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Objectives To create awareness of single RHDV2 infections and cases of death despite immunisation with RHDV2-specific vaccine. Materials and methods Retrospective case series of four companion rabbits. Patient signalment, vaccination history, pathology and molecular examinations were obtained from the medical records. Results The animals died peracutely or with nonspecific symptoms like apathy and inappetence. Pathological examination indicated and molecular biological findings confirmed RHDV2 infection in four animals. Several partner animals died at the same time under the same circumstances. Clinical significance This is the first report of fatalities in RHDV2-vaccinated companion rabbits due to rabbit haemorrhagic disease virus 2 infection with documented case and vaccination history. Veterinarians should be aware of possible single fatal RHDV2 infections despite vaccination, should initiate the clarification of suspected cases and inform vaccine manufacturers and competent authorities.

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 naive rabbit populations in Australia in 1996 provides a unique dataset with which to validate phylodynamic models. By comparing the results obtained for RHDV1 with our current understanding of the 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 trajectory in the first few months after the virus release, followed by a more gradual increase. The phylodynamic analyses with a birth-death tree prior 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 RHDV1 in Australia. Furthermore, the analyses that took into account the 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 geographic and within outbreak coverage to adequately infer some of the model parameters. In general, while these Bayesian analyses proved to be greatly informative in some regards, we caution that their interpretation may not be straight forward and recommend further research in evaluating the robustness of these models to assumption violations and sensitivity to sampling regimes.

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.

Viral hemorrhagic disease of rabbits is an acute, highly contagious disease characterized by the phenomena of hemorrhagic diathesis in all organs, especially in the lungs and liver. The causative agent of viral haemorrhagic disease of rabbits is a virus of haemorrhagic disease of rabbits, belonging to the family Caliciviridae, genus Lagovirus. Currently, there are 4 genogroups of lagoviruses, two pathogenic: GI1 (GI1a-GI1d) and GI2, and two non-pathogenic: GI3 and GI4. The greatest danger to rabbits in the Russian Federation is posed by viruses of the GI1 genotype. The virulence of these viruses for rabbits is extremely high, the incubation period is 48-72 hours. Clinically, the disease is almost not manifested. Mortality can reach 100%. For the prevention of HBV in the Russian Federation, inactivated tissue vaccines are used, which are a suspension of the liver of rabbits infected with virulent strains of the rabbit hemorrhagic disease virus. Currently, in veterinary practice, subunit recombinant vaccines based on virus proteins obtained in the baculovirus gene expression system are increasingly used. The authors obtained the recombinant VP60 virus of rabbit hemorrhagic disease of the GI1 genotype in the baculovirus gene expression system and studied its antigenic and immunogenic activity for rabbits. It was found that the recombinant capsid protein VP60 of the hemorrhagic disease virus, administered to rabbits at a dose of 50 pg, causes the synthesis of specific antibodies in animals, detected by enzyme immunoassay and in the hemagglutination inhibition reaction, and protects 80% of animals during control infection with the virulent strain «Voronezh-87» at a dose of 1000 LD 50. These data indicate the possibility of using this protein as a specific component of a subunit vaccine against rabbit hemorrhagic disease caused by strains of the GI1 gene group.

Relevance . Rabbit haemorrhagic disease virus type 2 (RHDV-2, GI2,) was first detected in 2010 in France and quickly spread across еuropean countries. Unlike strains of the first genogroup, RHDV GI2 also affects rabbits under 2 months old, as well as various types of hares. In addition, RHDV GI2 causes the death of rabbits vaccinated with vaccines based on type 1 hemorrhagic disease viruses. The rabbit hemorrhagic disease virus 2 (RHDV-2) was detected during an outbreak of the disease, characterized by the sudden death of rabbits of different age groups, in the summer of 2019 in a private farm in the Tula region, Russia. The purpose of this study was to detect and obtain the molecular genetic characteristics of the pathogen. Methods. The ELISA was used for the detection of RHDV antigen in samples of pathological material from dead animals. The presence of RHDV RNA was determined by the method of reverse transcription/polymerase chain reaction (RT/PCR) using pair of specific primers for the regions of the VP1 and VP60 genes. The Sanger sequencing reaction was performed and obtained nucleotide sequences were analyzed. Results . Antigen and RNA of the rabbit haemorrhagic disease virus were detected by ELISA and RT-PCR in laboratory studies of the liver of dead animals. Analysis of the nucleotide sequence of a 470 base pair region of the genome, containing the 5 ‘terminal region of the VP1 gene and the 3’ terminal region of the VP60 gene, showed that the detected virus belongs to RHDV-2. Phylogenetic analysis revealed that the closest to it were the sequences of RHDV-2 from the USA, France and Poland isolated in the 2016–2020. The VP60 amino acid sequence of detected virus contained the A46T substitution (alanine for threonine at position 46), which can affect the conformation of the capsid protein and, as a consequence, the antigenic structure of the virus.

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.

Objectives To report clinical features, gross post mortem and histopathological findings from an investigation into sudden or unexpected death in rabbits that was undertaken during an outbreak of rabbit haemorrhagic disease. Materials and methods Using a standard protocol, veterinarians were invited to submit case histories and results of their post mortem examination of pet rabbits that died unexpectedly. Histopathological examination of heart, lungs, liver, spleen and kidney samples was collated with macroscopic appearance and clinical details. Results Hepatocellular necrosis, characteristic of rabbit haemorrhagic disease, was observed in 185 of 300 (62%) submissions, often accompanied by glomerular thrombosis and changes in other organs. Evidence of rabbit haemorrhagic disease was not apparent on histopathology in 113 of 300 (38%) rabbits. Gross post mortem examination by veterinary practitioners did not always reflect reported histopathological changes. No macroscopic abnormalities were seen in 78/185 (42%) of rabbit haemorrhagic disease cases. Rapid death and death of other rabbits in the household were common features of rabbit haemorrhagic disease. Ante mortem clinical signs included anorexia, collapse, lethargy, seizures, icterus, bleeding from the mouth, dyspnoea, hypothermia, pyrexia, bradycardia or poor blood clotting. Clinical importance Rabbit haemorrhagic disease can be suspected from a history of sudden death, especially if multiple rabbits are affected. There is not always macroscopic evidence of the disease but histopathology is useful to support or refute a diagnosis of rabbit haemorrhagic disease and provide information about other causes of death.

