Kenzo Yonemitsu

High Prevalence of Hepatitis E Virus in Wild Boar (Sus scrofa) in Yamaguchi Prefecture, Japan

Abstract Hepatitis E virus (HEV) causes a food- and water-borne disease in humans, and Japanese wild boar (Sus scrofa leucomystax) meat is one of the most important sources of infection in Japan. We tested 113 serum samples from wild boar captured in Shimonoseki City, Yamaguchi Prefecture, Japan from 2010 to 2012. Serum samples were tested by enzyme-linked immunosorbent assay (ELISA) using virus-like particles as antigen and nested reverse-transcription PCR (RT-PCR). Anti-HEV IgG antibodies were detected in 47 of the 113 wild boar serum samples (42%), and HEV RNA was detected in five samples (4%). Sequence analysis showed that the five HEV isolates belonged to genotype 4, forming a cluster with a previous isolate from a human hepatitis E case in this region in 2011. These results indicate that wild boar in this region are infected with potentially pathogenic HEV at a high prevalence.

Ferret hepatitis E virus infection in Japan.

We examined 85 fecal samples from pet ferrets in 10 animal hospitals in Japan for the detection of ferret hepatitis E virus (HEV) RNA. We found that 6 (7.1%) of the samples were positive for ferret HEV RNA. Phylogenetic analysis based on the partial ORF1 indicated that these ferret HEV strains were clearly separated from the Netherlands strains and were divided into 2 distinct clusters. These results suggest that ferret HEV is genetically diverse, and since ferrets are not indigenous to Japan, ferret HEV has been introduced into Japan through importation.

Epidemiological study of relapsing fever borreliae detected in Haemaphysalis ticks and wild animals in the western part of Japan

The genus Borrelia comprises arthropod-borne bacteria, which are infectious agents in vertebrates. They are mainly transmitted by ixodid or argasid ticks. In Hokkaido, Japan, Borrelia spp. were found in deer and Haemaphysalis ticks between 2011 and 2013; however, the study was limited to a particular area. Therefore, in the present study, we conducted large-scale surveillance of ticks and wild animals in the western part of the main island of Japan. We collected 6,407 host-seeking ticks from two regions and 1,598 larvae obtained from 32 engorged female ticks and examined them to elucidate transovarial transmission. In addition, we examined whole blood samples from 190 wild boars and 276 sika deer, as well as sera from 120 wild raccoons. We detected Borrelia spp. in Haemaphysalis flava, Haemaphysalis megaspinosa, Haemaphysalis kitaokai, Haemaphysalis longicornis, and Haemaphysalis formosensis. In addition, we isolated a strain from H. megaspinosa using Barbour-Stoenner-Kelly medium. The minimum infection rate of ticks was less than 5%. Transovarial transmission was observed in H. kitaokai. Phylogenetic analysis of the isolated strain and DNA fragments amplified from ticks identified at least four bacterial genotypes, which corresponded to the tick species detected. Bacteria were detected in 8.4%, 15%, and 0.8% of wild boars, sika deer, and raccoons, respectively. In this study, we found seasonal differences in the prevalence of bacterial genotypes in sika deer during the winter and summer. The tick activity season corresponds to the season with a high prevalence of animals. The present study suggests that a particular bacterial genotype detected in this study are defined by a particular tick species in which they are present.

Simple and specific method for detection of antibodies against hepatitis E virus in mammalian species

Hepatitis E virus (HEV) is the causative agent of hepatitis E, a food- and water-borne disease. In developed countries, consumption of meats from pigs, wild boars and deer is a major source of infection. Although HEV and HEV-related viruses have been detected in many animal species, their zoonotic potential and prevalence has not been completely understood. To detect anti-HEV antibody in mammalian species, a simple enzyme-linked immunosorbent assay (ELISA) was established using extract from cells expressing HEV capsid protein and protein A/G as an antigen and a reagent for detection of antibody. Absorbance in the ELISA was compared with those in our previous ELISA using VLPs and anti-swine antibody, suggesting that newly established ELISA was similarly specific and sensitive as the previous ELISA. Seroprevalence of HEV infection among wild boars was examined in Yamaguchi Prefecture, confirming that 111 of 364 wild boars (30.5%) were positive for anti-HEV antibody. Next, this ELISA was applied to humans, dogs, cats, ferrets, raccoons and masked palm civets in Japan, and anti-HEV antibodies were detected in humans, ferrets, dogs and cats. This ELISA is thus useful for serological surveys and comparison of HEV infection among various mammals, including humans.

