Outline of the Completed Research

Molecular Biology

Antigenic analysis of Japanese equine influenza vaccine strains and recent epidemic strains by using horse antisera (2012-2014)

 The protection induced by an equine influenza (EI) vaccine strain depends on its antigenic relatedness to the challenge virus. Despite the World Organisation for Animal Health (OIE) recommendation to include both Florida sublineage clade 1 (Fc1) and clade 2 (Fc2) viruses in EI vaccines, Japanese vaccines have yet to be updated to include an Fc2 virus. Here, we evaluated the efficacy of antibodies raised against Japanese vaccine strains in neutralizing recently isolated strains. Antiserum raised against Japanese vaccine strain A/equine/Ibaraki/1/2007 cross-neutralized the recent Fc1 strains isolated in the US at a titer similar to the homologous one. In contrast, antiserum raised against Japanese vaccine strain A/equine/La Plata/1993 exhibited poor cross-neutralizing activity against the Fc2 viruses isolated recently in Ireland and the UK, which have an alanine to valine substitution at position 144 of antigenic site A on the hemagglutinin gene. In contrast, this antiserum exhibited good cross-neutralizing activity against Fc2 viruses without the mutation. This suggests that the efficacy of the Japanese vaccine against some of the recent Fc2 viruses is suboptimal.

Characterization of interferon gamma synthesis by equine herpesvirus type 1 (EHV-1) specific lymphocytes in horses vaccinated with a modified live virus vaccine containing a glycoprotein E deletion mutant of EHV-1 (2011-2013)

 Cellular immunity has been considered to be important for the control of equine herpesvirus type 1 (EHV-1) infection. The aim of this study was to characterize interferon gamma (IFNγ) synthesis by EHV-1 specific lymphocytes in horses vaccinated with a modified live virus (MLV) vaccine containing a glycoprotein E deletion mutant of EHV-1. Thoroughbred horses aged 1 year received the MLV vaccine once or twice at a 4-week interval. The IFNγ mRNA expression by EHV-1 specific lymphocytes significantly increased 1 week after the first vaccination (6- to 36-fold increase). The increased expression of IFNγ mRNA decreased the following week (30 to 80% decrease). In 2 out of 4 horses that were given 2 doses of the vaccine, about 2-fold increase of the IFNγ mRNA expression was observed 1 week after the second vaccination. This increment also decreased the following week. Seven weeks after the first vaccination, the IFNγ mRNA expression began to increase in all horses, and the expression level remained significantly higher than that observed before the vaccination until 12 weeks after the first vaccination. The vaccinated horses were challenged with EHV-1 at 12 weeks after the first vaccination. From day 5 to day 8 post-infection, all challenged horses developed pyrexia (≧39.0℃), which was not distinguishable from that observed in non-vaccinated horses. The IFNγ mRNA expression in the vaccinated horses decreased 1 week after the challenge infection. Therefore it was considered that the suppression of cellular immunity might lead to the development of clinical symptoms of EHV-1 infection.

Development of a serological test which can distinguish horses inoculated with a modified live vaccine of equine herpesvirus type-1 and those infected with EHV-1 naturally (2010-2013) (Joint research conducted with Yamaguchi University)

 A modified live vaccine (MLV) for equine herpesvirus type-1 (EHV-1) was developed by the Equine Research Institute, and is under preparation for a clinical use. After introduction of the MLV into the field, a currently used ELISA for EHV-1 may not be available because the antigen used in the ELISA may react with the antibodies induced by the MLV immunization. To overcome the problem, we developed the novel ELISA which can distinguish the horses inoculated with the MLV and those infected with EHV-1 naturally. Because the MLV lacks glycoprotein E gene, we focused on a major epitope region of the gE protein and developed the ELISA using a synthetic peptide of the region as antigen. The gE-peptide-ELISA detected the antibodies induced by EHV-1-infection specifically, and did not react with antibodies induced by MLV immunization. This ELISA, in combination with the ELISA using an extract of EHV-1-infected cells as antigen, enables us to distinguish the horses inoculated with the MLV and those infected with EHV-1 naturally. Furthermore, the peptide ELISA using glycoprotein G of equine herpesvirus type-4 (EHV-4) was also developed, and found to be useful in detecting EHV-4-infected horses specifically.

