Performance of a microfluidic immunofluorescence assay kit for equine influenza virus antigen detection.

Equine influenza virus (EIV) infection is one of the most important respiratory diseases in the equine industry around the world. Rapid diagnosis, facilitated by point-of-care testing, is essential to implement movement restrictions and control disease outbreaks. This study evaluated a microfluidic immunofluorescence assay kit, which detects influenza virus and SARS-CoV-2 antigens in human specimens with a 12 min turnaround time, for its potential use in detecting EIV. The microfluidic immunofluorescence assay kit succeeded in detecting 11 EIV strains. Using the real-time reverse transcription polymerase chain reaction as a reference assay, the microfluidic immunofluorescence assay kit showed a sensitivity of 60.7% when evaluating nasopharyngeal swab samples of three horses experimentally infected with EIV. Comparing with the other two rapid antigen detection kits based on immunochromatography and silver amplification immunochromatography, the microfluidic immunofluorescence assay kit exhibited higher sensitivity than the former assay (53.6%) and the same sensitivity as the latter (60.7%). The microfluidic immunofluorescence assay kit did not detect nine non-EIV viruses including one equine coronavirus strain and seven bacteria, suggesting a high specificity for EIV antigens. Similar to other rapid antigen detection kits, the microfluidic immunofluorescence assay kit could be an effective diagnostic tool to detect EIV in the field.

Insight into the population dynamics of pathogenic bacteria causing grapevine crown gall in snowfall areas: snow cover protects the proliferation of pathogenic bacteria.

Grapevine crown gall (GCG) is a significant bacterial disease caused by tumorigenic Allorhizobium vitis (TAV) and is prevalent worldwide. TAV infects grapevines through wounds such as freezing injuries. Although grapevines typically avoid being wounded under snow cover, GCG occurs in many commercial vineyards in snowy regions. This study investigated the TAV population in GCG gall tissues, grapevine skins, and snow on grapevine skins from six infected vineyards located in Hokkaido, Japan, an area known for heavy snowfall. TAV was isolated not only from gall tissues but also from skins and snow on skins throughout the year. Hierarchical Bayesian model (HBM) analysis revealed that the number of TAV cells in gall tissues was affected by cultivar and low temperature, while those in skins were affected by location and low temperature. Additionally, Bayesian changepoint detection (BCD) showed that the number of TAV cells in gall and skin tissues increased during winter, including the snowfall season. Furthermore, the TAV population in grapevine skins under the snow was significantly higher than those above the snow, indicating that TAV under the snow is protected by the snow and can survive well during the snowfall season. This study highlights the ability of TAV to overwinter on/in galls and skins under the snow and act as inoculum for the next season.

Prevalence of serum and salivary virus-neutralizing antibodies against equine coronavirus in four riding stables in Japan.

To assess the prevalence of equine coronavirus infection in riding horses, virus-neutralizing tests were performed on serum and saliva samples collected at four facilities in Japan. Seropositivity rates ranged from 79.2% to 94.6%, suggesting widespread circulation of the virus in these populations. Antibody prevalence in saliva samples from two facilities that had experienced outbreaks in the previous year (67.6% and 71.4%) was significantly higher than at the other facilities without reported outbreaks (41.7% and 45.2%, P<0.05). The presence of salivary antibodies in a high proportion of horses is therefore suggestive of recent exposure to the virus.

Comparison of 4 agar gel immunodiffusion kits for serologic detection of equine infectious anemia virus antibodies.

Using 85 sera collected from horses that had been experimentally infected with equine infectious anemia virus (EIAV) and 200 field sera collected from racehorses in Japan, we compared 4 agar gel immunodiffusion (AGID) kits for serologic detection of EIAV antibodies from Idexx, VMRD, IDvet, and the National Engineering Research Center of Veterinary Biologics, China (NECVB). The positive control lines were sufficiently clear in all kits for evaluation to be made, with slight differences in sharpness: NECVB was the sharpest, followed by VMRD, IDvet, and Idexx. The test results for all 285 samples agreed among the 4 kits, with 62 positives and 223 negatives. The sensitivities and specificities of VMRD, IDvet, and NECVB compared with the Idexx kit were 100%, and the kappa coefficient values between the kits were 1.0 for all combinations. We concluded that the testing capacity of these 4 kits was virtually identical.

