Phylogenetic Analysis of European Brown Hare Syndrome Virus Strains from Poland (1992–2004)

European brown hare syndrome (EBHS) is lethal to several species of free-living hares worldwide. The genetic characterization of its virus (EBHSV) strains in European circulation and epidemiological knowledge of EBHSV infections is not yet complete. The study determined the nucleotide sequences of the genomes of EBHSV strains from Poland and analyzed their genetic and phylogenetic relationships to a group of hare lagoviruses. The genome of five virus strains detected in Poland between 1992 and 2004 was obtained by RT-PCR and sequencing of the obtained amplicons. The genetic relationships of the EBHSV strains were analyzed using the full genome and VP60 gene sequences. Additionally, the amino acid sequence of the VP60 gene was analyzed to identify mutations specific to recognized EBHSV subgroups. Partial amplification of the virus open reading frame (ORF)1 and ORF2 regions obtained nearly complete nucleotide genome sequences of the EBHSV strains. Phylogenetic analysis placed them in a GII.1 cluster with other European strains related to nonpathogenic hare caliciviruses. VP60 gene analysis allocated these EBHSV strains to the G1.2, G2.2–2.3 or G3 virus genetic groups. The amino acid sequence differences in the entire genome ranged from 1.1 to 2.6%. Compared to a reference French EBHSV-GD strain, 22 variable amino acid sites were identified in the VP60 region of the Polish strains, but only six were in VP10. Single amino acid changes appeared in different sequence positions among Polish and other European virus strains from different genetic groups, as well as in VP10 sequences of nonpathogenic hare caliciviruses. The results of the study showed a high genetic homogeneity of EBHSV strains from Poland despite their different location occurrence and initial detection times. These strains are also phylogenetically closely related to other EBHSV strains circulating in Europe, likely confirming the slow evolutionary dynamics of this lagovirus species.

Microbial metagenomic approach uncovers the first rabbit haemorrhagic disease virus genome in Sub-Saharan Africa

Rabbit Haemorrhagic Disease (RHD) causes high morbidity and mortality in rabbits and hares. Here, we report the first genomic characterization of lagovirus GI.2 virus in domestic rabbits from sub-Saharan Africa. We used an unbiased microbial metagenomic Next Generation Sequencing (mNGS) approach to diagnose the pathogen causing the suspected outbreak of RHD in Ibadan, Nigeria. The liver, spleen, and lung samples of five rabbits from an outbreak in 2 farms were analyzed. The mNGS revealed one full and two partial RHDV2 genomes on both farms. Phylogenetic analysis showed close clustering with RHDV2 lineages from Europe (98.6% similarity with RHDV2 in the Netherlands, and 99.1 to 100% identity with RHDV2 in Germany), suggesting potential importation. Subsequently, all the samples were confirmed by RHDV virus-specific RT-PCR targeting the VP60 gene with the expected band size of 398 bp for the five rabbits sampled. Our findings highlight the need for increased genomic surveillance of RHDV2 to track its origin, understand its diversity and to inform public health policy in Nigeria, and Sub-Saharan Africa.

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.

Rabbit Hemorrhagic Disease Virus Isolated from Diseased Alpine Musk Deer (Moschus sifanicus)

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.

Genotyping of rabbit hemorrhagic disease virus detected in diseased rabbits in Egyptian Provinces by VP60 sequencing

Background and Aim: Rabbit hemorrhagic disease (RHD) is an economically important disorder of rabbits, where infection results in severe losses to the meat and fur industries. Our goal was to characterize the RHD virus (RHDV) strains currently circulating in different regions of Egypt. Materials and Methods: Fifty rabbits suspected of harboring RHDV from 15 Egyptian governorates were evaluated. Diseased rabbits were identified by clinical signs and postmortem lesions. RHDV was confirmed through hemagglutination assay (HA) and polymerase chain reaction (PCR). Partial sequencing of the VP60 gene was performed for genotyping. Results: From 50 rabbits, we identified 16 cases of RHDV (32%) by HA and PCR, including seven males and nine females. We identified two distinct genotypes through sequencing of an amplified fragment of the virus VP 60 gene. One group is composed of those circulating primarily in upper Egypt, which is closely related to the classical G3-G5 virus strains, and the second group, circulating predominantly in lower Egypt, was more closely related to the RHDV2 variant. The overall nucleotide sequence identity ranged from 78.4% to 100%, and identity with the vaccine strains ranged from 78.8% to 91.1%. Conclusion: Our results constitute important documentation of RHDV strains currently circulating in Egypt. The findings suggest that there may be a limit to the effectiveness of currently applied vaccine strains as this formulation may not cover all circulating strains. A wider investigation that includes both domestic and wild rabbits will be needed to identify appropriate control measures for this disease.

Detection and characterisation of rabbit haemorrhagic disease virus strains circulating in Egypt

Rabbit haemorrhagic disease (RHD) is a highly virulent viral disease of Oryctolagus cuniculus which threatens the rabbit population in Egypt and worldwide. The etiological agent is the rabbit haemorrhagic disease virus (RHDV), a member of the family Caliciviridae. The aim of this study was to identify the possible evolutionary changes of the currently circulating RHDV in Egypt. Twenty suspected cases were collected from outbreaks that occurred in non-vaccinated rabbit flocks during 2015 and 2016. Liver homogenate samples were investigated for detection and identification of circulating RHDV using haemagglutination (HA) and reverse transcriptase polymerase chain reaction. Further characterisation of selected five viral strains was performed by nucleotide sequencing of VP60 gene. All twenty tested samples were haemagglutinin positive and VP60 gene was amplified. Based on nucleotide sequence analysis, four isolates were identified as classical RHDV strains, while one isolate was assigned as RHDVa variant strain but with the same HA pattern. The new RHDVb variant was not identified.

Evaluation of the usefulness of laboratory diagnostic methods in RHD outbreak

The field outbreak of RHD that occurred late summer 2012 on a small-scale rabbit-rearing operation in Poland and the usefulness of techniques for RHD virus diagnosis are described. During the epizootic, the overall mortality rate of rabbits older than two months was 77%. Eight liver specimens collected from dead unvaccinated rabbits (aged 3-5 months) underwent virological examinations. RHDV specific antigen was detected in two out of eight liver homogenates by haemagglutination (HA) test and ELISA, one of the two being negative in HA assay. However the presence of genetic material of RHD virus was confirmed by RT-PCR and real-time RT-PCR in all liver samples tested. Based on antigen reactivity in ELISA and sequencing of PCR amplicons of the VP60 gene, the RHDVa subtype strain was identified as the cause of infection. The partial genome sequence of a field isolate (STR 2012), comprising the C-terminus of the polymerase gene and the full capsid protein gene, revealed 91% nucleotide homology to reference FRG89 RHDV isolate and 97% to strain Triptis representing the RHDVa variant. Serological evidence of an RHD outbreak in the STR rabbit-rearing operation was confirmed in a serum sample collected from an unvaccinated surviving rabbit. A cross-reactivity examination of RHDV positive serum revealed a decrease in HI titre against the STR 2012 field antigen, and a decrease in the RHDVa control antigen as compared to classic RHDV.