Equine Coronavirus Infection Research Articles

Equine coronavirus infection and replication in equine intestinal enteroids

In this study, equine intestinal enteroids (EIEs) were generated from the duodenum, jejunum, and ileum and inoculated with equine coronavirus (ECoV) to investigate their suitability as in vitro models with which to study ECoV infection. Immunohistochemistry revealed that the EIEs were composed of various cell types expressed in vivo in the intestinal epithelium. Quantitative reverse-transcription PCR (qRT-PCR) and virus titration showed that ECoV had infected and replicated in the EIEs. These results were corroborated by electron microscopy. This study suggests that EIEs can be novel in vitro tools for studying the interaction between equine intestinal epithelium and ECoV.

Equine coronavirus infection and replication in equine intestinal enteroids

Equine coronavirus infection and replication in equine intestinal enteroids

Risk factors associated with an outbreak of equine coronavirus at a large farm in North Carolina.

Equine coronavirus (ECoV) leads to outbreaks with variable morbidity and mortality. Few previous reports of risk factors for infection are available in the literature. To describe unique clinical findings and risk factors for infection and development of clinical disease. 135 horses on a farm affected by ECoV outbreak. Retrospective cohort study. Data obtained included age, breed, gender, activity level, housing, and feed at the onset of the outbreak. Factors were evaluated for assessment of risk of infection using simple logistic regression or Fisher's exact test. Significance was set at p ≤ 0.05. Forty-three of 54 (79.6%) horses tested on the farm were positive on fecal PCR for ECoV, and 17 horses (12.6%) developed clinical signs consistent with ECoV. Out of 17 horses in which the presence or absence of signs of colic was noted, 6 of 17 (35.3%) showed signs of colic. Three of these horses had small colon impactions, 2 of which required surgical intervention. Significant risk factors for having positive PCR results included being primarily stalled (OR 167.1, 95% CI 26.4-1719), housing next to a positive horse (OR 7.5, 95% CI 3.1-19.0), being in work (OR 26.9, 95% CI 4.6-281.9), being fed rationed hay vs. ad libitum (OR 1,558, 95% CI 130.8-15,593), and being fed alfalfa hay (OR 1,558, 95% CI 130.8-15,593). This report describes risk factors for ECoV infection many of which were associated with intensive management of show horses. Clinicians should be aware that clinical signs vary and can include severe colic.

Serosurveillance of equine coronavirus infection among Thoroughbreds in Japan.

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. To understand the epidemiology and to evaluate the potential risk of ECoV infection to the horse industry in Japan. Longitudinal. The virus-neutralisation (VN) test was performed using sera collected three times a year at 4months intervals from 161 yearlings and at 6-7months 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. 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. Clinical records did not cover the period during horses' absence from the training centre. 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.

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.

Evaluation of the potential risk to horse racing in Japan of equine coronavirus infection

Evaluation of the potential risk to horse racing in Japan of equine coronavirus infection

Equine Coronavirus-Associated Colitis in Horses: A Retrospective Study

Equine coronavirus (ECoV) is a known cause of fever, anorexia, and lethargy in adult horses. Although there are multiple reports of ECoV outbreaks, less is known about the clinical presentation of individual horses during a nonoutbreak situation. The purpose of this study was to describe the clinical presentation of horses diagnosed with ECoV infection that were not associated with an outbreak. Medical records of all horses admitted to Washington State University, Veterinary Teaching Hospital, during an 8-year period were reviewed (2010–2018). The five horses included in this study were older than 1 year of age, were diagnosed with colitis, tested positive for ECoV using real-time polymerase chain reaction, and were negative to other enteric pathogens. Interestingly, 4 of 5 horses had moderate to severe diarrhea, 3 had abnormal large colon ultrasonography, 2 had transient ventricular tachycardia and 2 had clinicopathologic evidence of liver dysfunction. ECoV should be included as a differential diagnosis for individual horses presenting with anorexia, fever, lethargy, and colitis. Early identification of ECoV cases is key to implement appropriate biosecurity measures to prevent the potential spread of this disease.

Development and Validation of a S1 Protein-Based ELISA for the Specific Detection of Antibodies against Equine Coronavirus.

