Hiroshi Bannai

Toxoplasma gondii Cyclophilin 18-Mediated Production of Nitric Oxide Induces Bradyzoite Conversion in a CCR5-Dependent Manner

ABSTRACT Toxoplasma gondii modulates pro- and anti-inflammatory responses to regulate parasite multiplication and host survival. Pressure from the immune response causes the conversion of tachyzoites into slowly dividing bradyzoites. The regulatory mechanisms involved in this switch are poorly understood. The aim of this study was to investigate the immunomodulatory role of T. gondii cyclophilin 18 (TgCyp18) in macrophages and the consequences of the cellular responses on the conversion machinery. Recombinant TgCyp18 induced the production of nitric oxide (NO), interleukin-12 (IL-12), and tumor necrosis factor alpha through its binding with cysteine-cysteine chemokine receptor 5 (CCR5) and the production of gamma interferon and IL-6 in a CCR5-independent manner. Interestingly, the treatment of macrophages with TgCyp18 resulted in the inhibition of parasite growth and an enhancement of the conversion into bradyzoites via NO in a CCR5-dependent manner. In conclusion, T. gondii possesses sophisticated mechanisms to manipulate host cell responses in a TgCyp18-mediated process.

The potential impact of a single amino‐acid substitution on the efficacy of equine influenza vaccines

REASONS FOR PERFORMING STUDY The protection induced by an equine influenza (EI) vaccine strain depends on its antigenic relatedness to the challenge virus. Although the World Organisation for Animal Health (OIE) recommend that both Florida sublineage clade 1 (Fc1) and clade 2 (Fc2) viruses should be included in EI vaccines, Japanese EI vaccines have not, thus far, been updated to include a Fc2 virus. OBJECTIVES To evaluate the efficacy of antibodies raised against Japanese EI vaccine strains in the neutralisation of recent Fc2 viruses. STUDY DESIGN Antigenic analysis. METHODS Virus neutralisation tests were performed using antisera from experimentally infected horses and from horses that had received a primary course of the currently available vaccines. RESULTS Antiserum raised against the Japanese EI vaccine strain, A/equine/La Plata/1993, exhibited poor cross-neutralising activity against the Fc2 viruses isolated recently in Ireland and the UK, which have the substitution of alanine to valine at position 144 in antigenic site A of the haemagglutinin gene. In contrast, the antiserum exhibited good cross-neutralising activity against the Fc2 viruses without the substitution. This finding was supported in experiments with antisera collected from vaccinated horses. CONCLUSIONS This suggests that the efficacy of the Japanese EI vaccine for some of the recent Fc2 viruses is suboptimal and that vaccines should be updated in accordance with the OIE recommendations.

Efficacy of a single intravenous dose of the neuraminidase inhibitor peramivir in the treatment of equine influenza

Equine influenza A virus (EIV) of the H3N8 subtype is an important pathogen causing acute respiratory disease in horses. Peramivir is a selective inhibitor of the influenza virus neuraminidase (NA). The characteristics of peramivir are not only its capacity for parenteral administration, but also its strong affinity for NA and slow off-rate from the NA-peramivir complex, suggesting that it could lead to a prolonged inhibitory effect and thus allow a lower dosing frequency. The aims of this study were to evaluate the inhibitory efficacy of peramivir against the NA activities of EIV in vitro and the treatment efficacy of a single intravenous dose of peramivir in horses experimentally infected with EIV. Peramivir inhibited the activities of NA from the seven contemporary EIV strains in vitro, with 50% inhibitory concentrations ranging from 0.10 to 0.20 nmol/L. Horses treated with a single IV dose of peramivir (3,000 mg/600 mL/animal, 7.8-9.3mg/kg of bodyweight) showed significantly milder clinical signs (pyrexia, nasal discharge and cough) with a shorter duration than control horses injected with normal saline. Moreover, the mean duration of virus shedding for the horses treated with peramivir was significantly shorter than for the control horses. These findings suggested that a single IV administration of peramivir had good potential for the treatment of equine influenza, and may help to limit the spread of the disease in the horse population.

Development and evaluation of a reverse transcription loop-mediated isothermal amplification assay for H3N8 equine influenza virus.

Reverse transcription loop-mediated isothermal amplification (RT-LAMP) was applied to the detection of equine influenza virus (EIV). Because equine influenza is caused currently by EIV of the H3H8 subtype, the RT-LAMP primer set was designed to target the hemagglutinin gene of this subtype. The detection limit of the RT-LAMP assay was a virus dilution of 10(-5); which was 10(3) times more sensitive than the Espline Influenza A&B-N test and 10 times more sensitive than a reverse transcription polymerase chain reaction (RT-PCR) assay. The specificity of the RT-LAMP assay was examined by using several equine pathogens and nasal swabs collected from horses with fever in 2010 after EIV was eradicated in Japan. No cross-reactions were observed. Using 100 nasal swabs collected from horses with fever during an EIV outbreak in 2007, the RT-LAMP assay detected EIV in 52 samples, whereas the Espline test and the RT-PCR assay detected EIV in only 17 and 41 samples, respectively. These results indicate that the RT-LAMP assay is specific for EIV and more sensitive than the Espline test and the RT-PCR assay. Because it provides high sensitivity and ease of manipulation without the need for a thermal cycler or gel electrophoresis, the RT-LAMP assay should be applicable for laboratory diagnosis of EIV.