Antigenic comparison of H3N8 equine influenza viruses belonging to Florida sublineage clade 1 between vaccine strains and North American strains isolated in 2021-2022.

Equine influenza virus strains of Florida sublineage clade 1 (Fc1) have been circulating in North America. In this study, virus neutralization assays were performed to evaluate antigenic differences between Fc1 vaccine strains and North American Fc1 strains isolated in 2021-2022, using equine antisera against A/equine/South Africa/4/2003 (a vaccine strain recommended by the World Organisation for Animal Health) and A/equine/Ibaraki/1/2007 (a Japanese vaccine strain). Antibody titers against four North American Fc1 strains isolated in 2021-2022 were comparable to those against the homologous vaccine strains. These results suggest that current Fc1 vaccine strains are effective against North American strains from 2021-2022.

Surveillance of Getah virus in mosquitoes and racehorses from 2016 to 2019 at a training center in Ibaraki Prefecture, Japan, a site of several previous Getah virus outbreaks.

Mosquitoes and EDTA-treated blood samples from febrile racehorses were investigated for Getah virus infection from 2016 to 2019 at the Miho Training Center, where several outbreaks of Getah virus have occurred. We collected 5557 mosquitoes and 331 blood samples from febrile horses in this study. The most frequently captured mosquito species was Culex tritaeniorhynchus (51.9%), followed by Aedes vexans nipponii (14.2%) and Anopheles sinensis (11.2%). Getah virus was detected in mosquitoes (Aedes vexans nipponii) in 2016 (strain 16-0810-26) but not in 2017-2019. Six of 74 febrile horses in 2016 and one of 69 in 2019 tested positive for Getah virus; none of the horses tested positive in 2017 or 2018. Phylogenetic and sequence analysis showed that strain 16-0810-26 was closely related to strains that had been isolated from horses and a pig around the training center in 2014-2016 but have not been detected in samples collected at the training center since 2017. In contrast, the strain isolated from the infected horse in 2019 (19-I-703) was genetically distinct from the strains isolated from horses and a pig in 2014-2016 and was more closely related to a strain isolated in 1978 at the training center. The source of strain 19-I-703 is unclear, but the virus was not detected in other horses sampled in 2019. In summary, we found that the distribution of mosquito species present at the training center had not changed significantly since 1979, and although a small outbreak of Getah virus infection occurred among horses at the training center in 2016, limited Getah virus activity was detected in mosquitoes and horses at the training center from 2017 to 2019.

Experimental challenge of horses after prime-boost immunization with a modified live equid alphaherpesvirus 1 vaccine administered by two different routes.

The immune response and protective efficacy of a modified equid alphaherpesvirus 1 (EHV-1) vaccine administered by two different routes were tested in horses. Horses that received intramuscular (IM) priming and an intranasal (IN) booster with a 28-day interval (IM-IN group [n = 6]), IN priming and IM booster (IN-IM group [n = 5]), or no vaccination (control group [n = 6]) were challenged with EHV-1 strain 10-I-224 28 days after the second vaccination. Both vaccinated groups had significantly higher serum virus-neutralizing titers than the control group, with increased levels of serum IgGa, IgGb, and IgA antibodies (p < 0.05). The nasal antibody response was dominated by the IgGa and IgGb subclasses in both vaccinated groups, with no IgA antibody response. After challenge infection, three of six control horses were pyretic for 1-4 days post-inoculation (dpi), whereas none in the vaccinated groups were pyretic during this period. The number of horses that were pyretic at 5-10 dpi was 4 out of 6 for the controls, 3 out of 6 for the IM-IN group, and 2 out of 5 for the IN-IM group. Nasal virus replication in the IN-IM group (3-4 dpi) and IM-IN group (3 dpi) was significantly lower than in the control group (p < 0.05). All of the control horses showed viremia, whereas two horses in the IM-IN group and one in the IN-IM group did not. In conclusion, although IM-IN or IN-IM vaccination did not elicit a mucosal IgA response, it provided partial protection at a level similar to that of the conventional program, likely due to systemic antibodies and mucosal IgG subclass responses.

