Equine Coronavirus Research Articles

Antibody response to equine coronavirus in horses inoculated with a bovine coronavirus vaccine.

A vaccine for equine coronavirus (ECoV) is so far unavailable. Bovine coronavirus (BCoV) is antigenically related to ECoV; it is therefore possible that BCoV vaccine will induce antibodies against ECoV in horses. This study investigated antibody response to ECoV in horses inoculated with BCoV vaccine. Virus neutralization tests showed that antibody titers against ECoV increased in all six horses tested at 14 days post inoculation, although the antibody titers were lower against ECoV than against BCoV. This study showed that BCoV vaccine provides horses with antibodies against ECoV to some extent. It is unclear whether antibodies provided by BCoV vaccine are effective against ECoV, and therefore ECoV challenge studies are needed to evaluate efficacy of the vaccine in the future.

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．

Multiple molecular detection of respiratory viruses and associated signs of airway inflammation in racehorses.

BackgroundThe potential involvement of viruses in inflammatory airway disease (IAD) was previously investigated through either serology or PCR from nasopharyngeal swabs (NS). The aims of this study were to determine the prevalence and incidence of viral genome detection by qPCR in the equine airways, and their association with respiratory clinical signs.MethodsBoth NS and tracheal washes (TW) were collected monthly on 52 Standardbred racehorses at training, over 27 consecutive months (581 samples). Equid herpesviruses (EHV)-1, −4, −2 and −5, equine rhinitis virus-A and -B (ERBV), equine adenovirus-1 and −2, equine coronavirus and equine influenza virus were systematically investigated in both NS and TW. Nasal discharge, coughing, tracheal mucus score and TW neutrophil proportions were simultaneously recorded.ResultsGenome for 7/10 viruses were detected at least once throughout the study; up to 4 different viruses being also concomitantly detected. Monthly incidence in TW was respectively 27.9% (EHV-5), 24.8% (EHV-2), 7.1% (ERBV), 3.8% (EHV-4), 1.9% (EAdV1) and 0.2% (EHV-1; ERAV). Neither agreement nor correlation between NS and TW was found for respectively genome detection and viral loads. Detection of viral genome in NS was not associated with any clinical sign. Coughing was significantly associated with TW detection of EHV-2 DNA (OR 3.1; P = 0.01) and ERBV RNA (OR 5.3; P < 0.001). Detection of EHV-2 DNA in TW was also significantly associated with excess tracheal mucus (OR 2.1; P = 0.02).ConclusionsDetection and quantification of EHV-2 and ERBV by qPCR in TW, but not in NS, should be considered when investigating horses with IAD.Electronic supplementary materialThe online version of this article (doi:10.1186/s12985-016-0657-5) contains supplementary material, which is available to authorized users.

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.

Low prevalence of equine coronavirus in foals in the largest thoroughbred horse breeding region of Japan, 2012-2014.

BackgroundEquine coronavirus (ECoV) is considered to be a diarrheic pathogen in foals. In central Kentucky in the United States, it has been shown that approximately 30 % of thoroughbred foals are infected with ECoV and thus it is considered widely prevalent. In contrast, the epidemiology of ECoV and its relationship to diarrhea in foals are poorly understood in Japan. We investigated ECoV in rectal swabs collected from thoroughbred foals in Japan.ResultsWe collected 337 rectal swabs from 307 diarrheic foals in the Hidaka district of Hokkaido, the largest thoroughbred horse breeding region in Japan, between 2012 and 2014. In addition, 120 rectal swabs were collected from 120 healthy foals in 2012. These samples were tested by reverse transcription loop-mediated isothermal amplification and a real-time reverse transcription-polymerase chain reaction. All samples collected from diarrheic foals were negative, and only three samples (2.5 %) collected from healthy foals were positive for ECoV. Compared with central Kentucky, ECoV is not prevalent among thoroughbred foals in the Hidaka district of Hokkaido.ConclusionECoV is not prevalent and was not related to diarrhea in thoroughbred foals in the Hidaka district of Hokkaido between 2012 and 2014.