Multi-strain, host-disease dynamics describe a system where multiple strains of a pathogen compete for susceptible individuals of a single host. The theoretical properties of these systems have been well studied, but there are few empirical studies in wildlife hosts. We examined the impacts of two novel strains of rabbit haemorrhagic disease virus (RHDV) recently introduced into Australia, one inadvertently (RHDV2) and one deliberately for rabbit biocontrol (RHDV-K5), by analysing long-term monitoring data for introduced European rabbits (Oryctolagus cuniculus) from 18 sites throughout Australia. We examined population-level impacts using rabbit spotlight counts pre- and post-arrival of the two strains. We also analysed serological data to determine potential interactions among the introduced and existing field strains of RHDV, as well as a pre-existing benign strain of calicivirus (RCV-A1). Serological analyses suggested that RHDV2 arrived in Australia during spring 2014 and spread rapidly through the Australian rabbit population within two years. Following the establishment of RHDV2, rabbit abundance was reduced by an average of 60%, with impacts most pronounced in southern and western Australia. In contrast, the deliberate release of RHDV-K5 had little impact on rabbit populations. Although RHDV2 has spread rapidly throughout Australia, our serological analyses do not support the observation that RHDV2 is rapidly replacing existing field strains of RHDV, as was previously reported in Australia and Europe. Nevertheless, RHDV2 has negatively impacted the ability of RHDV and RCV-A1 to spread within rabbit populations, most likely due to its ability to infect juvenile rabbits, thereby removing them from the pool of susceptible individuals available to be infected by competing strains. Synthesis and applications. The impact of the release of a novel strain of rabbit haemorrhagic disease virus (RHDV-K5) for rabbit biocontrol in Australia has been suppressed by the emergence of a competing strain, RHDV2. Hence, the success of further releases of similar RHDV strains for rabbit biocontrol appear doubtful. Despite this, RHDV2 has suppressed rabbit abundances by an average of 60%, with impacts most pronounced in southern and western Australia. Whether the incursion of RHDV2 leads to the competitive exclusion of other endemic RHDV strains remains to be resolved. However, the existence of partial cross-immunity could allow some level of coexistence between RHDV2 and RHDV strains, at least in the medium term.

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.

Background Rabbit haemorrhagic disease virus (RHDV) is a Lagovirus, a subgroup of the family Caliciviridae. RHDV2 is a variant first described in France in 2010, and has since spread globally. It has been reported in several Lagomorph species (rabbits, hares, and their relatives) as well as other mammals including voles and shrews. The disease has raised international concerns for its potential impact on population abundance trajectories, particularly as 25% of Lagomorphs are currently Red-Listed by the International Union for the Conservation of Nature (IUCN). The Irish hare (Lepus timidus hibernicus) is a subspecies of the mountain hare, L. timidus, and is endemic to Ireland, making it an Evolutionarily Significant Unit of intrinsic value. Case presentation The first case of RHDV2 was detected in a wild Irish hare in July 2019. The individual exhibited atypical neurological behaviour (running in circles) prior to death. On necropsy, pink tinged foam was seen in the trachea and congestion was noted in the lungs, but there was no evidence of haemorrhages in any other organ. Both the liver and spleen were tested by reverse transcription real time qPCR confirming high levels of RHDV2 RNA. Histopathology confirmed multifocal necrotising hepatitis. Conclusion The Irish hare is susceptible to RHDV2 infection. Further investigation is warranted to explore the clinical, epidemiological, and population biology implications.

The diversity of lagoviruses (Caliciviridae) in Australia has increased considerably in recent years. By the end of 2017, five variants from three viral genotypes were present in populations of Australian rabbits, while prior to 2014 only two variants were known. To understand the evolutionary interactions among these lagovirus variants, we monitored their geographical distribution and relative incidence over time in a continental-scale competition study. Within 3 years of the incursion of rabbit haemorrhagic disease virus 2 (RHDV2, denoted genotype GI.1bP-GI.2 [polymerase genotype]P-[capsid genotype]) into Australia, two novel recombinant lagovirus variants emerged: RHDV2-4e (genotype GI.4eP-GI.2) in New South Wales and RHDV2-4c (genotype GI.4cP-GI.2) in Victoria. Although both novel recombinants contain non-structural genes related to those from benign, rabbit-specific, enterotropic viruses, these variants were recovered from the livers of both rabbits and hares that had died acutely. This suggests that the determinants of host and tissue tropism for lagoviruses are associated with the structural genes, and that tropism is intricately connected with pathogenicity. Phylogenetic analyses demonstrated that the RHDV2-4c recombinant emerged independently on multiple occasions, with five distinct lineages observed. Both the new RHDV2-4e and -4c recombinant variants replaced the previous dominant parental RHDV2 (genotype GI.1bP-GI.2) in their respective geographical areas, despite sharing an identical or near-identical (i.e. single amino acid change) VP60 major capsid protein with the parental virus. This suggests that the observed replacement by these recombinants was not driven by antigenic variation in VP60, implicating the non-structural genes as key drivers of epidemiological fitness. Molecular clock estimates place the RHDV2-4e recombination event in early to mid-2015, while the five RHDV2-4c recombination events occurred from late 2015 through to early 2017. The emergence of at least six viable recombinant variants within a 2-year period highlights the high frequency of these events, detectable only through intensive surveillance, and demonstrates the importance of recombination in lagovirus evolution.

