猪δ冠状病毒HeN10株的分离鉴定及致病性研究

doi:10.16431/j.cnki.1671-7236.2021.08.031

摘要/Abstract

摘要： 本研究旨在获得猪δ冠状病毒（Porcine deltacoronavirus，PDCoV）地方分离株，并对其生物学特性和致病性进行初步研究。使用猪睾丸（ST）细胞分离来自河南某猪场PDCoV阳性病料中的病毒，并通过观察细胞病变效应（cytopathic effect，CPE）、反转录PCR、间接免疫荧光试验（indirect immunofluorescence assay，IFA）、电镜观察、S基因序列分析等方法进行鉴定，同时评价分离毒株在仔猪上的致病性。结果显示，阳性病料接种ST细胞后，病毒稳定增殖并连续传代至第10代；自第4代开始，感染细胞发生CPE，呈圆缩、拉网、碎裂、脱落状态；IFA鉴定为PDCoV阳性，且电镜观察可见带有刺突的冠状病毒粒子。将分离到的PDCoV命名为HeN10，其S基因序列与中国SD株相似性最高，达99.8%，与国内报道毒株CHN-JS-2017株和CHN-HeB1-2017株等处于同一分支。将HeN10株以5×10^6.3 TCID₅₀/头的剂量口服感染5头10日龄健康易感仔猪，可导致所有感染仔猪发生水样腹泻。本研究成功分离了PDCoV HeN10株，其与SD株等国内流行毒株处于同一分支，攻毒后可引起仔猪典型腹泻症状，可为下一步诊断方法及疫苗相关研究奠定一定的基础。

关键词: 猪δ冠状病毒(PDCoV); 分离鉴定; 序列分析; 致病性

Abstract: The purpose of this study was to obtain clinical isolates of Porcine deltacoronavirus (PDCoV) and investigate the biological characteristics and pathogenicity of the coronavirus.The swine testicle (ST) cells were used for PDCoV isolation with the PDCoV positive samples obtained clinically from a pig farm in Henan province.The isolated virus was identified by cytopathic effect (CPE) observation, RT-PCR, indirect immunofluorescence (IFA), electron microscopy, and sequence analysis of spike (S) gene, in addition, the pathogenicity was also evaluated using piglets.The PDCoV in the ST cell was successively passaged for 10 passages and confirmed by RT-PCR.The CPE (cell shrinkage, net pulling, fragmentation, and shedding) could be observed in the ST cells from the 4th passage.The isolated virus were identified by IFA, and typical crown shaped viral particles were observed by negative staining in electron microscope, and then the strain of PDCoV was named HeN10.The S gene of HeN10 had the highest sequence identity with China SD strain (99.8%), within the same branch of domestic prevalent strains phylogenetically, such as CHN-JS-2017 and CHN-HeB1-2017.The results of animal test showed that the virus (oral inoculation at a dose of 5×10^6.3 TCID₅₀/piglet) was highly pathogenic by causing watery diarrhea in 10-day-old piglets (5/5).Overall, the PDCoV HeN10 strain was successfully isolated, it showed high sequence identity with domestic prevalent strains such as SD, and also showed high pathogenicity in piglets, which providing a foundation for the development of diagnostic methods and vaccines.

Key words: Porcine deltacoronavirus (PDCoV); isolation and identification; sequence analysis; pathogenicity

中图分类号:

S852.65⁺1

汪志艳, 孙玉洁, 徐鑫, 黄柏成, 田克恭. 猪δ冠状病毒HeN10株的分离鉴定及致病性研究[J]. 中国畜牧兽医, 2021, 48(8): 2982-2989.

WANG Zhiyan, SUN Yujie, XU Xin, HUANG Baicheng, TIAN Kegong. Isolation,Identification and Pathogenicity of Porcine Deltacoronavirus Strain HeN10[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(8): 2982-2989.

