猪丁型冠状病毒CH/GX/1468B/2017的分离鉴定及全基因组序列分析

本研究于广西某猪场采集疑似感染猪丁型冠状病毒（porcine deltacoronavirus，PDCoV）的腹泻仔猪小肠及其内容物进行病毒分离，并通过细胞病变（CPE）、RT-PCR、间接免疫荧光（IFA）和全基因序列测定分析对分离的病毒进行鉴定。结果显示，试验成功分离到1株PDCoV，命名为CH/GX/1468B/2017（简称PDCoV 1468B）。该毒株可稳定有效地在LLC-PK细胞生长增殖，并引起典型CPE；该毒株已在LLC-PK细胞连续传代15代，病毒滴度随着传代次数的增加逐渐提高并稳定在10^8.10TCID₅₀/mL以上。该毒株全基因组序列长25 399 nt；与23个GenBank中登录的参考毒株的全基因组序列比对显示，核苷酸同源性为97.2%~99.4%，其中PDCoV 1468B分离毒株与Vietnam/Binh21/2015株同源性最高，为99.4%。全基因组系统进化分析显示，PDCoV 1468B分离毒株属于Ⅱ群，与东南亚国家PDCoV毒株亲缘关系密切，处于同一进化分支。综上所述，本研究成功分离出PDCoV 1468B株并进行了全基因序列分析，为进一步开展PDCoV致病性等生物学特性研究及疫苗研制奠定了基础，同时可为PDCoV的遗传进化提供数据支持。

Isolation,Identification and Complete Genome Sequence Analysis of Porcine Deltacoronavirus Strain CH/GX/1468B/2017

The small intestine tissue and its contents of diarrhea piglets from porcine deltacoronavirus (PDCoV) suspected case were collected for virus isolation in the study,the isolated virus were identified by typical cytopathic effect (CPE),RT-PCR,IFA and genomic sequence analysis.The results showed that a PDCoV strain named CH/GX/1468B/2017 (PDCoV 1468B) was isolated from piglets.The strain could grow and proliferate steadily and effectively in LLC-PK cells with typical cytopathic changes.The strain has been passaged for 15 generations in LLC-PK cells.The infectious titers of the viruses gradually increasing and stabilized above 10^8.10 TCID₅₀/mL.The complete genome sequence of PDCoV 1468B was 25 399 nt in length,excluding the poly(A) tail.Genomic analysis showed that PDCoV 1468B shared 97.2% to 99.4% nucleotide identity with other 23 reference PDCoV strains (GenBank),and the highest sequence identity (99.4%) was found with Vietnam/Binh21/2015 (Vietnamese strain).Genome-wide phylogenetic analysis showed that the PDCoV 1468B strain belonged to group Ⅱ and was closely related to the PDCoV strain in Southeast Asian countries which belonged to the same evolutionary branch.In summary,PDCoV 1468B strain was successfully isolated and sequenced,which would lay a foundation for further research on biological characteristics of PDCoV,such as pathogenicity and vaccine development,and provided data support for genetic evolution of PDCoV.