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.

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 urbanised 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. This article is protected by copyright. All rights reserved.

BACKGROUND Fast-spreading diseases affecting wildlife populations threaten biodiversity. Two caliciviruses, Lagovirus europaeus/GI.1 and Lagovirus europaeus/GI.2, caused rabbit haemorrhagic disease virus (RHDV) in wild rabbits. Despite having different characteristics, these variants spread quickly, posing a threat to wild rabbit populations. METHODS In this study, we conducted a thorough review of the scientific literature and reports of international organisations of first detections of both variants of RHDV in the Euro-Mediterranean region. We concentrated on this area to avoid bias due to intentional human introductions. RESULTS The estimated mean spread rate of GI.2 was higher than that of GI.1 (GI.2: 479 km/year, range: 47-7346; GI.1: 330 km/year, 37-6248). These differences were not statistically significant. This lack of difference may be due to the interactions between each variant's virulence characteristics. Humans may have a dominant effect on their spread. Potential limitations associated with the observational process could have hindered our ability to identify statistical differences. CONCLUSIONS The lack of difference in the spread patterns of the two variants could be due to a biological cause, human facilitation or a lack of statistical power. Adapting protocols to detect diseases in wildlife using homogeneous criteria will be indispensable in the coming years.

Rabbit Haemorrhagic Disease (RHD) causes high morbidity and mortality in rabbits and hares. Here, we report the first genomic characterization of Rabbit Hemorrhagic Disease Virus (RHDV) from sub-Saharan Africa. While suspected, only a single PCR finding without sequence confirmation or characterization has been reported. Here, we used a microbial metagenomic approach to confirm and characterize pathogens causing the suspected outbreak of RHD in Ibadan, Nigeria. On the 25th September 2020, the liver, spleen, and lung samples of five rabbits from an outbreak in 2 farms in Ibadan, Nigeria, were analyzed for the vp60 gene of RHDV by RT-PCR. Subsequently, Next Generation Sequencing on 1st of October revealed one full and two partial RHDV2 genomes on both farms. Phylogenetic analysis showed close clustering with RHDV2 lineages from Europe, in particular, 98.6% similarity with RHDV2 in the Netherlands, and 99.1 to 100% identity with RHDV2 in Germany, suggesting potential importation from Europe. The detection of twelve unique mutations in RHDV2 sequences from the Ibadan outbreak compared to other RHDV2 sequences in the same clade suggests significant genetic diversity of the GI.2 strains in Nigeria. This highlights the need to further understand the genetic diversity of Lagoviruses to, inform vaccine development, and for accurate tracking, monitoring, and control of outbreaks in Africa.

Rabbit Haemorrhagic Disease (RHD) is a significant 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 suggest 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, and control, including active case finding, expedient vaccine dissemination, and risk communications. It shows that Singapore, an urbanised city-state, without a significant lagomorph population, could still encounter emerging diseases such as rabbit haemorrhagic disease. Given its social impact on rabbit owners, the National Parks Board Singapore and the private veterinarians worked together to communicate and urge the adoption of biosecurity measures and assuage the concerns of rabbit owners.

African swine fever (ASF) is an acute, highly contagious and deadly viral haemorrhagic disease of domestic pigs caused by African swine fever virus (ASFV). In ASF endemic countries, there are an increasing number of reports on circulating ASFV strains with different levels of virulence causing a broad range of clinical symptoms in susceptible animals. Tanzania, where ASFV is endemic since 2001, recorded several outbreaks including symptomatic and asymptomatic cases between 2015 and 2017. We collected 35 clinical samples from four outbreaks for diagnostic confirmation and sequenced the partial B646L (p72), the full E183L (p54) gene, the central variable region of the B602L gene and the intergenic region between the I73R and I329L genes to characterize molecularly the new ASFV isolates and analyse their relatedness with previously reported Tanzanian and foreign isolates. We detected ASFV in 21 samples, 15 from symptomatic and six from asymptomatic pigs. Phylogenetic analyses based on the partial p72 gene and the complete p54 (E183L) genes revealed that the ASFVs in samples from symptomatic pigs belonged to genotypes II and those in samples from asymptomatic pigs belonged to genotype IX. The CVR profiles of the p72 genotype II and genotype IX isolates differed between each other and from previously published Tanzanian sequences. The sequence analysis of the intergenic region between the I73R and I329L for the 2017 genotype II isolates showed the absence of one GGAATATATA motif in those isolates. This study showed the simultaneous circulation of two different ASFV genotypes with different levels of pathogenicity in Tanzania. Since the existence of sub-clinically infected pigs may contribute to the persistence of the virus, our findings suggest continuous surveillance and characterization of ASFV isolates in disease-endemic regions.

