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Rabbit hemorrhagic disease (RHD) causes lethal fulminant hepatitis in rabbits. Two different genotypes (GI.1 and GI.2) responsible for RHD are reported. GI.2 was first detected in France in 2010 and subsequently spread to other countries in Europe. In April 2020, GI.2 was detected in China. In this study, we report a novel recombinant strain of fatal rabbit hemorrhagic disease virus 2 (RHDV2 or GI.2) detected from domestic rabbits in three provinces in China in 2020-2021. Full-length genomic analysis has revealed that the recombinant virus contained an RHDV2 capsid gene and nonstructural genes from an unclassified lagovirus genotype. This type of virus emerged and circulated throughout China within a year after the initial detection of the original RHDV2. Compared with the original strain, the new virus showed a longer infected time and lower mortality rate but almost the same viral load at the moribund stage of infection. This might have resulted in high virus contamination in the environment, facilitating virus transmission. As the consequences of the presence of novel recombinant strains are unpredictable, the circulation of the novel variant in the population should be carefully monitored in China. [ABSTRACT FROM AUTHOR]

Rabbit haemorrhagic disease virus (RHDV) GI.2 has caused significant declines in the abundance of wild European rabbits (Oryctolagus cuniculus), contributing to the species being recently classified as "endangered" in its native range. The epidemiology of this virus is still poorly understood despite its relevance for domestic and wild rabbits. During a longitudinal capture-mark-recapture (CMR) study of wild Iberian rabbits, O. c. algirus, in a semiextensive breeding enclosure, an outbreak of RHDV GI.2 took place in January-February 2022, allowing us to estimate key epidemiological parameters of a natural outbreak. From April 2021 to July 2022, 340 rabbits were captured 466 times and individually identified, and some were vaccinated against myxoma virus (MYXV) and/or RHDV GI.2. Sera were collected and tested for IgG specific for MYXV and RHDV GI.2, and data were analyzed using multievent CMR models. During six weeks in January-February 2022, an estimated 81.0% (CI95 77.1-84.3%) of the population died. Intensive aboveground searches could recover 189 carcasses (50.5% of the estimated mortality, CI95 41.8-63.4%), with RHDV GI.2 detected in 6/7 tested. Apparent RHDV GI.2 seroprevalence rose from 15.4% (CI95 8.0-27.5%) in January 2022 to 87.9% (CI95 72.7-95.2%) in February 2022. The apparent mortality of RHDV GI.2-seropositive rabbits during the outbreak was estimated as null, while for seronegative rabbits, it was 76.0% (CI95 53.8-90.3%). Among the seronegative rabbits, mortality was higher in unvaccinated (100%) than in recently vaccinated (60.0 ± 16.6%) and in females (100%) than in males (52.0 ± 17.1%). Infected carcasses in the burrows might explain the medium-term disease persistence in the population following the outbreak. Rabbits with antibodies at the cutoff for seropositivity were fully protected from fatal infection. Females had a higher fatality rate than males, underscoring the impact of RHDV GI.2 on the population dynamics of this endangered species. [ABSTRACT FROM AUTHOR]

Rabbit hemorrhagic disease virus 2 (RHDV2 or Lagovirus europaeus GI.2) is spreading across North America. This has enabled submissions of lagomorphs for testing to veterinary diagnostic laboratories (VDLs). The liver is currently the gold-standard sample type for testing by RT-qPCR. VDL clients usually seek alternate diagnostic approaches that permit simpler and faster sample collection with less risk of environmental contamination; there is also a necessity for a sample type that can be collected from live animals. Therefore, the goal of this study was to optimize and evaluate an RT-qPCR assay on rectal swabs collected from a group of carcasses of leporids of different species that were submitted to a VDL during an RHDV2 outbreak. A total of 130 carcasses were tested both by liver tissue and rectal swab RT-qPCR. The results of the liver samples were considered the gold standard, and 73 carcasses tested positive and 57 carcasses tested negative in liver. Out of the 73 liver RT-qPCR-positive carcasses, 64 tested positive and 9 tested negative on the rectal RT-qPCR. All 57 liver RT-qPCR-negative carcasses tested negative on the rectal RT-qPCR. The sensitivity and specificity of the rectal RT-qPCR were 88% and 100%, respectively, most likely due to significantly (p < 0.001) lower viral loads in the rectal swabs (median Ct: 27.03) compared to the liver samples (median Ct: 12.69). Despite being more than 4 logs less sensitive, RT-qPCRs from rectal swabs can be used to screen leporid carcasses for the presence of RHDV2 RNA. [ABSTRACT FROM AUTHOR]

