一株牛副流感病毒3型毒株的分离鉴定与全基因序列分析

Isolation,identification and complete genome analysis of a bovine parainfluenza virus type 3

目的采集内蒙古通辽地区某牛场牛鼻拭子样品,PCR检测鼻拭子样品中牛副流感病毒3型(BPIV3)感染情况,研究阳性鼻拭子样品中BPIV3毒株的全长基因组和遗传进化。方法将PCR检测结果呈BPIV3阳性且测序正确的鼻拭子样品接种至MDBK细胞,连续传代5次,然后通过RT-PCR对分离株进行鉴定,并对其进行基因组全长测定及同源性和遗传进化分析。结果成功分离到一株BPIV3毒株,PCR检测结果为阳性,将其命名为NMTL-21。该毒株基因组全长为15454 bp,Karber法结果测得,BPIV3 NMTL-21的半数细胞培养物感染剂量为10^(-7) TCID50/100μL。基于基因组全长序列的同源性和遗传进化分析结果显示,NMTL-21与2015年分离自黑龙江省大庆市的NX49株同源性最高,达99.6%;与2013年分离自北京的XJA13株同源性为99.5%。遗传进化分析结果显示,该毒株为基因C型。结论从病牛鼻拭子样品中成功分离到一株能够致细胞病变的BPIV3-C型毒株,可为进一步开展BPIV3研究提供理论参考和实验材料。

Objective To detect and isolate BPIV3 strains from nasal swab samples of diarrhea calves in Tongliao,Inner Mongolia by PCR and perform the complete genome sequencing and phylogenetic evolutionary tree analysis.Methods The nasal swab samples detected positive for BPIV3 by PCR were incubated with MDBK cells and passaged for 5 times to isolate the BPIV3 strains.Subsequently,the BPIV3 isolate was identified through PCR and the complete genome was sequenced and analyzed for phylogenetic evolutionary tree.Results A BPIV3 strain,designated as NMTL-21,was successfully isolated from a nasal swab of infected cattle,with a positive result in PCR testing.The total genome length of this strain is 15454 bp.According to the Karber method,the half-maximal tissue culture infectious dose(TCID_(50))for BPIV3 NMTL-21 isolate was determined to be 10^(-7)TCID_(50)/100μL.Homology and genetic evolution analysis based on the full-length genome sequence revealed that NMTL-21 shares the highest homology with the NX49 strain isolated from Daqing City,Heilongjiang Province in 2015,with a homology of 99.6%.Additionally,it exhibits 99.5%homology with the XJA13 strain isolated from Beijing in 2013.Genetic evolution analysis indicates that this strain belongs to genotype C.Conclusion A BPIV3 strain,classified under genotype C,was effectively isolated from a nasal swab obtained from infected cattle.This finding offers valuable theoretical insights and experimental resources for the control and development of vaccines targeting BPIV3.