Complete genome analysis of equine coronavirus isolated in Japan.

Equine coronavirus has been responsible for several outbreaks of disease in the United States and Japan. Only one complete genome sequence (NC99 isolated in the US) had been reported for this pathogenic RNA virus. Here, we report the complete genome sequences of three equine coronaviruses isolated in 2009 and 2012 in Japan. The genome sequences of Tokachi09, Obihiro12-1 and Obihiro12-2 were 30,782, 30,916 and 30,916 nucleotides in length, respectively, excluding the 3’-poly (A) tails. All three isolates were genetically similar to NC99 (98.2–98.7 %), but deletions and insertions were observed in the genes nsp3 of ORF1a, NS2 and p4.7.Electronic supplementary materialThe online version of this article (doi:10.1007/s00705-015-2565-1) contains supplementary material, which is available to authorized users.

Prevalence of equine coronavirus in nasal secretions from horses with fever and upper respiratory tract infection

Veterinary RecordVolume 177, Issue 11 p. 289-289 Research Prevalence of equine coronavirus in nasal secretions from horses with fever and upper respiratory tract infection N. Pusterla DVM, Corresponding Author N....

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.

First detection of equine coronavirus (ECoV) in Europe

Equine coronavirus (ECoV) is involved mainly in enteric infections. Following the recent description of ECoV in 2000, this study reports for the first time the presence of ECoV in France and, on a wider scale, in Europe. ECoV was molecularly detected from diarrheic and respiratory specimens. Sequencing and phylogenetic analyses demonstrated that European strains are most closely related to the reference North American strain (ECoV-NC99) than the Asian strain (ECoV-Tokachi09).

Infectious agents associated with diarrhoea in neonatal foals in central Kentucky: A comprehensive molecular study

SummaryReasons for performing studyDiarrhoea caused by infectious agents is common in foals but there is no comprehensive molecular work‐up of the relative prevalence of common agents and appearance of coinfections.ObjectivesTo determine the prevalence of 9 infectious agents in gastrointestinal (GI)‐diseased and healthy foals with ages ranging from 1 to 20 weeks of age and to what degree coinfections are associated with clinical signs of GI disease.Study designRetrospective controlled observational study.MethodsThe population consisted of 88 Thoroughbred foals aged 2 days to 17 weeks born on 32 different studfarms in Kentucky. Healthy (n = 37) and GI‐diseased (n = 51) foals were identified based on clinical presentation. Faecal samples were analysed for 9 infectious agents by real‐time PCR: equine rotavirus, equine coronavirus, Clostridium difficile toxins A & B, Neorickettsia risticii, Clostridium perfringens alpha toxin, Lawsonia intracellularis, Rhodococcus equi, Cryptosporidium spp., and Salmonella spp. Salmonella was also cultured from overnight selenite enrichment broth.ResultsThe prevalence of infectious pathogens under study was between 0% (Lawsonia intracellularis) and 34.6% (equine rotavirus). The overall prevalence for any infectious agent was 63.2% in the GI‐diseased group and 43.2% in the healthy group. Coinfections were significantly more frequent in the sick group (15 monoinfections vs. 22 coinfections) than in the healthy group (12 vs. 4, respectively, P = 0.0002). Six of the 8 infectious agents were associated with the GI‐diseased group, the other 2 were not (equine coronavirus and R. equi).ConclusionsThe use of panels rather than individual tests in combination with quantitative toxin gene analysis enables detection of coinfections significantly associated with risk of disease. Several infectious diseases previously not tested for or considered unimportant were found at high prevalence and require further investigation.