Isolation and phylogenetic analysis of canine distemper virus among domestic dogs in Vietnam

Canine distemper virus (CDV) is one of the most serious pathogens found in many species of carnivores, including domestic dogs. In this study, hemagglutinin (H) genes were detected in five domestic Vietnamese dogs with diarrhea, and two CDVs were successfully isolated from dogs positive for H genes. The complete genome of one isolate, CDV/dog/HCM/33/140816, was determined. Phylogenetic analysis showed that all Vietnamese CDVs belonged to the Asia-1 genotype. In addition, the H proteins of Vietnamese CDV strains were the most homologous to those of Chinese CDVs (98.4% to 99.3% identity). These results indicated that the Asia-1 genotype of CDV was the predominant genotype circulating among the domestic dog population in Vietnam and that transboundary transmission of CDV has occurred between Vietnam and China.

Detection of novel ferret coronaviruses and evidence of recombination among ferret coronaviruses.

In an epidemiological study of ferret coronaviruses (FRCoVs), novel FRCoV strains (Saitama-1 and Aichi-1) were detected by reverse transcription-polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of partial RNA-dependent RNA polymerase (RdRp) genes. Phylogenetic analysis indicated that these strains belonged to different clusters from other FRCoV strains. Next, the nucleotide sequence of the 3′-terminal region of Saitama-1 (8271 bases) strain was determined and compared with those of the other FRCoVs, indicating that the Saitama-1 strain differed from the previously reported MSU-1 and MSU-2 strains in the regions encoding spike (S) protein, nucleocapsid, and open reading frame 7b. Furthermore, the results of SimPlot analysis indicated that FRCoV (MSU-2 strain) emerged via a recombination event of S protein between the MSU-1 and Saitama-1 strains. This mechanism is similar to that responsible for the emergence of type II feline coronavirus. This information will be useful for understanding the pathogenesis of FRCoV in ferrets.

Getah virus epizootic among wild boars in Japan around 2012

In 2014, an outbreak of Getah virus (GETV) infection occurred in Japan in a horse population that was inoculated with a vaccine against GETV. In this study, we investigated the seroprevalence of GETV infection among wild boars in Japan. Interestingly, the highest rate of anti-GETV-positive wild boars was observed in 2013, which gradually decreased during 2014–2016. The results suggested that GETV spread among wild boars around 2012, resulting in the 2014 outbreak.

The genetic diversity of D-loop sequences in eastern bent-winged bats (Miniopterus fuliginosus) living in Wakayama Prefecture, Japan

The eastern bent-winged bat (Miniopterus fuliginosus) is an insectivorous bat that lives in the caves, throughout Japan [11]. The bats aggregate in cave in populations of tens to thousands of individuals. We examined the mitochondrial D-loop sequences of bats in Wakayama, Japan, and divided them into 35 haplotypes. The sequences of 3 haplotypes in Wakayama were the same as those of 10 Miniopterus fuliginosus individuals living in China. Given the substitution rate of the D-loop region, we speculated that the bats had moved between Japan and China within the last 16,000 years. We could not determine how the bats crossed the sea; however, it is possible that the bats undergo dynamic movement widely throughout East Asia.

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.

Characterization of canine coronavirus spread among domestic dogs in Vietnam

Canine coronavirus (CCoV) is an important pathogen that causes enteritis in dogs, but there is no information on CCoV infection in Vietnam. To examine the prevalence of CCoV infection among Vietnamese dogs, 201 serum samples were analyzed by virus-neutralization (VN) test. The results showed that antibody against CCoV-II was present in 87 dogs (43.3%). To detect genes of CCoV, fecal samples collected from 30 diarrheic and 50 healthy dogs were examinated by RT-PCR, confirming that 2 diarrheic dogs and 5 healthy dogs were positive for CCoV. Nucleotide sequences of N-terminal region of spike (S) gene indicated that CCoV strains were divided into two subgenotypes, CCoV-IIa and -IIb, respectively. Furthemore, we succeeded in isolating CCoV/dog/HCM47/2015, the isolate was plaque-purified three times, and 3’-terminal one-third of the genome was analyzed. Interestingly, the plaque-purified virus had a large deletion in ORF3abc and E genes (1,165 nt), and a short deletion in ORF7b gene (60 nt), suggesting that these regions are not necessary for in vitro replication of CCoV. Next, the antigenicity between the isolated CCoV-IIb and the other CCoV-IIa was compared by VN test, revealing that antigenicty of the isolated CCoV is equal or higher than that of the other CCoV. In summary, two subgenotypes of CCoV-II are spreading among Vietnamese dogs. The isolated virus with a large deletion after in vitro passage may be useful for the development of vaccine, owing to its antigenicity and efficient viral growth in vitro.