Infectivity and pathogenesis of influenza A virus (H3N8) (2009-2011)

 Since equine influenza A virus (H3N8) was transmitted to dogs in the United States in 2004, the causative virus, a canine influenza A virus (CIV), has become widespread in dogs. In this study, we investigated (1) whether EIV-infected horses could transmit EIV to dogs and (2) whether CIV-infected dogs could transmit CIV to horses in experimental conditions.
(1) Three pairs of a horse inoculated with EIV and a healthy dog were kept together in individual stalls for 15 days. All horses showed typical clinical signs and seroconversion. All dogs showed seroconversion but antibody titers were low and one of three dogs did not show virus shedding. (2) Three pairs of a dog inoculated with CIV and a healthy horse were kept together in individual stalls for 15 days. Although all CIV-infected dogs showed clinical signs, virus shedding, and seroconversion, no paired horses showed any clinical signs, virus shedding or seroconversion. These findings may indicate that a single dog infected with CIV is not sufficient to constitute a source of CIV infection in horses.

Study on the capability of transmission of African horse sickness virus by Japanese midges (Joint research conducted with National Institute of Animal Health) (2009-2011)

 To evaluate the capability of Japanese midges for transmission of African horse sickness virus (AHSV), we have developed highly sensitive real-time RT-PCR that can detect all nine serotypes of AHSV with the almost same sensitivities. Phylogenetic analysis revealed that Culicoides imicola, a major vector of AHSV in Africa, is closely related in some Japanese midge species. It has been shown that AHSV could be propagated in some level in some Japanese midge species, which have been captured by light trap and fed with blood containing AHSV, but growth level in Japanese midges seemed to be much lower than that in Culicoides imicola reported in literatures.

Development of ELISA and real-time RT-PCR for the diagnosis of equine coronavirus infection (Joint research conducted with National Institute of Animal Health) (2009-2011)

 In 2004 and 2009, outbreaks of equine coronavirus infection have occurred among draft horses in a racecourse in Hokkaido, Japan. To investigate the epidemiology and the pathogenicity of the virus, we have developed the diagnostic tests. For serological diagnosis, we have developed ELISA using the nucleocapsid protein of equine coronavirus, which was expressed by E. coli expression system, as antigen. For genetic diagnosis, we have developed real-time RT-PCR that can distinguish equine corona virus and closely related bovine corona virus

Nucleotide sequence analysis of neuropathogenic and grouping markers of equine herpesvirus type 1 isolates in Japan (2009-2010)

 The aim of this study was to investigate the prevalence of the neuropathogenic genotype among equine herpesvirus type 1 (EHV-1) isolates in Japan, and to group the isolates based on the nucleotide sequence polymorphism of the open reading frame (ORF) 68, which can be used as the primary marker for grouping field isolates into 6 major groups (Groups 1 to 6). The EHV-1 isolates collected between 1989 and 2010 were analyzed. As a result, the prevalence of the neuropathogenic genotype in Japan was lower than those previously reported in USA, Argentina, France and Germany. The ORF68 nucleotide sequence analysis revealed that most Japanese isolates belong to Group 1, which is minority in both North America and Europe. However, since 2003, the isolates belonging to Group 5, which has been observed mainly in North America, has been increasing in Japan.