A long‐term active epidemiological surveillance programme was conducted to determine seroprevalence to myxoma virus (MYXV), infection prevalence and spatiotemporal patterns and factors associated with MYXV circulation in wild rabbits (Oryctolagus cuniculus) in Spanish Mediterranean ecosystems. A total of 2376 animals were sampled over four study periods: 2009–2012 (P1), 2012–2015 (P2), 2015–2018 (P3) and 2018–2021 (P4). Antibodies against MYXV were detected by a commercial indirect ELISA in 59.9% (1424/2376; 95% CI: 58.0–61.9) of wild rabbits. At least one seropositive animal was detected on 131 (96.3%) of 136 game estates sampled. MYXV infection was confirmed by PCR in 94 of 1063 (8.8%; 95% CI: 7.3–10.7) wild rabbits. Circulation of the novel recombinant MYXV (ha‐MYXV) was not found in wild rabbits analysed during P4. Five statistically significant spatiotemporal clusters of high MYXV seroprevalence were identified using a Bernoulli model: one in P2 and four in P3. A generalized linear mixed model (GLMM) analysis identified sampling season (autumn), age (adult and juvenile), outbreaks of myxomatosis in the month prior to sampling, mean annual temperature, humidity and seropositivity to rabbit haemorrhagic disease virus as factors potentially linked with MYXV seropositivity. GLMM analysis identified outbreaks of myxomatosis in the month prior to sampling, MYXV seropositivity and presence of lesions compatible with myxomatosis as factors associated with MYXV infection. The results indicate high exposure, widespread but non‐homogeneous distribution, and endemic circulation of MYXV in wild rabbit populations in southern Spain during the last decade. Prevalence of antibodies against MYXV showed fluctuations both within the year and over the study periods, revealing variations in the immunity of wild rabbit populations in Mediterranean ecosystems that could increase the risk of MYXV re‐emergence in immunologically naïve populations. The present study highlights the importance of long‐term surveillance to better understand the epidemiology of MYXV in wild lagomorphs. [ABSTRACT FROM AUTHOR]

Estimation of the diagnostic performance of serological tests often relies on another test assumed as a reference or on samples of known infection status, yet both are seldom available for emerging pathogens in wildlife. Longitudinal disease serological data can be analysed through multi‐event capture–mark–recapture (MECMR) models accounting for the uncertainty in state assignment, allowing us to estimate epidemiological parameters such as incidence and mortality. We hypothesized that by estimating the uncertainty in state assignment, MECMR models estimate the diagnostic performance of serological tests for rabbit haemorrhagic disease virus (RHDV) and myxoma virus (MYXV). We evaluated this hypothesis on longitudinal serological data of three tests of RHDV and one test of MYXV in two populations of the European rabbit (Oryctolagus cuniculus algirus). First, we selected the optimal cut‐off threshold for each test using finite mixture models, a reference method not relying on reference tests or samples. Second, we used MECMR models to compare the diagnostic sensitivity (Se) and specificity (Sp) of the three tests for RHDV. Third, we compared the estimates of diagnostic performance by MECMR and finite mixture models across a range of cut‐off values. The MECMR models showed that the RHDV test employing GI.2 antigens (Se: 100%) outperformed two tests employing GI.1 antigens (Se: 21.7% ± 8.6% and 8.7% ± 5.9%). At their selected cut‐offs (2.0 for RHDV GI.2 and 2.4 for MYXV), the estimates of Se and Sp were concordant between the MECMR and finite mixture models. Over the duration of the study (May 2018 to September 2020), the monthly survival of European rabbits seropositive for MYXV was significantly higher than that of seronegative rabbits (82.7% ± 4.9% versus 61.5% ± 12.7%) at the non‐fenced site. We conclude that MECMR models can reliably estimate the diagnostic performance of serological tests for RHDV and MYXV in European rabbits. This conclusion could extend to other diagnostic tests and host‒pathogen systems. Longitudinal disease surveillance data analysed through MECMR models allow the validation of diagnostic tests for emerging pathogens in novel host species while simultaneously estimating epidemiological parameters. [ABSTRACT FROM AUTHOR]

