Detections of Rabbit Hemorrhagic Disease Virus 2 (RHDV2) Following the 2020 Outbreak in Wild Lagomorphs across the Western United States.

Rabbit hemorrhagic disease virus 2 (RHDV2) is a highly infectious, often fatal viral disease that affects both domestic and wild lagomorph species. In the United States (U.S.), the virus first was detected in wild lagomorph populations in the southwest in March 2020 and has continued to be detected in native North American lagomorph species over several years. The susceptibility of host species and exact mechanisms of environmental transmission across the U.S. landscape remain poorly understood. Our study aims to increase the understanding of RHDV2 in wild lagomorph populations by providing a history of detection. We present and summarize results from all RHDV2-suspect wild lagomorph morbidity and mortality samples submitted for diagnostic testing in the U.S. from March 2020 to March 2024. Samples were submitted from 916 wild lagomorphs across eight native North American species in 14 western states, of which 313 (34.2%) tested positive by RHDV2 RT-qPCR. Detections of RHDV2 in pygmy rabbits (Brachylagus idahoensis) and riparian brush rabbits (Sylvilagus bachmani riparius) suggest that the risk to threatened and endangered species warrants more attention. Continuing to investigate wild lagomorph morbidity and mortality events and tracking RHDV2 detections over time can help inform on disease epidemiology and wild lagomorph population trends.

Neglected Spleen Transcriptional Profile Reveals Inflammatory Disorder Conferred by Rabbit Hemorrhagic Disease Virus 2 Infection.

Rabbit hemorrhagic disease (RHD) is an acute fatal disease caused by the rabbit hemorrhagic disease virus (RHDV). Since the first outbreaks of type 2 RHDV (RHDV2) in April 2020 in China, the persistence of this virus in the rabbit population has caused substantial economic losses in rabbit husbandry. Previous failures in preventing RHDV2 prompted us to further investigate the immune mechanisms underlying the virus's pathogenicity, particularly concerning the spleen, a vital component of the mononuclear phagocyte system (MPS). For this, a previous RHDV2 isolate, CHN/SC2020, was utilized to challenge naive adult rabbits. Then, the splenic transcriptome was determined by RNA-Seq. This study showed that the infected adult rabbits had 3148 differentially expressed genes (DEGs), which were associated with disease, signal transduction, cellular processes, and cytokine signaling categories. Of these, 100 upregulated DEGs were involved in inflammatory factors such as IL1α, IL-6, and IL-8. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that these DEGs were significantly enriched in the cytokine-cytokine receptor interaction signaling pathway, which may play a vital role in CHN/SC2020 infection. At the same time, proinflammatory cytokines and chemokines were significantly increased in the spleen at the late stages of infection. These findings suggested that RHDV2 (CHN/SC2020) might induce dysregulation of the cytokine network and compromise splenic immunity against viral infection, which expanded our understanding of RHDV2 pathogenicity.

First Detection and Circulation of RHDV2 in New Zealand.

Rabbit haemorrhage disease virus 2 (RHDV2) is a highly pathogenic lagovirus that causes lethal disease in rabbits and hares (lagomorphs). Since its first detection in Europe in 2010, RHDV2 has spread worldwide and has been detected in over 35 countries so far. Here, we provide the first detailed report of the detection and subsequent circulation of RHDV2 in New Zealand. RHDV2 was first detected in New Zealand in 2018, with positive samples retrospectively identified in December 2017. Subsequent time-resolved phylogenetic analysis suggested a single introduction into the North Island between March and November 2016. Genetic analysis identified a GI.3P-GI.2 variant supporting a non-Australian origin for the incursion; however, more accurate identification of the source of the incursion remains challenging due to the wide global distribution of the GI.3P-GI.2 variant. Furthermore, our analysis suggests the spread of the virus between the North and South Islands of New Zealand at least twice, dated to mid-2017 and around 2018. Further phylogenetic analysis also revealed a strong phylogeographic pattern. So far, no recombination events with endemic benign New Zealand rabbit caliciviruses have been identified. This study highlights the need for further research and surveillance to monitor the distribution and diversity of lagoviruses in New Zealand and to detect incursions of novel variants.