Study on the non-structural protein 1 antibody-based immunoassay for the differential diagnosis of West Nile virus infections from Japanese encephalitis in horses (2006-2009)

 West Nile virus (WNV) and Japanese encephalitis virus (JEV) are included in the Japanese encephalitis (JE) serological group of the Flavivirus genus of the Flaviviridae family. There is a possibility that WNV may be introduced into Japan. Since the clinical features of WNV infection are similar to those caused by JEV, the differential diagnosis of WNV from JEV infections is necessary. Although neutralization test provides the highest specificity among currently available serodiagnostic tests, cross-reactivity among members of the JE serological group can affect a differential diagnosis, making it difficult to differentiate between WNV and JEV infections. In this study, we have established two immunoassays to detect antibody in horse sera against non-structural protein 1 (NS1) of WNV. First, we generated a monoclonal antibody specific for the nonstructural protein 1 (NS1) of WNV and established an epitope-blocking enzyme-linked immunosorbent assay (ELISA) that can differentiate WNV from JEV infections in horse sera. However, the ELISA occasionally produce nonspecific reactions, thus making it difficult to reliably measure low levels of specific antibodies. Next, to minimize nonspecific reactions, we introduced the principle of antibody-mediated complement-dependent cytotoxicity (CDC) into an antibody assay and demonstrated the utility of this CDC assay for differentiating WNV from JEV infections in horses. We examined 2,400 racehorse sera collected between 2006 and 2008 in two JRA Training Centers using ELISA and CDC assays and found no evidence of invasion of WNV into racehorse population in Japan.

Safety evaluation and experiments to study effects of live vaccine against equine rhinopneumonitis: Experimental research necessary when applying for approval to manufacture live vaccine (2006-2008)
(Joint research conducted with Nippon Institute for Biological Science and Research Institute for Animal Science in Biochemistry)

 Research necessary when applying for approval to manufacture live vaccine against equine rhinopneumonitis was conducted in this study. The research involved tests on the cohabitation infectivity, pathogenic reversion potential and reactivation potential of live vaccine, and tests on the comparative effects of live vaccine with existing commercial inactive vaccines. In every case, significant results were obtained with a view to applying for approval to manufacture the vaccine. Toxicology and storage stability tests were conducted by the Nippon Institute for Biological Science, while safety tests and field tests on the vaccine were conducted by the Research Institute for Animal Science in Biochemistry. In each case, significant results were obtained with a view to applying for approval to manufacture the vaccine.

Development of serological diagnosis methods for equine coronavirus infection (2006-2008)
(Joint research conducted with National Institute of Animal Health)

 The aim of this study was to develop a neutralization test and an ELISA method for equine coronavirus. It was confirmed that the equine coronavirus NC99 strain propagates well in Vero cells, accompanied by CPE. As a result, a neutralization test for equine coronavirus using Vero cells was established. Meanwhile, equine coronavirus NP genes were detected from the fecal samples of febrile horses at a draft-horse racecourse in Hokkaido, where a mass outbreak had occurred. The expressed protein obtained by combining these genes with E. coli showed specific reaction to equine coronavirus immune rabbit serum. Therefore, it is expected that an ELISA method for detecting antibodies in infected horses can be developed by using this expressed protein as an antigen.

Investigation of gene regions useful for genetic diagnosis of vesicular stomatitis virus and their application to diagnosis (2006-2008)
(Joint research conducted with National Institute of Animal Health)

 The aim of this study was to develop a new method of genetic diagnosis for vesicular stomatitis and to develop a quick method of testing antibodies allowing a large number of samples to be processed. PCR-ELOSA was developed as a method that enables genes of the two serotypes of vesicular stomatitis virus (the New Jersey type and the Indiana type) to be detected quickly and efficiently. In addition, monoclonal antibodies were used to develop a competitive ELISA method for detecting serum antibodies against New Jersey type virus, irrespective of the species of animal.

