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Rabbit hemorrhagic disease virus 2/genotype GI.2 (RHDV2/GI.2; Caliciviridae , Lagovirus ) causes a highly contagious disease with hepatic necrosis and disseminated intravascular coagulation in several Leporidae species. RHDV2 was first detected in European rabbits ( Oryctolagus cuniculus ) in France in 2010 and has since spread widely. We gather here data on viral detections reported in various countries and affected species, and discuss pathology, genetic differences, and novel diagnostic aspects. RHDV2 has been detected almost globally, with cases reported in Europe, Africa, Oceania, Asia, and North America as of 2023. Since 2020, large scale outbreaks have occurred in the United States and Mexico and, at the same time, cases have been reported for the first time in previously unaffected countries, such as China, Japan, Singapore, and South Africa, among others. Detections have been notified in domestic and wild European rabbits, hares and jackrabbits ( Lepus spp.), several species of cottontail and brush rabbits ( Sylvilagus spp.), pygmy rabbits ( Brachylagus idahoensis ), and red rock rabbits ( Pronolagus spp.). RHDV2 has also been detected in a few non-lagomorph species. Detection of RHDV2 causing RHD in Sylvilagus spp. and Leporidae species other than those in the genera Oryctolagus and Lepus is very novel. The global spread of this fast-evolving RNA virus into previously unexploited geographic areas increases the likelihood of host range expansion as new species are exposed; animals may also be infected by nonpathogenic caliciviruses that are disseminated by almost all species, and with which genetic recombination may occur., Competing Interests: Declaration of conflicting interestsThe authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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

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

Rabbit hemorrhagic disease virus 2 (RHDV2) causes an often-fatal disease of rabbits that has resulted in outbreaks in rabbitries in Europe, Africa, Australia, and Asia. RHD has historically been characterized as a foreign animal disease in the United States. In July 2019, RHDV2 was detected in rabbits on Orcas Island along the northwestern coast of Washington (WA) State following reports of deaths in multiple feral and domestic rabbits. We document and highlight here the unique clinical presentation and gross and histologic lesions observed in this recent WA outbreak. Affected rabbits died without premonitory signs or displayed hyporexia and/or lethargy for ≤1 d prior to death. The most consistent pathologic finding was random, multifocal hepatocellular necrosis, often with concurrent multifocal-to-diffuse splenic necrosis. The lack of significant clinical signs in conjunction with the random distribution of hepatic necrosis in the WA outbreak contrasts with previous reports of RHDV2 disease progression.

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., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The aim of this study was the molecular epidemiology of independently introduced RHDV2 strains in Poland. The nucleotide sequences of RHDV2 diagnosed in domestic rabbits in 2018 in the voivodeships of Swietokrzyskie (strain PIN), Malopolskie (strain LIB) and Mazowieckie (strain WAK), and RHDVa from 2015 (strain F77-3) recognized in wild rabbits in Kujawsko-Pomorskie voivodeship were compared to the genome sequences of the first native RHDV2 strains from 2016-2017. The reference sequences available in public databases, the representative for a classical RHDV (G1-G5 genogroups), RHDVa (G6), non-pathogenic caliciviruses (RCV, GI.3 and GI.4) as well as original and recombinant RHDV2 isolates were included for this analysis. Nucleotide sequence similarity among the most distanced RHDV2 strains isolated in Poland in 2018 was from 92.3% to 98.2% in the genome sequence encoding ORF1, ORF2 and 3'UTR, between 94.8-98.7% in the VP60 gene and between 91.3-98.1% in non-structural proteins (NSP) region. The diversity between three RHDV2 and RHDVa from 2015 was up to 16.3% in the VP60 region. Similarities are shown for the VP60 tree within the RHDV2 group, however, the nucleotide analysis of NSP region revealed the differences between older and new native RHDV2 strains. The Polish RHDV2 isolates from 2016-2017 clustered together with RHDV G1/RHDV2 recombinants, first identified in the Iberian Peninsula in 2012, while all strains from 2018 are close to the original RHDV2. The F77-3 strain clustered to well supported RHDVa (G6) genetic group, together with other Polish and European RHDVa isolates. Based on the results of phylogenetic characterization of RHDV2 strains detected in Poland between 2016-2018 and the chronology of their emergence it can be concluded that RHDV2 strains of 2018 and RHDV2 strains of 2016-2017 were introduced independently thus confirming their different origin and simultaneous pathway of spreading.

Rabbit hemorrhagic disease virus (RHDV) is the causative agent of rabbit hemorrhagic disease (RHD), and its infection results in mortality of 70-90% in farmed and wild rabbits. RHDV is thought to replicate strictly in rabbits. However, there are also reports showing that gene segments from the RHDV genome or antibodies against RHDV have been detected in other animals. Here, we report the detection and isolation of a RHDV from diseased Alpine musk deer ( Moschus sifanicus ). The clinical manifestations in those deer were sudden death without clinical signs and hemorrhage in the internal organs. To identify the potential causative agents of the disease, we used sequence independent single primer amplification (SISPA) to detect gene segments from viruses in the tissue samples collected from the dead deer. From the obtained sequences, we identified some gene fragments showing very high nucleotide sequence similarity with RHDV genome. Furthermore, we identified caliciviral particles using an electron microscope in the samples. The new virus was designated as RHDV GS/YZ. We then designed primers based on the genome sequence of an RHDV strain CD/China to amplify and sequence the whole genome of the virus. The genome of the virus was determined to be 7437 nucleotides in length, sharing the highest genome sequence identity of 98.7% with a Chinese rabbit strain HB. The virus was assigned to the G2 genotype of RHDVs according to the phylogenetic analyses based on both the full-length genome and VP60 gene sequences. Animal experiments showed that GS/YZ infection in rabbits resulted in the macroscopic and microscopic lesions similar to that caused by the other RHDVs. This is the first report of RHDV isolated from Alpine musk deer, and our findings extended the epidemiology and host range of RHDV.