Vaccination against Rabbit Hemorrhagic Disease Virus 2 (RHDV2) Using a Baculovirus Recombinant Vaccine Provides Durable Immunity in Rabbits.

Rabbit hemorrhagic disease virus 2 (RHDV2) emerged in the United States in 2018 and has spread in both domestic and wild rabbits nationwide. The virus has a high mortality rate and can spread rapidly once introduced in a rabbit population. Vaccination against RHDV2 provides the best protection against disease and should be considered by all rabbit owners. Here, we investigate the duration of immunity provided by vaccination with the Medgene Platform conditionally licensed commercial vaccine 6 months following the initial series. Rabbits received either the vaccination or a placebo and were challenged with RHDV2 6 months later. All vaccinated rabbits survived challenge whereas 18/19 non-vaccinated controls succumbed to infection within 10 or fewer days post-challenge. These results demonstrate lasting immunity following vaccination with the Medgene RHDV2 vaccine.

Coding-complete genome sequences of rabbit hemorrhagic disease virus type 2 detected in 2023 in Washington State indicate a divergent incursion into the United States.

Three rabbit hemorrhagic disease virus type 2 (RHDV2) coding-complete genome sequences were obtained from domestic and wild rabbits in Washington State in June and July 2023. These three RHDV2 sequences are <82% identical to previous RHDV2 sequences in North America and likely indicate a discrete incursion.

Serological characterisation of Lagovirus virus-like particles originating from native and mutated VP60 of rabbit haemorrhagic disease virus 2 and European brown hare syndrome virus.

Introduction: Since lagoviruses cannot be cultivated in vitro, using expression systems is an alternative and promising way of producing diagnostic viral antigens. It opens up their use as active immunogens for vaccine production. Material and Methods: Virus-like particles (VLPs) were produced in a baculovirus expression system in Spodoptera frugiperda 9 (Sf9) insect cells based on wild-type and mutated variants of the virus capsid VP60 protein from a Polish strain of European brown hare syndrome virus (EBHSV) and wild-type and mutated versions of this protein from a Polish strain of rabbit haemorrhagic disease virus 2 (RHDV2). The mutations were the substitution of an arginylglycylaspartic acid (Arg-Gly-Asp/RGD) motif in the P2 subdomain and, in the S or P2 domain, the substitution of three lysines. The VLPs were purified with sucrose gradient ultracentrifugation. Results: Protein production was confirmed by Western blot analysis using rabbit or hare sera and ELISA tests with different types of monoclonal antibody. The haemagglutination properties of some VLPs were also evaluated. Electron microscopy of wild-type EBHSV, wild-type RHDV2 and the four VP60 variants produced in this experiment revealed the formation of characteristic VLP structures. Conclusion: For the first time, mutated VLPs of RHDV2 with an RGD motif in the VP60 sequence were obtained, which could potentially be used to deliver cargo to eukaryotic cells. Virus-like particles based on the VP60 proteins of EBHSV and RHDV with a three-lysine substitution in the S or P2 domains were also obtained. Potential exists for VLPs of EBHSV and RHDV2 as vaccine candidates.

Recombination between non-structural and structural genes as a mechanism of selection in lagoviruses: The evolutionary dead-end of an RHDV2 isolated from European hare.

