猪流行性腹泻病毒JX16株的分离鉴定及体外传代对其毒力的影响

doi:10.16431/j.cnki.1671-7236.2019.06.026

Abstract

Abstract:

In order to clarify the characteristics of the porcine epidemic diarrhea virus (PEDV) mutant strain in Shandong,Vero cells were used for virus isolation and in vitro subculture of diarrhea materials from a pig farm in Juxian.The virus sequence and characteristic analysis were performed by optical microscopy and ultrathin section observation,RT-PCR detection,indirect immunofluorescence assay,S gene sequence evolution analysis,titer determination and pathogenicity test,and explored the change of cell shedding time after poisoning.Optical microscopy showed that the virus infected with Vero cells could cause typical syncytium lesions.Ultrathin sections of the cells showed that the virions were mostly dense cores,which caused the mitochondria of the host cells to swell and dissolve.The results of RT-PCR and indirect immunofluorescence assay confirmed that the isolated virus of JX16 belonged to PEDV.The strain was finally confirmed to be a variant strain by sequencing. In vitro subculture to 100 generations,as the virus generation increased,its adaptability to cells continued to increase.The cell shedding time was gradually shortened from 48 to 17 h after virus infected.The virus titer was gradually increased from 10^5.3TCID₅₀/mL at the 6th generation to 10^7.5TCID₅₀/mL at the 100th generation.The virulence test showed that the virulence of the virus gradually decreased with the increase of the generation,and the 40th generation had no pathogenicity to the piglets.This article built the foundation for the identification of the pathogenic genes and the development of vaccines of PEDV mutants.

Key words: porcine epidemic diarrhea virus(PEDV); passage culture; pathogenicity

CLC Number:

S852.6

TANG Rongzhi, CHEN Zhi, JIAO Anqi, ZHAO Lu, YU Jiang, ZHANG Yuyu, SUN Wenbo, ZHANG Shujin, ZENG Hao, PENG Jun, WU Jiaqiang. Isolation and Identification of Porcine Epidemic Diarrhea Virus JX16 Strain and Effect of Passage Culture in vitro on Its Virulence[J]. , 2019, 46(6): 1783-1791.

