摘要
【目的】明确猪流行性腹泻病毒(PEDV)变异株的遗传进化关系及变异株各亚型间的抗原性差异位点,为PEDV新型疫苗及诊断试剂盒的研发打下基础。【方法】将采集的3份PEDV阳性仔猪肠道组织样品接种至非洲绿猴肾细胞(Vero)上进行PEDV分离,分别采用间接免疫荧光试验(IFA)、Western blotting、RT-PCR和电镜观察进行鉴定,通过TCID_(50)测定病毒滴度及绘制病毒体外增殖动态曲线;将分离毒株基因组分成33段进行RT-PCR扩增,使用Lasergene中的SeqMan拼接序列,然后以MEGA 7.0进行遗传进化分析,并以Protean中的Jameson-Wolf算法进行抗原性分析。【结果】其中1份PEDV阳性仔猪肠道组织样品在Vero细胞上盲传至第6代时出现典型的细胞病变,将其命名为CH-HK-2021毒株;IFA和Western blotting鉴定结果表明CH-HK-2021毒株能与小鼠抗PEDV N蛋白单克隆抗体发生特异性反应,且RT-PCR能扩增获得预期的目的条带,证实分离毒株即为PEDV。CH-HK-2021毒株的直径在80~120 nm,且病毒粒子表面带有刺突样的形状,属于冠状病毒;其在Vero细胞上能稳定传代,目前已传至100代。CH-HK-2021毒株在感染Vero细胞24 h后,其病毒滴度达最高值,为10^(5.6)TCID_(50)/mL。CH-HK-2021毒株全基因组除去poly(A)尾结构为28034 bp,与PEDV参考株在全基因组水平上的核苷酸序列相似性为96.0%~98.9%,与参考毒株S基因核苷酸序列的相似性为93.1%~99.0%,其中与变异株CH/JX/01(KX058031)的核苷酸序列相似性最高,与经典毒株AVCT12(LC053455)的核苷酸序列相似性最低。CH-HK-2021毒株属于G2a变异株,为我国当前的流行毒株。G2a毒株与G2b变异株在S基因上存在42个核苷酸差异位点,在S蛋白上则存在6处抗原性差异位点,且主要的差异位点位于S2亚基上。【结论】分离获得的CH-HK-2021毒株属于PEDV G2a变异株,为我国当前的流行毒株,与PEDV经典毒株的亲缘关系相对较远。G2a毒株和G2b变异株在S2亚基上存在多处抗原性差异位点,故推测S2亚基是导致G2a毒株与G2b变异株抗原性差异的主要原因。
【Object】To explore genetic evolution relationship of variant porcine epidemic diarrhea virus(PEDV)and antigenic differential sites among variant strain subtypes,so as to lay a foundation for the development of novel vaccines and diagnostic kits.【Method】Three PEDV-positive porcine intestinal samples were inoculated on to confluent Vero cells to isolate PEDV.Virus identification was performed by indirect fluorescence assay(IFA),Western blotting,RT-PCR and whole genome sequencing and electron microscopic observation;virus titer was determined by TCID_(50) and the in vitvo proliferation dynamin curve of the virus was drawn.The genome of the isolated strain was divided into 33 segments for RT-PCR amplification,and the SeqMan of Lasergene was used to splice sequences.Then the genetic evolution analysis was performed with MEGA 7.0,and the antigenicity analysis was performed with Jameson-Wolf algorithm in Protean.【Result】Typical cytopathic effect appeared in one PEDV-positive porcine intestinal sample in Vero cells when it was blindly passaged to the 6thgeneration and the sample was designated as CH-HK-2021.IFA and Western blotting results showed that the strain CH-HK-2021 could react with PEDV N monoclonal antibody and expected reads were obtained through RT-PCR amplification,which demonstrated this virus was PEDV.Diameter of strain CH-HK-2021 was 80-120nm and the surface of the virus particles were in spike-like shape,indicating it was coronavirus.The strain could be stably propagated in Vero cells,and it has been passaged to 100thgeneration.After 24 h of infecting the Vero cells,virus titer of strain CH-HK-2021 reached the highest,10^(5.6)TCID_(50)/mL.The size whole genome of strain CH-HK-2021 not including poly(A)tail was 28034 bp,with a similarity of 96.0%-98.9%with nucleotide sequence of the PEDV reference strain and a similarity of 93.1%-99.0%with S-base nucleotide sequence of the reference strain.The strain had the highest similarity with nucleotide sequence of variant strain CH/JX/01(KX058031)and the lowest similarity with nucleotide sequence of classical strain AVCT12(LC053455).Strain CH-HK-2021 was a subtype of G2a and it is spreading in China.Strain G2a and variant strain G2b had 42 nucleotide differential sites in S gene and 6 antigenic differential sites;and main differential sites located in subunit S2.【Conclusion】Strain CH-HK-2021 obtained through isolation was subtype variant strain of PEDV G2a,and it is spreading in China.The strain has relatively distant relationship with the classical PEDV strain.G2a stain and G2b variant strain have multiple antigenicity differential sites on S2 subunit,and it is speculated that S2 subunit may be the main reason of the difference in antigenicity between G2a and G2b strains.
作者
彭棋
张雪
赫文龙
范宝超
王丹丹
刘茂军
李彬
PENG Qi;ZHANG Xue;HE Wen-long;FAN Bao-chao;WANG Dan-dan;LIU Mao-jun;LI Bin(Institute of Veterinary Medicine,Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biological Engineering and Technology,Ministry of Agriculture and Rural Affairs,Nanjing,Jiangsu 210014,China;College of Veterinary Medicine,Nanjing Agricultural University,Nanjing,Jiangsu 210095,China)
出处
《南方农业学报》
CAS
CSCD
北大核心
2022年第8期2077-2087,共11页
Journal of Southern Agriculture
基金
国家重点研发计划项目(2021YFD1801104)
国家自然科学基金项目(32272996,32202823)
中国博士后科学基金项目(2022M711398)。
关键词
猪流行性腹泻病毒(PEDV)
变异株
分离鉴定
遗传进化分析
抗原性
porcine epidemic diarrhea virus(PEDV)
variant strain
isolation and identification
genetic evolution analysis
antigenicity