The genus Lagovirus, belonging to the family Caliciviridae, emerged around the 1980s. It includes highly pathogenic species, rabbit hemorrhagic disease virus (RHDV/GI.1) and European brown hare syndrome virus (EBHSV/GII.1), which cause fatal hepatitis, and nonpathogenic viruses with enteric tropism, rabbit calicivirus (RCV/GI.3,4) and hare calicivirus (HaCV/GII.2). Lagoviruses have evolved along two independent genetic lineages: GI (RHDV and RCV) in rabbits and GII (EBHSV and HaCV) in hares. To be emphasized is that genomes of lagoviruses, like other caliciviruses, are highly conserved at RdRp-VP60 junctions, favoring intergenotypic recombination events at this point. The recombination between an RCV (genotype GI.3), donor of non-structural (NS) genes, and an unknown virus, donor of structural (S) genes, likely led to the emergence of a new lagovirus in the European rabbit, called RHDV type 2 (GI.2), identified in Europe in 2010. New RHDV2 intergenotypic recombinants isolated in rabbits in Europe and Australia originated from similar events between RHDV2 (GI.2) and RHDV (GI.1) or RCV (GI.3,4). RHDV2 (GI.2) rapidly spread worldwide, replacing RHDV and showing several lagomorph species as secondary hosts. The recombination events in RHDV2 viruses have led to a number of viruses with very different combinations of NS and S genes. Recombinant RHDV2 with NS genes from hare lineage (GII) was recently identified in the European hare. This study investigated the first RHDV2 (GI.2) identified in Italy in European hare (RHDV2_Bg12), demonstrating that it was a new virus that originated from the recombination between RHDV2, as an S-gene donor and a hare lagovirus, not yet identified but presumably nonpathogenic, as an NS gene donor. When rabbits were inoculated with RHDV2_Bg12, neither deaths nor seroconversions were recorded, demonstrating that RHDV2_Bg12 cannot infect the rabbit. Furthermore, despite intensive and continuous field surveillance, RHDV2_Bg12 has never again been identified in either hares or rabbits in Italy or elsewhere. This result showed that the host specificity of lagoviruses can depend not only on S genes, as expected until today, but potentially also on some species-specific NS gene sequences. Therefore, because RHDV2 (GI.2) infects several lagomorphs, which in turn probably harbor several specific nonpathogenic lagoviruses, the possibility of new speciation, especially in those other than rabbits, is real. RHDV2 Bg_12 demonstrated this, although the attempt apparently failed.

First detection and molecular characterization of rabbit hemorrhagic disease virus (RHDV) in Algeria.

Since the first detection of rabbit hemorrhagic disease (RHD), the rabbit hemorrhagic disease virus (RHDV) has been responsible for high morbidity and mortality worldwide, both in domestic and in wild rabbits. Despite the apparent control of RHD in rabbitries through vaccination, several studies highlighted the rapid evolution of RHDV by recombination, which may facilitate the emergence of new pathogenic strains. The aim of this study was to confirm the presence and characterize RHDV in Algeria. For this, rabbit samples were collected in the north of Algeria, between 2018 and 2021, from small farms where the virus was suspected after the sudden death of a high number of rabbits, and from healthy hunted wild rabbits. The domestic rabbits revealed clinical signs and lesions that were suggestive of RHD. RT-PCR showed that 79.31% of the domestic rabbit samples were positive for RHDV, while in 20.69%, including the hunted rabbits, the virus was not detected. Phylogenetic analysis of the Algerian strains allowed the confirmation and identification as GI.2 (RHDV2), and showed a close relation to GI.3P-GI.2 recombinant strains, suggesting a potential introduction from other countries, with an older strain potentially originated from neighboring Tunisia, while more recent isolates grouped with strains from North America. Our study reports for the first time the presence of GI.2 (RHDV2) in Algeria with multiple routes of introduction. Consequently, we propose that RHDV control in Algeria should be based on epidemiological surveys in association with an adequate prophylactic program.

Understanding rabbit owners' willingness to engage in disease prevention behaviors.

Rabbit hemorrhagic disease virus 2 (RHDV2) is a fatal, highly contagious pathogen that infects wild and domestic lagomorphs (rabbits and hares). RHDV2 is an important cause of disease in pet and companion rabbits, has resulted in economic losses for the commercial rabbit industry, and has caused declines of wild lagomorph populations. It is essential for domestic rabbit owners to engage in appropriate actions (e.g., using effective disinfectants, creating secure barriers between domestic and wild rabbits) to protect the health and welfare of their rabbits and reduce the risk of human-mediated spread of RHDV2. Thus, we investigated rabbit owners' stated willingness to engage in nine commonly recommended biosecurity practices and their support for seven potential government-implemented management actions. We administered an online survey to 1790 rabbit owners in the United States between April and August 2021. Respondents were likely to engage in all biosecurity measures and were supportive of most management actions that could be implemented by government agencies. Respondents' willingness to engage in and support biosecurity measures was positively correlated with their perceptions of the importance of biosecurity, risk perceptions pertaining to the impact of RHDV2 on lagomorphs and rabbit-related industries, knowledge of RHDV2, and trust in government to manage RHDV2. Respondents' motivations for owning rabbits, husbandry behaviors, and demographic characteristics also influenced their willingness to engage in or support biosecurity measures. Engaging domestic rabbit owners in collaborative biosecurity measures is critical for protecting domestic rabbit health and preventing potential spillover between domestic and free-roaming lagomorphs, as there are still many uncertainties about how RHDV2 is spreading across the United States and the world. Implementing outreach strategies that communicate the importance and effectiveness of biosecurity practices in protecting rabbit welfare, rabbit-related activities, and wild lagomorph populations may increase the likelihood of rabbit owners adopting biosecurity measures.

