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.

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.

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.

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.

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.

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.

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.

Comparative analysis of rabbit hemorrhagic disease virus strains originating from outbreaks in the Russian Federation.

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.

White and red blood cell picture in rabbits experimentally infected with strains of the rabbit haemorrhagic disease (RHD) virus without or with variable haemagglutination capacity.

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: ZD, 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.

Complete genome sequence of two rabbit hemorrhagic disease virus variant b isolates detected on the Iberian Peninsula.

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.

Detection of RHDVa on the Iberian Peninsula: isolation of an RHDVa strain from a Spanish rabbitry.

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.

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.

Emergence of a new lagovirus related to Rabbit Haemorrhagic Disease Virus.

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.

Real time PCR detection of rabbit haemorrhagic disease virus in rabbits infected with different European strains of RHDV.

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.

Lymphocytes T and B in rabbits infected with RHD virus.

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.

Variant rabbit hemorrhagic disease virus in young rabbits, Spain.

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.

Rabbit haemorrhagic disease: applying Occam's razor to competing hypotheses.

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.

Phylogenetic analysis of rabbit hemorrhagic disease virus in China and the antigenic variation of new strains.

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.

Lymphocyte subpopulations and apoptosis of immune cells in rabbits experimentally infected with a strain of the RHD virus having a variable haemagglutination capacity.

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.