Serosurveillance of equine coronavirus infection among Thoroughbreds in Japan.

BACKGROUND: Equine coronavirus (ECoV) causes fever, lethargy, anorexia and gastrointestinal signs in horses. There has been limited information about the prevalence and seasonality of ECoV among Thoroughbreds in Japan. OBJECTIVES: To understand the epidemiology and to evaluate the potential risk of ECoV infection to the horse industry in Japan. STUDY DESIGN: Longitudinal. METHODS: The virus-neutralisation (VN) test was performed using sera collected three times a year at 4 months intervals from 161 yearlings and at 6-7 months intervals from 181 active racehorses in Japan in 2017-2018, 2018-2019 and 2019-2020. VN titre ≥1:8 was defined as seropositive, and ≥4-fold increase in titres between paired sera was regarded as indicative of infection. RESULTS: The VN test showed that 44.1% (71/161) of yearlings were seropositive in August, when they first entered the yearling farm. The infection rate was significantly higher between August and December (60.9%, 98/161) than between December and the following April (5.6%, 9/161; p = 0.002). Among the racehorses, it was significantly higher between November and the following May (15.5%, 28/181) than between the preceding April/May and November (0%; p = 0.02). The morbidity rates during the estimated periods of viral exposure were 39.2% in the yearlings and 4% in the racehorses. No horses showed any severe clinical signs. MAIN LIMITATIONS: Clinical records did not cover the period during horses' absence from the training centre. CONCLUSIONS: ECoV was substantially prevalent in Thoroughbred yearlings and racehorses in Japan, and there was a difference in epizootic pattern between these populations in terms of predominant periods of infection. ECoV infection was considered to be responsible for some of the pyretic cases in the yearlings. However, no diseased horses were severely affected in either population, suggesting that the potential risk of ECoV infection to the horse industry in Japan is low.

The survival rate of patients with burns induced by explosions was significantly higher than that of common burn cases: A nationwide observational study using the Japan Trauma Data Bank.

BACKGROUNDS: Blast injuries have a variety of mechanisms, with some cases resulting in immediate death and others resulting in burns as a fourth type of blast injury when the energy of the explosion is relatively low. We reported in 2020, as an incidental result, that burns caused by explosions had a higher survival rate than usual burns caused by other mechanisms. The present study confirmed whether or not burns caused by explosions had higher survival rates than those caused by other mechanisms using the Japan Trauma Data Bank (JTDB), a leading nationwide trauma registry in Japan. METHODS: Burn patients registered to the JTDB database from January 2004 to March 2019 were analyzed retrospectively. The 338,744 patients registered to the JTDB database published in 2021 were identified. After exclusion, 7127 patients met the criteria for inclusion in this study. Logistic regression analyses were conducted for in-hospital survival rates using patients with burns, including cases complicated by usual trauma and burned patients without usual trauma. The survival rates by External burn grade AIS98 were compared between the explosion group and other cause groups using burn cases without usual trauma. RESULTS: The cause of the explosion significantly influenced the survival according to logistic regression analyses using burn groups with and without usual trauma. For AIS 4 and 5, we found significant differences between the explosion group and other cause groups in survival rates among burn cases without usual trauma. CONCLUSION: The survival rate of patients with burns induced by explosions was higher than that of common burn cases according to analyses based on a burn grade of AIS98 among burn cases without common trauma. Multivariate analyses also showed that explosion burns had a significantly better outcome than those induced by other causes.

Study of certain easily available biochemical markers in prognostication in severe traumatic brain injury requiring surgery.