Australia is known for its long history of using biocontrol agents, such as myxoma virus (MYXV) and rabbit haemorrhagic disease virus (RHDV), to manage wild European rabbit populations. Interestingly, while undertaking RHDV surveillance of rabbits that were found dead, we observed that approximately 40% of samples were negative for RHDV. To investigate whether other infectious agents are responsible for killing rabbits in Australia, we subjected a subset of these RHDV‐negative liver samples to metatranscriptomic sequencing. In addition, we investigated whether the host transcriptome data could provide additional differentiation between likely infectious versus non‐infectious causes of death. We identified transcripts from several Clostridia species, Pasteurella multocida, Pseudomonas spp., and Eimeria stiedae, in liver samples of several rabbits that had died suddenly, all of which are known to infect rabbits and are capable of causing disease and mortality. In addition, we identified Hepatitis E virus and Cyniclomyces yeast in some samples, both of which are not usually associated with severe disease. In one‐third of the sequenced total liver RNAs, no infectious agent could be identified. While metatranscriptomic sequencing cannot provide definitive evidence of causation, additional host transcriptome analysis provided further insights to distinguish between pathogenic microbes and commensals or environmental contaminants. Interestingly, three samples where no pathogen could be identified showed evidence of up‐regulated host immune responses, while immune response pathways were not up‐regulated when E. stiedae, Pseudomonas, or yeast were detected. In summary, although no new putative rabbit pathogens were identified, this study provides a robust workflow for future investigations into rabbit mortality events. [ABSTRACT FROM AUTHOR]

Rabbit haemorrhagic disease virus 2 (RHDV2) is now the dominant calicivirus circulating in wild rabbit populations in Australia. This study compared the infection and case fatality rates of RHDV2 and two RHDVs in wild rabbits, as well as their ability to overcome immunity to the respective other strains. Wild rabbits were allocated to groups either blindly or based on pre‐screening for RHDV/RHDV2 antibodies at capture. Rabbits were monitored regularly until their death or humane killing at 7 days post infection. Liver and eyeball samples were collected for lagovirus testing and aging rabbits, respectively. At capture, rabbits showed high seroprevalence to RHDV2 but not to RHDV. In RHDV/RHDV2 seronegative rabbits at capture, infection rates were highest in those inoculated with RHDV2 (81.8%, 18 out of 22), followed by K5 (53.8%, seven out of 13) and CZECH (40.0%, two out of five), but these differences were not statistically significant. In rabbits with previous exposure to RHDV2 at capture, infection rates were highest when inoculated with K5 (59.6%, 31 out of 52) followed by CZECH (46.0%, 23 out of 50), with infection rates higher in younger rabbits for both viruses. In RHDV/RHDV2 seronegative rabbits at capture, case fatality rates were highest for those inoculated with K5 (71.4%), followed by RHDV2 (50.0%) and CZECH (50.0%). In rabbits with previous exposure to RHDV2 at capture, case fatality rates were highest in rabbits inoculated with K5 (12.9%) followed by CZECH (8.7%), with no case fatalities following RHDV2 inoculation. Case fatality rates did not differ significantly between inoculums in either serostatus group at capture. Based on multivariable modelling, time to death post RHDV inoculation increased in rabbits with recent RHDV2 exposure compared with seronegative rabbits and with age. The results suggest that RHDV2 may cause higher mortalities than other variants in seronegative rabbit populations but that K5 may be more effective in reducing rabbit populations in an RHDV2‐dominant landscape. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rabbit haemorrhagic disease (RHD) is a highly contagious viral disease affecting lagomorphs. The first documented cases of RHD in Singapore occurred in adult pet European rabbits in September 2020. Singapore subsequently declared the outbreak resolved in December 2020. Epidemiological investigations ruled out introductions via importation of infected rabbits and contaminated feed. The source could not be definitively determined. However, the findings suggested that the incident involved both inter‐ and intra‐household transmission and veterinary clinic‐household transmission. This incident demonstrated the importance of sustained application of biosecurity measures, epidemiological investigations including active case finding, control measures such as expedient vaccine dissemination and risk communications. It showed that even without a wild lagomorph population, an urbanized city‐state like Singapore could still encounter emerging diseases such as RHD. Given its social impact on rabbit owners, the National Parks Board, Singapore and private veterinarians worked together to communicate with rabbit owners in order to urge them to adopt biosecurity measures and to address their concerns. [ABSTRACT FROM AUTHOR]

