摘要
目的了解吉林省2006年和2008年急性弛缓性麻痹(Acute Flaccid Paralysis,AFP)病例和健康人群中分离到的柯萨奇病毒A组4型(Coxsackievirus Group A Type4,CVA4)的基因特征,探索特定血清型肠道病毒(Enterovirus,EV)与AFP病例之间的关系。方法 从75例AFP病例和60例边境地区健康人群中采集原始粪便标本,使用人横纹肌肉瘤(Human Rhabdom yosarcom a,RD)细胞和人喉癌(Human Laryngeal Carcinoma,Hep-2)上皮细胞进行病毒分离,阳性标本用EV组合血清进行中和试验鉴定血清型,未能定型的标本用EVVP4-VP2蛋白编码区引物通过逆转录-聚合酶链反应(Reverse Transcription-Polymerase Chain Reaction,RT-PCR)进行扩增,扩增阳性标本通过基因序列分析鉴定血清型。从中挑选CVA4扩增全长VP1编码区片段并进行核苷酸序列测定和分析,测序结果利用Bioedit软件进行核苷酸和氨基酸同源性分析,用Mega软件建立基因进化树,进行基因亲缘关系分析。结果使用RD细胞从AFP病例粪便标本中分离到5株CVA4,从健康人群粪便标本中分离到4株CVA4。这9株病毒用EV组合血清均无法定型,经过VP4-VP2测序的方法 鉴定为CVA4。9株病毒VP1编码区的核苷酸和氨基酸同源性分别为88.3%～100%和98%～100%,与CVA4原型株CVA4/HighPoint(海波因特)/USA(美国)/1950的核苷酸和氨基酸同源性分别为83.3%～85.6%和97.7%～98.6%。其中4株来自AFP病例的病毒具有高度的基因同源性(99.6%～100%),这4株病毒与来自健康人群的病毒比较,核苷酸和氨基酸同源性分别为88.3%～88.9%和98.0%～98.6%。在基因进化树上显示,这4株来自AFP病例的CVA4构成一个独立的分支。结论基因进化分析表明,4株来自AFP病例的毒株VP1编码区的基因特征,与来自健康人群的毒株的基因特征显著不同。
Objective To study the genetic characteristic of coxsackievirus group A type 4 （CVA4）, isolated in 2006 and 2008, try to find the relationship between acute flaccid paralysis （AFP） and characteristic of CVA4 strains. Methods Stool specimens were collected from 75 AFP cases and 60 healthy people living in border area, then virus isolation was performed by inoculating in human rhabdomyosarcoma （RD） cell and human laryngeal carcinoma （Hep-2） cell. Positive isolates were identified with neutralization test and VPa-VP2 gene sequence analysis, CAV4 stains were then selected to amplify the complete VP1 coding region and analyze the gene characteristic. The homogeneity was analyzed by the BioEdit sequence alignment editor and the phylogenetic dendrogram was constructed by the Mega software. Results Five CVA4 viruses were isolated from the AFP cases and four were isolated from healthy population by inoculating RD ceils. They were untypable by neutralization test and the serotype was obtained by VP4-VP2 gene sequence. The homology of the nucleotide sequences in VP1 region among the nine Jilin CVA4 strains was 88.3%-100%, and which of the amino acid sequences was 98.0%-100%. Compared with the prototype strain of CVA4 （Strain High Point/USM1948）, the homologies of nucleotide sequences and amino acid sequences were 83.3%-85.6% and 97.7%-98.6% respectively. Among the nine virus strain, four CVA4 virus which were from the AFP cases has high VP1 gene sequence homology （99.6%-100% ）, compared with the four viruses from healthy population, the homologies of nucleotide sequences and amino acid sequences were 88.3%-88.9% and 98.0%-98.6% respectively. Phylogenetic dendrogram showed that the four CVA4 strains from the AFP cases constructed an independent evolution lineage. Conclusion Four of CVA4 virus strains from AFP cases were found genetically significant different from the strains isolated from healthy population.
出处
《中国疫苗和免疫》
CAS
2012年第6期531-535,565,共6页
Chinese Journal of Vaccines and Immunization
基金
吉林省卫生厅科学研究课题"急性弛缓性麻痹病例中肠道病毒的研究"(编号:20082086)
关键词
急性弛缓性麻痹病例
健康人群
柯萨奇病毒A组4型
基因特征
Acute flaccid paralysis cases
Health papulation
Coxsackieviurs group A type 4
Genetic characteristic