Background: This study aimed to identify easily available prognostic factors in severe traumatic brain injury (TBI) patients undergoing craniotomy. Methods: We retrospectively analyzed the clinical characteristics (age, sex, Glasgow coma scale score, cause of TBI, and oral antithrombotic drug use), laboratory parameters (hemoglobin, sodium, C-reactive protein, D-dimer, activated partial thromboplastin time, prothrombin time-international normalized ratio, and glucose-potassium [GP] ratio), and neuroradiological findings of 132 patients who underwent craniotomy for severe TBI in our hospital between January 2015 and December 2021. The patients were divided into two groups: Those with fatal clinical outcomes and those with non-fatal clinical outcomes, and compared between the two groups. Results: The patients comprised 79 (59.8%) male and 53 (40.2%) female patients. Their mean age was 67 ± 17 years (range, 16-94 years). Computed tomography revealed acute subdural hematoma in 108 (81.8%) patients, acute epidural hematoma in 31 (23.5%), traumatic brain contusion in 39 (29.5%), and traumatic subarachnoid hemorrhage in 62 (47.0%). All 132 patients underwent craniotomy, and 41 eventually died. There were significant differences in the D-dimer, GP ratio, and optic nerve sheath diameter between the groups (all P < 0.01). Multivariate logistic regression analysis showed elevated GP ratio and D-dimer were associated with the death group (P < 0.01, P < 0.01, respectively). A GP ratio of >42 was the optimal cutoff value for the prediction of a fatal outcome of TBI (sensitivity, 85.4%; specificity, 51.1%). Conclusion: The GP ratio and D-dimer were significantly associated with poor outcomes of TBI. A GP ratio of >42 could be a predictor of a fatal outcome of TBI.

Seroconversion of anti-Getah virus antibody among Japanese native Noma horses around 2012.

Getah virus (GETV), an arthropod-borne virus transmitted by mosquitoes, has been isolated from several animals. GETV infection in horses shows clinical signs such as fever, rash, and edema in the leg. Noma horses are one of the eight Japanese native horses. The present study aimed to clarify the occurrence of GETV infection in Noma horses. Serum samples collected from Noma horses were analyzed using a virus neutralization test and enzyme-linked immunosorbent assay and showed that the anti-GETV antibody titers in the samples collected in 2017 were significantly higher than those collected in 2012. We concluded that a seroconversion of anti-GETV antibodies was occurred in the Noma horse population around 2012, providing evidence of the GETV epidemic in Japan circa 2012.

Protective efficacy of a reverse genetics-derived inactivated vaccine against equine influenza virus in horses.

Updating vaccine strains is essential to control equine influenza. We evaluated the protective efficacy of an inactivated equine influenza vaccine derived from viruses generated by reverse genetics (RG) in horses in an experimental viral challenge study. Wild-type (WT) virus (A/equine/Tipperary/1/2019) and virus generated by RG (consisting of hemagglutinin and neuraminidase genes from A/equine/Tipperary/1/2019 and six other genes from high-growth A/Puerto Rico/8/34) were inactivated by formalin for vaccine use. Twelve 1-year-old naïve horses with no antibodies against equine influenza virus were assigned to three groups (each n = 4): control, WT, and RG. They were vaccinated twice, 4 weeks apart, and were challenged with A/equine/Tipperary/1/2019 2 weeks after the second vaccination. All four horses in the control group and one horse in the WT group had pyrexia for multiple days and respiratory illness, and one horse in the RG group had pyrexia for 2 days without respiratory illness. The mean rectal temperatures and the mean concentrations of serum amyloid A in the WT and RG groups were significantly lower than those in the control group, with no significant differences between them. The WT and RG vaccines significantly reduced viral shedding relative to the control. The protective efficacy of the RG-derived inactivated vaccine against equine influenza virus is comparable to that of the vaccine derived from WT viruses in horses. The RG technique can make it easy to update equine influenza vaccine strains.

Distribution of equine coronavirus RNA in the intestinal and respiratory tracts of experimentally infected horses.

