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.

Genetic Diversity and Epidemiological Significance of Wild Boar HEV-3 Strains Circulating in Poland.

The wild boar is the most important reservoir of zoonotic HEV-3 strains among different wildlife species. The aim of the study was subtype identification of wild boar HEV-3 strains circulating in Poland. Wild boar liver was used in the study in the form of homogenates prepared from 57 samples positive for HEV in a real-time RT-PCR. These samples were collected from juvenile and adult wild boars hunted in the jurisdictions of different Regional Directorates of State Forests (RDSF) across Poland. Subtype identification of detected HEV strains was based on a phylogenetic analysis of the most conserved HEV ORF2 genome fragment. Out of 57 tested samples, consensus HEV ORF2 sequences of 348 bp were obtained for 45 strains. Nineteen strains were identified and belonged to the HEV gt 3a and 3i subtypes, whereas 26 were not assigned to any virus subtype. HEV gt 3i strains prevailed in the Polish wild boar population, 16 of such were identified, and they were significantly more often observed in the RDSF Katowice area (χ2 = 28.6, p = 0.027 (<0.05)) compared to other regions of the country. Circulation of 3a strains was limited only to the RDSF Gdansk territory (χ2 = 48, p = 0.000 (<0.05)). The virus strains detected in the Polish population of wild boars representing previously identified HEV subtypes in wild boars, pigs, or humans in Europe are of epidemiological importance for public health.

Wild Boar as a Sylvatic Reservoir of Hepatitis E Virus in Poland: A Cross-Sectional Population Study.

The most important wildlife species in the epidemiology of hepatitis E virus (HEV) infections are wild boars, which are also the main reservoir of the virus in a sylvatic environment. The aim of the study was a serological and molecular assessment of the prevalence of HEV infections in wild boars in Poland. In total, 470 pairs of samples (wild boar blood and livers) and 433 samples of faeces were tested. An ELISA (ID.vet, France) was used for serological analysis. For the detection of HEV RNA, real-time (RT)-qPCR was employed. The presence of specific anti-HEV IgG antibodies was found in 232 (49.4%; 95%CI: 44.7-54%) sera, with regional differences observed in the seroprevalence of infections. HEV RNA was detected in 57 (12.1%, 95%CI: 9.3-15.4%) livers and in 27 (6.2%, 95%CI: 4.1-8.9%) faecal samples, with the viral load ranging from 1.4 to 1.7 × 1011 G.C./g and 38 to 9.3 × 107 G.C./mL, respectively. A correlation between serological and molecular results of testing of wild boars infected with HEV was shown. HEV infections in wild boars appeared to be common in Poland.

Detection of hepatitis E virus (rabbit genotype) in farmed rabbits entering the food chain.

Hepatitis E virus (HEV) infects humans and many animal species. The rabbit HEV has been found in farmed, wild and pet rabbits as well as in human patients suggesting zoonotic transmission. Although the routes of human infection with rabbit strains are unclear a foodborne transmission is suggested especially when asymptomatically infected animals could enter the food chain. The aims of the study were an evaluation of the prevalence of HEV infections in slaughtered rabbits, identification of the virus genotype(s) and assessment of their genetic relatedness to other zoonotic HEV strains. A pair of blood and liver samples (n = 482) were collected from meat rabbits of different breeds slaughtered at the age of 2.8 to 6 months. The animals originated from 20 small-scale and 4 large-scale commercial farms operating in Poland. The presence of anti-HEV antibodies in animals was detected by the use of a recomWell HEV IgG (human) ELISA kit (Mikrogen Diagnostik) adapted to rabbit sera. The isolation of HEV and sample process control virus (feline calicivirus) RNA from homogenates of liver destined for food and virus-positive sera was performed using a QIAamp® Viral RNA Mini Kit (Qiagen). A one-step real-time reverse transcription PCR method containing a target-specific internal amplification control was used for detection of HEV. The (sub)genotype of detected rabbit HEV strains was identified based on sequence analysis of the ORF2 and ORF2/3 virus genome fragments. Anti-HEV antibodies were detected in 29 (6%) out of 482 rabbit sera samples collected from animals raised only on the small-scale rabbit farms. Four sera were also positive for HEV RNA. Viral RNA was detected in 72 (14.9%) animal livers. Analysing ELISA and PCR results using Student's t-test, there were significant differences observed in the frequency of HEV infections between rabbits from small-scale and commercial farms (t = 2.675, p = 0.015 < 0.05 for ELISA and t = 2.705, p = 0.014 < 0.05 for PCR). All detected virus strains were identified as HEV gt3 ra subtype. The results of this study provide data on the occurrence of HEV infections in rabbits entering the food chain, suggesting that a risk of foodborne HEV infection due to consumption of contaminated meat and liver exists. In this light, the presence of rabbit HEV in food animals is pertinent as an issue of food safety and the surveillance of these animals for emerging or re-emerging viruses.

Occurrence of norovirus and hepatitis A virus in wild mussels collected from the Baltic Sea.

The aim of the study was to define the occurrence of human noroviruses of genogroup I and II (NoV GI and NoV GII) and hepatitis A virus (HAV) in the Baltic Sea mussels. The shellfish samples were taken at the sampling sites located on the Polish coast. In total, 120 shellfish were tested as pooled samples using RT-PCR and hybridisation with virus specific probes. NoV GI was detected in 22 (18.3%), NoV GII in 28 (23.3%), and HAV in 9 (7.5%) of the shellfish. The nucleotide sequence analysis of the detected NoV GII strains showed a 97.3-99.3% similarity to GII.4 virus strain. This is the first report describing the NoV and HAV occurrence in wild Baltic mussels and their possible role as bioindicators of seawater contamination with human enteric viruses.