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

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

Ectoparasites play an important role in virus transmission among vertebrates. Little, however, is known about the nature of those viruses that pass between invertebrates and vertebrates. In Australia, flies and fleas support the mechanical transmission of two viral biological controls against wild rabbits-rabbit hemorrhagic disease virus (RHDV) and myxoma virus. We compared virome compositions in rabbits and these ectoparasites, sequencing total RNA from multiple tissues and gut contents of wild rabbits, fleas collected from these rabbits, and flies trapped sympatrically. Meta-transcriptomic analyses identified 50 novel viruses from multiple RNA virus families. Rabbits and their ectoparasites were characterized by markedly different viromes, with virus abundance greatest in flies. Although viral contigs from six virus families/groups were found in both rabbits and ectoparasites, they clustered in distinct host-dependent lineages. A novel calicivirus and a picornavirus detected in rabbit cecal content were vertebrate specific; the newly detected calicivirus was distinct from known rabbit caliciviruses, while the picornavirus clustered with sapeloviruses. Several picobirnaviruses were also identified that fell in diverse phylogenetic positions, compatible with the idea that they are associated with bacteria. Further comparative analysis revealed that the remaining viruses found in rabbits, and all those from ectoparasites, were likely associated with invertebrates, plants, and coinfecting endosymbionts. While no full genomes of vertebrate-associated viruses were detected in ectoparasites, small numbers of reads from rabbit astrovirus, RHDV, and other lagoviruses were present in flies. This supports a role for flies in the mechanical transmission of RHDV, while their involvement in astrovirus transmission merits additional exploration. IMPORTANCE Ectoparasites play an important role in the transmission of many vertebrate-infecting viruses, including Zika and dengue viruses. Although it is becoming increasingly clear that invertebrate species harbor substantial virus diversity, it is unclear how many of the viruses carried by invertebrates have the potential to infect vertebrate species. We used the European rabbit ( Oryctolagus cuniculus ) as a model species to compare virome compositions in a vertebrate host and known associated ectoparasite mechanical vectors, in this case, fleas and blowflies. In particular, we aimed to infer the extent of viral transfer between these distinct types of host. Our analysis revealed that despite extensive viral diversity in both rabbits and associated ectoparasites, and the close interaction of these vertebrate and invertebrate species, biological viral transmission from ectoparasites to vertebrate species is rare. We did, however, find evidence to support the idea of a role of blowflies in transmitting viruses without active replication in the insect., (Copyright © 2020 Mahar et al.)

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

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

Rabbit haemorrhagic disease virus (RHDV) type 2 (GI.2/RHDV2/b) is an emerging pathogen in wild rabbits and in domestic rabbits vaccinated against RHDV (GI.1). Here we report the genome sequence of a contemporary RHDV2 isolate from the Netherlands and investigate the immunogenicity of virus-like particles (VLPs) produced in insect cells. RHDV2 RNA was isolated from the liver of a naturally infected wild rabbit and the complete viral genome sequence was assembled from sequenced RT-PCR products. Phylogenetic analysis based on the VP60 capsid gene demonstrated that the RHDV2 NL2016 isolate clustered with other contemporary RHDV2 strains. The VP60 gene was cloned in a baculovirus expression vector to produce VLPs in Sf9 insect cells. Density-gradient purified RHDV2 VLPs were visualized by transmission electron microscopy as spherical particles of around 30 nm in diameter with a morphology resembling authentic RHDV. Immunization of rabbits with RHDV2 VLPs resulted in high production of serum antibodies against VP60, and the production of cytokines (IFN-γ and IL-4) was significantly elevated in the immunized rabbits compared to the control group. The results demonstrate that the recombinant RHDV2 VLPs are highly immunogenic and may find applications in serological detection assays and might be further developed as a vaccine candidate to protect domestic rabbits against RHDV2 infection., Competing Interests: The authors declare no conflict of interest.

The rabbit caliciviruses Lagovirus europaeus GI.1 and GI.2 both cause acute necrotizing hepatitis in European rabbits ( Oryctolagus cuniculus ). Whilst GI.2 is highly virulent in both young and adult rabbits, rabbits younger than eight weeks of age are highly resistant to disease caused by GI.1, although they are still permissive to infection and viral replication. To investigate the underlying mechanism(s) of this age related resistance to GI.1, we compared liver transcriptomes of young rabbits infected with GI.1 to those of adult rabbits infected with GI.1 and young rabbits infected with GI.2. Our data suggest that kittens have constitutively heightened innate immune responses compared to adult rabbits, particularly associated with increased expression of major histocompatibility class II molecules and activity of natural killer cells, macrophages, and cholangiocytes. This enables them to respond more rapidly to GI.1 infection than adult rabbits and thus limit virus-induced pathology. In contrast, these responses were not fully developed during GI.2 infection. We speculate that the observed downregulation of multiple genes associated with innate immunity in kittens during GI.2 infection may be due to virally-mediated immunomodulation, permitting fatal disease to develop. Our study provides insight into the fundamental host⁻pathogen interactions responsible for the differences in age-related susceptibility, which likely plays a critical role in defining the success of GI.2 in outcompeting GI.1 in the field.