Diagnosis of a Rabbit Hemorrhagic Disease Virus 2 (RHDV2) and the Humoral Immune Protection Effect of VP60 Vaccine.

Rabbit hemorrhagic disease (RHD) is known as rabbit plague and hemorrhagic pneumonia. It is an acute, septic, and highly fatal infectious disease caused by the Lagovirus rabbit hemorrhagic disease virus (RHDV) in the family Caliciviridae that infects wild and domestic rabbits and hares (lagomorphs). At present, RHDV2 has caused huge economic losses to the commercial rabbit trade and led to a decline in the number of wild lagomorphs worldwide. We performed a necropsy and pathological observations on five dead rabbits on a rabbit farm in Tai'an, China. The results were highly similar to the clinical and pathological changes of typical RHD. RHDV2 strain was isolated and identified by RT-PCR, and partial gene sequencing and genetic evolution analysis were carried out. There were significant differences in genetic characteristics and antigenicity between RHDV2 and classical RHDV strain, and the vaccine prepared with the RHDV strain cannot effectively prevent rabbit infection with RHDV2. Therefore, we evaluated the protective efficacy of a novel rabbit hemorrhagic virus baculovirus vector inactivated vaccine (VP60) in clinical application by animal regression experiment. The result showed that VP60 could effectively induce humoral immunity in rabbits. The vaccine itself had no significant effect on the health status of rabbits. This study suggested that the clinical application of VP60 may provide new ideas for preventing the spread of RHD2.

Comparison of magnetic bead and rapid swab RNA extraction methods for detecting rabbit hemorrhagic disease virus 2 in rabbit liver samples.

We compared a bead RNA extraction method with a one-tube method that required only a heat block and ice. RNA was first extracted from liver samples from nine rabbits dying from rabbit hemorrhagic disease virus 2 (RHDV2) using magnetic beads, and RT-PCR was used to detect RHDV2 sequence. Following freezing, RNA was extracted a second time using the SwiftX™ Swabs Viral RNA Extraction Reagent. RHDV2 was detected in all nine samples. Cycle threshold values were higher in the RT-PCR following SwiftX extraction (mean: 3.79), indicating that the second extraction method resulted in approximately a 1 log10 reduction in sensitivity. A second freeze-thaw for the samples and less tissue extracted using SwiftX may have contributed additionally to the loss in sensitivity.

Comparison of the Impact between Classical and Novel Strains of Rabbit Haemorrhagic Disease on Wild Rabbit Populations in Spain.

The outbreaks of two strains of rabbit haemorrhagic disease (RHD) (GI.1 and GI.2) in the Iberian Peninsula have caused substantial economic losses in commercial rabbitries and have affected the conservation of rabbit-sensitive predators due to the dramatic decline of their natural populations. However, the assessment of the impact of both RHD strains on wild rabbit populations has been limited to a few small-scale studies. Little is known about the overall impact within its native range. In this study, we described and compared the effects of GI.1 and GI.2 countrywide by using time series of hunting bag data widely available across the country and compared their trend during the first eight years after the first outbreak of GI.1 (i.e., 1998) and GI.2 (i.e., 2011), respectively. We used Gaussian generalised additive models (GAM) with the number of hunted rabbits as the response variable and year as the predictor to evaluate the non-linear temporal dynamics of the population at the national and regional community levels. The first GI.1 caused a population decline of around 53%, affecting most Spanish regional communities where the disease occurred. The positive trend observed after GI.1 in Spain ended with the initial outbreak of GI.2, which did not appear to cause a national population decline. In contrast, we found significant variability in the rabbit population trend among regional communities, where some increased, and others decreased. Such a disparity is unlikely to be explained by a single factor; rather, it appears to result from several factors, such as climatic conditions, host resistance improvement, virulence attenuation, or population density. Our study suggests that a national comprehensive hunting bag series could aid in elucidating the differences in the impact of emerging diseases on a large scale. Future research should focus on national longitudinal serological studies to shed light on the immunological status of rabbit populations in different regions to better understand the evolution of RHD strains and the resistance gained by the wild populations.

