Prevalence and genotype/subtype distribution of hepatitis E virus (HEV) among wild boars in Japan: Identification of a genotype 5 HEV strain.

To further investigate the prevalence of hepatitis E virus (HEV) infection and characterize HEV genomes among Japanese wild boars (Sus scrofa leucomystax), 1880 boars captured in 17 prefectures in Japan from 2013 to 2019 were studied. Overall, anti-HEV IgG was detected in 8.9 % and HEV RNA was detected in 3.9 % of boars, which was comparable with our previous studies during 2003-2013 (10.3 % and 3.5 %, respectively). Among 74 boar HEV strains obtained from infected boars in the present study, 50 (68 %) were classified into genotype 3 (3a and 3b), 23 (31 %) were classified into genotype 4 (4i), and the remaining strain (wbJGF_19-1) was classified into genotype 5. The wbGF_19-1 strain shared 92.7 % identity over the entire genome with the prototype genotype 5 strain (JBOAR135-Shiz09). The identification of the second genotype 5 HEV strain in a place that is located only 100 km from the site at which JBOAR135-Shiz09 was identified, suggests that genotype 5 HEV circulates within a relatively close range in Japan. Genetically similar HEV strains forming a clade were identified from wild boars living in each area during the observation period of 11-13 years, although the nucleotide sequence changed gradually, accounting for up to 3.4-3.6 % within the 412-nucleotide ORF2 sequence. Eight groups of boars with a cluster of HEV infections were observed, consisting of two, three or four infected offspring, presumably born to the same mother or offspring with their mother. These results suggest that wild boars continue to be important reservoirs for HEV infection in humans in Japan.

Surveillance of Shiga toxin-producing Escherichia coli and Campylobacter spp. in wild Japanese deer (Cervus nippon) and boar (Sus scrofa).

Increasing game meat consumption in Japan requires the dissemination of safety information regarding the presence of human pathogens in game animals. Health information regarding the suitability of these animals as a meat source is not widely available. In this study, we aimed to evaluate the safety of game meat and detect potential human pathogens in wild deer (Cervus nippon) and boar (Sus scrofa) in Japan. Fecal samples from 305 wild deer and 248 boars of Yamaguchi, Kagoshima, and Tochigi prefectures collected monthly for 2 years were examined for the prevalence of Shiga toxin-producing Escherichia coli (STEC) and Campylobacter spp. STEC was isolated from 51 deer consistently throughout the year and from three boars; O-antigen genotype O146, the expression of stx2b, and eaeA absence (n=33) were the major characteristics of our STEC isolates. Other serotypes included the medically important O157, stx2b or stx2c, and eaeA-positive (n=4) and O26, stx1a, and eaeA-positive strains (n=1). Campylobacter spp. were isolated from 17 deer and 31 boars. Campylobacter hyointestinalis was the most common species isolated from 17 deer and 25 boars, whereas Campylobacter lanienae and Campylobacter coli were isolated from three and two boars, respectively. Seasonal trends for the isolation of these bacteria were not significant. This study demonstrates that wild game animals carry human pathogens; therefore, detailed knowledge of the safe handling of game meat is needed to prevent foodborne infections.

Detection of a novel tick-borne flavivirus and its serological surveillance.

Tick-borne encephalitis virus (TBEV), a flavivirus that causes severe neurological symptoms in humans, has been found in Hokkaido, Japan. In the present study, we detected sequences from a novel tick-borne flavivirus, designated Yamaguchi virus (YGV), in liver and serum samples obtained from a wild boar in the Yamaguchi prefecture, Japan. Phylogenetic analysis revealed that YGV belongs to the TBEV complex and is closely related to Langat virus (LGTV). YGV was also detected by specific RT-PCR from 20 in 378 pools of ticks (2923 ticks) collected in Yamaguchi and Wakayama prefectures and from seven in 46 wild boar captured in Wakayama. The major ticks infected with YGV belong to the genus Haemaphysalis. Unfortunately, YGV could not be isolated from any samples from the RT-PCR positive wild boar or ticks. Therefore, ELISA for detection of antibodies against YGV was established using LGTV, and surveillance was performed among wild boar in 10 different prefectures on Honshu Island, the main island of Japan. The results showed that the seroprevalence of tick-borne flavivirus infection in the Wakayama and Hyogo prefectures of western Japan was significantly higher than that in the other prefectures, while antibodies against tick-borne flavivirus were not detected in any wild boar in the Tochigi prefecture in the eastern part of Japan. In addition, wild raccoons or masked palm civets in the Hyogo prefecture did not possess detectable antibodies against tick-borne flaviviruses. In conclusion, YGV appears to be maintained primarily among wild boar and ticks in the western part of Japan. YGV is the second flavivirus (after Japanese encephalitis virus) shown to be circulating on Honshu Island in Japan.

