摘要
为从猪体内分离猪源牛病毒性腹泻病毒(BVDV)并了解其分子特征与致病性,本研究基于疑似患猪瘟仔猪病料样品进行BVDV的MDBK细胞分离培养、荧光定量RT-PCR(RT-qPCR)和间接免疫荧光试验(IFA)检测,并对分离病毒的5'-UTR和E0基因进行PCR扩增、测序、同源性与遗传进化分析,在分离病毒细胞培养物纯净性检测后将其以肌肉注射和滴鼻方式接种实验组仔猪,采用流式细胞术、RT-qPCR、ELISA分别检测感染猪淋巴细胞数量、外周血和主要脏器中BVDV 5'-UTR基因的转录与抗体水平,并观察猪临床表现。结果显示,分离病毒经细胞盲传15代均未引起细胞病变,RT-q PCR检测出现扩增曲线,IFA检测出现典型的特异性胞浆内黄绿色荧光,与BVDV-1处于同一进化分支,其5'-UTR、E0基因与牛源BVDV-1 VEDEVAC疫苗株相应基因序列的同源性最高(98.2%与98.7%),将分离病毒命名为BVDV FQ株。经检测FQ株第15代细胞培养物纯净性较好,将其感染猪后第7 d实验组猪淋巴细胞数量下降至最低值,并极显著低于对照组仔猪(P<0.01),此时4/5猪外周血检测到FQ株5'-UTR基因m RNA的转录水平,3/5猪出现腹泻等临床症状,感染后21 d仔猪可检出BVDV抗体,脾脏、肠系膜淋巴结、肠道组织、肺脏、肝脏、肾脏等主要脏器均可检出FQ株5'-UTR基因mRNA的转录水平。上述结果表明猪源BVDV FQ株属于BVDV-1基因型,可能起源于牛源BVDV,可明显降低感染猪的淋巴细胞数量,对猪具有一定致病性。本研究为深入开展猪源BVDV病原学研究提供了参考。
To isolate bovine viral diarrhea virus(BVDV),from pigs and understand its molecular characteristics and pathogenicity,this study carried out virus isolation and culture by MDBK cells from suspected classical swine fever piglet samples,followed by BVDV RT-qPCR and indirect immunofluorescence assay(IFA)detection,amplification and sequencing of the 5'-UTR and E0 genes,and phylogenetic analysis.After confirming the purity of the isolated viral cell culture,the virus was inoculated into experimental piglets via intramuscular injection and intranasal administration.The lymphocyte counts of piglet,BVDV 5'-UTR gene transcription in peripheral blood and major organs,and antibody level of piglet were detected by the flow cytometry,RT-qPCR,and ELISA,respectively.The clinical manifestations of the pigs were observed.The results showed that the isolated strains caused no cytopathic effects(CPE)after 15 blind passages in cell culture.RT-qPCR revealed amplification curves,and IFA displayed typical specific yellow-green fluorescence in the cytoplas.Phylogenetic analysis placed the isolate within the BVDV-1 branch,showing the highest sequence homology with the bovine BVDV-1 VEDEVAC vaccine strain in the 5'-UTR and E0 genes(98.2%and 98.7%,respectively).The strain was designated as BVDV FQ.The 15th-passage viral culture exhibited good purity.After infection of piglets,the lymphocyte counts in the experimental group decreased to the lowest level by day 7 post infection(dpi)and were significantly lower than the control group(P<0.01).At this time,FQ strain 5'-UTR gene mRNA was detected in the peripheral blood of 4/5 pigs,and 3/5 exhibited clinical symptoms such as diarrhea.BVDV antibodies were detectable by 21dpi,and FQ strain 5'-UTR mRNA transcription level was present in all major organs examined such as spleen,mesenteric lymph nodes,intestinal tissue,lungs,liver,and kidneys.These findings indicate that the porcine BVDV FQ strain belongs to the BVDV-1 genotype and may have originated from the bovine BVDV source.The virus significantly reduces the number of lymphocytes in infected pigs and exhibits a certain degree of pathogenicity.This study provides a reference for further research on the etiology and pathogenesis of porcine-origin BVDV.
作者
徐磊
曾亮明
张体银
陶洁
廖惠珍
林祚贵
魏梅蕊
吴宏正
王育顺
朱国强
黄瑜
XU Lei;ZENG Liang-ming;ZHANG Ti-yin;TAO Jie;LIAO Hui-zhen;LIN Zuo-gui;WEI Mei-rui;WU Hong-zheng;WANG Yu-shun;ZHU Guo-qiang;HUANG Yu(College of Animal Science and Technology,Fujian Vocational College of Agriculture,Fuzhou 350119,China;Zhaofenghua Biotechnology(Fuzhou)Co.,Ltd.,Fuzhou 350014,China;Technology Center of Fuzhou Customs,Fuzhou 350001,China;Institute of Animal Husbandry and Veterinary Medicine,Shanghai Academy of Agricultural Science,Shanghai 201106,China;Agriculture and Rural Affairs Bureau of Fuzhou City Changle District,Fuzhou 350200,China;College of Veterinary Medicine,Yangzhou University,Yangzhou 225009,China;Institute of Animal Husbandry and Veterinary Medicine,Fujian Academy of Agricultural Sciences,Fuzhou 350013,China)
出处
《中国预防兽医学报》
北大核心
2025年第9期972-978,共7页
Chinese Journal of Preventive Veterinary Medicine
基金
福建省科技计划项目(2023N0025、2025N0020、2025S0012)
福州市科技计划项目(2024-G-001)
福建农业职业技术学院科研创新平台课题(2024PT5-2)
扬州市级计划政策引导类计划(国际科技合作)项目(YZ2023261)。
关键词
猪源BVDV
分离鉴定
遗传进化分析
淋巴细胞
致病性
porcine BVDV
isolation and identification
phylogenetic analysis
lymphocyte
pathogenicity