Equine coronavirus (ECoV) causes pyrexia, anorexia, lethargy, and sometimes diarrhoea. Infected horses excrete the virus in their faeces, and ECoV is also detected in nasal samples from febrile horses. However, details about ECoV infection sites in the intestinal and respiratory tracts are lacking. To identify the ECoV infection sites in the intestinal and respiratory tracts, we performed an experimental infection study and analysed intestinal and respiratory samples collected from four infected horses at 3, 5, 7, and 14 days post-inoculation (dpi) by real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and in situ hybridization (ISH). Two horses became febrile, but the other two did not. None of the horses had diarrhoea or respiratory signs, and severe cases were not observed in this study. None of the horses showed obvious abnormalities in their intestinal or respiratory tracts. Real-time RT-PCR and ISH showed that ECoV RNA was present throughout the intestinal tract, and ECoV-positive cells were mainly detected on the surface of the intestine. In one horse showing viremia at 3 dpi, ECoV RNA was detected in the lung by real-time RT-PCR, but not by ISH. This suggests that the lung cells themselves were not infected with ECoV and that real-time RT-PCR detected viremia in the lung. The other three horses were positive for ECoV RNA in nasal swabs but were negative in the trachea and lung by real-time RT-PCR and ISH. This study suggests that ECoV broadly infects the intestinal tract and is less likely to infect the respiratory tract.

Antibody Responses to a Reverse Genetics-Derived Bivalent Inactivated Equine Influenza Vaccine in Thoroughbred Horses.

Updating vaccine strains is important to control equine influenza (EI). Previously, we reported that a monovalent inactivated EI vaccine derived from a virus generated by reverse genetics (RG) elicited immunogenicity in horses. In the present study, we compared antibody responses to a bivalent inactivated EI vaccine generated by RG and a commercially available bivalent inactivated EI (CO) vaccine derived from wild-type equine influenza viruses in Thoroughbred horses. The CO vaccine contained A/equine/Ibaraki/1/2007 (Florida sub-lineage clade 1) and A/equine/Yokohama/aq13/2010 (Florida sub-lineage clade 2) as vaccine strains. We generated two RG viruses possessing the hemagglutinin and neuraminidase genes from A/equine/Ibaraki/1/2007 or A/equine/Yokohama/aq13/2010. These viruses were inactivated by formalin, and the hemagglutinin titer of the RG vaccine was adjusted to be the same as that of the CO vaccine. Sixteen unvaccinated yearlings (7 for the RG vaccine group and 9 for the CO vaccine group) received two doses of a primary vaccination course four weeks apart. Thirty-two vaccinated adult horses (18 in the RG-vaccinated group and 14 in the CO vaccine group) received a single dose of a booster vaccination. The patterns of hemagglutination inhibition antibody response to the primary and booster vaccinations were similar for the RG and CO groups in unvaccinated yearlings and vaccinated adult horses. These results suggest that a bivalent vaccine derived from RG viruses elicits equivalent immunogenicity to that elicited by a CO vaccine derived from wild-type viruses. RG viruses can, therefore, be used in multivalent as well as monovalent vaccines for horses.

Growth properties and immunogenicity of a virus generated by reverse genetics for an inactivated equine influenza vaccine.