RHDVb has become the dominant RHDV on the Iberian Peninsula. A better understanding of its pathogenicity is required to aid control measures. Thus, the clinical course, humoral immune response, viraemia and kinetics of RHDV-N11 (a Spanish RHDVb isolate) infection in different tissues at both viral RNA and protein levels were studied in experimentally infected young and adult rabbits. The case fatality rate differed between the two age groups, with 21% of kits succumbing while no deaths were observed in adults. Fever and viremia were strongly associated with death, which occurred 48 h post infection (PI) too fast for an effective humoral immune response to be mounted. A significant effect on the number of viral RNA copies with regard to the variables age, tissue and time PI (p < 0.0001 in all cases) was detected. Histological lesions in infected rabbits were consistently more frequent and severe in liver and spleen and additionally intestine in kits, these tissues containing the highest levels of viral RNA and protein. Although no adults showed lesions or virus antigen in intestine, both kits and adults maintained steady viral RNA levels from days 1 to 7 PI in this organ. Analysis revealed the fecal route as the main dissemination route of RHDV-N11. Subclinically infected rabbits had detectable viral RNA in their faeces for up to seven days and thus may play an important role spreading the virus. This study allows a better understanding of the transmission of this virus and improvement of the control strategies for this disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

European rabbits (Oryctolagus cuniculus) are severely affected by rabbit haemorrhagic disease (RHD). Caused by a lagovirus, the disease leads to losses in the rabbit industry and has implications for wildlife conservation. Past RHD outbreaks have been caused by GI.1/RHDV genotype viruses. A new virus belonging to the GI.2/RHDV2/b genotype emerged in 2010, quickly spreading and replacing the former in several countries; however, limited data are available on its pathogenicity and epidemiological factors. The present work extends these issues and evaluates cross-protection between both genotypes. Ninety-four and 88 domestic rabbits were challenged with GI.2/RHDV2/b and GI.1b/RHDV variant isolates, respectively. Cross-protection was determined by a second challenge on survivors with the corresponding strain. Mortality by GI.2/RHDV2/b was highly variable due to unknown individual factors, whereas mortality by GI.1b/RHDV was associated with age. Mortality in rabbits < 4 weeks old was 84%, higher than previously reported. Cross-protection was not identical between the two viruses because the ratio of mortality rate ratios for the first and second challenges was 3.80 ± 2.68 times higher for GI.2/RHDV2/b than it was for GI.1b/RHDV. Rabbit susceptibility to GI.2/RHDV2/b varied greatly and appeared to be modulated by the innate functionality of the immune response and/or its prompt activation by other pathogens. GI.1b/RHDV pathogenicity appeared to be associated with undetermined age-related factors. These results suggest that GI.2/RHDV2/b may interact with other pathogens at the population level but does not satisfactorily explain the GI.1b/RHDV virus's quick replacement., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Rabbit hemorrhagic disease virus 2 (RHDV2; Lagovirus GI.2) is a pathogenic calicivirus that affects European rabbits ( Oryctolagus cuniculus ) and various hare ( Lepus ) species. GI.2 was first detected in France in 2010 and subsequently caused epidemics in wild and domestic lagomorph populations throughout Europe. In May 2015, GI.2 was detected in Australia. Within 18 months of its initial detection, GI.2 had spread to all Australian states and territories and rapidly became the dominant circulating strain, replacing Rabbit hemorrhagic disease virus (RHDV/GI.1) in mainland Australia. Reconstruction of the evolutionary history of 127 Australian GI.2 isolates revealed that the virus arrived in Australia at least several months before its initial description and likely circulated unnoticed in wild rabbit populations in the east of the continent prior to its detection. GI.2 sequences isolated from five hares clustered with sequences from sympatric rabbit populations sampled contemporaneously, indicating multiple spillover events into hares rather than an adaptation of the Australian GI.2 to a new host. Since the presence of GI.2 in Australia may have wide-ranging consequences for rabbit biocontrol, particularly with the release of the novel biocontrol agent GI.1a/RHDVa-K5 in March 2017, ongoing surveillance is critical to understanding the interactions of the various lagoviruses in Australia and their impact on host populations. IMPORTANCE This study describes the spread and distribution of Rabbit hemorrhagic disease virus 2 (GI.2) in Australia since its first detection in May 2015. Within the first 18 months following its detection, RHDV2 spread from east to west across the continent and became the dominant strain in all mainland states of Australia. This has important implications for pest animal management and for owners of pet and farmed rabbits, as there currently is no effective vaccine available in Australia for GI.2. The closely related RHDV (GI.1) is used to control overabundant wild rabbits, a serious environmental and agricultural pest in this country, and it is currently unclear how the widespread circulation of GI.2 will impact ongoing targeted wild rabbit management operations., (Copyright © 2018 American Society for Microbiology.)

Since its emergence, variant RHDV (RHDVb/RHDV2) has spread throughout the Iberian Peninsula aided by the apparent lack of cross protection provided by classic (genogroup 1; G1) strain derived vaccines. In addition to RHDVb, full-length genome sequencing of RHDV strains has recently revealed the circulation of recombinant viruses on the Iberian Peninsula. These recombinant viruses contain the RHDVb structural protein encoding sequences and the non-structural coding regions of either pathogenic RHDV-G1 strains or non-pathogenic (np) rabbit caliciviruses. The aim of the work was twofold: firstly to validate a diagnostic real time RT-PCR developed in 2012 for the detection of RHDVb strains and secondly, to design a conventional RT-PCR for the differentiation of RHDVb strains from RHDVb recombinants by subsequent sequencing of the amplicon., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Rabbit hemorrhagic disease (RHD) is a veterinary disease that affects the European rabbit and has a significant economic and ecological negative impact. In Portugal, rabbit hemorrhagic disease virus (RHDV) was reported in 1989 and still causes enzootic outbreaks. Several recombination events have been detected in RHDV strains, including in the first reported outbreak. Here we describe the occurrence of recombination in RHDV strains recovered from rabbit and Iberian hare samples collected in the mid-1990s in Portugal. Characterization of full genomic sequences revealed the existence of a single recombination breakpoint at the boundary of the non-structural and the structural encoding regions, further supporting the importance of this region as a recombination hotspot in lagoviruses. Phylogenetic analysis showed that in the structural region, the recombinant strains were similar to pathogenic G1 strains, but in the non-structural region they formed a new group that diverged ~13% from known strains. No further reports of such group exist, but this recombination event was also detected in an Iberian hare that was associated with the earliest species jump in RHDV. Our results highlight the importance of the characterization of full genomes to disclose RHDV evolution and show that lagoviruses' diversity has been significantly undersampled.

