Isolation, identification, and phylogenetic analysis of an equine coronavirus strain

Abstract:

Equine coronavirus (ECoV) is an emerging virus attacking the gastrointestinal tract in horses, and the infected adult horses mainly present fever, abdominal pain, and diarrhea. In 1975, ECoV infection first appeared in the United States, and since then it has been prevalent in many countries and regions. Only one recombinant strain of ECoV was isolated from the small intestine of a donkey experiencing diarrhea in Shandong Province, China. [Objective] Understanding the genetic composition, genetic relationship, and biological characteristics of ECoV strains in China can lay a foundation for unveiling the epidemic status and genetic evolution trend of ECoV and provide materials for the research and development of products for the prevention and control of ECoV. [Methods] RT-PCR was employed to detect the stool samples from a horse experiencing diarrhea in Huangpi District, Wuhan City, Hubei Province. The virus was isolated from the positive samples and verified by indirect immunofluorescence assay (IFA) with monoclonal antibodies targeting the S1 protein of ECoV. Based on the whole genome sequencing results of the isolate, the phylogenetic analysis and sequence alignments of the whole genome, N gene, and NS2 gene were performed. [Results] An ECoV strain was successfully isolated and named ECoV-JL. Transmission electron microscopy (TEM) showed that the isolated virus particles were spheroidal and had a capsule membrane and a typical spiroid structure of coronaviruses. The tissue culture infectious dose 50% (TCID₅₀) of ECoV-JL reached a peak of 10^6.16 TCID₅₀/mL 72 h post infection in HRT-18 cells. ECoV-JL strains could infect three human cell lines: HRT-18 (human ileocecal cancer cells), Caco-2 (human colorectal adenocarcinoma cells), and Huh7 (human liver cancer cells). The genome sequence of ECoV-JL and the ECoV genome sequences in GenBank showed the similarity within the range of 97.9%–99.0%. ECoV-JL was in a separate branch of the phylogenetic tree and far related to other strains, which indicated that ECoV-JL might be derived from recombination mutations. The NS2 gene presented more mutations, and the difference in NS2 gene was the main reason for the poor homology between ECoV-JL and other strains. [Conclusion] We isolated and identified an ECoV strain from the stool samples of horses with diarrhea and named it ECOV-JL. The study about the biological characteristics and phylogenetic relationship of this strain reflected the characteristics of the epidemic strains in Hubei Province, providing a clue for the epidemic status and evolution trend of ECoV in China.