The viral biocontrol agents Myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV1), released in 1950 and 1996 respectively, are the only control tools to have resulted in significant and lasting landscape-scale suppression of rabbit populations in Australia. Multiple conservation benefits and significant economic savings have resulted from the long-term and widespread reductions in rabbit numbers and impacts. In an effort to ‘boost’ rabbit biocontrol, an additional variant of RHDV1 ('K5') was recently released nationwide to counteract the decreasing effectiveness of both RHDV1 and MYXV that results from the evolutionary ‘arms race’ between viruses and their hosts. Two years prior to the K5 release, an exotic RHDV strain (RHDV2) appeared in Australia. The commercially available vaccine used to protect pet and farmed rabbits against the officially released K5 was ineffective against the exotic RHDV2, resulting in numerous deaths of domestic rabbits. This created substantial confusion about which strain was released as a biocontrol tool, as well as renewed concerns amongst pet rabbit owners and rabbit farmers about the use of viruses as lethal rabbit control tools in general. Ongoing effective control of wild rabbits in Australia is absolutely essential to protect the substantial conservation gains made by the long-term suppression of rabbit numbers over the past decades, and there is currently no alternative population control tool to achieve this at the required landscape scale. Vaccine formulations need updating to protect non-target farmed and pet rabbits from circulating field variants, including RHDV2, and to increase public acceptance for the ongoing use of viral biocontrol for feral rabbit populations.

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 was presented to the clinics; 8/11 rabbits died within 48 hours of presentation, while the remaining two rabbits had recovered after prolonged hospitalisation 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.

Áreas de pesquisa: Infectious Diseases ; Veterinary Sciences 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 info:eu-repo/semantics/publishedVersion

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.

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.

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.

The Red fox (Vulpes vulpes) has established large populations in Australia’s urban and rural areas since its introduction following European settlement. The cryptic and highly adaptable nature of foxes allows them to invade cities and live among humans whilst remaining largely unnoticed. Urban living and access to anthropogenic food resources also influence fox ecology. Urban foxes grow larger, live at higher densities, and are more social than their rural counterparts. These ecological changes in urban red foxes are likely to impact the pathogens that they harbour, and foxes could pose a disease risk to humans and other species that share these urban spaces. To investigate this possibility, we used a meta-transcriptomic approach to characterise the virome of urban and rural foxes across the Greater Sydney region in Australia. Urban and rural foxes differed significantly in virome composition, with rural foxes harbouring a greater abundance of viruses compared to their urban counterparts. We identified ten potentially novel vertebrate-associated viruses in both urban and rural foxes, some of which are related to viruses associated with disease in domestic species and humans. These included members of the Astroviridae, Picobirnaviridae, Hepeviridae, and Picornaviridae as well as rabbit haemorrhagic disease virus-2. This study sheds light on the viruses carried by urban and rural foxes and emphasises the need for greater genomic surveillance of foxes and other invasive species at the human–wildlife interface.

Rabbit haemorrhagic disease (RHD) is an acute fatal disease caused by the Lagovirus rabbit haemorrhagic disease virus (RHDV), which was first reported in 1984 in China. Strains of two different genotypes (GI.1a and GI.1c) have been detected in China to date. In 2010, a new RHDV variant with a unique genetic and antigenic profile was identified in France, designated RHDV2, which rapidly spread throughout continental Europe and nearby islands. Here, we report the first outbreak of RHD induced by RHDV2 (GI.2) in rabbit farms in the Sichuan province of China. We conducted haemagglutination tests and phylogenetic analysis of the new RHDV isolate SC2020/04, which was identified as a non‐haemagglutinating strain belonging to the RHDV2 (GI.2) genogroup. Considering the serious risk of RHDV2 to the Chinese rabbit industry, the circulation of RHDV2 in the population should be carefully monitored in China., The first outbreak of rabbit hemorrhagic disease induced by rabbit hemorrhagic disease virus 2 (GI.2) in China.

The Red fox (Vulpes vulpes) has established large populations in Australia’s urban and rural areas since its introduction following European settlement. Foxes’ cryptic and highly adaptable nature allows them to invade cities and live among humans while remaining largely unnoticed. Urban living and access to anthropogenic food resources also influences fox ecology. Urban foxes grow larger, live at higher densities and are more social than their rural counterparts. These ecological changes in urban red foxes are likely to impact the pathogens that they harbour, and foxes could pose a disease risk to humans and other species that share these urban spaces. To assess this possibility, we used a meta-transcriptomic approach to characterise the viromes of urban and rural foxes across the Greater Sydney region in Australia. Urban and rural foxes differed significantly in virome composition, with rural foxes harbouring a greater abundance of viruses compared to their urban counterparts. In contrast, urban fox viromes comprised a greater diversity of viruses compared to rural foxes. We identified nine potentially novel vertebrate-associated viruses in both urban and rural foxes, some of which are related to viruses associated with disease in domestic species and humans. These included members of theAstroviridae, Picobirnaviridae, HepeviridaeandPicornaviridaeas well as rabbit haemorrhagic disease virus-2 (RHDV2). This study sheds light on the viruses carried by urban and rural foxes and emphasises the need for greater genomic surveillance of foxes and other invasive species at the human-wildlife interface.ImportanceUrbanisation of wild environments is increasing as human populations continue to expand. Remnant pockets of natural environments and other green spaces in urban landscapes provide invasive wildlife such as red foxes with refuges within urban areas, where they thrive on the food resources provisioned by humans. Close contact between humans, domestic species and foxes likely increases the risk of novel pathogen emergence. Indeed, the vast majority of emerging infectious diseases in humans originate in wild animals. Here, we explored potential differences in viromes between urban fox invaders and their rural counterparts. Viromes of foxes and their ectoparasites comprise a diversity of viruses including those from theAstroviridae, Picobirnaviridae, Hepeviridae, CaliciviridaeandPicornaviridae. Microbial surveillance in foxes and other urban wildlife is vital for monitoring viral emergence and for the prevention of infectious diseases.