The Czech v351 strain of rabbit hemorrhagic disease virus (RHDV1) is used in Australia and New Zealand as a biological control agent for rabbits, which are important and damaging introduced vertebrate pests in these countries. However, nonpathogenic rabbit caliciviruses (RCVs) can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with effective rabbit biocontrol. Antibodies that cross-reacted against RHDV antigens were found in wild rabbits before the release of RHDV1 in New Zealand in 1997, suggesting that nonpathogenic RCVs were already present in New Zealand. The aim of this study was to confirm the presence of nonpathogenic RCV in New Zealand and describe its geographical distribution. RCV and RHDV antibody assays were used to screen serum samples from 350 wild rabbits from 14 locations in New Zealand. The serological survey indicated that both RCV and RHDV are widespread in New Zealand wild rabbits, with antibodies detected in 10 out of 14 and 12 out of 14 populations, respectively. Two closely related RCV strains were identified in the duodenal tissue from a New Zealand wild rabbit (RCV Gore-425A and RCV Gore-425B). Both variants are most closely related to Australian RCV strains, but with 88% nucleotide identity, they are genetically distinct. Phylogenetic analysis revealed that the New Zealand RCV strains fall within the genetic diversity of the Australian RCV isolates, indicating a relatively recent movement of RCVs between Australia and New Zealand. IMPORTANCE Wild rabbits are important and damaging introduced vertebrate pests in Australia and New Zealand. Although RHDV1 is used as a biological control agent, some nonpathogenic RCVs can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with its effectiveness for rabbit control. The presence of nonpathogenic RCVs in New Zealand wild rabbits has been long hypothesized, but earlier attempts to isolate a New Zealand RCV strain have been unsuccessful. Therefore, it is important to determine if such nonpathogenic viruses exist in New Zealand rabbits, especially considering the proposed introduction of new RHDV strains into New Zealand as biocontrols., (Copyright © 2017 American Society for Microbiology.)

Rabbit hemorrhagic disease is an acute fatal highly contagious viral infectious disease that causes high losses among rabbitries. The disease was first reported in China in 1984 and later on in Saudi Arabia in 1996. The aim of this study was to investigate the emergence and pathogenicity of new rabbit hemorrhagic disease virus (RHDV) strains in Saudi Arabia. The pathogenicity was confirmed by inoculation in susceptible rabbits. Three RHDV strains were detected by reverse transcriptase polymerase chain reaction (RT-PCR) using primers targeting VP60 capsid protein gene in infected rabbitries during 2012 and 2013. These strains clustered into two genetically distinct genogroups related to year of isolation (G2 and G3). All new Saudi Arabia viruses clustered with the European strains, while the old strains clustered with strains from China and America. Based on amino acids and nucleotide sequences, the Saudi Arabia strains (RHD/1/SA/2012, RHD/2/SA/2012, and RHD/3/SA /2013) had high identity with Mexico89, Ca11-ITA, and 00-13,FRA virus; on the other hand, there was a relatively high identity with Bahrain strain. The evolutionary relationship of Saudi RHDVs strains revealed significant nucleotides and amino acid substitutions in hypervariable region E, suggesting the emergence of new RHDVs circulating in Saudi Arabia rabbitries. These antigenic changes represented by the antigenic index might be a potential cause of vaccination failure and raises the need to review the vaccination strategies against RHD.

The aim of the study was to establish if haemagglutination of rabbit haemorrhagic disease virus (RHDV) affects haematological picture of peripheral blood in rabbits and the pathogenicity of the virus. The study analyzed white and red blood cell picture in rabbits experimentally infected with two non-haemagglutinating (HA-) RHDV strains (Frankfurt and Asturias) and one strain with variable haemagglutination capacity (HA+/-) (Hagenow). Studies with HA- and HA +/- are rare and relate only to 4 HA- strains (2 RHDV: BLA and Rainham; 2 RHDVa: Pv97 and 9905) and 1 HA+/- RHDV strain: ŻD, where less changes in haematological indices and less pathogenicity were observed. We found that changes caused by HA- Frankfurt strain were related to the number of neutrophils and thrombocytes, while in HA- strain Asturias, in thrombocytes and leukocytes. Changes evoked by HA+/- Hagenow strain pertained to the number of eosinophils, thrombocytes, leukocytes, monocytes, and concentration of hemoglobin. Mortality caused by the Frankfurt strain was 100% between 36 and 48 h post infection (p.i.), while that caused by Asturias strain was 100% between 24 and 36 h p.i., and that observed in case of Hagenow strain was 90% between 36 and 48 h p.i. The changes in haematological picture caused by the HA- and HA+/- RHDV strains were less intensive than those found in case of the HA+ RHDV strains, which cannot be confirmed for pathogenicity, and is not in line with the existing hypothesis suggesting higher pathogenicity in HA+ viruses.

