Characterization of a coronavirus isolated from a diarrheic foal

Abstract

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.

FIG. 1

Coronavirus particles (80 to 120 nm in diameter) identified in infected HRT cell supernatant fluids. Note the presence on virion surfaces of an outer layer of large club-shaped peplomers (arrow) and an inner layer of short peplomers (arrowhead).

FIG. 2

Comparison of N protein amino acid sequences of ECV strain NC99 and published sequences of BCV Mebus, BCV F15, and HCV OC43 (–16). Amino acid sequence differences are shown for BCV strains and HCV OC43. –, position where an amino acid is missing; …, similar amino acids.

FIG. 3

Phylogenetic relationship of ECV NC99 and other avian and mammalian coronaviruses based on a comparison of N protein sequences (2, 3, 6, 14, 15, 16, 20, 24). Amino acid sequences were aligned using the CLUSTAL method, and phylogenetic trees were constructed using the neighbor-joining method. Analyses were done using the MegAlign application of the Lasergene software package.