References

[1] AYUDHYA S N,ASSAVACHEEP P,THANAWONGNUWECH R.One world-one health:The threat of emerging swine diseases.An Asian perspective[J].Transboundary and Emerging Diseases,2012,59(S1):9-17.
[2] PENSAERT M B,DE BOUCK P.A new coronavirus-like particle associated with diarrhea in swine[J].Archives of Virology,1978,58(3):243-247.
[3] SUN R Q,CAI R J,CHEN Y Q,et al.Outbreak of porcine epidemic diarrhea in suckling piglets,China[J].Emerging Infectious Diseases,2012,18(1):161-163.
[4] CHEN J F,SUN D B,WANG C B,et al.Molecular characterization and phylogenetic analysis of membrane protein genes of porcine epidemic diarrhea virus isolates in China[J].Virus Genes,2008,36(2):355-364.
[5] KOCHERHANS R,BRIDGEN A,ACKERMANN M,et al.Completion of the porcine epidemic diarrhoea coronavirus (PEDV) genome sequence[J].Virus Genes,2001,23(2):137-144.
[6] HOFMANN M,WYLER R,HOFMANN M,et al.Propagation of the virus of porcine epidemic diarrhea in cell culture[J].Journal of Clinical Microbiology,1988,26(11):2235-2239.
[7] KUSANAGI K,KUWAHARA H,KATOH T,et al.Isolation and serial propagation of porcine epidemic diarrhea virus in cell cultures and partial characterization of the isolate[J].Journal of Veterinary Medical Science,1992,54(2):313-318.
[8] KWEON C H,KWON B J,JUNG T S,et al.Isolation of porcine epidemic diarrhea virus (PEDV) in Korea[J].Korean Journal Veterinary Research,1993,33(2):249-254.
[9] 任敏,朱礼倩,焦海宏,等.基因2型牛病毒性腹泻病毒新疆及山东分离株的鉴定[J].中国动物传染病学报,2009,17(4):20-25. REN M,ZHU L Q,JIAO H H,et al.Identification of bovine viral diarrhea virus genotype 2 isolates of XJ-04 and SD-06[J].Chinese Journal of Animal Infectious Diseases,2009,17(4):20-25.(in Chinese)
[10] 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,identification and complete genome sequence analysis of porcine epidemic diarrhea virus CH/GX/2015/750A[J].China Animal Husbandry & Veterinary Medicine,2018,45(1):178-188.(in Chinese)
[11] SUN D,FENG L,SHI H,et al.Identification of two novel B cell epitopes on porcine epidemic diarrhea virus spike protein[J].Veterinary Microbiology,2008,131(1):73-81.
[12] 毛黎红,张双翔,周碧君,等.6株猪流行性腹泻病毒贵州株S基因序列分析[J].中国畜牧兽医,2017,44(6):1621-1629. MAO L H,ZHANG S X,ZHOU B J,et al.Sequence analysis of gene of 6 strains of porcine epidemic diarrhea virus from Guizhou[J].China Animal Husbandry & Veterinary Medicine,2017,44(6):1621-1629.(in Chinese)
[13] 曾旭健,包银莉.我国PEDV毒株结构蛋白遗传变异情况分析[J].猪业科学,2018,35(10):86-87. ZENG X J,BAO Y L.Analysis of genetic variation of structural proteins of PEDV strains in China[J].Swine Industry Science,2018,35(10):86-87.(in Chinese)
[14] 谢荣辉,赵灵燕,倪柏锋,等.浙江省猪流行性腹泻病毒S1基因克隆与序列分析[J].中国畜牧兽医,2018,45(6):1668-1676. XIE R H,ZHAO L Y,NI B F,et al.Cloning and sequence analysis of S1 gene of porcine epidemic diarrhea virus from Zhejiang province[J].China Animal Husbandry & Veterinary Medicine,2018,45(6):1668-1676.(in Chinese)
[15] 陈淑琴,钱炳旭,赵峰,等.猪流行性腹泻病毒3种结构蛋白的原核表达与免疫原性分析[J].扬州大学学报(农业与生命科学版),2018,39(2):70-73. CHEN S Q,QIAN B X,ZHAO F,et al.Prokaryotic expression and immunogenicity identification of three structure proteins of porcine epidemic diarrhea virus[J].Journal of Yangzhou University (Agricultural and Life Science Edition),2018,39(2):70-73.(in Chinese)
[16] 吴白,苏玮玮,鞠德财,等.猪流行性腹泻病毒SD201604株的分离鉴定及致病性研究[J].中国畜牧兽医,2018,45(11):3229-3236. WU B,SU W W,JU D C,et al.Isolation,identification and pathogenicity evaluation of porcine epidemic diarrhea virus SD201604 strain[J].China Animal Husbandry & Veterinary Medicine,2018,45(11):3229-3236.(in Chinese)
[17] OKDA F,LIU X,SINGREY A,et al.Development of an indirect ELISA,blocking ELISA,fluorescent microsphere immunoassay and fluorescent focus neutralization assay for serologic evaluation of exposure to North American strains of porcine epidemic diarrhea virus[J].BMC Veterinary Research,2015,11(1):180-194.
[18] 林忠武.猪流行性腹泻的流行特点及其防治措施[J].福建畜牧兽医,2011,33(4):14-15. LIN Z W.Epidemiological characteristics of porcine epidemic diarrhea and its control measures[J].Fujian Journal of Animal Husbandry and Veterinary Medicine,2011,33(4):14-15.(in Chinese)
[19] 陈建飞,冯力,时洪艳,等.猪流行性腹泻疫苗的研究[J].猪业科学,2010,27(12):50-51. CHEN J F,FENG L,SHI H Y,et al.Study on porcine epidemic diarrhea vaccine[J].Swine Industry Science,2010,27(12):50-51.(in Chinese)
[20] 苏运芳,孙彦刚,刘运超,等.猪流行性腹泻疫苗研究进展[J].畜牧与兽医,2016,48(2):134-138. SU Y F,SUN Y G,LIU Y C,et al.Advance in porcine epidemic diarrhea vaccines[J].Animal Husbandry and Veterinary Medicine,2016,48(2):134-138.(in Chinese)
[21] 胡鸿惠,南文金,龚中贵,等.猪流行性腹泻弱毒疫苗研究进展[J].安徽农业科学,2016,44(26):69-72. HU H H,NAN W J,GONG Z G,et al.Research progress of porcine epidemic diarrhea attenuated virus vaccine[J].Journal of Anhui Agricultural Sciences,2016,44(26):69-72.(in Chinese)
[22] 李娇,王文秀,谢金文,等.我国猪流行性腹泻疫苗研究与应用现状[J].养猪,2018,1:110-112. LI J,WANG W X,XIE J W,et al.Current status of research and application of porcine epidemic diarrhea vaccine in China[J].Swine Production,2018,1:110-112.(in Chinese)
[23] 刘政龙,王金良,沈志强.猪流行性腹泻病毒研究进展[J].中国畜牧兽医,2015,42(9):2506-2511. LIU Z L,WANG J L,SHEN Z Q.Research progress on porcine epidemic diarrhea virus[J]. China Animal Husbandry & Veterinary Medicine,2015,42(9):2506-2511.(in Chinese)
[24] GAO Y,KOU Q,GE X,et al.Phylogenetic analysis of porcine epidemic diarrhea virus field strains prevailing recently in China[J].Archives of Virology,2013,158(3):711-715.

Isolation and Identification of Porcine Epidemic Diarrhea Virus JX16 Strain and Effect of Passage Culture in vitro on Its Virulence

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