Epidemic of Equine Coronavirus at Obihiro Racecourse, Hokkaido, Japan in 2012

Equine coronavirus (ECoV) outbreaks have occurred three times at Obihiro Racecourse in Hokkaido, Japan. The third ECoV outbreak occurred between late February and early April 2012. The main clinical signs of affected horses were anorexia, pyrexia and leucopenia; gastrointestinal disease was observed in about 10% of affected horses. Two ECoV strains were isolated from diarrheal samples. All paired sera (9/9) collected from febrile horses showed seroconversion by neutralization test. Sequence and phylogenetic analysis of the ECoV isolated showed that putative amino acid sequences in S and N genes were highly conserved among ECoV strains. In contrast, sequences of the region coding 4.7 kDa non-structural protein (p 4.7) differed among the strains. Because of the diversity of the p4.7 region, this region should be useful for epidemiological investigation of ECoV.

Emerging outbreaks associated with equine coronavirus in adult horses

The purpose of this study was to describe clinical, hematological and fecal PCR results from 161 horses involved in outbreaks associated with ECoV. The outbreaks happened at four separate boarding facilities between November 2011 and April 2012 in the States of CA, TX, WI and MA. Following the molecular detection of ECoV in the feces from the initial index cases, the remaining herdmates were closely observed for the development of clinical signs. Fecal samples were collected from sick and healthy horses for the PCR detection of ECoV. All four outbreaks involved primarily adult horses. Fifty-nine horses developed clinical signs with 12–16 sick horses per outbreak. The main clinical signs reported were anorexia, lethargy and fever. Four horses from 3 different outbreaks were euthanized or died due to rapid progression of clinical signs. The cause of death could not be determined with necropsy evaluation in 2 horses, while septicemia secondary to gastrointestinal translocation was suspected in 2 horses. Blood work was available from 10 horses with clinical disease and common hematological abnormalities were leucopenia due to neutropenia and/or lymphopenia. Feces were available for ECoV testing by real-time PCR from 44 and 96 sick and healthy horses, respectively. 38/44 (86%) horses with abnormal clinical signs tested PCR positive for ECoV, while 89/96 (93%) healthy horses tested PCR negative for ECoV. The overall agreement between clinical status and PCR detection of ECoV was 91%. The study results suggest that ECoV is associated with self-limiting clinical and hematological abnormalities in adult horses.

Equine coronavirus, a possible cause for adult horse enteric disease outbreaks

Equine coronavirus, a possible cause for adult horse enteric disease outbreaks

Emerging outbreaks associated with equine coronavirus in adult horses

Equine proliferative enteropathy (EPE) is an emerging enteric disease of foals caused by Lawsonia intracellularis. Affected foals, generally less than one year of age, display lethargy, anorexia, fever, weight loss, peripheral edema, diarrhea and colic. Transmission of L. intracellularis is thought to occur through the ingestion of feed or water contaminated with L. intracellularis-infected feces from free-living or domestic animals. Lawsonia intracellularis has been detected by PCR in the feces of a variety of domestic and wild animals. Recently, the authors found that 7.5% of fecal samples and 27% of serum samples from cottontail rabbits from a farm with endemic occurrence of EPE tested positive for L. intracellularis by PCR and serology, respectively. Of interest was that on this farm, a large population of rabbits lived in the hay barn and directly contaminated hay fed to the horses. The goal of this study was to determine if feces from rabbits experimentally infected with L. intracellularis could be the source of infection for naive weanling foals. Two 9-week-old New Zealand white rabbits were experimentally infected with 2.5 x 109 L. intracellularis of equine origin via nasogastric intubation, while two rabbits served as uninfected controls. Eight weanling foals randomly assigned to one of two groups (infected and control) received daily feces from the infected or control rabbits mixed with feed or water. All rabbits and foals were monitored daily for the development of clinical abnormalities and were weighed once weekly for the duration of the study. Feceswere collected every day to every other day for the quantitative molecular detection of L. intracellularis via real-time PCR. Blood was collected weekly for the measurement of concentrations of total solids and serologic analysis. None of the infected rabbits or foals developed any clinical signs or hypoproteinemia compatible with PE. Onset of fecal shedding of L. intracellularis was detected by PCR on days 3 and 9 post-challenge in the 2 infected rabbits. The duration of fecal shedding was 6 and 9 days for the 2 infected rabbits. All infected foals began to shed L. intracellularis between days 10 and 14 post-infection, and fecal shedding lasted between 4 and 10 days. Feces and rectal swabs indicated that control rabbits and control foals, respectively, remained PCR negative for L. intracellularis throughout the entire study period. A humoral immune response was detected in all infected rabbits and foals. This study represents the first report documenting the successful feco-oral transmission of L. intracellularis using infectious fecal material from rabbits. Lagomorphs may represent an effective reservoir/amplifier host for L. intracellularis due to their large population, their close contact to horses, their short reproductive cycle and their world-wide distribution.