Since the first introduction of rabbit hemorrhagic disease (RHD) in 1986, disease outbreaks have been continuously reported in different regions of Russia. Despite extensive vaccination, sporadic RHD cases are still reported. Here, we examine eleven RHDV strains originating from disease outbreaks occurring between 2003 and 2012 and one widely used vaccine strain. Notable phenotypic and genetic heterogeneity among RHDV strains was observed. The RHDV strains Tambov-2010, Perm-2010, Manihino-09 showed different hemagglutinating activity (HA) at 4 °C and room temperature. While all RHDV field strains were identified as hemagglutinating virulent viruses of the RHDVa variant, the vaccine strain was assigned as a "classical" RHDV. These data indicate that since 2003, RHDVa has become the predominant variant circulating in Russia.

To investigate the genetic variability and evolution of rabbit hemorrhagic disease virus (RHDV) strains in China, VP60 gene sequences of eight new isolates collected from farms with RHD occurrences in China between 2009 and 2014 were analyzed, and compared with the reference sequence of the vaccine strain WF/China/2007. We conducted a comprehensive analysis of the Chinese RHDV strains, including hemagglutination tests, western blot and immunosassays of capsid proteins, and phylogenetic analysis, and identified a new distinct antigenic variant. Specifically, strain HB/2014 collected in North China was identified as a non-hemagglutinating strain, and belongs to the original RHDV (G1-G5) group. The other seven isolates were classified in genogroup G6 (RHDVa), which was widely distributed across China before 2014, and was thought to replace the earlier groups. Antigenic characterization of the VP60 genes revealed a large degree of nucleotide sequence divergence between HB/2014 and the other Chinese strains. However, the current vaccine showed complete cross-protection against HB/2014 challenge in inoculated rabbits. Collectively, these data provide new tools and insight for further understanding the molecular evolution of RHDV in China., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Rabbit haemorrhagic disease virus 2 (RHDV2) is widespread in several countries of Western Europe, but it has not been introduced to other continents. However, between late 2014 and early 2015, the presence of RHDV2 was confirmed outside of the European continent, in the Azores, initially in the islands of Graciosa, Flores, S. Jorge and Terceira. In this study we report the subsequent detection of RHDV2 in wild rabbits from the islands of Faial, St. Maria and S. Miguel, and display the necropsy and microscopic examination data obtained, which showed lesions similar to those induced by classical strains of RHDV, with severe affection of lungs and liver. We also disclose the result of a genetic investigation carried out with RHDV2 positive samples from wild rabbits found dead in the seven islands. Partial vp60 sequences were amplified from 27 tissue samples. Nucleotide analysis showed that the Azorean strains are closely related to each other, sharing a high genetic identity (>99.15%). None of the obtained sequences were identical to any RHDV2 sequence publically known, hampering a clue for the source of the outbreaks. However, Bayesian and maximum likelihood phylogenetic analyses disclosed that Azorean strains are more closely related to a few strains from Southern Portugal than with any others presently known. In the analysed region comprising the terminal 942 nucleotides of the vp60 gene, four new single nucleotide polymorphisms (SNP) were identified. Based on the present data, these four SNPs, which are unique in the strains from Azores, may constitute putative molecular geographic markers for Azorean RHDV2 strains, if they persist in the future. One of these variations is a non-synonymous substitution that involves the replacement of one amino acid in a hypervariable region of the capsid protein., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A specific real time RT-PCR for the detection of RHDV2 was developed and validated using RHDV and RHDV2 RNA preparations from positive field samples. The system was designed to amplify a 127 nucleotide-long RNA region located within the vp60 gene, based on the alignment of six sequences originated in Portugal, obtained in our laboratory, and 11 sequences from France and Italy. The primers and probe target sequences are highly conserved in the vast majority of the RHDV2 sequences presently known. In the sequences showing variability, only one mismatch is found per strain, usually outlying the 3' end of the primer or probe hybridization sequences. The specificity of the method was demonstrated in vitro with a panel of common rabbit pathogens. Standardization was performed with RNA transcripts obtained from a recombinant plasmid harboring the target sequence. The method was able to detected nine RNA molecules with an efficiency of 99.4% and a R(2) value of 1. Repeatability and reproducibility of the method were very high, with coefficients of variation lower than 2.40%. The assay was proven a valuable tool to diagnose most of RDVH2 circulating strains, and may be also useful to monitor viral loads, and consequently, disease progression and vaccination efficacy., (Copyright © 2015 Elsevier B.V. All rights reserved.)

We report the complete genome sequences of two isolates (RHDV-N11 and CBVal16) of variant rabbit hemorrhagic disease virus (RHDVb). Isolate N11 was detected in young domestic animals during a rabbit hemorrhagic disease (RHD) outbreak that occurred in 2011 on a rabbit farm in Navarra, Spain, while CBVal16 was isolated from a wild rabbit found dead in Valpaços, Northern Portugal, a year later. The viral sequences reported show 84.8-85.1 % and 78.3-78.5 % identity to RHDVAst/89 and RCV-A1 MIC-07, representative members of the pathogenic genogroup 1 RHDV and apathogenic rabbit calicivirus, respectively. In comparison with other RHDV isolates belonging to the previously known genogroups 1-6, RHDVb shows marked phenotypic differences, as it causes disease preferentially in young rabbits under 40 days of age and shows modified red blood cell agglutination profiles as well as antigenic differences that allow this variant to escape protection by the currently available vaccines.