Status of Infectious Diseases in Free-Ranging European Brown Hares (Lepus europaeus) Found Dead between 2017 and 2020 in Schleswig-Holstein, Germany.

The European brown hare (Lepus europaeus) is a quite adaptable species, but populations have been decreasing for several decades in different countries, including Germany. To investigate infectious diseases as possible influences on observed population decline in the German federal state Schleswig-Holstein, 118 deceased free-ranging European brown hares were collected between 2017 and 2020 and underwent detailed postmortem examination with extended sampling. Infectious diseases were a major cause of death (34.7%). The number of juveniles found exceeded the adult ones. The main pathomorphological findings were hepatitis (32.8%), pneumonia (22.2%), nephritis (19.1%), liver necrosis (12.9%), and enteritis (40.7%). An unusual main finding was steatitis (20.9%) of unknown origin. Animals were mainly emaciated and showed high infection rates with Eimeria spp. (91.3%) and Trichostrongylus spp. (36.2%). European Brown Hare Syndrome Virus reached an epidemic status with few fatal infections (4.2%) and high seroprevalence (64.9%), whereas the prevalence of Rabbit Haemorrhagic Disease Virus 2 was very low (0.8%) in hares in Schleswig-Holstein. Pathogens such as Yersinia pseudotuberculosis (5.9%), Pasteurella multocida (0.8%), and Staphylococcus aureus (3.4%) only caused sporadic deaths. This study illustrates the wide distribution of various infectious pathogens with high mortality and even zoonotic potential. Infectious diseases need to be considered as an important influence on population dynamics in Schleswig-Holstein.

Immunological Cross-Protection between Different Rabbit Hemorrhagic Disease Viruses-Implications for Rabbit Biocontrol and Vaccine Development.

The use of rabbit hemorrhagic disease virus (RHDV) as a biocontrol agent to control feral rabbit populations in Australia, in combination with circulating endemic strains, provides a unique environment to observe the interactions between different lagoviruses competing for the same host. Following the arrival of RHDV2 (GI.2) in Australia, it became necessary to investigate the potential for immunological cross-protection between different variants, and the implications of this for biocontrol programs and vaccine development. Laboratory rabbits of various immune status-(1) rabbits with no detectable immunity against RHDV; (2) rabbits with experimentally acquired immunity after laboratory challenge; (3) rabbits immunised with a GI.2-specific or a multivalent RHDV inactivated virus prototype vaccine; or (4) rabbits with naturally acquired immunity-were challenged with one of three different RHDV variants (GI.1c, GI.1a or GI.2). The degree of cross-protection observed in immune rabbits was associated with the variant used for challenge, infectious dose of the virus and age, or time since acquisition of the immunity, at challenge. The immune status of feral rabbit populations should be determined prior to intentional RHDV release because of the high survival proportions in rabbits with pre-existing immunity. In addition, to protect domestic rabbits in Australia, a multivalent RHDV vaccine should be considered because of the limited cross-protection observed in rabbits given monovalent vaccines.

Establishment of a duplex TaqMan RT-PCR for the differential detection of RHDV GI.1 and GI.2.