Isolation of an equine coronavirus from adult horses with pyrogenic and enteric disease and its antigenic and genomic characterization in comparison with the NC99 strain

A new equine coronavirus was isolated from the feces of adult horses with pyrogenic and enteric disease. The disease outbreak was mainly observed among 2- to 4-year-old horses living in stables of a draft-horse racetrack in Japan. On comparing the isolated virus (isolate Tokachi09) with the equine coronavirus NC99 strain, no significant differences were observed in several biological properties such as hemagglutinating activity, antigenicity (in indirect immunofluorescence and neutralization tests), and one-step growth (in cell culture). The sequences of the nucleocapsid and spike genes of isolate Tokachi09 showed identical size (1341 and 4092 nucleotides, 446 and 1363 amino acids, respectively) and high similarity (98.0% and 99.0% at the nucleotides, 97.3% and 99.0% at the amino acids, respectively) to those of strain NC99. However, the isolate had a 185-nucleotide deletion from four bases after the 3′-terminal end of the spike gene, resulting in the absence of the open reading frame predicted to encode a 4.7-kDa nonstructural protein in strain NC99. These results suggest that the 4.7-kDa nonstructural protein is not essential for viral replication, at least in cell culture, and that the Japanese strain probably originated from a different lineage to the North American strain. This is the first equine coronavirus to be isolated from adult horses with pyrogenic and enteric disease.

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．

Coronavirus Diversity, Phylogeny and Interspecies Jumping

The SARS epidemic has boosted interest in research on coronavirus biodiversity and genomics. Before 2003, there were only 10 coronaviruses with complete genomes available. After the SARS epidemic, up to December 2008, there was an addition of 16 coronaviruses with complete genomes sequenced. These include two human coronaviruses (human coronavirus NL63 and human coronavirus HKU1), 10 other mammalian coronaviruses [bat SARS coronavirus, bat coronavirus (bat-CoV) HKU2, bat-CoV HKU4, bat-CoV HKU5, bat-CoV HKU8, bat-CoV HKU9, bat-CoV 512/2005, bat-CoV 1A, equine coronavirus, and beluga whale coronavirus] and four avian coronaviruses (turkey coronavirus, bulbul coronavirus HKU11, thrush coronavirus HKU12, and munia coronavirus HKU13). Two novel subgroups in group 2 coronavirus (groups 2c and 2d) and two novel subgroups in group 3 coronavirus (groups 3b and 3c) have been proposed. The diversity of coronaviruses is a result of the infidelity of RNA-dependent RNA polymerase, high frequency of homologous RNA recombination, and the large genomes of coronaviruses. Among all hosts, the diversity of coronaviruses is most evidenced in bats and birds, which may be a result of their species diversity, ability to fly, environmental pressures, and habits of roosting and flocking. The present evidence supports that bat coronaviruses are the gene pools of group 1 and 2 coronaviruses, whereas bird coronaviruses are the gene pools of group 3 coronaviruses. With the increasing number of coronaviruses, more and more closely related coronaviruses from distantly related animals have been observed, which were results of recent interspecies jumping and may be the cause of disastrous outbreaks of zoonotic diseases.