Establishment of IgM-captured ELISA test on Japanese encephalitis (2005-2006)

 Ever since it entered the USA in 1999, the danger of West Nile Virus (WNV) being introduced into Japan has been increasing. In a previous research project, we established WNV-IgM captured ELISA, using WNV as an antigen, as a diagnostic method for WNV infected horses, and incorporated this in a pathological appraisal manual based on the Prevention Manual of WNV infection in Japan. However, although WNV has similar antigenicity to Japanese Encephalitis Virus (JEV), its cross-reactivity with IgM ELISA has yet to be studied. In this study, therefore, we first established a technique for IgM ELISA using JEV as an antigen (JEV- IgM captured ELISA), then studied cross-reactivity with JEV and WNV-IgM ELISA using the serum of horses infected by both viruses.

 1. We studied the reproductivity of JEV and WNV in Vero cells and C6/36 cells. As a result, both viruses showed high reproductivity in C6/36 cells, and it was indicated that the virus titers would peak on the 4th to 5th day after inoculation. In view of these results, we decided to retrieve and inactivate the supernatant in this period and prepare an antigen for IgM ELISA.
 2. Using the post-serum of two horses experimentally infected with WNV, we studied cross-reactivity with JEV and WNV-IgM ELISA. As a result, horse No.1, which showed high reactivity to WNV antigens, also showed positivity to JEV antigens due to cross-reaction on the 14th day, when antibody titers reach their peak. In horse No.2, which had weak reactivity to WNV, reactivity to JEV remained negative, however.
 3. We studied cross-reactivity with JEV and WNV-IgM ELISA using the serum of JEV experimentally infected horses and eight naturally infected horses. As a result, antibody titers against the homogenous JEV antigens showed higher values than those against WNV antigens in all serum samples.
 4. We studied reactivity with JEV and WNV-IgM captured ELISA in the serum of yearlings in the Hidaka region both before and after inoculation with JEV vaccine (140 samples and 30 samples, respectively). As a result, reactivity to both types of ELISA was negative in all serum samples. From this result, it was shown that, as with WNV-IgM, JEV-IgM captured ELISA also does not detect antibodies in vaccinated horses. Meanwhile, the rate of appearance of non-specific reactions was estimated to be no more than 1% (as 0 of 140 samples tested positive). Next, to study the status of JEV infection among outdoor horses, we studied JEV and WNV-IgM captured ELISA reactivity of racehorse serum sampled at the Ritto Training Center during the periodic testing in November 2005 (70 samples obtained from horses inoculated regularly with JEV vaccine). As a result, all of the samples tested this time were negative for both types of ELISA.

Epidemiological surveillance of equine infectious disease in breeding areas (2004-2006)

 As horseracing becomes progressively more global, we carry out continuous epidemiological surveillance in breeding areas in response to contagions of equine infectious diseases not only overseas but also in Japan.

 1. Equine arteritis; As a result of antibody tests conducted on a total of 499 horses, it was confirmed that unvaccinated horses were antibody negative and that there was no rise in the antibody titers of vaccinated horses.
 2. Influenza: On conducting antibody tests for a total of 497 yearlings, all except one vaccinated imported horse were negative.
 3. Equine rhinopneumonitis; Cohabitant yearlings in farms that had suffered a spate of EHV-1 miscarriages had a high rate of antibody positivity towards equine herpes virus type 1 (EHV-1). The possibility was suggested that pregnant mares and cohabitant yearlings play a part in the transmission and propagation of EHV-1 inside farms.
 4. Rotavirus infection; The rotavirus positive rate of diarrheic stools over three years was 56%, and the majority of diarrhea over several months after birth was due to rotavirus infection.
 5. Rhodococcus equi infection; We showed that cytological diagnosis using bronchoalveolar lavage is useful as a test method for this disease.
 6. Strangles, equine protozoal myeloencephalitis (EPM); In both cases, no incidences or antibody positive horses were observed during the study period.

 Equine arteritis and equine influenza have not been introduced. Equine rhinopneumonitis, rotavirus infection and Rhodococcus equi infection occurred continuously, but there was no incidence of strangles, and no horses were found to be antibody positive to EPM.