Lagovirus europaeus GI.1 (RHDV-rabbit haemorrhagic disease virus) and GI.2 (RHDV2-rabbit haemorrhagic disease virus 2), family Caliciviridae, genus Lagovirus, are etiological factors of the rabbit haemorrhagic disease (RHD). This small RNA virus is a great model for tracking the variability and evolution of RNA viruses, because it uses an RNA-dependent RNA polymerase (RdRp) to replicate its own genetic material. This polymerase determines the fidelity and the rates of replication and mutation of the virus, conditioning its adaptation to the environment and even to a new host, and thus influencing evolution of the virus. The aim of this study was to determine the genetic variability and phylogenetic relationships of 105 Lagovirus europaeus strains with different genotypes based on the RdRp gene. The strains came from around the world in the years of 1987–2017. The aforementioned group of 105 strains included 14 strains whose RdRp sequences were obtained and analysed in this study, and the rest were retrieved from GenBank: 74 strains classified as genotype GI.1 (RHDV), 14 as GI.2 (RHDV2), 2 strains of Lagovirus europaeus not assigned to any genotype, and a MRCV strain, the sequences of which were collected from GenBank. Among the 14 strains whose RdRp sequences were obtained in this study, the highest variability was presented in the Austrian 237 strain from 2004. The genetic distance between the Austrian 237 strain and the remaining thirteen analysed strains ranged from 0.117 to 0.123 (from 11.7% to 12.3% nucleotide substitutions). The lowest variability, however, was recorded for Hungarian, Czech and Austrian strains. On the phylogenetic tree, the 14 analysed strains were allocated into GI.1c (G2), GI.1d (G3-G5) and GI.1a (RHDVa). Analysis of the genetic variability of the 105 strains of Lagovirus europaeus indicated a growing genetic distance between the strains, both in time and location. Phylogenetic analysis showed a division of the strains into seven groups, dictated by the chronology, geographical location and evolutionary events in the history of the virus, such as mutations and recombinations.

The introduced European rabbit (Oryctolagus cuniculus) is one of Australia’s most damaging invasive alien species, both in terms of ecological and economic impact. Biological control of rabbits using the myxoma and rabbit haemorrhagic disease viruses has been undertaken in Australia since the mid-1950s, and locally varying genetic resistance to these biocontrol viruses has been reported. The efficacy of biocontrol agents may be influenced, among several factors, by the genetic background of rabbit populations. Therefore, understanding the invasion process of rabbits in Australia, and their resultant population structure, remains crucial for enhancing future rabbit management strategies. Using reduced-representation sequencing techniques we genotyped 18 Australian rabbit populations at 7617 SNP loci and show that Australia’s invasive rabbits form three broad geographic clusters representing different ancestral lineages, along with a number of highly localised, strongly differentiated lineages. This molecular data supports a history of multiple independent rabbit introductions across the continent followed by regional dispersal, and the resulting patchwork genetic structure may contribute to variation across the country in rabbit resistance to the viral biocontrols. Our study highlights the importance of using genome-wide molecular information to better understand the historical establishment process of invasive species as this may ultimately influence genetic variabilty, disease resistance and the efficacy of biocontrol agents.

Rabbit haemorrhagic disease (RHD) is a viral disease that affects the European rabbit. RHD was detected in 1984 in China and rapidly disseminated worldwide causing a severe decline in wild rabbit populations. The aetiological agent, rabbit haemorrhagic disease virus (RHDV), is an RNA virus of the family Caliciviridae, genus Lagovirus. Pathogenic (G1-G6 or variants GI.1a-GI.1d) and non-pathogenic strains (GI.4) have been characterized. In 2010, a new variant of RHDV, RHDV2/RHDVb/GI.2, was detected in France. GI.2 arrived to the Iberian Peninsula in 2011, and several recombination events were reported. Here, we sequenced full genomes of 19 samples collected in Portugal between 2014 and 2016. New GI.2 recombinant strains were detected, including triple recombinants. These recombinants possess a non-structural protein p16 related to a non-pathogenic strain. Evolutionary analyses were conducted on GI.2 VP60 sequences. Estimated time to the most recent common ancestor (tMRCA) suggests an emergence of GI.2 in July 2008, not distant from its first detection in 2010. This is the first study on GI.2 evolution and highlights the need of continued monitoring and characterization of complete genome sequences when studying lagoviruses' evolution.

Rabbit haemorrhagic disease (RHD) is a highly infectious, often fatal, disease of rabbits and it is commonly found throughout the UK. RHD is caused by rabbit haemorrhagic disease virus (RHDV), also known as rabbit calicivirus (RCV). Since 2010, a new virus variant (RHDV2/RHDVb) emerged in Europe, and was identified in the UK in 2014; this new variant has now replaced the original virus in many countries. This article discusses the diagnosis and prevention of RHDV2 and the resources, such as diagnostic laboratories and vaccines, currently available in the UK.

Robinia pseudoacacia flower, a common component in traditional Chinese medicine, has long been well-known for its high pharmaceutical value. This study aimed to assess the immunopotentiating effects of Taishan Robinia Pseudoacacia polysaccharides (TRPPS) in rabbits inoculated with a rabbit haemorrhagic disease virus (RHDV) inactivated vaccine. The rabbits were administered with the RHDV vaccine in conjunction with varying concentrations of TRPPS, and their blood samples were collected at different time points to analyze the ratio and number of blood lymphocytes. In addition, sera were prepared and analyzed to determine the overall antibody titer and the level of IL-2, a cytokine commonly used as an indicator of immune activity. The various TRPPS-supplemented vaccines were shown to be more effective in enhancing the immune functions of the inoculated rabbits compared to their polysaccharide-free counterpart, with 200 mg/mL of TRPPS exhibiting the most pronounced benefits that were comparable to those of propolis. In addition, the TRPPS-supplemented RHDV inactivated vaccines could significantly improve the survival rates of the immunized rabbits against RHDV infection. Our studies offered convincing experimental evidence for the development of TRPPS as a new type of plant-derived immunopotentiator.