Equine coronavirus (ECoV) is considered to be involved in enteric diseases in foals. Recently, several outbreaks of ECoV infection have also been reported in adult horses from the USA, France and Japan. Epidemiological studies of ECoV infection are still limited, and the seroprevalence of ECoV infection in Europe is unknown. In this study, an indirect enzyme-linked immunosorbent assay (ELISA) method utilizing ECoV spike S1 protein was developed in two formats, and further validated by analyzing 27 paired serum samples (acute and convalescent sera) from horses involved in an ECoV outbreak and 1084 sera of horses with unknown ECoV exposure. Both formats showed high diagnostic accuracy compared to virus neutralization (VN) assay. Receiver-operating characteristic (ROC) analyses were performed to determine the best cut-off values for both ELISA formats, assuming a test specificity of 99%. Employing the developed ELISA method, we detected seroconversion in 70.4% of horses from an ECoV outbreak. Among the 1084 horse sera, seropositivity varied from 25.9% (young horses) to 82.8% (adult horses) in Dutch horse populations. Further, sera of Icelandic horses were included in this study and a significant number of sera (62%) were found to be positive. Overall, the results demonstrated that the ECoV S1-based ELISA has reliable diagnostic performance compared to the VN assay and is a useful assay to support seroconversion in horses involved with ECoV outbreaks and to estimate ECoV seroprevalence in populations of horses.

Clinical presentation, diagnostic findings, and outcome of adult horses with equine coronavirus infection at a veterinary teaching hospital: 33 cases (2012–2018)

Equine coronavirus (ECoV) is a recently described enteric virus with worldwide outbreaks; however, there are little data available on clinical presentation, diagnosis, and outcome. The study objective was to document case management of ECoV in adult horses presented to a referral hospital and compare to a cohort of horses that tested negative for ECoV. A retrospective case series was performed based on positive real-time quantitative PCR results for ECoV on faeces from horses treated at the UC Davis Veterinary Medical Teaching Hospital from 1 March 2012 to 31 March 2018. Horses negative for ECoV were matched to the ECoV-positive group as controls. Data collected included signalment, history, exam findings, diagnostics, treatment, and follow-up.Thirty-three horses (median age, 11 years; range, 2–37 years) tested ECoV-positive, including three horses with co-infections. Presenting complaints for ECoV-infected horses included historic fevers (n = 25/30; 83%), anorexia (n = 14/30; 47%), and colic (n = 13/30; 43%). ECoV-positive horses had significantly lower white blood cell (median, 3.0 × 109/L; range, 0.68–16.2 × 109/L), neutrophil (median, 1.26 × 109/L; range, 0.15–14.4 × 109/L), and lymphocyte (median, 0.86 × 109/L; range, 0.42–3.47 × 109/L) counts than ECoV-negative horses. Electrolyte and metabolic derangements and scant faeces were common. Twenty-seven horses were hospitalised for a median of 5 days (range, 0.5–14 days), with 26/27 (96%) horses surviving to discharge. ECoV infection should be a differential diagnosis for adult horses with fever, colic, anorexia, and leukopenia. The disease has a low mortality rate, but horses may require intensive care to resolve severe leukopenia, systemic inflammation, and metabolic disturbances.

Enteric coronavirus infection in adult horses

A new enteric virus of adult horses, equine coronavirus (ECoV), has recently been recognized. It is associated with fever, lethargy, anorexia, and less frequently, colic and diarrhea. This enteric virus is transmitted via the feco-oral route and horses become infected by ingesting fecally contaminated feed and water. Various outbreaks have been reported since 2010 from Japan, Europe and the USA. While the clinical signs are fairly non-specific, lymphopenia and neutropenia are often seen. Specific diagnosis is made by the detection of ECoV in feces by either quantitative real-time PCR, electron microscopy or antigen-capture ELISA. Supportive treatment is usually required, as most infections are self-limiting. However, rare complications, such as endotoxemia, septicemia and hyperammonemia-associated encephalopathy, have been reported, and have been related to the loss of barrier function at the intestinal mucosa. This review article will focus on the latest information pertaining to the virus, epidemiology, clinical signs, diagnosis, pathology, treatment and prevention of ECoV infection in adult horses.