The Lagovirus rabbit hemorrhagic disease virus (RHDV), a member of the family Caliciviridae, severely affects European rabbit (Oryctolagus cuniculus) populations by causing rabbit hemorrhagic disease (RHD). RHDV is subdivided in six genogroups but, more recently, a new RHDV variant with a unique genetic and antigenic profile emerged. We performed a study in rabbits found dead in the field during 2013 and 2014 in Portugal to determine the prevalence of this new variant versus the classical RHDV. Fifty-seven liver samples were screened for the presence of RHDV and positive samples were genotyped. All cases of RHDV infection were caused by the new variant. The only former genogroup circulating in Portugal, G1, was not detected. We hence conclude that the new RHDV variant is replacing G1 in Portugal, probably due to a selective advantage. This sudden and rapid replacement emphasizes the necessity of continued monitoring of wild rabbit populations.

The genetic relationships between 10 rabbit hemorrhagic disease strains collected in Portugal between 2006 and 2013, originated in the mainland and Azorean islands, were investigated based on the vp60 gene variability. A genetic diversity ranging from 2% to 13% was determined among the 10-vp60 complete sequences revealing a significant level of genetic heterogeneity between same strains. Phylogenetic Bayesian analysis showed that the Portuguese RHDV strains fell within different genogroups, namely G1, G5 and G6. Interestingly, all strains obtained from Azores, where RHDV was first detected in 1988, belong to G5 genogroup. G5 strains, that were not identified in the continent so far, seem to be the dominant group in these Atlantic islands. G1-related strains belonging to the Iberian group 3 (n=3) and G6 (RHDVa) strains (n=2) were identified among the samples originated in mainland which were collected between 2006 and 2008. Although the presence of G1 and G6 in Portugal had been shown before, our data refines the time of circulation of these strains until at least 2008. In summary, this study revises the epidemiological information of RHDV in Portugal since it reports for the first time the presence of G5 strains in Azores and demonstrates the circulation of G1 and G6 strains in mainland Portugal until the late 2000s., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The release of myxoma virus (MYXV) and Rabbit Haemorrhagic Disease Virus (RHDV) in Australia with the aim of controlling overabundant rabbits has provided a unique opportunity to study the initial spread and establishment of emerging pathogens, as well as their co-evolution with their mammalian hosts. In contrast to MYXV, which attenuated shortly after its introduction, rapid attenuation of RHDV has not been observed. By studying the change in virulence of recent field isolates at a single field site we show, for the first time, that RHDV virulence has increased through time, likely because of selection to overcome developing genetic resistance in Australian wild rabbits. High virulence also appears to be favoured as rabbit carcasses, rather than diseased animals, are the likely source of mechanical insect transmission. These findings not only help elucidate the co-evolutionary interaction between rabbits and RHDV, but reveal some of the key factors shaping virulence evolution., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Rabbit hemorrhagic disease is caused by a calicivirus, rabbit hemorrhagic disease virus (RHDV), which is responsible for high mortality in domestic and wild European rabbits (Oryctolagus cuniculus). RHDV strains were sequenced from wild European rabbits (Oryctolagus cuniculus algirus) collected in the Azorean island of Pico, Portugal. Phylogenetic analyses showed that the Pico RHDV strains diverge from all of the others described so far, but cluster with the genogroups 1-5 (G1-G5). The genetic distance between the Pico RHDV sequences and each G1, G2 and G3-G5 genogroup (~0.08) is compatible with an RHDV introduction at least 17 years ago. Our results show that in Pico, RHDV is the outcome of an independent evolution from the original RHDV strain that appeared in its European rabbit population. These are the first sequences of RHDV obtained in the subspecies O. c. algirus, outside of its original region, the Iberian Peninsula. Furthermore, we discuss the risk of rabbit translocations from the Azores to the Iberian Peninsula, where the rabbit wild populations are suffering high mortalities.

Rabbit hemorrhagic disease outbreaks in young rabbits have been recently observed in Spain. In this study we have tracked the spread of variant RHDV in samples collected from rabbit farms over a period of more than one year using RT-PCR and antigen-capture ELISA. The isolates were sequenced and compared to classic and variant RHDV strains and phylogenetic analyses were conducted. Mortalities have been observed in kits as young as 11 days. More than 50% of the dead rabbits had been previously vaccinated against RHDV using commercially available inactivated vaccines indicating a putative lack of protection against the variant RHDV. The large majority of the studied outbreaks (94.5%) in Spanish farms during 2012 were due to variant RHDV and only 3 out of the 55 farms were affected by classic RHDV. The data demonstrates that the variant RHDV has spread through a large number of Spanish provinces in a relatively short period of time, largely replacing the previously predominant G1 RHDV genotypes. Considering the lack of efficient vaccines against the variant RHDV strains strict disease control and greater vigilance measures should be put in place., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Rabbit haemorrhagic disease virus (RHDV), genus Lagovirus, family Caliciviridae, causes a large number of deaths in wild and domestic adult European rabbits (Oryctolagus cuniculus). The first documented outbreak dates from 1984 in China, but the virus rapidly dispersed worldwide. In 1997, an antigenic variant was detected in Italy and designated RHDVa. Despite causing symptoms similar to those caused by classic RHDV strains, marked antigenic and genetic differences exist. In some parts of Europe, RHDVa is replacing classic strains. Here, we report the presence of RHDVa on the Iberian Peninsula, where this variant was thought not to contribute to viral diversity.