Mange in rabbits is a very important parasitic disease causing high losses. The disease is caused mainly by Sarcoptes scabiei, Psoroptes cuniculi, Cheyletiella parasitovorax, and Notoedres cati. Body mange and ear mange are the most common forms of this disease in rabbits. Animals can get mite infestation through direct contact with infected animals or contaminated fomites. This infestation is characterized by zoonotic nature and public health burden. The skin affection is characterized by pruritus, alopecia, severe cachexia, and sometimes death. Infestation is diagnosed mainly by skin scraping and microscopic examination. Control measures mainly depend on the use of different types of systemic and topical acaricides and the use of natural products and supportive elements. Vaccine is not commercially available and is still under investigation. Accordingly, this review article was designed to shed the light on the mange disease in rabbits in terms of mite's infestation and susceptibility, clinical manifestations, zoonosis, diagnosis, and control strategies. [ABSTRACT FROM AUTHOR]

Myxomatosis is an emergent disease in the Iberian hare (Lepus granatensis). In this species, the disease is caused by a natural recombinant virus (ha‐myxoma virus [MYXV]) identified for the first time in 2018 and has since been responsible for a large number of outbreaks in Spain and Portugal. The ha‐MYXV, which harbours a 2.8 Kb insert‐disrupting gene M009L, can also infect and cause disease in wild and domestic rabbits, despite being less frequently identified in rabbits. During the laboratory investigations of wild leporids found dead in Portugal carried out within the scope of a Nacional Surveillance Plan (Dispatch 4757/17, MAFDR), co‐infection events by classic (MYXV) and naturally recombinant (ha‐MYXV) strains were detected in both one Iberian hare and one European wild rabbit (Oryctolagus cuniculus algirus). These two cases were initially detected by a multiplex qPCR detection of MYXV and ha‐MYXV and subsequently confirmed by conventional PCR and sequencing of the M009L gene, which contains an ha‐MYXV‐specific insertion. To our knowledge, this is the first documented report of co‐infection by classic MYXV and ha‐MYXV strains either in Iberian hare or in European wild rabbit. It is also the first report of infection of an Iberian hare by a classic MYXV strain. These findings highlight the continuous evolution of the MYXV and the frequent host range changes that justify the nonstop monitoring of the sanitary condition of wild Leporidae populations in the Iberian Peninsula. [ABSTRACT FROM AUTHOR]

Rabbit haemorrhagic disease virus (RHDV) is associated with high morbidity and mortality in the European rabbit (Oryctolagus cuniculus). In 2010, a genetically distinct RHDV named RHDV2 emerged in Europe and spread to many other regions, including North America in 2016. Prior to this study it was unknown if eastern cottontails (ECT(s); Sylvilagus floridanus), one of the most common wild lagomorphs in the United States, were susceptible to RHDV2. In this study, 10 wild‐caught ECTs and 10 New Zealand white rabbits (NZWR(s); O. cuniculus) were each inoculated orally with either RHDV (RHDVa/GI.1a; n = 5 per species) or RHDV2 (a recombinant GI.1bP‐GI.2; n = 5 per species) and monitored for the development of disease. Three of the five ECTs that were infected with RHDV2 developed disease consistent with RHD and died at 4 and 6 days post‐inoculation (DPI). The RHDV major capsid protein/antigen (VP60) was detected in the livers of three ECTs infected with RHDV2, but none was detected in the ECTs infected with RHDV. Additionally, RHD viral RNA was detected in the liver, spleen, intestine and blood of ECTs infected with RHDV2, but not in the ECTs infected with RHDV. RHD viral RNA was detected in urine, oral swabs and rectal swabs in at least two of five ECTs infected with RHDV2. One ECT inoculated with RHDV2 seroconverted and developed a high antibody titre by the end of the experimental period (21 DPI). ECTs inoculated with the classic RHDV did not seroconvert. In comparison, NZWRs inoculated with RHDV2 exhibited high mortality (five of five) at 2 DPI and four of five NZWRs inoculated with RHDV either died or were euthanized at 2 DPI indicating both of these viruses were highly pathogenic to this species. This experiment indicates that ECTs are susceptible to RHDV2 and can shed viral RNA, thereby suggesting this species could be involved in the epidemiology of this virus. [ABSTRACT FROM AUTHOR]