参考文献

[1] WANG L,HAYES J,SARVER C,et al.Porcine deltacoronavirus:Histological lesions and genetic characterization[J].Archives of Virology,2016,161(1):171-175.
[2] MARTHALER D,RAYMOND L,JIANG Y,et al.Rapid detection,complete genome sequencing,and phylogenetic analysis of Porcine deltacoronavirus[J].Emerging Infectious Diseases,2014,20(8):1347-1350.
[3] LI G,CHEN Q,HARMON K M,et al.Full-length genome sequence of Porcine deltacoronavirus strain USA/IA/2014/8734[J].Genome Announcements,2014,2(2):e00278-14.
[4] MA Y,ZHANG Y,LIANG X,et al.Origin,evolution,and virulence of Porcine deltacoronaviruses in the United States[J].mBio,2015,6(2):e00064.
[5] JUNG K,HU H,SAIF L J.Porcine deltacoronavirus infection:Etiology,cell culture for virus isolation and propagation,molecular epidemiology and pathogenesis[J].Virus Research,2016,226:50-59.
[6] WOO P C,LAU S K,LAM C S,et al.Discovery of seven novel mammalian and avian coronaviruses in the genus Deltacoronavirus supports bat coronaviruses as the gene source of Alphacoronavirus and Betacoronavirus and avian coronaviruses as the gene source of Gammacoronavirus and Deltacoronavirus[J].Journal of Virology,2012,86(7):3995-4008.
[7] WANG L,BYRUM B,ZHANG Y.Detection and genetic characterization of deltacoronavirus in pigs,Ohio,USA,2014[J].Emerging Infectious Diseases,2014,20(7):1227-1230.
[8] JANETANAKIT T,LUMYAI M,BUNPAPONG N,et al.Porcine deltacoronavirus,Thailand,2015[J].Emerging Infectious Diseases,2016,22(4):757-759.
[9] LEE J H,CHUNG H C,NGUYEN V G,et al.Detection and phylogenetic analysis of Porcine deltacoronavirus in Korean swine farms,2015[J].Transboundary and Emerging Diseases,2016,63(3):248-252.
[10] LORSIRIGOOL A,SAENG-CHUTO K,TEMEE-YASEN G,et al.The first detection and full-length genome sequence of Porcine deltacoronavirus isolated in Lao PDR[J].Archives of Virology,2016,161(10):2909-2911.
[11] SUZUKI T,SHIBAHARA T,IMAI N,et al.Genetic characterization and pathogenicity of Japanese Porcine deltacoronavirus[J].Infection,Genetics and Evolution,2018,61:176-182.
[12] SONG D,ZHOU X,PENG Q,et al.Newly emerged Porcine deltacoronavirus associated with diarrhoea in swine in China:Identification,prevalence and full-length genome sequence analysis[J].Transboundary and Emerging Diseases,2015,62(6):575-580.
[13] MAI K,FENG J,CHEN G,et al.The detection and phylogenetic analysis of Porcine deltacoronavirus from Guangdong province in Southern China[J].Transboundary and Emerging Diseases,2018,65(1):166-173.
[14] ZHANG Y,CHENG Y,XING G,et al.Detection and spike gene characterization in Porcine deltacoronavirus in China during 2016-2018[J].Infection,Genetics and Evolution,2019,73:151-158.
[15] ZHANG H,LIANG Q,LI B,et al.Prevalence,phylogenetic and evolutionary analysis of Porcine deltacoronavirus in Henan province,China[J].Preventive Veterinary Medicine,2019,166:8-15.
[16] DONG N,FANG L,ZENG S,et al.Porcine deltacoronavirus in Mainland China[J].Emerging Infectious Diseases,2015,21(12):2254-2255.
[17] MARTHALER D,JIANG Y,COLLINS J,et al.Complete genome sequence of strain SDCV/USA/Illinois121/2014,a Porcine deltacoronavirus from the United States[J].Genome Announcements,2014,2(2):e00218-14.
[18] CHEN Q,GAUGER P,STAFNE M,et al.Pathogenicity and pathogenesis of a United States Porcine deltacoronavirus cell culture isolate in 5-day-old neonatal piglets[J].Virology,2015,482:51-59.
[19] LIU B J,ZUO Y Z,GU W Y,et al.Isolation and phylogenetic analysis of Porcine deltacoronavirus from pigs with diarrhoea in Hebei province,China[J].Transboundary and Emerging Diseases,2018,65(3):874-882.
[20] 陈建飞,王潇博,焦贺勋,等.国内首株猪德尔塔冠状病毒(Porcine deltacoronavirus)的分离鉴定[J].中国预防兽医学报,2016,38(3):171-174. CHEN J F,WANG X B,JIAO H X,et al.Isolation and identification of the first Porcine deltacoronavirus strain in China[J].Chinese Journal of Preventive Veterinary Medicine,2016,38(3):171-174.(in Chinese)
[21] DONG N,FANG L,YANG H,et al.Isolation,genomic characterization,and pathogenicity of a Chinese Porcine deltacoronavirus strain CHN-HN-2014[J].Veterinary Microbiology,2016,196:98-106.
[22] 张坤,何启盖.猪流行性腹泻病毒、猪传染性胃肠炎病毒和猪A群轮状病毒多重RT-PCR检测方法的建立及临床应用[J].畜牧兽医学报,2010,41(8):1001-1005. ZHANG K,HE Q G.Establishment and clinical application of a multiplex reverse transcription-PCR for detection of Porcine epidemic diarrhea virus,porcine Transmissible gastroenteritis virus and Porcine group A rotavirus[J].Acta Veterinaria et Zootechnica Sinica,2010,41(8):1001-1005.(in Chinese)
[23] HU H,JUNG K,VLASOVA A N,et al.Isolation and characterization of Porcine deltacoronavirus from pigs with diarrhea in the United States[J].Journal of Clinical Microbiology,2015,53(5):1537-1548.
[24] SHIRATO K,MATSUYAMA S,UJIKE M,et al.Role of proteases in the release of Porcine epidemic diarrhea virus from infected cells[J].Journal of Virology,2011,85(15):7872-7880.
[25] WICHT O,LI W,WILLEMS L,et al.Proteolytic activation of the Porcine epidemic diarrhea coronavirus spike fusion protein by trypsin in cell culture[J].Journal of Virology,2014,88(14):7952-7961.
[26] GRAHAM R L,BARIC R S.Recombination,reservoirs,and the modular spike:Mechanisms of coronavirus cross-species transmission[J].Journal of Virology,2010,84(7):3134-3146.
[27] HODGES K,GILL R.Infectious diarrhea:Cellular and molecular mechanisms[J].Gut Microbes,2010,1(1):4-21.
[28] JUNG K,HU H,EYERLY B,et al.Pathogenicity of 2 Porcine deltacoronavirus strains in gnotobiotic pigs[J].Emerging Infectious Diseases,2015,21(4):650-654.
[29] XU Z,ZHONG H,ZHOU Q,et al.A highly pathogenic strain of Porcine deltacoronavirus caused watery diarrhea in newborn piglets[J].Virologica Sinica,2018,33(2):131-141.
[30] ZHANG M J,LIU D J,LIU X L,et al.Genomic characterization and pathogenicity of Porcine deltacoronavirus strain CHN-HG-2017 from China[J].Archives of Virology,2019,164(2):413-425.
[31] WU J L,MAI K J,LI D,et al.Expression profile analysis of 5-day-old neonatal piglets infected with Porcine deltacoronavirus[J].BMC Veterinary Research,2019,15(1):117.