3株人鼻病毒地方株的分离鉴定与VP4基因序列分析

Isolation and Identification of Three Strains of the Human Rhinovirus and Sequencing of the VP4 Gene

人鼻病毒(Human rhinovirus,HRV)是小儿呼吸道感染的重要病原体,具有多种基因型,定期对HRV进行病毒分离与基因序列分型,对了解病毒的变异情况具有重要价值。为了探讨HRV在安徽省蚌埠市上呼吸道感染患者咽拭子样本中的分离情况,并进行分离株HRV的衣壳蛋白(VP)4基因序列分析,本研究选择安徽省蚌埠医学院第一附属医院诊断为上呼吸道感染患者的64例咽拭子标本,采用定量聚合酶链式反应(quantitative polymerase chain reaction,qPCR)检测HRV感染情况。将检测阳性标本感染H1-HeLa细胞进行病毒分离,对成功分离的HRV地方株进行HRV VP4基因的逆转录-聚合酶链反应(Reverse transcription-polymerase chain reaction,RT-PCR)扩增,并采用MEGA软件进行序列分析。结果显示:qPCR检出HRV阳性6例,从中分离到3株HRV地方株。基因序列分析显示3株HRV地方株分别为HRV A型、HRV B型、HRV C型病毒株(NCBI识别序列号分别为MK820682、MK848595、MN146019)。MK820682株与HRV A2型参考株X02316株(奥地利株)属于同一分支;MK848595株与HRV B型参考株KY093602株(马来西亚株)属于同一分支;MN146019株单独处于一个分支。本研究成功从安徽省蚌埠市分离出3株HRV地方株,分别为HRV A型、HRV B型、HRV C型地方株,说明当地HRV存在频繁变异情况,要加强对当地的HRV防控管理。

Human rhinoviruses(HRVs)are important pathogens of respiratory infections in children and have multiple genotypes. Isolation and gene sequencing of HRVs are of great value in understanding variations in HRVs. To study the isolation of HRVs in throat-swab samples from patients with upper respiratory tract infections(URTIs)in The First Affiliated Hospital of Bengbu Medical College,Bengbu City,China and to undertake sequencing of the VP4 gene of isolated HRV strains,sixty-four throat-swab samples from patients with URTIs diagnosed in sentinel hospitals in Bengbu City,China were selected. Quantitative polymerase chain reaction(qPCR)was used to detect HRV infection. Positive samples were infected with H1-HeLa cells for virus isolation. The VP4 gene of HRVs was amplified by reverse transcription-PCR. The sequences of the VP4 gene of isolated local strains of HRVs were analyzed by MEGA software. Results showed that six HRV-positive samples were detected by qPCR,and three local strains of HRVs were isolated. Gene sequencing showed that the three local strains were A,B,and C(NCBI identification ID:MK820682,MK848595 and MN146019,respectively). The MK820682 strain belonged to the same branch as the HRV A2 reference strain X02316(Austrian strain). The MK848595 strain belonged to the same branch as the HRV B-type reference strain KY093602(Malaysia strain). The MN146019 strain was in a single branch. Three local strains of HRVs were isolated in Bengbu City,China:HRV A,HRV B,and HRV C,and these data suggested frequent variation of local HRVs,and that local prevention and control of HRVs is needed.