Rabbit haemorrhagic disease virus type 2 (RHDV2) causes a severe systemic disease with hepatic necrosis. Differently from classic RHDV, which affects only European rabbits (Oryctolagus cuniculus), RHDV2 can affect many leporid species, including hares (Lepus spp.) and cottontail rabbits (Sylvilagus spp.). RHDV2 emerged in Europe in 2010 and spread worldwide. During the last 5 years, there have been multiple outbreaks in North America since the first known event in 2016 in Quebec, Canada, including several detections in British Columbia, Canada, between 2018 and 2019, Washington State and Ohio, USA, in 2018 and 2019, and New York, USA, in 2020. However, the most widespread outbreak commenced in March 2020 in the southwestern USA and Mexico. In California, RHDV2 spread widely across several southern counties between 2020 and 2021, and the aim of this study was to report and characterize these early events of viral incursion and circulation within the state. Domestic and wild lagomorphs (n = 81) collected between August 2020 and February 2021 in California with a suspicion of RHDV2 infection were tested by reverse transcription quantitative real‐time PCR on the liver, and histology and immunohistochemistry for pan‐lagovirus were performed on liver sections. In addition, whole genome sequencing from 12 cases was performed. During this period, 33/81 lagomorphs including 24/59 domestic rabbits (O. cuniculus), 3/16 desert cottontail rabbits (Sylvilagus audubonii), and 6/6 black‐tailed jackrabbits (Lepus californicus) tested positive. All RHDV2‐positive animals had hepatic necrosis typical of pathogenic lagovirus infection, and the antigen was detected in sections from individuals of the three species. The 12 California sequences were closely related (98.9%–99.95%) to each other, and also very similar (99.0%–99.4%) to sequences obtained in other southwestern states during the 2020–2021 outbreak; however, they were less similar to strains obtained in New York in 2020 (96.7%–96.9%) and Quebec in 2016 (92.4%–92.6%), suggesting that those events could be related to different viral incursions. The California sequences were more similar (98.6%–98.7%) to a strain collected in British Columbia in 2018, which suggests that that event could have been related to the 2020 outbreak in the southwestern USA. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Rabbit haemorrhagic disease virus (RHDV) is highly pathogenic to European rabbits. Until recently, only one serotype of RHDV was known, GI.1/RHDV. RHDV2/GI.2 is a novel virus that has rapidly spread and become the dominant pathogenic calicivirus in wild rabbits worldwide. It is speculated that RHDV2 has three competitive advantages over RHDV: (a) the ability to partially overcome immunity to other variants; (b) the ability to clinically infect young rabbits; and (c) a wider host range. These differences would be expected to influence virus transmission dynamics. We used markers of recent infection (IgM/IgA antibodies) to investigate virus transmission dynamics pre and post the arrival of RHDV2. Our data set contained over 3,900 rabbits sampled across a 7-year period at 12 Australian sites. Following the arrival of RHDV2, seasonal peaks in IgM and IgA seropositivity shifted forward one season, from winter to autumn and spring to winter, respectively. Contrary to predictions, we found only weak effects of rabbit age, seropositivity to non-pathogenic calicivirus RCV-A1 and population abundance on IgM/IgA seropositivity. Our results demonstrate that RHDV2 enters rabbit populations shortly after the commencement of annual breeding cycles. Upon entering, the population RHDV2 undergoes extensive replication in young rabbits, causing clinical disease, high virus shedding, mortality and the creation of virus-laden carcasses. This results in high virus contamination in the environment, furthering the transmission of RHDV2 and initiating outbreaks, whilst simultaneously removing the susceptible cohort required for the effective transmission of RHDV. Although RHDV may enter the population at the same time point, it is sub-clinical in young rabbits, causing minimal virus shedding and low environmental contamination. Our results demonstrate a major shift in epidemiological patterns in virus transmission, providing the first evidence that RHDV2's ability to clinically infect young rabbits is a key competitive advantage in the field. [ABSTRACT FROM AUTHOR]

