Yutaka Terada

Phylogenetic relationships of the benign Theileria species in cattle and Asian buffalo based on the major piroplasm surface protein (p33/34) gene sequences.

In order to examine the taxonomic relationship of Theileria sp. of Asian buffalo to the benign Theileria spp. of cattle, we sequenced and compared the major piroplasm protein (p33/34) genes of these parasites. The two consensus sequences determined for the buffalo parasite were of the same length (852 bp) and showed >80% identity with the sequences of the homologous genes (849 bp) in the cattle parasites. Alignment of the inferred aa sequences with those of Theileria sergenti and Theileria buffeli predicted that there is an insertion of a single residue at the N-terminus in the inferred polypeptide of the buffalo parasite. Phylogenetic analyses based on the aa sequences suggested that Theileria sp. of the Asian buffalo should be classified within the benign Theileria parasite group as a separate species from the cattle parasites. Based on this, we propose a rearrangement of the currently used classification for the benign Theileria species in cattle and Asian buffalo.

Molecular phylogenetic studies on Theileria parasites based on small subunit ribosomal RNA gene sequences

Classification of Theileria parasites of south-east Asian countries is still ambiguous due to the lack of basic studies, especially their molecular genetic information. In this study, we included 6 known species and 14 unclassified Theileria parasite isolates: Theileria annulata, Theileria parva, Theileria taurotragi, Theileria sergenti, Theileria buffeli, Theileria types Sable, Theileria types A, B, B1, B2, C, D, E, F, G, G1, Theileria type Medan (Indonesia), Theileria type Ipoh (Malaysia) and Theileria type Thong Song (Thailand). Small subunit ribosomal RNA (srRNA) nucleotide sequence data were collected by PCR, cloning and dideoxy sequencing. The srRNA nucleotide sequences were aligned and analyzed by distance methods, maximum parsimony algorithms and maximum likelihood methods to construct phylogenetic trees. Bootstrap analysis was used to test the strength of the different phylogenetic reconstructions. The data indicated that all of the tree-building methods gave very similar results. This study identified two groups of Theileria, the pathogenic and benign groups, which are strongly supported by bootstrap analysis. The analysis also indicated that three subgroups (A, B and C) were generated within the benign Theileria group whereas the classification of Theileria type D and Thong Song is questionable. However, more basic information such as life cycle differences, vectors, modes of transmission, virulent and genetic/sexual compatability is essential for clearer taxonomic definition of the benign Theileria parasites.

Emergence of pathogenic coronaviruses in cats by homologous recombination between feline and canine coronaviruses.

Type II feline coronavirus (FCoV) emerged via double recombination between type I FCoV and type II canine coronavirus (CCoV). In this study, two type I FCoVs, three type II FCoVs and ten type II CCoVs were genetically compared. The results showed that three Japanese type II FCoVs, M91-267, KUK-H/L and Tokyo/cat/130627, also emerged by homologous recombination between type I FCoV and type II CCoV and their parent viruses were genetically different from one another. In addition, the 3′-terminal recombination sites of M91-267, KUK-H/L and Tokyo/cat/130627 were different from one another within the genes encoding membrane and spike proteins, and the 5′-terminal recombination sites were also located at different regions of ORF1. These results indicate that at least three Japanese type II FCoVs emerged independently. Sera from a cat experimentally infected with type I FCoV was unable to neutralize type II CCoV infection, indicating that cats persistently infected with type I FCoV may be superinfected with type II CCoV. Our previous study reported that few Japanese cats have antibody against type II FCoV. All of these observations suggest that type II FCoV emerged inside the cat body and is unable to readily spread among cats, indicating that these recombination events for emergence of pathogenic coronaviruses occur frequently.

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.

Genetic characterization of coronaviruses from domestic ferrets, Japan.

We detected ferret coronaviruses in 44 (55.7%) of 79 pet ferrets tested in Japan and classified the viruses into 2 genotypes on the basis of genotype-specific PCR. Our results show that 2 ferret coronaviruses that cause feline infectious peritonitis–like disease and epizootic catarrhal enteritis are enzootic among ferrets in Japan.

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.

Identification of a major immunogenic region of equine herpesvirus-1 glycoprotein E and its application to enzyme-linked immunosorbent assay.

