus GI.1) induces a contagious and highly lethal hemorrhagic disease in rabbits. In 2010 a new genotype of lagovirus (GI.2), emerged in Europe, infecting wild and domestic population of rabbits and hares. CASE PRESENTATION: We describe the infection with a GI.2 strain, "Bremerhaven-17", in captive mountain hares (Lepus timidus) in a zoo facility in Germany. Postmortem examination revealed RHD-like lesions including necrotizing hepatitis. RT-qPCR and AG-ELISA confirmed presence of GI.2. Recombination and phylogenetic analysis grouped the identified strain with other GI.2 strains, sharing nucleotide identity of 91-99%. CONCLUSION: Our findings confirm that mountain hares are susceptible to GI.2 infection, due to a past recombination event facilitating virus spillover from sympatric rabbits.

Rabbit Hemorrhagic Disease Virus 2 (RHDV2; GI.2) Is Replacing Endemic Strains of RHDV in the Australian Landscape within 18 Months of Its Arrival.

Rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) is a pathogenic calicivirus that affects European rabbits (Oryctolagus cuniculus) and various hare (Lepus) species. GI.2 was first detected in France in 2010 and subsequently caused epidemics in wild and domestic lagomorph populations throughout Europe. In May 2015, GI.2 was detected in Australia. Within 18 months of its initial detection, GI.2 had spread to all Australian states and territories and rapidly became the dominant circulating strain, replacing Rabbit hemorrhagic disease virus (RHDV/GI.1) in mainland Australia. Reconstruction of the evolutionary history of 127 Australian GI.2 isolates revealed that the virus arrived in Australia at least several months before its initial description and likely circulated unnoticed in wild rabbit populations in the east of the continent prior to its detection. GI.2 sequences isolated from five hares clustered with sequences from sympatric rabbit populations sampled contemporaneously, indicating multiple spillover events into hares rather than an adaptation of the Australian GI.2 to a new host. Since the presence of GI.2 in Australia may have wide-ranging consequences for rabbit biocontrol, particularly with the release of the novel biocontrol agent GI.1a/RHDVa-K5 in March 2017, ongoing surveillance is critical to understanding the interactions of the various lagoviruses in Australia and their impact on host populations.IMPORTANCE This study describes the spread and distribution of Rabbit hemorrhagic disease virus 2 (GI.2) in Australia since its first detection in May 2015. Within the first 18 months following its detection, RHDV2 spread from east to west across the continent and became the dominant strain in all mainland states of Australia. This has important implications for pest animal management and for owners of pet and farmed rabbits, as there currently is no effective vaccine available in Australia for GI.2. The closely related RHDV (GI.1) is used to control overabundant wild rabbits, a serious environmental and agricultural pest in this country, and it is currently unclear how the widespread circulation of GI.2 will impact ongoing targeted wild rabbit management operations.

Genetic characterization and phylogenetic analysis of rabbit hemorrhagic disease virus isolated in Tunisia from 2015 to 2018.

Two distinct genotypes responsible for rabbit hemorrhagic disease (RHD) are reported, GI.1 (RHDV) and GI.2 (RHDV2). Vaccines based on these two genotypes are only partially cross-protective. Hence, knowing which genotype is circulating is important for appropriate control measures. We have investigated 25 field samples isolated between 2015 and 2018 from rabbits with clinical signs of RHD. Only GI.2 (RHDV2) is currently circulating in Tunisia. All Tunisian samples were grouped together with typical genotypic and phenotypic mutations. Therefore, we recommend initiating an extensive preventive vaccination program based on GI.2 vaccines in addition to a regular monitoring of the circulating lagoviruses.

Bioinformatics analysis of capsid protein of different subtypes rabbit hemorrhagic disease virus.