北海道における馬コロナウイルスの血清学的調査

馬コロナウイルス（ECoV）の血清学的調査を，1989〜2009年に北海道十勝管内で採材した馬の血清560検体について中和試験を用いて実施した結果，各年の抗体陽性率は2004年を除き84.4〜100％と高く，1989年には十勝管内の馬群にECoVが広く浸潤していたと考えられた．また，過去にECoV病の流行がみられた競馬場在厩馬6頭分18検体の血清について，2株のECoVと牛コロナウイルスを抗原に用いて中和試験を実施した結果，ECoVを抗原とした方が抗体陽性率と抗体価が高くなった．ECoVを抗原に使用した場合でもウイルス株が異なれば抗体価も異なる検体がみられたため，ECoVの株間でも抗原性は異なるものと考えられた．

Development of an equine coronavirus–specific enzyme-linked immunosorbent assay to determine serologic responses in naturally infected horses

Equine coronavirus (EqCoV) infection has been documented in most reports through quantitative qPCR analysis of feces and viral genome sequencing. Although qPCR is used to detect antigen during the acute disease phase, there is no equine-specific antibody test available to study EqCoV seroprevalence in various horse populations. We developed an enzyme-linked immunosorbent assay (ELISA) targeting antibodies to the spike (S) protein of EqCoV and validated its use, using acute and convalescent sera from 83 adult horses involved in 6 outbreaks. The EqCoV S protein-based ELISA was able to reliably detect antibodies to EqCoV in naturally infected horses. The greatest seroconversion rate was observed in horses with clinical signs compatible with EqCoV infection and EqCoV qPCR detection in feces. The EqCoV S protein-based ELISA could be used effectively for seroepidemiologic studies in order to better characterize the overall infection rate of EqCoV in various horse populations.

Equine coronavirus: An emerging enteric virus of adult horses.

SummaryEquine coronavirus (ECoV) is an emerging virus associated clinically and epidemiologically with fever, depression, anorexia and less frequently colic and diarrhoea in adult horses. Sporadic cases and outbreaks have been reported with increased frequency since 2010 from Japan, the USA and more recently from Europe. A faeco‐oral transmission route is suspected and clinical or asymptomatic infected horses appear to be responsible for direct and indirect transmission of ECoV. A presumptive clinical diagnosis of ECoV infection may be suggested by clinical presentation, haematological abnormalities such as leucopenia due to lymphopenia and/or neutropenia. Confirmation of ECoV infection is provided by specific ECoV nucleic acid detection in faeces by quantitative PCR (qPCR) or demonstration of coronavirus antigen by immunohistochemistry or electron microscopy in intestinal biopsy material obtained ante or post mortem. The disease is generally self‐limiting and horses typically recover with symptomatic supportive care. Complications associated with disruption of the gastrointestinal barrier have been reported in some infected horses and include endotoxaemia, septicaemia and hyperammonaemia‐associated encephalopathy. Although specific immunoprophylactic measures have been shown to be effective in disease prevention for closely‐related coronaviruses such as bovine coronavirus (BCoV), such strategies have yet not been investigated for horses and disease prevention is limited to basic biosecurity protocols. This article reviews current knowledge concerning the aetiology, epidemiology, clinical signs, diagnosis, pathology, treatment and prevention of ECoV infection in adult horses.

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

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 101.8copies/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.

Disease associated with equine coronavirus infection and high case fatality rate.

BackgroundEquine coronavirus (ECoV) is associated with clinical disease in adult horses. Outbreaks are associated with a low case fatality rate and a small number of animals with signs of encephalopathic disease are described.ObjectivesThe aim of this study is to describe the epidemiological and clinical features of two outbreaks of ECoV infection that were associated with an high case fatality rate.Animals14 miniature horses and 1 miniature donkey testing fecal positive for ECoV from two related disease outbreaks.MethodsRetrospective study describing the epidemiological findings, clinicopathological findings, and fecal viral load from affected horses.ResultsIn EcoV positive horses, 27% (4/15) of the animals died or were euthanized. Severe hyperammonemia (677 μmol/L, reference range ≤60 μmol/L) was identified in one animal with signs of encephalopathic disease that subsequently died. Fecal viral load (ECoV genome equivalents per gram of feces) was significantly higher in the nonsurvivors compared to animals that survived (P = .02).Conclusions and Clinical ImportanceEquine coronavirus had a higher case fatality rate in this group of miniature horses than previously reported in other outbreaks of varying breeds. Hyperammonemia could contribute to signs of encephalopathic disease, and the fecal viral load might be of prognostic value in affected horses.