Since summer 2010, numerous cases of Rabbit Haemorrhagic Disease (RHD) have been reported in north-western France both in rabbitries, affecting RHD-vaccinated rabbits, and in wild populations. We demonstrate that the aetiological agent was a lagovirus phylogenetically distinct from other lagoviruses and which presents a unique antigenic profile. Experimental results show that the disease differs from RHD in terms of disease duration, mortality rates, higher occurrence of subacute/chronic forms and that partial cross-protection occurs between RHDV and the new RHDV variant, designated RHDV2. These data support the hypothesis that RHDV2 is a new member of the Lagovirus genus. A molecular epidemiology study detected RHDV2 in France a few months before the first recorded cases and revealed that one year after its discovery it had spread throughout the country and had almost replaced RHDV strains. RHDV2 was detected in continental Italy in June 2011, then four months later in Sardinia.

The objective of this study was to analyse the genetic variability and phylogenetic analysis of six strains of rabbit haemorrhagic disease virus (RHDV), including four Czech strains (CAMPV-351, CAMPV-561, CAMPV-562, CAMPV-558) and two French strains (Fr-1, Fr-2), on the basis of a fragment of the VP60 capsid structural protein-coding gene N-terminal region. The results of our own studies were compared to 26 RHDV strains obtained from GenBank. The analysis of the genetic variability of six RHDV strains indicated that the CAMPV-561 strain is the most genetically variable. Less variable were the Fr-1 and Fr-2 strains, while the least variable was CAMPV-351. In turn, the genetic distance among the six analysed strains and 26 strains obtained from GenBank was the greatest for CAMPV-351 and Whn/China [11.3 % according to the observed divergence (OD) method and 12.2 % according to the maximum likelihood (ML) method], while it was the lowest for CAMPV-351 and FRG (0.8 % in both the OD and ML methods). In turn, the scale of the genetic distances among the six analysed strains and five RHDVa strains (99-05, NY-01, Whn/China, Triptis, Iowa2000) ranged from 9.3-10.3 % in the OD method to 10.3-13.7 % in the ML method. The image of phylogenetic dependencies generated for the strains analysed and those obtained from GenBank revealed their distribution to be in five genetic groups (G1-G5), whereas the analysed strains were included in genetic groups 2 and 3.

The aim of this study was to determine the differences in immunological response of animals infected with different antigenic variants of the virus--three haemagglutinating (Vt97, Triptis, Hartmannsdorf) and two non-haemagglutinating (Pv97, 9905 RHDVa). The specific immunological response was measured by the dynamics of changes in the amount of lymphocytes T (with CD5+, CD4+, CD8+, CD25+ receptor) and B (with CD19+ receptor). The study showed differences in immunogenicity of the analysed RHDV antigenic variants, which allowed them to be divided into groups of: more immunogenic strains, including non-haemagglutinating 9905 RHDVa and haemagglutinating Vt97 and Triptis variants; and less immunogenic strains, including the haemagglutinating Hartmannsdorf variant and the non-haemagglutinating Pv97 variant. Such a result may indicate that the agglutination capacity of red blood cells might not be a factor impacting the number of T and B lymphocytes.

The paper concerns the use of a novel, very effective diagnostic method, a real-time PCR for diagnosis of a viral agent causing viral haemorrhagic disease in rabbits - RHDV. Until now, the method was widely used for detecting many different viruses, both DNA, and RNA, but as far as RHDV is concerned, there are not many records of such use. This study aimed at the detection of 17 different strains from different European regions, differing in biological features and mortality. The study confirmed that real-time PCR is an applicable and effective method for diagnosis of RHDV, irrespective of the stains' features.

Outbreaks of rabbit hemorrhagic disease have occurred recently in young rabbits on farms on the Iberian Peninsula where rabbits were previously vaccinated. Investigation identified a rabbit hemorrhagic disease virus variant genetically related to apathogenic rabbit caliciviruses. Improved antivirus strategies are needed to slow the spread of this pathogen.

This study aimed to investigate rabbit hemorrhagic disease virus (RHDV) in China. VP60 sequences of five RHDVs collected by our team, as well as those of 16 other published Chinese RHDV strains, were analyzed. Polygenic analysis using MEGA 4 software showed that 20 of the 21 Chinese strains could be clustered in the RHDVa subgroup, and WX/China/1984 was different from them. The Chinese RHDV strains were further classified into four subgroups, CH1 to CH4. Subgroup CH1, represented by the WX/China/1984 strain, was not prevalent in China after the first RHDV epidemic strain was reported. The CH2, CH3, and CH4 subgroups were far different from the CH1 subgroup, formed three separate clusters, and were distributed according to the time the strains were collected. Recently collected strains formed a new subgroup (CH4), represented by new RHDV varieties identified by challenging immunized rabbits and by comparison of genomic sequences. The present work is the first comprehensive analysis of Chinese RHDV and reveals a new RHDV variation that should be carefully monitored.

Rabbit haemorrhagic disease virus (RHDV) is a highly virulent lagovirus endemic in Europe and Australasian populations of the European rabbit, Oryctolagus cuniculus. It has also caused several unexplained disease outbreaks in domestic European rabbits in North America. Non-pathogenic spread of RHDV leading to persistent infection which later reactivated has recently been proposed as the cause of overt disease and death of a pet rabbit in Canada, the first confirmed case of Rabbit haemorrhagic disease in that country. We suggest that there is little evidence to support non-pathogenic spread of virulent RHDV, some evidence that is contradictory, and evidence to support a simpler alternative hypothesis. RHDV can be spread over long distances between sparse rabbit populations by fomites or flying insects. Although highly pathogenic, RHDV can be limited in its spread within rabbit populations, or its presence masked by closely related but non-pathogenic lagoviruses which can provide protection against acute disease. In the absence of any evidence from clinical studies to support reactivation of persistent RHDV infection, the simpler explanation seems more likely to be correct., (© 2012 Blackwell Publishing Ltd.)