Getah Virus Infection among Racehorses, Japan, 2014.

An outbreak of Getah virus infection occurred among racehorses in Japan during September and October 2014. Of 49 febrile horses tested by reverse transcription PCR, 25 were positive for Getah virus. Viruses detected in 2014 were phylogenetically different from the virus isolated in Japan in 1978.

No evidence of horizontal infection in horses kept in close contact with dogs experimentally infected with canine influenza A virus (H3N8)

BackgroundSince equine influenza A virus (H3N8) was transmitted to dogs in the United States in 2004, the causative virus, which is called canine influenza A virus (CIV), has become widespread in dogs. To date, it has remained unclear whether or not CIV-infected dogs could transmit CIV to horses. To address this, we tested whether or not close contact between horses and dogs experimentally infected with CIV would result in its interspecies transmission.MethodsThree pairs of animals consisting of a dog inoculated with CIV (108.3 egg infectious dose50/dog) and a healthy horse were kept together in individual stalls for 15 consecutive days. During the study, all the dogs and horses were clinically observed. Virus titres in nasal swab extracts and serological responses were also evaluated. In addition, all the animals were subjected to a gross pathological examination after euthanasia.ResultsAll three dogs inoculated with CIV exhibited clinical signs including, pyrexia, cough, nasal discharge, virus shedding and seroconversion. Gross pathology revealed lung consolidations in all the dogs, and Streptococcus equi subsp. zooepidemicus was isolated from the lesions. Meanwhile, none of the paired horses showed any clinical signs, virus shedding or seroconversion. Moreover, gross pathology revealed no lesions in the respiratory tracts including the lungs of the horses.ConclusionsThese findings may indicate that a single dog infected with CIV is not sufficient to constitute a source of CIV infection in horses.

Antibody response in vaccinated pregnant mares to recent G3BP[12] and G14P[12] equine rotaviruses

BackgroundBoth the G3P[12] and the G14P[12] type of equine group A rotavirus (RVA) have recently become predominant in many countries, including Japan. G3 types are classified further into G3A and G3B. The G3A viruses have been circulating in Europe, Australia, and Argentina, and the G3B viruses have been circulating in Japan. However, only an inactivated vaccine containing a single G3BP[12] strain is commercially available in Japan. To assess the efficacy of the current vaccine against recently circulating equine RVA strains, we examined antibody responses in pregnant mares to recent G3BP[12] and G14P[12] strains by virus neutralization test.FindingsAfter vaccination in five pregnant mares, the geometric mean serum titers of virus-neutralizing antibody to recent G3BP[12] strains increased 5.3- to 7.0-fold and were similar to that against homologous vaccine strain. Moreover, antibody titers to recent G14P[12] strains were also increased 3.0- to 3.5-fold.ConclusionsThese results suggest that inoculation of mares with the current vaccine should provide foals with virus-neutralizing antibodies against not only the G3BP[12] but also the G14P[12] RVA strain via the colostrum.

Rapid detection of equine coronavirus by reverse transcription loop-mediated isothermal amplification

Abstract A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for the rapid detection of equine coronavirus (ECoV). This assay was conducted at 60°C for 40min. Specificity of the RT-LAMP assay was confirmed using several equine intestinal and respiratory pathogens in addition to ECoV. The novel assay failed to cross-react with the other pathogens tested, suggesting it is highly specific for ECoV. Using artificially synthesized ECoV RNA, the 50% detection limit of the RT-LAMP assay was 101.8 copies/reaction. This is a 50-fold greater sensitivity than conventional reverse transcription polymerase chain reaction (RT-PCR) assays, but a 4-fold lower sensitivity than quantitative RT-PCR (qRT-PCR) assays. Eighty-two fecal samples collected during ECoV outbreaks were analyzed. ECoV was detected in 59 samples using the RT-LAMP assay, and in 30 and 65 samples using RT-PCR or qRT-PCR assays, respectively. Although the RT-LAMP assay is less sensitive than qRT-PCR techniques, it can be performed without the need for expensive equipment. Thus, the RT-LAMP assay might be suitable for large-scale surveillance and diagnosis of ECoV infection in laboratories with limited resources.

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.

Programmed Cell Death 5 from Toxoplasma gondii: a secreted molecule that exerts a pro-apoptotic effect on host cells.

Although parasite-infected host cells become resistant to apoptosis, uninfected bystander cells undergo apoptosis during Toxoplasma gondii infection. The Programmed Cell Death 5 (TgPDCD5) gene, a homologue of the human apoptosis-related molecule, was cloned from a T. gondii full-length cDNA database and subsequently characterized. The native TgPDCD5 was located in the cytosol and also detected in the secreted fraction. Immuno-electron microscopic analysis showed TgPDCD5 was primarily located close to the rhoptries or vesicle-like structures near the surface membrane of the parasite. Studies using recombinant TgPDCD5 (rTgPDCD5) demonstrated that host cells internalize the molecule in a heparan sulfate proteoglycan-binding motif-dependent manner. Furthermore, the addition of rTgPDCD5 to culture medium resulted in the enhancement of host-cell apoptosis triggered by etoposide in macrophage cell line J774A.1 and leukemic cell line HL-60 cells. Additionally, rTgPDCD5 induced apoptosis in J774A.1 cells in the presence of IFN-gamma. This report is the first to identify a parasitic molecule of T. gondii that has a pro-apoptotic effect on host cells.