Experimental inoculation of equine coronavirus into Japanese draft horses.

Recently, outbreaks associated with equine coronavirus (ECoV) have occurred in Japan and the United States. While ECoV is likely to be pathogenic to horses, it has not been shown that experimental inoculation of horses with ECoV produces clinical signs of disease. In this study, we inoculated three Japanese draft horses with an ECoV-positive diarrheic fecal sample to confirm infection after inoculation and to investigate the clinical course and virus shedding patterns of ECoV. Virus neutralization tests showed that all three horses became infected with ECoV. Two of the three horses developed clinical signs similar to those observed during ECoV outbreaks, including fever, anorexia, and gastrointestinal dysfunction. All horses excreted a large amount of virus into their feces for more than 9 days after inoculation regardless of the presence or absence of clinical signs, which suggests that feces are an important source of ECoV infection. ECoV was also detected in nasal swabs from all horses, suggesting that respiratory transmission of ECoV may occur. Both symptomatic horses developed viremia, while the asymptomatic horse did not. White blood cell counts and serum amyloid A concentrations changed relative to the clinical condition of the inoculated horses; these may be useful markers for monitoring the clinical status of horses infected with ECoV. This is the first report of induction of clinical signs of ECoV infection in horses by experimental inoculation. These clinical and virological findings should aid further investigation of the pathogenesis of ECoV.

ばんえい競馬場在厩󠄃馬に流行した馬コロナウイルス感染を疑う事例

北海道帯広市のばんえい競馬場で2004年12月から翌年3月，191頭の重種馬（在厩馬の約3割）に発熱と食欲不振の症状がみられた．発症馬21頭中14頭の発症時と回復時の組血清で牛コロナウイルスに対する中和抗体価の有意な上昇が認められ，発症馬の糞便から馬コロナウイルスNC99株の遺伝子ときわめて相同性の高い遺伝子が検出された．一方発症馬の鼻汁と糞便からコロナウイルス以外の既知病原体と遺伝子は検出されず，発症馬21頭の血清学的検査で馬ヘルペスウイルス1型，馬インフルエンザウイルス，ゲタウイルス，馬ロタウイルス，馬鼻炎Aウイルス，馬アデノウイルス，馬動脈炎ウイルス，腺疫，馬パラチフスに対する有意な抗体価の上昇は認められなかった．以上のことから今回集団発生した疾病は馬コロナウイルスの感染により起こされたことが疑われた．

Equine coronavirus induces apoptosis in cultured cells

Equine coronavirus (ECoV) was first isolated from a diarrheic foal and was found genetically similar to group II coronaviruses. However, its pathological characteristics were not adequately investigated. In our preliminary in vitro investigation, ECoV-induced cell death was observed in bovine kidney-derived MDBK cells. Based on this finding, we investigated whether the ECoV-induced CPE was apoptosis. Following ECoV infection, MDBK cells showed morphological changes such as cell rounding and detachment from the culture surface. Moreover, syncytium formation was observed as the other type of cytopathic effect in ECoV infection. Morphologic and biochemical features of apoptosis, such as nuclear fragmentation and DNA ladder formation, were also detected in ECoV-infected cells. Moreover, as is commonly observed in coronavirus infection in other animals, the activities of effecter caspases – caspase-3/7 – and initiator caspases – caspase-8 and caspase-9 – that are representative factors in the death receptor-mediated apoptotic pathway and mitochondrial apoptotic pathway, respectively, were increased in ECoV-infected MDBK cells. Therefore, it was suggested that ECoV can induce apoptosis in MDBK cells via a caspase-dependent pathway. Apoptotic death of infected cells is detrimental because it causes cell and tissue destruction and inflammatory responses. Although the pathological characteristics of ECoV are largely unknown, apoptosis may be the pathological basis of lesions of the digestive system in ECoV infection.