Equine Coronavirus Research Articles

Coronaviruses: Molecular and Cellular Biology.

‘The knowledge contained in the book demonstrates how quickly and relatively efficiently the worldwide research community can meld together into a giant collaborative effort and concentrate its resources to rapidly begin to understand and solve problems, in this case a health problem.’ Coronaviruses cause respiratory and gastrointestinal diseases in various vertebrates, including minor respiratory diseases in humans. Because of that fact, focused research on these viruses was limited to a few dedicated, but talented, research groups. However, that changed in 2003, when a new coronavirus emerged, designated as severe acute respiratory syndrome corona-like virus (SARS-CoV), which caused a life-threatening respiratory disease characterized as an acute respiratory distress syndrome with a fatality rate of almost 10%. Suddenly, the world’s researchers devoted countless hours to attempting to identify, characterize and develop

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.

Protection from Infection with Severe Acute Respiratory Syndrome Coronavirus in a Chinese Hamster Model by Equine Neutralizing F(ab′)2

To warrant potential clinical testing, the equine anti-severe acute respiratory syndrome coronavirus (SARS-CoV) F(ab')(2) requires evaluation in as many animal models as possible. In this study, we established a new animal model, the Chinese hamster, susceptible to SARS-CoV infection. SARS-CoV could propagate effectively and sustain high levels for 1 wk in animal lungs. All animals were protected from SARS-CoV infection in preventive settings. Further, when used therapeutically this antibody led to an approximately 4-log(10) decrease in viral burden in infected animal lungs. The pathological changes in lungs correlated closely with the dose of antibody administered. The excellent preventive and therapeutic roles of equine anti-SARS-CoV F(ab')(2) in several animal models, including the novel Chinese hamster model described in this study, have provided exciting data concerning its potential clinical study.

Genomic characterization of equine coronavirus

The complete genome sequence of the first equine coronavirus (ECoV) isolate, NC99 strain was accomplished by directly sequencing 11 overlapping fragments which were RT–PCR amplified from viral RNA. The ECoV genome is 30,992 nucleotides in length, excluding the polyA tail. Analysis of the sequence identified 11 open reading frames which encode two replicase polyproteins, five structural proteins (hemagglutinin esterase, spike, envelope, membrane, and nucleocapsid) and four accessory proteins (NS2, p4.7, p12.7, and I). The two replicase polyproteins are predicted to be proteolytically processed by three virus-encoded proteases into 16 non-structural proteins (nsp1–16). The ECoV nsp3 protein had considerable amino acid deletions and insertions compared to the nsp3 proteins of bovine coronavirus, human coronavirus OC43, and porcine hemagglutinating encephalomyelitis virus, three group 2 coronaviruses phylogenetically most closely related to ECoV. The structure of subgenomic mRNAs was analyzed by Northern blot analysis and sequencing of the leader–body junction in each sg mRNA.

Common RNA replication signals exist among group 2 coronaviruses: evidence for in vivo recombination between animal and human coronavius molecules

5′ and 3′ UTR sequences on the coronavirus genome are known to carry cis-acting elements for DI RNA replication and presumably also virus genome replication. 5′ UTR-adjacent coding sequences are also thought to harbor cis-acting elements. Here we have determined the 5′ UTR and adjacent 289-nt sequences, and 3′ UTR sequences, for six group 2 coronaviruses and have compared them to each other and to three previously reported group 2 members. Extensive regions of highly similar UTR sequences were found but small regions of divergence were also found indicating group 2 coronaviruses could be subdivided into those that are bovine coronavirus (BCoV)-like (BCoV, human respiratory coronavirus-OC43, human enteric coronavirus, porcine hemagglutinating encephalomyelitis virus, and equine coronavirus) and those that are murine hepatitis virus (MHV)-like (A59, 2, and JHM strains of MHV, puffinosis virus, and rat sialodacryoadenitis virus). The 3′ UTRs of BCoV and MHV have been previously shown to be interchangeable. Here, a reporter-containing BCoV DI RNA was shown to be replicated by all five BCoV-like helper viruses and by MHV-H2 (a human cell-adapted MHV strain), a representative of the MHV-like subgroup, demonstrating group 2 common 5′ and 3′ replication signaling elements. BCoV DI RNA, furthermore, acquired the leader of HCoV-OC43 by leader switching, demonstrating for the first time in vivo recombination between animal and human coronavirus molecules. These results indicate that common replication signaling elements exist among group 2 coronaviruses despite a two-cluster pattern within the group and imply there could exist a high potential for recombination among group members.

Characterization of a coronavirus isolated from a diarrheic foal.

A coronavirus was isolated from feces of a diarrheic foal and serially propagated in human rectal adenocarcinoma (HRT-18) cells. Antigenic and genomic characterizations of the virus (isolate NC99) were based on serological comparison with other avian and mammalian coronaviruses and sequence analysis of the nucleocapsid (N) protein gene. Indirect fluorescent-antibody assay procedures and virus neutralization assays demonstrated a close antigenic relationship with bovine coronavirus (BCV) and porcine hemagglutinating encephalomyelitis virus (mammalian group 2 coronaviruses). Using previously described BCV primers, the N protein gene of isolate NC99 was amplified by a reverse transcriptase PCR (RT-PCR) procedure. The RT-PCR product was cloned into pUC19 and sequenced; the complete N protein of NC99 (446 amino acids) was then compared with published N protein sequences of other avian and mammalian coronaviruses. A high degree of identity (89.0 to 90.1%) was observed between the N protein sequence of NC99 and published sequences of BCV (Mebus and F15 strains) and human coronavirus (strain OC43); only limited identity (<25%) was observed with group 1 and group 3 coronaviruses. Based on these findings, the virus has been tentatively identified as equine coronavirus (ECV). ECV NC99 was determined to have close antigenic and/or genetic relationships with mammalian group 2 coronaviruses, thus identifying it as a member of this coronavirus antigenic group.

Ulcerative duodenitis in foals.

Seven foals aged 18 days to 3 1/2 months had either single or multiple full-circumference segments or long antimesenteric bands of necrotizing duodenitis, sharply delineated from adjacent viable duodenum. Perforation of the necrotic wall had occurred in all foals, leading to acute fibrinous peritonitis. On the mucosal surface severe diffuse, acute inflammation and ulceration involved the anterior half of the duodenum. Two further foals, aged 28 and 30 days, had lesions that are believed to be a chronic form of this disease. Both foals had a thickened duodenal wall, with large areas of mucosa replaced by granulation tissue. In addition one had several strictures associated with firm adhesions between the duodenal serosa and adjacent structures, together with ascending cholangiohepatitis and pancreatitis. Eight foals had gastric ulcers that were considered to be of less significance than the duodenal lesions. No etiologic agent could be found by aerobic or anaerobic bacterial culturing, negative contrast electron microscopy for viruses, or immunofluorescence staining for equine herpesvirus 1, equine adenovirus, or equine coronavirus. The possible involvement of non-steroidal anti-inflammatory drugs is discussed.