BACKGROUND: Keeping vaccine strains up to date is the key to controlling equine influenza (EI). Viruses generated by reverse genetics (RG) are likely to be effective for quickly updating a vaccine strain. OBJECTIVES: To evaluate the growth properties of an RG virus in embryonated chicken eggs, and to evaluate antibody responses to a formalin-inactivated vaccine derived from the RG virus in Thoroughbred horses. STUDY DESIGN: In vitro and in vivo experiments. METHODS: Wild-type (WT) viruses (A/equine/Ibaraki/1/2007) or RG viruses (consisting of haemagglutinin [HA] and neuraminidase genes derived from A/equine/Ibaraki/1/2007 and the six other genes derived from high-growth A/Puerto Rico/8/34) were inoculated into embryonated chicken eggs, and the allantoic fluids were harvested at every 24 hours after inoculation. WT and RG viruses were inactivated by formalin for vaccine use. Ten unvaccinated yearlings (five each for WT or RG vaccine) received the first two doses of a primary vaccination course 4 weeks apart followed by their third dose 12 weeks later. Twenty vaccinated adult horses (10 each for WT or RG vaccine) received a single dose of a booster vaccination. RESULTS: The RG virus had high growth properties in embryonated chicken eggs. Unvaccinated yearlings responded poorly to the first vaccination, especially those that received the RG vaccine, but mounted better responses to the second and the third vaccinations, and maintained relatively high haemagglutination inhibition (HI) titres up to 28 weeks after the first vaccination. Vaccinated adult horses did not respond remarkably to the booster vaccination, but no horses showed titres below their pre-booster values even at 12 weeks after vaccination. The RG virus elicited immunogenicity in horses adequate for vaccine use. MAIN LIMITATIONS: No virus challenge study was performed. CONCLUSIONS: The RG viruses are useful as an EI vaccine strain, and quick updates of an EI vaccine strain can be achieved by using RG techniques.

Prediction of municipality-level winter wheat yield based on meteorological data using machine learning in Hokkaido, Japan.

This study analyzed meteorological constraints on winter wheat yield in the northern Japanese island, Hokkaido, and developed a machine learning model to predict municipality-level yields from meteorological data. Compared to most wheat producing areas, this island is characterized by wet climate owing to greater annual precipitation and abundant snowmelt water supply in spring. Based on yield statistics collected from 119 municipalities for 14 years (N = 1,516) and high-resolution surface meteorological data, correlation analyses showed that precipitation, daily minimum air temperature, and irradiance during the grain-filling period had significant effects on the yield throughout the island while the effect of snow depth in early winter and spring was dependent on sites. Using 10-d mean meteorological data within a certain period between seeding and harvest as predictor variables and one-year-leave-out cross-validation procedure, performance of machine learning models based on neural network (NN), random forest (RF), support vector machine regression (SVR), partial least squares regression (PLS), and cubist regression (CB) were compared to a multiple linear regression model (MLR) and a null model that returns an average yield of the municipality. The root mean square errors of PLS, SVR, and RF were 872, 982, and 1,024 kg ha-1 and were smaller than those of MLR (1,068 kg ha-1) and null model (1,035 kg ha-1). These models outperformed the controls in other metrics including Pearson's correlation coefficient and Nash-Sutcliffe efficiency. Variable importance analysis on PLS indicated that minimum air temperature and precipitation during the grain-filling period had major roles in the prediction and excluding predictors in this period (i.e. yield forecast with a longer lead-time) decreased forecast performance of the models. These results were consistent with our understanding of meteorological impacts on wheat yield, suggesting usefulness of explainable machine learning in meteorological crop yield prediction under wet climate.

Persistence of virus-neutralizing antibodies in horses inoculated with two doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals.

The antibody response in horses inoculated with 2 doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals (1 to 3 months) was evaluated with regard to the persistence of virus-neutralizing (VN) antibodies. The durations for which the geometric mean VN titers were maintained significantly higher than those before the first vaccination (P<0.05) were up to 5 months in horses that received the vaccination with a 1-month interval (n=17) and 7 months for those that received it with a 2-month (n=17) or 3-month interval (n=14 or 17). The vaccination program with the 2-month interval was the most effective in maintaining VN antibodies for a long duration with the smallest gap of antibody decline between the first and second vaccinations.

Decreased Virus-Neutralizing Antibodies Against Equine Herpesvirus type 1 In Nasal Secretions of Horses After 12-hour Transportation.

