广西地区牛肠道病毒E、F型的分离鉴定

doi:10.16431/j.cnki.1671-7236.2024.12.035

摘要/Abstract

摘要： 【目的】通过分离牛肠道病毒(BEV),了解广西地区BEV的传播途径和病原学特性，为疫情监测和疫苗研发提供数据支持，促进广西地区对该病毒的防控。【方法】本研究将BEV检测的阳性病料接种于胎牛肾细胞(MDBK)，经连续传代后，通过致细胞病变效应(CPE)、RT-PCR、间接免疫荧光试验(IFA)等鉴定分离毒株。对鉴定成功的毒株进行空斑纯化试验后测定毒价，并进行多步生长曲线试验。设计特异性引物扩增病毒全基因组序列，利用MegAlign软件分析分离毒株与参考毒株的核苷酸和氨基酸序列相似性，绘制病毒全基因组序列及VP1基因序列遗传进化树。【结果】细胞接种病料后第3代出现明显CPE,RT-PCR结果显示，扩增出大小为239 bp的目标条带。IFA结果显示，接种分离株的MDBK细胞内可观察到特异性绿色荧光。本研究分离出了2株BEV，命名为GXHC2317和GXWZ2317株。半数组织培养感染剂量(TCID₅₀)测定结果显示，毒株GXHC2317和GXWZ2317 TCID₅₀分别为10^7.47和10^5.75/0.1 mL。多步生长曲线试验结果显示，毒株GXHC2317和GXWZ2317在MDBK细胞中复制增殖良好，24 h后病毒复制能力达到峰值。相似性分析结果表明，GXHC2317株与参考毒株核苷酸和氨基酸序列相似性分别为69.5%～80.1%和81.1%～96.5%，与宁夏地区分离株NX-FY40氨基酸序列相似性最高。GXWZ2317株与参考毒株核苷酸和氨基酸序列相似性分别为69.3%～87.3%和80.3%～96.4%，与山东地区分离株SD-S67氨基酸序列相似性最高。病毒全基因组序列与VP1基因序列遗传进化树结果表明，GXHC2317株属于BEV E3亚型，GXWZ2317株属于BEV F1亚型。【结论】本研究从广西地区分离到BEV E3亚型和F1亚型，丰富了BEV在国内流行趋向调查及病原学研究，为广西地区BEV相关基础与应用研究奠定了病原学基础。

关键词: 牛肠道病毒(BEV); 分离鉴定; 基因分型

Abstract: 【Objective】 By isolating Bovine enterovirus (BEV),this study was aimed to understand its transmission routes and pathogenic characteristics in Guangxi,provide data support for epidemic monitoring and vaccine development,and further promoting the prevention and control of the virus in Guangxi.【Method】 The BEV-positive samples were inoculated into fetal bovine kidney cells (MDBK).After continuous passage,the isolated strains were identified through cytopathic effects (CPE),RT-PCR and indirect immunofluorescence assay (IFA).The identified strains were subjected to plaque purification,virus titer determination,and multi-step growth curve experiments.Specific primers were designed to amplify the full-length genome sequence of the virus.The nucleotide and amino acid similarity between the isolated strains and reference strains were analyzed using MegAlign software.Phylogenetic trees of the viral whole genome and VP1 gene sequences were constructed.【Result】 The third passage of inoculated cells showed significant CPE.RT-PCR results indicated the presence of target bands (239 bp),and IFA results showed that specific green fluorescence could be observed in MDBK cells inoculated with the isolated strains,confirming the isolation of two BEV strains,named GXHC2317 and GXWZ2317, respectively.50% tissue culture infective dose (TCID₅₀) results showed that the TCID₅₀ of strains GXHC2317 and GXWZ2317 were 10^7.47 and 10^5.75/0.1 mL,respectively.Multi-step growth curve experiments demonstrated that strains GXHC2317 and GXWZ2317 replicated well in MDBK cells,reaching peak viral replication capacity within 24 h.Similarity analysis showed that the nucleotide and amino acid sequence similarities between strain GXHC2317 and the reference strain were 69.5% to 80.1% and 81.1% to 96.5%,respectively.The amino acid sequence similarity between strain GXHC2317 and strain NX-FY40 isolated from Ningxia region was the highest.The nucleotide and amino acid sequence similarities between strain GXWZ2317 and reference strain were 69.3% to 87.3% and 80.3% to 96.4%,respectively,with the highest amino acid sequence similarity between GXWZ2317 strain and SD-S67 strain isolated from Shandong region.Phylogenetic analysis of the viral whole genome and VP1 gene sequences indicated that the GXHC2317 strain belonged to the BEV E3 subtype,and the GXWZ2317 strain belonged to the BEV F1 subtype.【Conclusion】 This study isolated BEV E3 and F1 subtypes from Guangxi,enriching the investigation of BEV epidemiology and pathogenesis in China.It laid a virological foundation for basic and applied research related to BEV in Guangxi.