A major immunogenic region of equine herpesvirus (EHV)-1 glycoprotein E (gE) was identified. Firstly, the various fragments of EHV-1 gE were expressed as fusion proteins with glutathione S-transferase (GST) in Escherichia coli and their antigenicities were compared by immunoblot analysis using sera from horses experimentally infected with EHV-1. Thirty-three amino acids of gE (a.a. 169-201) specifically and sensitively reacted with the antibodies induced by EHV-1 but not EHV-4 infection. The corresponding region of EHV-4 gE (a.a. 169-199) did not react with antibodies to EHV-1, indicating that this region is specific for each virus. In addition, when the antigenicities of three 20-mer synthetic peptides of EHV-1 gE, gE1(169-188), gE1(176-195) and gE1(182-201) were compared by enzyme-linked immunosorbent assay (ELISA), gE1(169-188) was found to contain a major B-cell epitope. ELISA using two synthetic peptides, gE1(169-188) and gG4(319-330), previously identified as the major EHV-4-specific epitope in gG, was developed and could specifically detect antibodies to EHV-1 and EHV-4, respectively. In Japan, the EHV-1 deleted in gE gene (EHV-1 ΔgE) virus is expected to be introduced in the field as a new modified live vaccine. This ELISA did not react with antibodies induced by inoculation with EHV-1 ΔgE, indicating that it is a useful method to differentiate between EHV-1 infection and EHV-1 ΔgE inoculation. In conclusion, the ELISA described herein, using synthetic peptides, is a simple method to distinguish between EHV-1 and EHV-4 infections and will be suitable as a vaccine marker after introduction of EHV-1 ΔgE into field horses.

Development and application of an indirect enzyme-linked immunosorbent assay for serological survey of Japanese encephalitis virus infection in dogs.

Japanese encephalitis virus (JEV) causes serious acute encephalitis in humans and horses. Although dogs are good sentinels for assessing the risk of JEV infection to humans, a virus neutralization test has been the only method available for measuring the levels of JEV antibody in dogs. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) using purified viral particles as an antigen, was developed for serological survey of JEV infection in dogs. In dogs inoculated experimentally with JEV, the ELISA detected anti-JEV IgM 3 days after infection, with IgM levels peaking 7 days after infection. Anti-JEV IgG was detected 14 days after infection and peaked on 21-28 days after infection. Virus neutralization titers correlated with anti-JEV immunoglobulins measured by the ELISA. To test the utility of the new assay, the seroprevalence of JEV infection among 102 dogs in Kyushu, Japan, was examined by IgG ELISA and by virus neutralization. The correlation coefficient between the IgG ELISA and virus neutralization was 0.813 (p<0.001); comparison of the IgG ELISA and virus neutralization showed a sensitivity and specificity of 82% and 98%, respectively. The IgG ELISA was used to survey dogs in Bangkok, Thailand and 51% of these dogs were found seropositive for JEV. These data suggest that in the capital city of Thailand, the risk of infection with JEV remains high.

Clearance of Theileria sergenti-infected bovine red blood cells in severe combined immune deficiency mice

Clearance of Theileria sergenti-infected bovine red blood cells (Bo-RBCs) from the blood circulation of severe combined immune deficiency (SCID) mice was studied to help understand the mechanisms of anemia developing in cattle infected with T. sergenti. For the clearance test, Bo-RBC samples having 2%, 58%, and 76% parasitemia and, as a control, parasite-free Bo-RBCs were prepared in the Bo-RBC-SCID mouse model. The T. sergenti-infected Bo-RBCs and the uninfected control Bo-RBCs were separately labeled with two, green and red, fluorescent dyes, mixed together, and injected intravenously into SCID mice. The blood samples collected at various time points were observed under a fluorescent microscope, and the numbers of green and red fluorescing RBCs were counted differentially to determine the clearance rates of T. sergenti-infected and uninfected Bo-RBCs. This test clearly demonstrated that the Bo-RBC samples having higher parasitemias were cleared faster from the blood circulation of SCID mice. The results suggest that the intravascular clearance system in SCID mice may have a mechanism by which T. sergenti-parasitized and non-parasitized Bo-RBCs are recognized and cleared differentially.

Canine distemper virus infection among wildlife before and after the epidemic

In 2007–2008, a canine distemper virus (CDV) epidemic occurred among wild animals in Wakayama Prefecture, Japan, and many mammals, including the wild boar and deer, were infected. In this study, CDV prevalence among wild animals was surveyed before and after the epidemic. At first, an enzyme-linked immunosorbent assay (ELISA) with horseradish peroxidase-conjugated protein A/G was established to detect CDV antibodies in many mammalian species. This established ELISA was available for testing dogs, raccoons and raccoon dogs as well as virus-neutralization test. Next, a serological survey of wild mammalians was conducted, and it was indicated that many wild mammalians, particularly raccoons, were infected with CDV during the epidemic, but few were infected before and after the epidemic. On the other hand, many raccoon dogs died during the epidemic, but CDV remained prevalent in the remaining population, and a small epidemic occurred in raccoon dogs in 2012–2013. These results indicated that the epidemic of 2007–2008 may have been intensified by transmission to raccoons.