This study evaluated the effects of 12-hour transportation on immune responses to equine herpesvirus type 1 (EHV-1) and type 4 (EHV-4). Possible replication of EHV-1 and EHV-4 was monitored by real-time PCR of nasal swabs and peripheral blood mononuclear cells (PBMCs), and changes in systemic and mucosal antibodies were investigated. Six healthy Thoroughbreds with transport experience were transported in commercial trucks, repeating the same three-hour route four times. Blood samples for cortisol measurement were taken before departure and every three hours. Nasal swabs, PBMCs, nasal wash and serum samples were collected before departure, at unloading, two and six days after arrival. Cortisol concentration increased significantly after three and six hours of transport (P < 0.05), confirming acute transport stress. However, no evidence of viral replication or lytic infection was observed, and serum virus neutralization (VN) titers for EHV-1 and EHV-4 were unchanged, except for one horse that showed a four-fold decrease in titer against EHV-1 after transportation. Urea and total IgA concentration in nasal washes increased significantly after transportation (P < 0.05), while total IgA/protein ratio was unchanged. A transient, ≥4-fold decrease in VN titers for EHV-1 in nasal wash concentrates was observed in four out of six horses after transportation (geometric mean titer declined from 202 to 57, P < 0.05), suggesting suppression of VN capacity in the nasal mucosa may contribute to susceptibility to EHV-1 after transportation. VN antibodies against EHV-4 in nasal secretion were not detected at any timepoint.

Antigenic differences between equine influenza virus vaccine strains and Florida sublineage clade 1 strains isolated in Europe in 2019.

From late 2018 to 2019, equine influenza virus (EIV) strains of Florida sublineage clade 1 (Fc1), which had until then been circulating mainly in the United States, suddenly spread across Europe causing many outbreaks, and Florida sublineage clade 2 (Fc2) strains, which had been circulating mainly in Europe, have not been detected in Europe since 2018. Since 2010, the World Organisation for Animal Health (OIE) has recommended that EIV vaccines contain an Fc1 strain that is like A/equine/South Africa/4/2003 or A/equine/Ohio/2003. Accordingly, Japanese vaccines contain A/equine/Ibaraki/1/2007 as the Fc1 strain. To evaluate the effectiveness of these vaccines against the Fc1 strains detected in Europe in 2019, we performed virus neutralization tests using horse antisera. Challenge viruses used were Irish strain A/equine/Tipperary/1/2019 and two recombinant viruses generated by reverse genetics. Recombinant viruses possessing hemagglutinin (HA) and neuraminidase (NA) derived from A/equine/Tipperary/1/2019 (rA/equine/Tipperary/1/2019) or British strain A/equine/Essex/1/2019 (rA/equine/Essex/1/2019) were generated. Equine antisera against A/equine/South Africa/2003 and A/equine/Ibaraki/2007 were produced by experimental infection. Antibody titers against A/equine/Tipperary/1/2019, rA/equine/Tipperary/1/2019, and rA/equine/Essex/1/2019 were 2.5- to 6.3-fold lower than those against the homologous vaccine strains A/equine/South Africa/4/2003 or A/equine/Ibaraki/2007. These results suggest that the ongoing evolution of the Fc1 viruses may impact on antigenicity and although antibodies against current vaccine strains neutralize the 2019 strains, ongoing surveillance is essential for optimum choice of candidate vaccine strains.

Outbreak of equine coronavirus infection among riding horses in Tokyo, Japan.

In 2020, an outbreak of equine coronavirus (ECoV) infection occurred among 41 horses at a riding stable in Tokyo, Japan. This stable had 16 Thoroughbreds and 25 horses of other breeds, including Andalusians, ponies and miniature horses. Fifteen horses (37 %) showed mild clinical signs such as fever, lethargy, anorexia and diarrhoea, and they recovered within 3 days of onset. A virus neutralization test showed that all 41 horses were infected with ECoV, signifying that 26 horses (63 %) were subclinical. The results suggest that subclinical horses played an important role as spreaders. A genome sequence analysis revealed that the lengths from genes p4.7 to p12.7 or NS2 in ECoV differed from those of ECoVs detected previously, suggesting that this outbreak was caused by a virus different from those that caused previous outbreaks among draughthorses in Japan. Among 30 horses that tested positive by real-time RT-PCR, ECoV shedding periods of non-Thoroughbreds were significantly longer than those of Thoroughbreds. The difference in shedding periods may indicate that some breeds excrete ECoV longer than other breeds and can contribute to the spread of ECoV.