Key words: Bovine enterovirus (BEV); isolation and identification; genetic typing

中图分类号:

S852.65⁺3

刘黄豪, 黄艳华, 罗宇航, 任同伟, 覃一峰, 韦祖樟, 欧阳康, 陈樱, 谢江, 李凤梅, 陈集成, 王小玲, 潘艳, 黄伟坚. 广西地区牛肠道病毒E、F型的分离鉴定[J]. 中国畜牧兽医, 2024, 51(12): 5489-5498.

LIU Huanghao, HUANG Yanhua, LUO Yuhang, REN Tongwei, QIN Yifeng, WEI Zuzhang, OUYANG Kang, CHEN Ying, XIE Jiang, LI Fengmei, CHEN Jicheng, WANG Xiaoling, PAN Yan, HUANG Weijian. Isolation and Identification of Bovine Enterovirus Types E and F in Guangxi[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(12): 5489-5498.

参考文献

[1] 常晓冉, 林倩, 钱明珠, 等.河南省牛肠道病毒的遗传变异分析[J].中国兽医学报, 2022, 42(10):1982-1987.CHANG X R，LIN Q，QIAN M Z，et al.Genetic variation of Bovine enterovirus strains from Henan province[J]. Chinese Journal of Veterinary Science，2022, 42(10):1982-1987.(in Chinese)
[2] MOLL T, DAVIS A D.Isolation and characterization of cytopathogenic Enteroviruses from cattle with respiratory disease[J].American Journal of Veterinary Research, 1959, 20:27-32.
[3] BLAS-MACHADO U, SALIKI J T, BOILEAU M J, et al.Fatal ulcerative and hemorrhagic typhlocolitis in a pregnant heifer associated with natural Bovine enterovirus type-1 infection[J].Veterinary Pathology, 2007, 44(1):110-115.
[4] 罗宇航, 乔成鹏, 朱鑫玥, 等.广西地区水牛E2型牛肠道病毒的分离和鉴定[J].中国兽医杂志, 2023, 59(1):53-58.LUO Y H，QIAO C P，ZHU X Y，et al.Isolation and identification of Bovine enterovirus E2 type from buffalo in Guangxi[J]. Chinese Journal of Veterinary Medicine，2023, 59(1):53-58.(in Chinese)
[5] KING A M Q, LEFKOWITZ E J, MUSHEGIAN A R, et al.Changes to taxonomy and the international code of virus classification and nomenclature ratified by the international committee on taxonomy of viruses (2018)[J]. Archives of Virology, 2018, 163(9):2601-2631.
[6] GVR S, YAPKIÇ O, YILMAZ A.Serological survey of Bovine enterovirus type 1 in different mammalian species in Turkey[J].Zoonoses Public Health, 2008, 55(2):106-111.
[7] 杨佳欣, 张涛, 淦雨洁, 等.攀西地区牛肠道病毒遗传变异分析[J].中国动物传染病学报, 2022, 30(4):130-134.YANG J X，ZHANG T，GAN Y J，et al.Analysis on the genetic variation of Bovine enterovirus in Panxi area[J]. Chinese Journal of Animal Infectious Diseases，2022，30(4):130-134.(in Chinese)
[8] 古丽巴哈尔·图尔荪, 张福慧, 张群, 等.新疆地区牛肠道病毒的病毒分离鉴定及毒株差异[J].中国兽医学报, 2022, 42(5):873-878.GULBAHAR·T，ZHANG F H，ZHANG Q，et al.Characterization of Bovine enterovirus strains and molecular difference from Xinjiang autonomous region[J]. Chinese Journal of Veterinary Science，2022, 42(5):873-878.(in Chinese)
[9] JI C, ZHANG Y, SUN R, et al.Isolation and identification of type F Bovine enterovirus from clinical cattle with diarrhoea[J].Viruses, 2021, 13(11):2217.
[10] KATAOKA C, SUZUKI T, KOTANI O, et al.The role of VP1 amino acid residue 145 of Enterovirus 71 in viral fitness and pathogenesis in a Cynomolgus monkey model[J].PLoS Pathogens, 2015, 11(7):e1005033.
[11] CAINE E A, MONCLA L H, RONDEROS M D, et al.A single mutation in the VP1 of Enterovirus 71 is responsible for increased virulence and neurotropism in adult interferon-deficient mice[J].Journal of Virology, 2016, 90(19):8592-8604.
[12] ZHU L, XING Z, GAI X, et al.Identification of a novel Enterovirus E isolates HY12 from cattle with severe respiratory and enteric diseases[J].PLoS One, 2014, 9(5):e97730.
[13] LI Y, CHANG J, WANG Q, et al.Isolation of two Chinese Bovine enteroviruses and sequence analysis of their complete genomes[J].Archives of Virology, 2012, 157(12):2369-2375.