Rabbit haemorrhagic disease virus (RHDV) induces a highly contagious and extremely lethal disease that fulfils many requirements of an animal model of fulminant hepatic failure (FHF); however, the pathogenesis of RHD has still not been fully elucidated. Cytokines play an important role in regulation of the immune response and pathogenesis of many diseases, including those caused by viral infections. Furthermore, recent studies indicate a role of the immune response, especially peripheral blood leukocytes (PBL), in the pathogenesis of RHD. Thus, in the present study we investigated the expression of IL-1Ra, IL-6, IL-8, IL-18, TNF-α and IFN-γ genes in PBL of RHDV-infected rabbits. We also compared the expression of genes encoding these cytokines in rabbits with different course of RHDV infection (in animals that died 36 h post infection or survived even over 60 h after infection). The study revealed increased expression of genes encoding pro-inflammatory cytokines: IL-6, IL-8, TNF-α, IFN-γ in PBL of RHDV-infected rabbits. Moreover, the level of cytokine gene expression depended on the course of RHD. Hence, the results obtained indicate the potential role of these cytokines in RHDV infection and their influence on the survival time of infected rabbits.

Rabbit haemorrhagic disease (RHD) is caused by a lagovirus affecting European rabbits (Oryctolagus cuniculus). Viral RNA is detected in tissues or faeces of convalescent rabbits, suggesting persistent infections; however, this RNA has not been shown to be related to infective viruses to date. In the present work, seven laboratory rabbits were challenged with the RHDV2/b virus variant. Viral RNA was individually detected by duplex qPCR in faeces collected for four weeks after infection, and the infective capacity of viral RNA excreted in the faeces of surviving rabbits was tested by challenging new rabbits with faecal inocula. As results, viral RNA was detected in faeces until the end of the assay. Viral RNA detected in the fourth week was infective only in the case of one rabbit that did not exhibit clear seroconversion, suggesting persistent infection as a result of an impaired immune response. Since the surviving rabbits were apparently healthy individuals, the importance of detecting carriers and the correct management of faeces to control RHD outbreaks in rabbitries are highlighted.

Proponents of the idea that dingoes protect mid-sized native mammals by suppressing introduced foxes and feral cats often use observations made on either side of Australia's dingo fence for support. However, many mid-sized mammals disappeared around 1900 when dingoes had increased, cats were present but foxes were yet to arrive, and before newly built fences to manage rabbits and dingoes were amalgamated to form the current dingo barrier fence. By concentrating on predation to explain small mammal losses, alternatives including the spread of introduced rabbits are ignored. This is despite rabbits being both severe environmental pests and the main prey of all three predators. Since rabbit haemorrhagic disease was introduced 20 years ago, rabbits have been less abundant and in that time several native mammals deemed to be protected by dingoes have expanded their range, even into dingo-free areas where cats and foxes are not suppressed. Historic and recent evidence therefore weakens the case that dingoes are of paramount importance in protecting small native mammals. Changes in dingo management, ostensibly to protect endangered native fauna, would be premature until all interacting factors are critically considered. A competitive predator model should not be applied without considering other equally plausible explanations.

Deciphering the genes involved in disease resistance is essential if we are to understand host-pathogen coevolutionary processes. The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus). During the first outbreaks of the disease, RHDV caused mortality rates of up to 97%. Recently, however, increased genetic resistance to RHDV has been reported. Here, we have aimed to identify genomic differences between rabbits that survived a natural infection with RHDV and those that died in the field using a genomewide next-generation sequencing (NGS) approach. We detected 72 SNPs corresponding to 133 genes associated with survival of a RHD infection. Most of the identified genes have known functions in virus infections and replication, immune responses or apoptosis, or have previously been found to be regulated during RHD. Some of the genes identified in experimental studies, however, did not seem to play a role under natural selection regimes, highlighting the importance of field studies to complement the genomic background of wildlife diseases. Our study provides a set of candidate markers as a tool for the future scanning of wild rabbits for their resistance to RHDV. This is important both for wild rabbit populations in southern Europe where RHD is regarded as a serious problem decimating the prey of endangered predator species and for assessing the success of currently planned RHDV variant biocontrol releases in Australia.

The rabbit haemorrhagic disease virus (RHDV) was imported into Australia in 1995 as a biocontrol agent to manage one of the most successful and devastating invasive species, the European rabbit (Oryctolagus cuniculus cuniculus). During the first disease outbreaks, RHDV caused mortality rates of up to 97% and reduced Australian rabbit numbers to very low levels. However, recently increased genetic resistance to RHDV and strong population growth has been reported. Major histocompatibility complex (MHC) class I immune genes are important for immune responses against viruses, and a high MHC variability is thought to be crucial in adaptive processes under pathogen-driven selection. We asked whether strong population bottlenecks and presumed genetic drift would have led to low MHC variability in wild Australian rabbits, and if the retained MHC variability was enough to explain the increased resistance against RHD. Despite the past bottlenecks we found a relatively high number of MHC class I sequences distributed over 2–4 loci. We identified positive selection on putative antigen-binding sites of the MHC. We detected evidence for RHDV-driven selection as one MHC supertype was negatively associated with RHD survival, fitting expectations of frequency-dependent selection. Gene duplication and pathogen-driven selection are possible (and likely) mechanisms that maintained the adaptive potential of MHC genes in Australian rabbits. Our findings not only contribute to a better understanding of the evolution of invasive species, they are also important in the light of planned future rabbit biocontrol in Australia., This study was funded by the Priority Programme of the German Science Foundation (DFG) ‘Host-parasite co-evolution—rapid reciprocal adaptation and its genetic basis’ (SPP 1399, PI: So 428/7-1).

