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
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摘要
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~(8.10)TCID_(50)/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。
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/1468 B/2017(PDCoV 1468 B) 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) TCID_(50)/mL.The complete genome sequence of PDCoV 1468 B was 25 399 nt in length,excluding the poly(A) tail.Genomic analysis showed that PDCoV 1468 B 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 1468 B 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 1468 B 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.
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/1468 B/2017(PDCoV 1468 B) 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) TCID_(50)/mL.The complete genome sequence of PDCoV 1468 B was 25 399 nt in length,excluding the poly(A) tail.Genomic analysis showed that PDCoV 1468 B 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 1468 B 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 1468 B 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.
引文
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[15] 郑丽,李秀丽,鄢明华,等.猪德尔塔冠状病毒TJ1株的分离鉴定及生物学特性分析[J].中国畜牧兽医,2018,45(1):219-224.ZHENG L,LI X L,YAN M H,et al.Isolation,identification and biological characteristics of porcine deltacoronavirus TJ1[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):219-224.(in Chinese)
[16] 卢冰霞,秦毅斌,何颖,等.2011—2014年广西猪流行性腹泻病毒检测及其M基因的序列分析[J].中国畜牧兽医,2015,42(3):549-557.LU B X,QIN Y B,HE Y,et al.Detection of porcine epidemic diarrhea virus from 2011 to 2014 in Guangxi province and sequence analysis of their M gene[J].China Animal Husbandry & Veterinary Medicine,2015,42(3):549-557.(in Chinese)
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[18] PAN Y,TIAN X,QIN P,et al.Discovery of a novel swine enteric alphacoronavirus (SeACoV) in Southern China[J].Veterinary Microbiology,2017,211:15-21.
[19] ZHOU P,FAN H,LAN T,et al.Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin[J].Nature,2018,556(7700):255-258.
[20] 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.
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[22] SINHA A,GAUGER P,ZHANG J,et al.PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine[J].Veterinary Microbiology,2015,179(3-4):296-298.
[23] THACHIL A,GERBER P F,XIAO C T,et al.Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies[J].PLoS One,2015,10(4):e0124363.
[24] ZHAI S L,WEI W K,LI X P,et al.Occurrence and sequence analysis of porcine deltacoronaviruses in Southern China[J].Virology Journal,2016,13:136.
[25] 刘宝京,左玉柱,罗尚星,等.猪丁型冠状病毒HB-BD株的分离与鉴定[J].病毒学报,2018,34(3):372-378.LIU B J,ZUO Y Z,LUO S X,et al.Isolation and identification of porcine deltacoronavirus HB-BD strain[J].Chinese Journal of Virology,2018,34(3):372-378.(in Chinese)
[26] 秦毅斌,卢冰霞,何颖,等.猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析[J].中国畜牧兽医,2018,45(1):178-188.QIN Y B,LU B X,HE Y,et al.Isolation and complete genome sequence analysis of porcine epidemic diarrhea virus strain CH/GX/2015/750A[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):178-188.(in Chinese)
[27] 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)
[2] 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.
[3] 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.
[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] 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.
[6] 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.
[7] 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.
[8] LEE S,LEE C.Complete genome characterization of Korean porcine deltacoronavirus strain KOR/KNU14-04/2014[J].Genome Announcements,2014,2(6):e01191.
[9] JANETANAKIT T,LUMYAI M,BUNPAPONG N,et al.Porcine deltacoronavirus,Thailand,2015[J].Emerging Infectious Diseases,2016,22(4):757-759.
[10] SAENG-CHUTO K,LORSIRIGOOL A,TEMEEYASEN G,et al.Different lineage of porcine deltacoronavirus in Thailand,Vietnam and Lao PDR in 2015[J].Transboundary and Emerging Diseases,2017,64(1):3-10.
[11] WANG Y W,YUE H,FANG W,et al.Complete genome sequence of porcine deltacoronavirus strain CH/Sichuan/S27/2012 from mainland China[J].Genome Announcements,2015,3(5):e00945.
[12] CHEN F,ZHU Y,WU M,et al.Full-length genome characterization of Chinese porcine deltacoronavirus strain CH/SXD1/2015[J].Genome Announcements,2015,3(5):e01284.
[13] DONG N,FANG L,ZENG S,et al.Porcine deltacoronavirus in mainland China[J].Emerging Infectious Diseases,2015,21(12):2254-2255.
[14] 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.
[15] 郑丽,李秀丽,鄢明华,等.猪德尔塔冠状病毒TJ1株的分离鉴定及生物学特性分析[J].中国畜牧兽医,2018,45(1):219-224.ZHENG L,LI X L,YAN M H,et al.Isolation,identification and biological characteristics of porcine deltacoronavirus TJ1[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):219-224.(in Chinese)
[16] 卢冰霞,秦毅斌,何颖,等.2011—2014年广西猪流行性腹泻病毒检测及其M基因的序列分析[J].中国畜牧兽医,2015,42(3):549-557.LU B X,QIN Y B,HE Y,et al.Detection of porcine epidemic diarrhea virus from 2011 to 2014 in Guangxi province and sequence analysis of their M gene[J].China Animal Husbandry & Veterinary Medicine,2015,42(3):549-557.(in Chinese)
[17] REED L J,MUENCH H.A simple method for estimating fifty percent endpoints[J].American Journal of Hygiene,1938,27:493-497.
[18] PAN Y,TIAN X,QIN P,et al.Discovery of a novel swine enteric alphacoronavirus (SeACoV) in Southern China[J].Veterinary Microbiology,2017,211:15-21.
[19] ZHOU P,FAN H,LAN T,et al.Fatal swine acute diarrhoea syndrome caused by an HKU2-related coronavirus of bat origin[J].Nature,2018,556(7700):255-258.
[20] 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.
[21] MCCLUSKEY B J,HALEY C,ROVIRA A,et al.Retrospective testing and case series study of porcine deltacoronavirus in U.S.swine herds[J].Preventive Veterinary Medicine,2016,123(1):185-191.
[22] SINHA A,GAUGER P,ZHANG J,et al.PCR-based retrospective evaluation of diagnostic samples for emergence of porcine deltacoronavirus in US swine[J].Veterinary Microbiology,2015,179(3-4):296-298.
[23] THACHIL A,GERBER P F,XIAO C T,et al.Development and application of an ELISA for the detection of porcine deltacoronavirus IgG antibodies[J].PLoS One,2015,10(4):e0124363.
[24] ZHAI S L,WEI W K,LI X P,et al.Occurrence and sequence analysis of porcine deltacoronaviruses in Southern China[J].Virology Journal,2016,13:136.
[25] 刘宝京,左玉柱,罗尚星,等.猪丁型冠状病毒HB-BD株的分离与鉴定[J].病毒学报,2018,34(3):372-378.LIU B J,ZUO Y Z,LUO S X,et al.Isolation and identification of porcine deltacoronavirus HB-BD strain[J].Chinese Journal of Virology,2018,34(3):372-378.(in Chinese)
[26] 秦毅斌,卢冰霞,何颖,等.猪流行性腹泻病毒CH/GX/2015/750A株的分离鉴定及全基因组序列分析[J].中国畜牧兽医,2018,45(1):178-188.QIN Y B,LU B X,HE Y,et al.Isolation and complete genome sequence analysis of porcine epidemic diarrhea virus strain CH/GX/2015/750A[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):178-188.(in Chinese)
[27] 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)
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