Amongst newly developed approaches to analyse molecular data, phylodynamic models are receiving much attention because of their potential to reveal changes to viral populations over short periods. This knowledge can be very important for understanding disease impacts. However, their accuracy needs to be fully understood, especially in relation to wildlife disease epidemiology, where sampling and prior knowledge may be limited. The release of the rabbit haemorrhagic disease virus (RHDV) as biological control in naïve rabbit populations in Australia in 1996 provides a unique data set with which to validate phylodynamic models. By comparing results obtained from RHDV sequence data with our current understanding of RHDV epidemiology in Australia, we evaluated the performances of these recently developed models. In line with our expectations, coalescent analyses detected a sharp increase in the virus population size in the first few months after release, followed by a more gradual increase. Phylodynamic analyses using a birth-death model generated effective reproductive number estimates (the average number of secondary infections per each infectious case, Re) larger than one for most of the epochs considered. However, the possible range of the initial Re included estimates lower than one despite the known rapid spread of RHDV in Australia. Furthermore, the analyses that accounted for geographical structuring failed to converge. We argue that the difficulties that we encountered most likely stem from the fact that the samples available from 1996 to 2014 were too sparse with respect to both geographic and within outbreak coverage to adequately infer some of the model parameters. In general, while these phylodynamic analyses proved to be greatly informative in some regards, we caution that their interpretation may not be straightforward. We recommend further research to evaluate the robustness of these models to assumption violations and sensitivity to sampling regimes. [ABSTRACT FROM AUTHOR]

The European rabbit (Oryctolagus cuniculus) is one of the most devastating invasive species in Australia. Since the 1950s, myxoma virus (MYXV) and rabbit haemorrhagic disease virus (RHDV) have been used to manage overabundant rabbit populations. Resistance to MYXV was observed within a few years of the release. More recently, resistance to lethal RHDV infection has also been reported, undermining the efficiency of landscape‐scale rabbit control. Previous studies suggest that genetic resistance to lethal RHDV infection may differ locally between populations, yet the mechanisms of genetic resistance remain poorly understood. Here, we used genotyping by sequencing (GBS) data representing a reduced representation of the genome, to investigate Australian rabbit populations. Our aims were to understand the relationship between populations and identify possible genomic signatures of selection for RHDV resistance. One population we investigated had previously been reported to show levels of resistance to lethal RHDV infection. This population was compared to three other populations with lower or no previously reported RHDV resistance. We identified a set of novel candidate genes that could be involved in host–pathogen interactions such as virus binding and infection processes. These genes did not overlap with previous studies on RHDV resistance carried out in different rabbit populations, suggesting that multiple mechanisms are feasible. These findings provide useful insights into the different potential mechanisms of genetic resistance to RHDV virus which will inform future functional studies in this area. [ABSTRACT FROM AUTHOR]

Viruses that affect lagomorphs have decades of reported history of spillover events. One of these viruses is the causative agent of the so‐called rabbit or 'lagomorph' haemorrhagic disease (e.g. Lagovirus europaeus/GI.1 and L. europaeus/GI.2). In particular, L. europaeus/GI.2 has shown a great capacity to recombine with existing lagoviruses. In fact, it has replaced the former GI.1 genotype in the wild, and recently, an increase on spillover events has been detected among several lagomorph species including European and North American species of hares. In this study, we report for the first time the infection of a wild Iberian hare with GI.2 (RHDV2/b), potential shedding and associated histopathological alterations. We identify the recombinant GI.4P‐GI.2 as causative of the infection and discuss plausible causes regarding the origin of the spillover event and its potential consequences for the Iberian hare wild populations, which is an endemic species of the Iberian Peninsula as well as an important game and prey species for many predators, including endangered species. [ABSTRACT FROM AUTHOR]

Rabbit haemorrhagic disease virus 2 (RHDV2) is a newly emerging Lagovirus belonging to the family Caliciviridae. After its first discovery in 2010 in France, this highly pathogenic virus rapidly spread to neighbouring countries and has become the dominant strain, replacing the classical RHDV strains. RHDV2 was first reported in North America in 2016 in Mont‐Joli, Quebec, Canada, and it was reported again in 2018 and 2019 on Vancouver Island and the southwest mainland of British Columbia (BC). The whole genome sequence of the RHDV2 Quebec isolate resembled the 2011 RHDV2‐N11 isolate from Navarra, Spain with 97% identity at the nucleotide level. The epidemiological investigation related to this outbreak involved three hobby farms and one personal residence in Quebec. In February 2018, high mortality was reported in a large colony of feral rabbits on the Vancouver Island University Campus, Nanaimo, BC. The virus identified showed only 93% identity to the Quebec RHDV2 isolate at the nucleotide level. Additional cases of RHDV2 on Vancouver Island and on the BC mainland affecting feral and captive domestic, and commercial rabbits were reported subsequently. Vaccination was recommended to control the outbreak and an inactivated bivalent vaccine was made available to the private veterinary practices. In June 2019, an isolated RHDV2 outbreak was reported in pet rabbits in an apartment building in Vancouver, BC. This virus showed only 97% identity to the RHDV2 isolates responsible for the BC outbreak in 2018 at the nucleotide level, suggesting that it was an independent incursion. The outbreak in BC killed a large number of feral European rabbits; however, there were no confirmed cases of RHD in native rabbit species in BC. [ABSTRACT FROM AUTHOR]