[14] 罗宇航, 刘黄豪, 李德政, 等.广西河池地区牛肠道病毒和牛冠状病毒流行病学调查及遗传变异分析[J].中国兽医杂志, 2023, 59(7):32-39.LUO Y H，LIU H H，LI D Z，et al.Epidemiological lnvestigation and genetic variation analysis of Bovine enterovirus and Bovine coronavirus in Hechi，Guangxi[J]. Chinese Journal of Veterinary Medicine，2023, 59(7):32-39.(in Chinese)
[15] LUO Y, LIU H, ZOU Y, et al.Molecular epidemiology of Bovine enteroviruses and genome characterization of two novel Bovine enterovirus strains in Guangxi, China[J]. Microbiology Spectrum, 2023, 11(2):e0378522.
[16] 陈俊贞, 李祯, 张成远, 等.E型牛肠道病毒新疆BL311株的分离鉴定及致病性分析[J].中国动物传染病学报, 1-11[2024-08-28].https://doi.org/10.19958/j.cnki.cn31-2031/s.20220707.004.CHEN J Z，LI Z，ZHANG C Y，et al.Isolation, identification and pathogenicity analysis of E-type Enterovirus BL311 strain in Xinjiang[J]. Chinese Journal of Animal Infectious Diseases, 1-11.https://doi.org/10.19958/j.cnki.cn31-2031/s.20220707.004.(in Chinese)
[17] MAŁACZEWSKA J, WRÓBEL M, KACZOREK-ŁUKOWSKA E, et al.Enterovirus E infects bovine peripheral blood mononuclear cells.Implications for pathogenesis?[J].Journal of Veterinary Research, 2023, 67(4):517-527.
[18] 李英利, 常继涛, 于力.牛肠道病毒的分离鉴定[J].中国预防兽医学报, 2011, 33(5):388-390.LI Y L，CHANG J T，YU L，Isolation and identification of Bovine enterovirus in China[J]. Chinese Journal of Preventive Veterinary Medicine, 2011，33(5):388-390.(in Chinese)
[19] 邢泽黎, 朱利塞, 王新平, 等.从吉林省肉牛场新发疫病分离出E种肠道病毒[J].中国兽医学报, 2014, 34(3):390-394.XING Z L，ZHU L S，WANG X P，et al.Identification of a Enterovirus E isolate from a cattle with respiratory diseases[J].Chinese Journal of Veterinary Science, 2014, 34(3):390-394.(in Chinese)
[20] 杨永宁.西宁市牦牛肠道病毒的流行病学调查与分析[J].中国动物保健, 2018, 20(11):22-23.YANG Y N，Epidemiological investigation and analysis of yak Enterovirus in Xining[J].China Animal Health，2018, 20(11):22-23.(in Chinese)
[21] 何欢.牦牛肠道病毒流行病学调查、分离鉴定及感染MDBK细胞的转录组学研究[D].成都：西南民族大学，2017.HE H.Epidemiological investigation, isolation and identification of yak Enterovirus and transcriptomics research of infection MDBK cells[D].Chengdu:Southwest Minzu University，2017.(in Chinese)
[22] 李欣, 胡俊英, 林倩, 等.山东省某地区牛、羊肠道病毒感染的流行病学调查[J].中国兽医学报, 2019, 39(2):223-227.LI X，HU J Y，LIN Q，et al.Epidemiological investigation on Enterovirus infections in cattle/sheep/goat herds in Shandong province[J]. Chinese Journal of Veterinary Science, 2019，39(2):223-227.(in Chinese)
[23] EARLE J A, SKUCE R A, FLEMING C S, et al.The complete nucleotide sequence of a Bovine enterovirus[J].Journal of General Virology, 1988, 69(Pt 2):253-63.
[24] 王位, 朱沛, 李占鸿, 等.2013—2022年云南地区猪肠病毒G型的分子流行病学调查[J].中国畜牧兽医, 2023, 50(12):5000-5012.WANG W，ZHU P，LI Z H，et al.Molecular epidemiological survey of Porcine enterovirus type G in Yunnan from 2013 to 2022[J]. China Animal Husbandry & Veterinary Medicine, 2023, 50(12):5000-5012.(in Chinese)
[25] 陈昌毅, 李根, 何红玲, 等.1株猫嵌杯病毒的分离鉴定及全基因序列分析[J].中国畜牧兽医, 2023, 50(9):3707-3718.CHEN C Y，LI G，HE H L，et al.Isolation and whole genome sequence analysis of a strain of Feline calicivirus[J]. China Animal Husbandry & Veterinary Medicine，2023，50(9):3707-3718.(in Chinese)
[26] 钱明珠, 胡俊英, 林倩, 等.河南省牛肠道病毒感染的流行病学调查与分析[J].中国兽医学报, 2021, 41(10):1937-1941.QIAN M Z, HU J Y，LIN Q，et al.Epidemiological investigation and analysis on Bovine enterovirus infection in Henan province[J]. Chinese Journal of Veterinary Science, 2021，41(10):1937-1941.(in Chinese)
[27] LUDWIG-BEGALL L F, MAUROY A, THIRY E.Norovirus recombinants:Recurrent in the field, recalcitrant in the lab-A scoping review of recombination and recombinant types of Noroviruses[J].Journal of General Virology, 2018, 99(8):970-988.

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