In order to determine the genetic variability of Polish RHD virus strains and to confirm the presence of genetic variant (RHDVa) subtype the partial nucleotide sequences of capsid protein gene, including two highly variable regions C and E, were examined. Phylogenetic analyses of 15 viral strains obtained over 18 years revealed the presence of three genetic groups. The oldest RHDV strains exhibit very close amino acid sequence similarity (98-99%) to the German FRG89 reference strain and most of European strains of the same period, as well as Chinese isolate from 1984. The HA-negative strains and isolates with variable reactivity in the HA test belong to the second subgroup and exhibit an intermediate level of variability (about 3%) in the analysed VP60 gene fragment. The most genetically variable strains (6-7%) clustered to RHDVa subtype. The analysis of nucleotides and amino acid sequences demonstrated three pairs of well conserved RHDV strains, isolated over 3, 6 and 10-year period.

The paper describes the immunological response in the matter of percentage of T cells (receptor CD5+) and subpopulations (Th with receptor CD4+, Tc/Ts with receptor CD8+, T with receptor CD25+) and B cells with receptor CD19+, as well as the percentage of apoptotic granulocytes and lymphocytes, in rabbits experimentally infected with the Hagenow strain of the RHD virus. The material chosen for the experiment is special, as among all strains of RHD virus, there are only two strains which carry the variable haemagglutination capacity of red cells. The results of the study show that the Hagenow strain gives an untypical picture of T and B lymphocytes, whereas the results in inducing apoptosis seems to corespond with previous data, confirming the inclusion of apoptosis from 4 h p.i. and the intensity of the phenomenon being higher in granulocytes.

Since its detection in China in 1984, rabbit haemorrhagic disease (RHD) has been the subject of numerous studies. Yet, the evolutionary origin of rabbit haemorrhagic disease virus (RHDV) is still under debate. For example, some aspects related to the epidemiology of the disease are still unknown, such as where the virus is hosted between RHD outbreaks. To detect the presence of RHDV in rabbit-sympatric micromammals, 51 rodents (29 Mus spretus and 22 Apodemus sylvaticus) and 31 rabbits (Oryctolagus cuniculus) from the same location in central Spain were analyzed. In those samples in which the virus was detected, a fragment of the VP60 protein gene from the RHDV capsid was sequenced and the phylogenetic relationships between them and other strains of RHDV in the Iberian Peninsula were analyzed. In total, five viral strains were identified in A. sylvaticus, M. spretus and O. cuniculus. All strains were found to be well supported within the clade of RHDV found in rabbits in the Iberian Peninsula. Moreover, one of the strains was found in all three species under study, which suggests the capability of RHDV to infect other mammals apart from the rabbit which have not yet been investigated. The transmission of the virus is discussed as well as its ecoepidemiological implications., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Four strains of RHDV assigned as haemagglutinating (Vt97 and Hartmannsdorf) and non-haemagglutinating (Pv97 and 9905) antigenic variants were examined for dynamic changes in the values of white and red blood cells indexes. The study showed differences among strains examined that were not depending on haemagglutination property.

Background: Rabbit haemorrhagic disease virus (RHDV) is a highly virulent calicivirus, first described in domestic rabbits in China in 1984. RHDV appears to be a mutant form of a benign virus that existed in Europe long before the first outbreak. In the Iberian Peninsula, the first epidemic in 1988 severely reduced the populations of autochthonous European wild rabbit. To examine the evolutionary history of RHDV in the Iberian Peninsula, we collected virus samples from wild rabbits and sequenced a fragment of the capsid protein gene VP60. These data together with available sequences from other Western European countries, were analyzed following Bayesian Markov chain Monte Carlo methods to infer their phylogenetic relationships, evolutionary rates and demographic history., Results: Evolutionary relationships of RHDV revealed three main lineages with significant phylogeographic structure. All lineages seem to have emerged at a common period of time, between ~1875 and ~1976. The Iberian Peninsula showed evidences of genetic isolation, probably due to geographic barriers to gene flow, and was also the region with the youngest MRCA.Overall, demographic analyses showed an initial increase and stabilization of the relative genetic diversity of RHDV, and a subsequent reduction in genetic diversity after the first epidemic breakout in 1984, which is compatible with a decline in effective population size., Conclusions: Results were consistent with the hypothesis that the current Iberian RHDV arose from a single infection between 1869 and 1955 (95% HPD), and rendered a temporal pattern of appearance and extinction of lineages. We propose that the rising positive selection pressure observed throughout the history of RHDV is likely mediated by the host immune system as a consequence of the genetic changes that rendered the virus virulent. Consequently, this relationship is suggested to condition RHDV demographic history.

The present paper refers to the cytometric analysis of lymphocytes T (with receptor CD5+), Th (with receptor CD4+), Tc/Ts (with receptor CD8+), lymphocytes CD25+ and lymphocytes B with receptor CD19+ in rabbits experimentally infected with strains of RHD virus--Rainham, Frankfurt and Asturias, not having haemagglutinogenic capacities, which makes them unique, as haemagglutinogenic capacity is a classic and typical property of most strains of this virus. The study was performed in the dynamic system, drawing blood samples from animals at hour 0, namely before the administration of the viral antigen, and then at 4, 8, 12, 24 and 36 h after the infection. The study indicated that Rainham and Asturias strains of RHD virus cause a similar amount of changes as the most immunogenic haemagglutinogenic strains CAMP V-561 and CAMP V-562 of the RHD virus do. In contrast, the Frankfurt strain of the RHD virus is characterised with 5-6-fold lower reactivity in this respect and is most similar to the least immunogenic haemagglutinogenic strain CAMP V-558 of the RHD virus.