BACKGROUND: Rabbit haemorrhagic disease (RHD) is a highly contagious and acute fatal hepatitis of the European rabbit (Oryctolagus cuniculus), caused by a calicivirus (genus Lagovirus). Up to 2010, all RHD viruses (RHDV) isolated belonged to one genotype. In 2010, a new genotype of RHDV (RHDV2/b, currently designated GI.2 based on phylogenetic analysis) emerged in France. The aim of this study was to develop a rapid, simple, specific and sensitive TaqMan real-time PCR assay for the classic strain of RHDV and RHDV2 detection. Specific primers and probes were designed for the VP60 gene of RHDV and RHDV2 within the conserved region of viral genome. RESULTS: This study was demonstrated to be highly specific for RHDV and RHDV2, without cross-reactions with other non-targeted viruses. The detection limit of this work was 102 copies of RHDV and RHDV2, respectively. The coefficient of variation of the assay was less than 5% for both intra-assay and inter-assay. The reproducibility of method was assessed using plasmids and the coefficient of variation obtained was 0.2-3.70. Of 79 clinical samples, 68 were positive samples (86.08%), of which 60 were classic RHDV variants (75.9%), 4 were co-infected (5.06%) and 8 were RHDV2 (10.12%), those results are more sensitivity compare with conventional RT-PCR RT-PCR. CONCLUSIONS: In conclusion, this duplex TaqMan RT-qPCR based on VP60 gene of RHDV and RHDV2 could be a valuable tool in diagnose and molecular epidemiological study of the RHDV and RHDV2.

Monitoring of Rabbit Hemorrhagic Disease Virus in European Wild Rabbit (Oryctolagus cuniculus) Populations by PCR Analysis of Rabbit Fecal Pellets.

European rabbits (Oryctolagus cuniculus) are affected by rabbit hemorrhagic disease (RHD), which is caused by a lagovirus responsible for significant mortality in European wild rabbit populations. Our study aimed to evaluate the potential for detecting viral RNA by duplex real-time PCR in rabbit fecal pellets collected in the field, as a noninvasive method to monitor RHD virus circulation in wild populations. To do this, monthly discoveries of rabbits that died from RHD and detection of viral RNA in fecal pellets were recorded in two enclosed populations of wild rabbits throughout a year. The results suggested a low performance of this procedure to monitor viral infection incidence and a weak concordance with monthly discoveries of rabbits that died from RHD. This poor association was probably due to the low amount of viral RNA in feces, the prolonged time of excretion after infection, and that the number of rabbits found dead from RHD does not necessarily correlate with RHD incidence. Nevertheless, this procedure may be a complementary noninvasive method to assist in determining the presence of RHD viruses in populations. Additional research is needed to determine the suitability of this methodology to perform epidemiologic surveys on wild populations of European rabbits and, especially, other European or North American lagomorph species affected by lagoviruses.

Changes in European wild rabbit population dynamics and the epidemiology of rabbit haemorrhagic disease in response to artificially increased viral transmission.

European wild rabbit (Oryctolagus cuniculus) populations are severely affected by rabbit haemorrhagic disease (RHD), currently aggravated by the spread of the new lagovirus serotype RHDV2 that replaced the classical RHDV strains (RHDV/RHDVa). This virus causes high mortality in both adult and young rabbits and to date, there is no management tool to effectively reduce its impact on wild rabbit populations. This hinders the success of common strategies, such as habitat management or restocking, in areas where rabbits are native. However, the present study, conducted on enclosed wild rabbit populations, showed that spreading RHDV2 on baits during breeding periods induced infection of young rabbits, reducing mortality rates, presumably due to maternal antibody protection. This reduced the young rabbit mortality hazard by a third, and more juvenile rabbits immune to RHDV2 were recruited into the adult breeding population. Young rabbits from populations in which the force of infection of RHDV2 was increased, however, exhibited considerably higher susceptibility to infection by RHDV than those from non-treated control populations. Since co-circulation of classical RHDVs was ruled out, differences in the type and degree of immunization, the level of cross-protection and/or other unknown factors, such as the circulation of undetected non-pathogenic lagoviruses, arose as possible explanations. This meant that although the present study demonstrated the possibility of successfully modulating the impact of RHD in wild populations, the epidemiological complexity of the situation where several lagoviruses circulate requires additional research to determine final applicability of the proposed method.

Down the Rabbit Hole: Domestic Rabbit Owners' Perceptions of Rabbit Hemorrhagic Disease Virus 2.