Myxomatosis is an emergent disease in the Iberian hare, having been considered a rabbit disease for decades. Genome sequencing of the strains obtained from Iberian hares with myxomatosis showed these to be distinct from the classical ones that circulated in rabbits since the virus introduction in Europe, in 1952. The main genomic difference in this natural recombinant hare myxoma virus (ha‐MYXV) is the presence of an additional 2.8 kb region disrupting the M009L gene and adding a set of genes homologous to the myxoma virus (MYXV) genes M060R, M061R, M064R, M065R and M066R originated in Poxviruses. After the emergence of this recombinant virus (ha‐MYXV) in hares, in the summer of 2019, the ha‐MYXV was not detected in rabbit surveys, suggesting an apparent species segregation with the MYXV classic strains persistently circulating in rabbits. Recently, a group of six unvaccinated European rabbits (Oryctolagus cuniculus cuniculus) from a backyard rabbitry in South Portugal developed signs of myxomatosis (anorexia, dyspnoea, oedema of eyelids, head, ears, external genitals and anus, and skin myxomas in the base of the ears). Five of them died within 24–48 hr of symptom onset. Molecular analysis revealed that only the recombinant MYXV was present. This is the first documented report of a recombinant hare myxoma virus in farm rabbits associated with high mortality, which increases the concern for the future of both the Iberian hare and wild rabbits and questions the safety of the rabbit industry. This highlights the urgent need to evaluate the efficacy of available vaccines against this new MYXV. [ABSTRACT FROM AUTHOR]

The Lagovirus genus comprises both pathogenic viruses as European brown hare syndrome virus (EBHSV‐ GII.1) and rabbit hemorrhagic disease viruses (RHDV‐GI.1 and RHDV2‐GI.2), that principally infect European brown hares (Lepus europeaus) and European rabbits (Oryctolagus cuniculus), respectively, causing severe necrotic hepatitis, spleen enlargement and disseminated haemorrhage. This genus includes also non‐pathogenic agents, such as rabbit calicivirus (RCV‐E1 – GI.3) and the non‐pathogenic hare Lagovirus, provisionally named hare calicivirus (HaCV – GII.2). The latter had been identified for the first time in 2012 in the gut contents and faeces of healthy young hares raised in a breeding farm. In this study, we further investigated the presence of HaCV by testing the intestinal tract of 621 wild hares collected between 2010 and 2018 in Northern and Central Italy, and in 2011 in Austria, Germany and Spain. These wild hares were found dead for causes other than EBHS or were healthy hares shot during the hunting season. Forty‐three out of 322 hare samples from Italy and 14 out of 299 samples from Austria and Germany were positive for HaCV–GII.2 by RT‐PCR using universal primers for lagoviruses and primers specific for HaCV. Sequence analysis of the full capsid protein gene conducted on 12 strains representative of different years and locations indicated that these viruses belong to the same, single cluster as the prototype strain initially identified at the hares' farm (HaCV_Bs12_1). The relatively high level of genetic variation (88% nt identity) within this cluster suggests HaCVs may have been circulating widely in Europe for some time. [ABSTRACT FROM AUTHOR]

Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp10025-33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN-γ) or the linker at the N-terminus (0.8 ng/ml IFN-γ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN-γ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy. [ABSTRACT FROM AUTHOR]

