一株马冠状病毒的分离鉴定及遗传演化分析

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

摘要:

马冠状病毒病是由马冠状病毒(equine coronavirus, ECoV)引起的一种马新发胃肠道病毒病，成年马感染后主要出现发烧、腹痛和腹泻等症状。1975年，马冠状病毒感染首次在美国出现，此后在多个国家和地区均有流行，此前我国仅从山东腹泻驴的小肠样品中分离得到了一株重组马冠状病毒。【目的】了解ECoV中国毒株的基因组成、亲缘关系以及生物学特性，可以为我国ECoV流行现状和遗传演化趋势提供依据，为ECoV防控产品的研发提供材料。【方法】对湖北省武汉市黄陂区腹泻马匹的粪便样品进行RT-PCR检测，对检测阳性样品进行病毒分离，并利用靶向ECoV S1蛋白的单克隆抗体通过间接免疫荧光试验(indirect immunofluorescence assay, IFA)对分离的病毒进行验证。根据ECoV-JL株全基因组测序结果，对全基因组、N基因和NS2基因进行了基因组系统发育分析和同源性比较。【结果】成功分离到一株ECoV，并命名为ECoV-JL。透射电镜(transmission electron microscopy, TEM)观察分离到的病毒颗粒呈球状，且具有囊膜和冠状病毒典型的纤突结构。该分离株感染HRT-18细胞72 h后病毒滴度可到达峰值，半数组织培养感染剂量(tissue culture infectious dose 50%, TCID₅₀)约为10^6.16 TCID₅₀/mL。ECoV-JL毒株可以在人回盲肠癌(human ileocecal cancer-18, HRT-18)细胞、人结直肠腺癌(human colorectal adenocarcinoma-2, Caco-2)细胞和人肝癌(human liver cancer cells, Huh7)细胞上稳定传代。ECoV-JL株与GenBank中现有的ECoV全基因组序列相似性为97.9%-99.0%，系统发育分析发现ECoV-JL株属于单独的演化分支，与其他毒株的亲缘关系较远，说明ECoV-JL株可能是重组变异而来，其中NS2基因突变较多，NS2基因编码的差异是造成ECoV-JL株与其他毒株同源性较差的主要原因。【结论】本研究从腹泻马的粪便样品中成功分离并鉴定了一株ECoV，将其命名为ECoV-JL株，对该毒株生物学特性和亲缘关系的研究反映了湖北地区流行毒株的特点，为我国ECoV流行现状和演化趋势提供重要依据。

关键词:马冠状病毒;分离鉴定;遗传演化分析

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.

Key words:equine coronavirus;isolation and identification;phylogenetic analysis