Rabbit hemorrhagic disease virus 2 (RHDV2) is a highly contagious pathogen that infects wild and domestic rabbits and hares (lagomorphs). Globally, RHDV2 has resulted in substantial economic losses for commercial rabbit trade and caused wild lagomorph population declines. Previous research on RHDV2 suggests that human-mediated movement of rabbits may contribute to the spread of RHDV2. We conducted the first survey of individuals who own or interact with domestic rabbits to identify their rabbit husbandry behaviors and knowledge of, and concerns about, RHDV2. In 2021, we surveyed 1807 rabbit owners, breeders, and rescue staff in the USA. Respondents had a high level of knowledge about RHDV2. Respondents believed RHDV2 posed a high risk to rabbit-related activities and were concerned about its economic and ecological impacts. Nearly half of respondents always kept their rabbits indoors, but 10.7% of respondents allowed their rabbits outside frequently on properties used by wild lagomorphs. Respondents with five rabbits or less were generally willing to vaccinate their rabbits, but respondents with larger herds argued that vaccines were cost prohibitive. Given respondents' concerns about RHDV2, communication about disease prevention should highlight the adverse ecological and economic consequences of RHDV2.

First detection of rabbit haemorrhagic disease virus (RHDV2) in Singapore.

Rabbit haemorrhagic disease (RHD) is a significant viral disease caused by infection with Rabbit haemorrhagic disease virus (RHDV). The first documented cases of RHDV in Singapore occurred in adult pet European rabbits (Oryctolagus cuniculus) in September 2020. Rabbits presented with acute hyporexia, lethargy, huddled posture, and varying degrees of pyrexia and tachypnoea. Clinical pathology consistently reflected markedly elevated alanine aminotransferase (ALT), aspartate aminotransferase (AST) and alkaline phosphatase (ALKP). Hepatic lobe torsion was ruled out using ultrasonography and colour Doppler studies in all patients. A total of 11 rabbits owned by 3 families were presented to the clinics; 8/11 rabbits died within 48 hr of presentation, while the remaining two rabbits had recovered after prolonged hospitalization and one rabbit was aclinical. Histopathology revealed acute, marked diffuse hepatocellular necrosis and degeneration, findings which were suggestive for RHDV infection and prompted the undertaking of further molecular diagnostics. Subsequent polymerase chain reaction of the liver samples detected RHDV RNA. Molecular characterization of viral genomes by whole genome sequencing revealed that the outbreak strain was of the genotype GI.2 (RHDV2/RHDVb). Nucleotide sequences of the VP60 gene were compared with various RHDV variants using phylogenetic analysis. The sample genome shared highest sequence identity with a GI.2-genotyped virus from GenBank (RHDV isolate Algarve 1 polyprotein and minor structural protein (VP10) genes, GenBank accession KF442961). The combination of clinical, histopathological, molecular and sequencing technologies enabled rapid detection and detailed genetic characterization of the RHDV virus causing the present outbreak for prompt implementation of disease control measures in Singapore. Further epidemiological investigations of potential virus introduction into Singapore are ongoing.

Spillover events of rabbit haemorrhagic disease virus 2 (recombinant GI.4P-GI.2) from Lagomorpha to Eurasian badger.

Rabbit haemorrhagic disease (RHD) is a major threat to domestic and wild European rabbits. Presently, in Europe, the disease is caused mainly by Rabbit haemorrhagic disease virus 2 (RHDV2/b or Lagovirus europaeus GI.2), the origin of which is still unclear, as no RHDV2 reservoir hosts were identified. After the RHDV2 emergence in 2010, viral RNA was detected in a few rodent species. Furthermore, RHDV2 was found to cause disease in some hare species resembling the disease in rabbits, evidencing the ability of the virus to cross the species barrier. In this study, through molecular, histopathologic, antigenic and morphological evidences, we demonstrate the presence and replication of RHDV2 in Eurasian badgers (Meles meles) found dead in the district of Santarém, Portugal, between March 2017 and January 2020. In these animals, we further classify the RHDV2 as a Lagovirus europaeus recombinant GI.4P-GI.2. Our results indicate that Meles meles is susceptible to RHDV2, developing systemic infection, and excreting the virus in the faeces. Given the high viral loads seen in several organs and matrices, we believe that transmission to the wild rabbit is likely. Furthermore, transmission electron microscopy data show the presence of calicivirus compatible virions in the nucleus of hepatocytes, which constitutes a paradigm shift for caliciviruses' replication cycle.