First Detection and Circulation of RHDV2 in New Zealand.

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

Clinical and pathologic findings in an outbreak in rabbits of natural infection by rabbit hemorrhagic disease virus 2 in the northwestern United States.

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.

Infectivity of rabbit haemorrhagic disease virus excreted in rabbit faecal pellets.

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.

Evidence of independent introductions of RHDV2 strains in Poland based on the genome analysis of viral isolates from 2016-2018.

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.

Retrospective Analysis Shows That Most RHDV GI.1 Strains Circulating Since the Late 1990s in France and Sweden Were Recombinant GI.3P-GI.1d Strains.

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.

Rabbit Hemorrhagic Disease Virus Isolated from Diseased Alpine Musk Deer (Moschussifanicus).

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 (Moschussifanicus). 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.

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

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

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

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

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

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

Immunogenicity in Rabbits of Virus-Like Particles from a Contemporary Rabbit Haemorrhagic Disease Virus Type 2 (GI.2/RHDV2/b) Isolated in The Netherlands.

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.

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

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

Robust Innate Immunity of Young Rabbits Mediates Resistance to Rabbit Hemorrhagic Disease Caused by Lagovirus Europaeus GI.1 But Not GI.2.

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.

Clinical course and pathogenicity of variant rabbit haemorrhagic disease virus in experimentally infected adult and kit rabbits: Significance towards control and spread.

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.

Rabbit haemorrhagic disease: Cross-protection and comparative pathogenicity of GI.2/RHDV2/b and GI.1b/RHDV lagoviruses in a challenge trial.

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 ±â€¯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.

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

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

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

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

Conventional and real time RT-PCR assays for the detection and differentiation of variant rabbit hemorrhagic disease virus (RHDVb) and its recombinants.

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.

Characterization of old RHDV strains by complete genome sequencing identifies a novel genetic group.

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.

Benign Rabbit Calicivirus in New Zealand.

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.

Emergence of new virulent rabbit hemorrhagic disease virus strains in Saudi Arabia.

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.