Positive rates of anti-acari-borne disease antibodies of rural inhabitants in Japan.

An assessment of acari (tick and mite) borne diseases was required to support development of risk management strategies in rural areas. To achieve this objective, blood samples were mainly collected from rural residents participating in hunting events. Out of 1,152 blood samples, 93 were positive against acari-borne pathogens from 12 prefectures in Japan. Urban areas had a lower rate of positive antibodies, whereas mountainous farming areas had a higher positive antibody prevalence. Residents of mountain areas were bitten by ticks or mites significantly more often than urban residents. Resident of mountain areas, including hunters, may necessary to be educated for prevention of akari-borne infectious diseases.

Identification and full-genome characterization of novel circoviruses in masked palm civets (Paguma larvata).

The family Circoviridae comprises a large group of small, circular, single-stranded DNA viruses and is classified into two genera: Circovirus and Cyclovirus. They have marked genetic diversity and a broad host range. In this study, three novel circovirus genomes were identified from wild-caught masked palm civets (Paguma larvata) in Japan and classified as a new species within the genus Circovirus based on the demarcation criteria of the International Committee on the Taxonomy of Viruses. Of note, the presence of two predicted introns at the 5'-terminus of the rep gene was suggested in the Paguma larvata circovirus genomes.

Identification and whole genome characterization of novel anelloviruses in masked palm civets (Paguma larvata): Segregation into four distinct clades.

The members of the family Anelloviridae are small and single-stranded DNA viruses with marked diversity in sequence and length, which ubiquitously infect many vertebrates, including mammals, birds and reptiles. The anelloviruses isolated from mammals are currently classified into 11 assigned and four proposed genera; some anelloviruses remain unassigned. The present study was conducted to identify anelloviruses in wild-caught masked palm civets (Paguma larvata) in Japan using a rolling-circle amplification method. Thirteen novel anellovirus strains were identified from 8 of 10 masked palm civets and their entire genomic sequences (2039-2535 nucleotides) were determined; they were classifiable into four distinct clades. Comparative analyses of all reported anelloviruses for which the entire or near-entire genomic sequences have been determined, including the 13 strains obtained in the present study, revealed that anelloviruses can provisionally be classified into 20 clades, which may correspond to 20 genera (including 11 assigned and four proposed genera) by a >70% amino acid sequence difference in open reading frame 1 (ORF1). This study suggested that novel anelloviruses of marked diversity are circulating in animals worldwide, and that the rolling-circle amplification method would be useful for identifying novel anelloviruses and other viruses with a circular DNA genome.

Influenza A virus infection in Japanese wild boars (Sus scrofa leucomystax).

Serum samples were collected from 385 wild boars between 2010 and 2013 to examine the seroprevalence of influenza A virus (IAV) in Japan. Antibodies against IAV were identified using a commercial kit in 13 wild boars (3.4%). To identify the serotypes, positive sera were examined by virus-neutralization test using representative serotypes and strains. Three wild boars in Yamaguchi and four in Tochigi showed the highest antibody titers against the pandemic H1N1 2009 virus and classical swine H1N1 virus strains, respectively. These data indicate that wild boars may have close contact with humans and domestic pigs and therefore that there is potential for IAVs to reassort in wild boars as they have been shown to do in pigs.