猪德尔塔冠状病毒CH7328株的致病性及全基因组序列分析

Pathogenicity and Whole Genome Sequence Analysis of Porcine Deltacoronavirus CH7328 Strain

为了解猪德尔塔冠状病毒(Porcine deltacoronavirus,PDCoV)CH7328株的致病性,并分析其基因变异和遗传演变规律,试验通过病毒培养、间接免疫荧光试验(IFA)、病毒生长曲线及仔猪致病性试验鉴定病毒的生长特性和致病性,并分析该毒株的全基因组序列。结果显示,感染PDCoV CH7328株的ST细胞出现肿大变圆、细胞呈碎玻璃状且拉丝、细胞集聚成簇和脱落等病变。IFA结果显示,PDCoV CH7328株主要分布在细胞质中。生长曲线结果显示,PDCoV CH7328株在6 h就已开始增殖,在36 h时达到峰值,对应的TCID_(50)为10^(-8.0)/mL,随后逐渐下降。仔猪致病性结果显示,攻毒组仔猪出现腹泻、脱水和厌食等临床症状,仔猪小肠明显变薄、透明而充盈,小肠绒毛严重脱落、萎缩或减少、肠绒毛细胞坏死等。粪便病原检测结果显示,在攻毒第2天便能检测到病毒,之后排毒持续增加,而正常对照仔猪无任何临床现象。全基因组测序及序列分析显示,PDCoV CH7328株的基因组全长为25420 bp,与国内GD株的相似性最高,达到99.9%;遗传演化结果显示,PDCoV CH7328株与国内HKU15-155、CHN-HN-2014和GD株处在同一进化分支,亲缘关系最近。本研究结果为中国PDCoV疫苗候选株的筛选及猪场疫病防控提供了参考数据和理论依据。

In order to understand the pathogenicity of Porcine deltacoronavirus(PDCoV)CH7328 strain,and analyze its genetic variation and genetic evolution,this test used virus culture,indirect immunofluorescence assay(IFA),virus growth curve and piglet pathogenicity test to identify the growth characteristics and pathogenicity of the virus,and analyze the entire genome sequence of the virus strain.The results showed that ST cells infected with PDCoV CH7328 strain showed swelling and rounding,the cells were broken glass-like and brushed,and the cells clustered and fell off.The results of the IFA test showed that the PDCoV CH7328 strain was mainly distributed in the cytoplasm.The growth curve results showed that the PDCoV CH7328 strain began to proliferate at 6 h,and reached a peak at 36 h,the corresponding TCID_(50) was 10^(-8.0)/mL,and then gradually decreased.The pathogenicity results of piglets showed that the piglets in challenge group showed clinical symptoms such as diarrhea,dehydration and anorexia,the small intestine was obviously thinner,transparent and full,and the small intestinal villi were severely shed,atrophy or reduced,and intestinal villi cells were necrotic.The results of fecal pathogen detection showed that the virus could be detected on the second day of the challenge,after which the detoxification continued to increase,while the normal control piglets did not have any clinical signs.Whole genome sequencing and sequence analysis showed that the full length of the genome of the PDCoV CH7328 strain was 25420 bp,which was the highest similar to the domestic GD strain,reaching 99.9%.The genetic evolution results showed that the PDCoV CH7328 strain was in the same evolutionary branch with domestic HKU15-155,CHN-HN-2014 and GD strains,and the genetic relationship was the closest.The results of this study provided a reference data and theoretical basis for the screening of PDCoV vaccine candidate strains and the prevention and control of pig farm diseases in my country.