Until the beginning of this decade, the genetic characterization of rabbit haemorrhagic disease virus (RHDV) from Iberian Peninsula had revealed the existence of two genogroups, G1 and sporadically G6. In 2010, the new emerging rabbit haemorrhagic disease variant, RHDV2 or RHDVb, was described in France, from where it has rapidly spread throughout Europe, including Iberian Peninsula countries. Nevertheless, although cases of rabbit haemorrhagic disease (RHD) have been reported in the Canary Islands, a Spanish archipelago located 100km off the coast of Morocco, no genetic characterization of RHDV had been carried out. Consequently, in order to identify the circulating RHDV strains in this archipelago, liver samples of six farm rabbits and fifteen wild rabbits were collected from several areas of the largest island, Tenerife, and analyzed for the presence of RHDV by antigen capture double antibody sandwich ELISA. In case of positive ELISA result, we amplified and sequenced two fragments of the vp60 gene, which were concatenated for phylogenetic purposes. The sequences analysis revealed the presence of RHDV2 in both farm and wild rabbits from several areas of Tenerife. This result constitutes the first finding of RHDV2 in the Canary Islands. These RHDV2 strains found in Tenerife shared two exclusive SNPs that have not been observed in the rest of RHDV2 strains. The identification of RHDV2 and the absence of classic RHDV strains in this study suggest that RHDV2 may be replacing classic strains in Tenerife, as has been also proposed in Iberian Peninsula, France and Azores. Given the proximity of the Canary Islands to the African continent, this result should raise awareness about a possible dispersal of RHDV2 from the Canary Islands to the North of Africa.

The lagovirus rabbit haemorrhagic disease virus (RHDV) has been circulating in Australia since the mid-1990s when it was released to control overabundant rabbit populations. In recent years, the viral diversity of different RHDVs in Australia has increased, and currently four different types of RHDV are known to be circulating. To allow for ongoing epidemiological studies and impact assessments of these viruses on Australian wild rabbit populations, it is essential that serological tools are updated. To this end, reference sera were produced against all four virulent RHDVs (RHDV, RHDV2 and two different strains of RHDVa) known to be present in Australia and tested in a series of available immunological assays originally developed for the prototype RHDV, to assess patterns of cross-reactivity and the usefulness of these assays to detect lagovirus antibodies, either in a generic or specific manner. Enzyme-linked immunosorbent assays (ELISAs) developed to detect antibody isotypes IgM, IgA and IgG were sufficiently cross-reactive to detect antibodies raised against all four virulent lagoviruses. For the more specific detection of antibodies to the antigenically more different RHDV2, a competition ELISA was adapted using RHDV2-specific monoclonal antibodies in combination with Australian viral antigen. Archival serum banks from a long-term rabbit monitoring site where rabbits were sampled quarterly over a period of 6 years were re-screened using this assay and revealed serological evidence for the arrival of RHDV2 in this population at least 5 months prior to its initial detection in Australia in a dead rabbit in May 2015. The serological methods and reference reagents described here will provide valuable tools to study presence, prevalence and impact of RHDV2 on Australian rabbit populations; however, the discrimination of different antigenic variants of RHDVs as well as mixed infections at the serological level remains challenging.

Viral haemorrhagic disease (VHD) and colibacillosis are common diseases in rabbits that cause economic losses worldwide. The effect of colibacillosis on the immune response of vaccinated rabbits against rabbit haemorrhagic disease virus (RHDV) was studied. Four groups (G1-G4) were included. G1 was the negative control group; G2 was the RHDV vaccine group; G3 was the E. coli-infected group; and G4 was the E. coli-infected + RHDV vaccine group. The E. coli infection and RHDV vaccination were simultaneously performed, with another previous infection, 3 days before vaccination. At 28 days post-vaccination (PV), the rabbits (G2-G4) were challenged intramuscularly with 0.5 ml of RHDV at a dose of 103 50% median lethal dose (LD50)/rabbit. The rabbits were observed for clinical signs, body weight gain and mortality rates. Tissue, blood, serum, and faecal samples and rectal swabs were collected at 3, 5, 7, 14, 21 and 28 days PV. Significant clinical signs and mortality and a decrease in BW were observed in the infected + RHDV vaccine group. On the 3rd day post-infection (PI), compared with all the other groups, the vaccinated group (G2) had significantly upregulated hepatic tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels; however, the infected + RHDV vaccine group had significantly higher intestinal levels of TNF-α and IL-6 than the other groups. Furthermore, E. coli infection in vaccinated rabbits led to immunosuppression, as shown by significant decreases (P

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.

BackgroundThe Tasmanian devil is an endangered carnivorous marsupial threatened by devil facial tumour disease (DFTD). While research on DFTD has been extensive, little is known about the viruses present in devils, and whether any of these are of potential conservation relevance for this endangered species.MethodsUsing both metagenomics based on virus-like particle (VLP) enrichment and sequence-independent amplification (VLP metagenomics), and meta-transcriptomics based on bulk RNA sequencing, we characterised and compared the faecal viromes of captive and wild Tasmanian devils.ResultsA total of 54 devil faecal samples collected from captive (n = 2) and wild (n = 4) populations were processed for virome characterisation using both approaches. We detected many novel, highly divergent viruses, including vertebrate viruses, bacteriophage and other dietary associated plant and insect viruses. In total, 18 new vertebrate viruses, including novel sapelovirus, astroviruses, bocaviruses, papillomaviruses and gammaherpesvirus were identified, as well as known mammalian pathogens including rabbit haemorrhagic disease virus 2 (RHDV2). Captive devils showed significantly lower levels of viral diversity than wild devils. Comparison of the two methodological approaches revealed substantial differences in the number and types of viruses detected, with meta-transcriptomics mainly identifying RNA viruses, and VLP metagenomics largely identifying DNA viruses.ConclusionThis study has greatly expanded our knowledge of eukaryotic viruses in the Tasmanian devil and provides important baseline information that will contribute to the conservation and captive management of this endangered species. In addition, our results showed that a combination of VLP metagenomics and meta-transcriptomics may be a more comprehensive approach to virome characterisation than either method alone.