Autogenous bone graft is gold standard in treating bone defects, but it might have difficulty in corporation and rejection reaction. This study is to compare the effectiveness among freeze-dried xenograft, freeze-dried allograft, hydroxyapatite xenograft, and demineralized bone matrix xenograft as bone graft to fill bone defect in femoral diaphysis of white rabbit. Thirty male New Zealand white rabbits were distributed into five groups. Bone defect was filled correspondingly with xenograft freeze-dried cortical bovine, allograft freeze-dried cortical New Zealand white rabbit, xenograft hydroxyapatite bovine, and xenograft demineralized bone matrix bovine. No graft was used in control group. VEGF, osteoblast, and woven bone were higher in allograft freeze-dried cortical New Zealand white rabbit (mean 5.6625 (p<0.05)) and xenograft demineralized bone matrix bovine (mean 5.2475 (p<0.05)) with calcification of woven bone was already seen in week 2 in the latter group. There was a decrease of woven bone (mean 4.685 (p<0.05)) fibrous tissue (mean 41.07 (p<0.05)) in xenograft demineralized bone matrix bovine. The Immunoglobulin-G was elevated in control and all study groups but not significantly (p=0.07855). Bone healing process in xenograft demineralized bone matrix bovine is more effective than in xenograft hydroxyapatite bovine, allograft freeze-dried New Zealand white rabbit, xenograft freeze-dried cortical bovine, and control. [ABSTRACT FROM AUTHOR]

We describe the extremely rapid worldwide spread of the Lagovirus europaeus/GI.2/RHDV2/b (henceforth GI.2), the causative infectious agent of the so‐called 'novel' rabbit haemorrhagic disease of the European rabbit (Oryctolagus cuniculus). We tracked down all novel confirmed detections of GI.2 between May 2010 and November 2018 by carrying out a two‐step in‐depth review. We suggest that such spread would not have been possible without anthropogenic involvement. Our results also point out the importance of reviewing and adapting the protocols of virus detection and management in order to control, mitigate and contain properly, not only GI.2, but also new viruses that may emerge in the future. [ABSTRACT FROM AUTHOR]

We report the first case of spontaneous syndactyly reported in a pet rabbit. Syndactyly only caused an atypical gait in the rabbit. The radiological study revealed bilateral 3rd and 4th metatarsal bones fused in its entire length preserving normal joint surfaces resembling syndactyly type Ia. The cause of this congenital malformation was unknown. [ABSTRACT FROM AUTHOR]

Summary: This work describes a simple and rapid test for field detection of the emerging rabbit pathogen RHDVb. The assay is specific for RHDVb, showing no cross‐reactivity with other RHDV types giving a specific result in under 10 min using rabbit liquid exudates or liver homogenate samples taken at necropsy. [ABSTRACT FROM AUTHOR]

This study aimed to evaluate the ability of a panel of lectins to inhibit the ability of Pasteurella multocida to adhere to and affect the rabbit respiratory epithelium. Nasal septa from rabbit fetuses were cultured with various lectins before the addition of P. multocida. The percentage of bacteria adhering to the epithelium was evaluated semiquantitatively by indirect immunoperoxidase (IIP) staining. The goblet cells (GCs) were counted in semithin sections stained with toluidine blue and served as the main morphological criterion to evaluate the inhibitory effect of the lectins. The lectins PNA, WGA, RCA120, and DBA significantly inhibited the adhesion of P. multocida to the ciliated epithelium P<0.05 and prevented the pathogen-induced increase in the number of GCs P<0.05 compared with those of positive control tissues. In addition, VVA, SJA, UEA I, DSL, SBA, and ECL significantly inhibited the increase in GCs compared with that of the control tissues. The results suggest that less aggressive therapeutic strategies, such as treatment with lectins, may represent alternative approaches to control bacterial respiratory infections. [ABSTRACT FROM AUTHOR]

A two-and-a-half-year-old giant lop-eared rabbit, weighing 5.1 kg, presented with a one-month history of intermittent right hind limb lameness.The limb locked in extension during hopping. On examination, a grade-2 medial patellar luxation of the right hind was diagnosed, with associated stifle joint swelling. Radiographic findings of the right stifle comprised periarticular osteophyte formation consistent with mild degenerative joint disease and joint effusion. Surgical correction involving right trochlear wedge recession sulcoplasty and lateral imbrication was carried out to stabilise the patella in the trochlear groove. The right hind limb lameness resolved, and the patella was stable at a 6-month postoperative examination. One year postoperatively, the right patella was luxating again concurrent with bilateral stifle effusions. Euthanasiawas performed twentymonths after surgery due to recurrent lameness in the right hind limb. [ABSTRACT FROM AUTHOR]