BACKGROUND: Rabbit Hemorrhagic Disease Virus (RHDV) belongs to the Caliciviridae family, is a highly lethal pathogen to rabbits. Increasing numbers of studies have demonstrated the existence of antigenic variation in RHDV, leading to the emergence of a new RHDV isolate (RHDVb). However, the underlying factors determining the emergence of the new RHDV and its unpredictable epidemiology remain unclear. To investigate these issues, we selected more than 184 partial and/or complete genome sequences of RHDV from GenBank and analyzed their phylogenetic relationships, divergence, and predicted protein modification sites. RESULTS: Phylogenetic analysis showed that classic RHDV isolates, RHDVa, and RHDVb formed different clades. It's interesting to note that RHDVa being more closely related to classic RHDV than RHDVb, while RHDVb had a closer genetic relationship to Rabbit Calicivirus (RCV) than to classic RHDV isolates. Moreover, divergence analysis suggested that the accumulation of amino acid (aa) changes might be a consequence of adaptive diversification of capsid protein (VP60) during the division between classical RHDV, RHDVa, RHDVb, and RCV. Notably, the prediction of N-glycosylation sites suggested that RHDVb subtypes had two unique N-glycosylation sites (aa 301, 362) but lacked three other N-glycosylation sites (aa 45, 308, 474) displayed in classic RHDV and RHDVa VP60 implying this divergence of N-glycosylation sites in RHDV might affect viral virulence. Analysis of phosphorylation sites also indicated that some phosphorylation sites in RHDVa and RHDVb differed from those in classic RHDV, potentially related to antigenic variation in RHDV. CONCLUSION: The genetic relationship between RHDVb and RCV was closer than classic RHDV isolates. Moreover, compared to RHDV and RHDVa, RHDVb had two unique N-glycosylation sites but lacked three sites, which might affect the virulence of RHDV. These results may provide new clues for further investigations of the origin of new types of RHDV and the mechanisms of genetic variation in RHDV.

Inclusion of an Arg-Gly-Asp receptor-recognition motif into the capsid protein of rabbit hemorrhagic disease virus enables culture of the virus in vitro.

The fact that rabbit hemorrhagic disease virus (RHDV), an important member of the Caliciviridae family, cannot be propagated in vitro has greatly impeded the progress of investigations into the mechanisms of pathogenesis, translation, and replication of this and related viruses. In this study, we have successfully bypassed this obstacle by constructing a mutant RHDV (mRHDV) by using a reverse genetics technique. By changing two amino acids (S305R,N307D), we produced a specific receptor-recognition motif (Arg-Gly-Asp; called RGD) on the surface of the RHDV capsid protein. mRHDV was recognized by the intrinsic membrane receptor (integrin) of the RK-13 cells, which then gained entry and proliferated as well as imparted apparent cytopathic effects. After 20 passages, the titers of RHDV reached 1 × 104.3 50% tissue culture infectious dose (TCID50)/ml at 72 h. Furthermore, mRHDV-infected rabbits showed typical rabbit plague symptoms and died within 48-72 h. After immunization with inactivated mRHDV, the rabbits survived wild-type RHDV infection, indicating that mRHDV could be a candidate virus strain for producing a vaccine against RHDV infection. In summary, this study offers a novel strategy for overcoming the challenges of proliferating RHDV in vitro Because virus uptake via specific membrane receptors, several of which specifically bind to the RGD peptide motif, is a common feature of host cells, we believe that this the strategy could also be applied to other RNA viruses that currently lack suitable cell lines for propagation such as hepatitis E virus and norovirus.

Detection and Circulation of a Novel Rabbit Hemorrhagic Disease Virus in Australia.

The highly virulent rabbit hemorrhagic disease virus (RHDV) has been widely used in Australia and New Zealand since the mid-1990s to control wild rabbits, an invasive vertebrate pest in these countries. In January 2014, an exotic RHDV was detected in Australia, and 8 additional outbreaks were reported in both domestic and wild rabbits in the 15 months following its detection. Full-length genomic analysis revealed that this virus is a recombinant containing an RHDVa capsid gene and nonstructural genes most closely related to nonpathogenic rabbit caliciviruses. Nationwide monitoring efforts need to be expanded to assess if the increasing number of different RHDV variants circulating in the Australian environment will affect biological control of rabbits. At the same time, updated vaccines and vaccination protocols are urgently needed to protect pet and farmed rabbits from these novel rabbit caliciviruses.

Full genome sequences are key to disclose RHDV2 emergence in the Macaronesian islands.

A recent publication by Carvalho et al. in "Virus Genes" (June 2017) reported the presence of the new variant of rabbit hemorrhagic disease virus (RHDV2) in the two larger islands of the archipelago of Madeira. Based on the capsid protein sequence, the authors suggested that the high sequence identity, along with the short time spanning between outbreaks, points to dissemination from Porto Santo to Madeira. By including information of the full RHDV2 genome of strains from Azores, Madeira, and the Canary Islands, we confirm the results obtained by Carvalho et al., but further show that several subtypes of RHDV2 circulate in these islands: non-recombinant RHDV2 in the Canary Islands, G1/RHDV2 in Azores, Porto Santo and Madeira, and NP/RHDV2 also in Madeira. Here we conclude that RHDV2 has been independently introduced in these archipelagos, and that in Madeira at least two independent introductions must have occurred. We provide additional information on the dynamics of RHDV2 in the Macaronesian archipelagos of Azores, Madeira, and the Canary Islands and highlight the importance of analyzing RHDV2 complete genome.