Copyright: © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/)., Rabbit haemorrhagic disease (RHD) is a highly contagious infectious disease of European wild and domestic rabbits. Rabbit haemorrhagic disease virus (RHDV, GI.1) emerged in 1986 in Europe, rapidly spreading all over the world. Several genotypes of RHDV have been recognised over time, but in 2010, a new virus (RHDV2/RHDVb, GI.2) emerged and progressively replaced the previous RHDV strains, due to the lack of cross-immunity conferred between RHDV and RHDV2. RHDV2 has a high mutation rate, similarly to the other calivirus and recombines with strains of RHDV and non-pathogenic calicivirus (GI.4), ensuring the continuous emergence of new field strains. Although this poses a threat to the already endangered European rabbit species, the available vaccines against RHDV2 and the compliance of biosafety measures seem to be controlling the infection in the rabbit industry Pet rabbits, especially when kept indoor, are considered at lower risk of infections, although RHDV2 and myxoma virus (MYXV) constitute a permanent threat due to transmission via insects. Vaccination against these viruses is therefore recommended every 6 months (myxomatosis) or annually (rabbit haemorrhagic disease). The combined immunization for myxomatosis and RHDV through a commercially available bivalent vaccine with RHDV antigen has been extensively used (Nobivac® Myxo-RHD, MSD, Kenilworth, NJ, USA). This vaccine however does not confer proper protection against the RHDV2, thus the need for a rabbit clinical vaccination protocol update. Here we report a clinical case of hepatitis and alteration of coagulation in a pet rabbit that had been vaccinated with the commercially available bivalent vaccine against RHDV and tested positive to RHDV2 after death. The animal developed a prolonged and atypical disease, compatible with RHD. The virus was identified to be an RHDV2 recombinant strain, with the structural backbone of RHDV2 (GI.2) and the non-structural genes of non-pathogenic-A1 strains (RCV-A1, GI.4). Although confirmation of the etiological agent was only made after death, the clinical signs and analytic data were very suggestive of RHD., Most of the field and laboratory work referred to in this manuscript was supported by the FCT—Fundação para a Ciência e Tecnologia IP, Grant SFRH/BD/137067/2018, Grant UIDB/00276/2020. (and Project Fight-two: PTDC/CVT-CVT/29062/2017-PT2020), by Fundo Florestal Permanente (Government of Portugal) in the scope of the Action Plan for the Control of Rabbit Viral Haemorrhagic Disease (+COELHO, Dispatch no. 4757/2017 of 31 May, ref no. 2017014300001) and by Interdisciplinary Research Centre on Animal Health, Faculty of Veterinary Medicine, University of Lisbon (CIISA, FMV-UL) (Portugal). Funding bodies played no direct role in the design or conclusion of the study.

The arrival of a new variant of rabbit haemorrhagic disease virus, known as RHDV2, has recently taken place in the native range of the European rabbit (Oryctolagus cuniculus), a keystone species which has undergone a sharp decline over the last sixty years as a consequence of certain harmful factors. Several works have noted the presence of this new variant in wild rabbit populations, and have in some cases recorded high mortality rates. However, little is known about the response to the arrival of this new virus variant at the population level. The goal of this work is therefore to show recent trends in 26 wild rabbit populations between 2010 (before the outbreak of the disease) and 2014 (after its onset) in two different ecosystems (woodland and agricultural areas), in order to test how their abundances changed over this period, which coincided with the spread of the RHDV2. Overall, our results showed that rabbit abundance was much lower in 2014 than in 2010, and that only 11.5% of the populations monitored proved to have a positive trend, that is, a higher abundance in 2014 than 2010. A positive correlation between rabbit abundance in 2010 and rabbit population trends was obtained, thus suggesting that the impact of the new variant on rabbit abundance is less evident in high density populations. Our results suggest that smaller rabbit populations are those most vulnerable to the outbreak of RHDV 2 and are therefore likely to decline sharply or even become extinct.

Rabbit haemorrhagic disease (RHD) is a highly morbid and mortal viral infection of European rabbits. This disease is one of the main causes of death in wild rabbits, and results in large economic losses in farms of rabbits worldwide. Although the first outbreak of this disease was noted in 1984, the pathogenesis of RHD and mechanisms of RHDV (rabbit haemorrhagic disease virus) pathogenecity have still not been fully elucidated. Recent studies indicate a role of the immune response, especially peripheral blood leukocytes (PBL), in the pathogenesis of this disease. Thus, in the present study we investigated the expression of IL-1β, IL-2, IL-10, TNF-β and GM-CSF genes in PBL of RHDV-infected rabbits. We also compared the expression of genes encoding these cytokines in rabbits with different course of RHDV infection (in animals that died 36h postinfection or survived until 60th h after infection). The study revealed that three (IL-10, TNF-β and GM-CSF) out of five investigated genes encoding cytokines showed increased expression in PBL of RHDV-infected rabbits, and the level of expression depended on the course of RHD. The results indicate the potential role of these cytokines in RHDV infection and their influence on the survival time of infected rabbits.