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目的分析2013-2014年杭州市季节性A(H3N2)流感病毒PB1基因及其编码蛋白的变异情况,揭示其分子特征与遗传进化趋势。方法对分离得到的78株流感病毒PB1基因进行扩增和测序,采用序列多重比对、突变位点比较分析和构建进化树等方法进行遗传进化分析。结果 78株流感病毒PB1基因的核苷酸序列和编码蛋白序列一致性分别为97.89%~100.00%和99.08%~100.00%,PB1-F2蛋白短肽的序列一致性为44.44%~100.00%;PB1蛋白氨基酸序列与参考株A/Texas/50/2012相比,在十多个位点均发生了氨基酸突变,其中V212M、R215K、E331D、A374S氨基酸突变频率较高;PB1-F2蛋白长度分析发现了5种不同的PB1-F2蛋白,其中3种为全长蛋白(90、87、79)aa,2种为截断型PB1-F2蛋白(52,24)aa;构建系统进化树发现78株H3N2毒株PB1基因及其编码蛋白均在同一个支系上,与同期流行季的疫苗株进化关系较近,第三个流行高峰(2014年6~10月)进化关系相对更集中。结论 2013-2014年季节性A(H3N2)流感病毒PB1蛋白氨基酸位点发生了部分重要突变,病毒PB1基因及其编码蛋白的遗传进化以及截断型PB1-F2蛋白的集中出现,可能促使病毒的复制能力和毒力发生变化,并最终导致其流行传播趋势的改变。
Objective To analyze the variation of the PB1 gene and the encoded protein of the seasonal influenza A (H3N2) influenza virus in Hangzhou from 2013 to 2014 and reveal its molecular characteristics and genetic evolution trend. Methods The 78 strains of influenza viruses isolated from PB1 were amplified and sequenced. The genetic evolution was analyzed by sequence multiple alignment, comparative analysis of mutation sites and construction of phylogenetic tree. Results The nucleotide sequence and the encoded protein sequence identity of PB1 gene of 78 strains of influenza viruses were 97.89% -100.00% and 99.08% -100.00%, respectively. The sequence identities of PB1-F2 protein short peptides were 44.44% -100.00%. PB1 Compared with the reference strain A / Texas / 50/2012, the amino acid sequence of the protein amino acid mutations occurred in more than ten loci, of which the mutation frequencies of V212M, R215K, E331D and A374S amino acids were higher; the length analysis of PB1-F2 protein revealed that Five different PB1-F2 proteins, of which three were full-length proteins (90,87,79) aa and two were truncated PB1-F2 proteins (52,24) aa. Construction of phylogenetic tree revealed 78 strains of H3N2 virulence The PB1 gene and its encoded protein were all in the same branch, which had close evolutionary relationship with the vaccine strains in the same epidemic season. The third peak of the epidemic (June to October in 2014) was relatively concentrated. Conclusion The amino acid sequence of the PB1 protein of seasonal A (H3N2) influenza virus from 2013 to 2014 was partially mutated. The genetic evolution of PB1 gene and its encoded protein and the concentration of truncated PB1-F2 protein may promote the replication of virus Capacity and virulence change, and ultimately lead to changes in the prevalence of its spread.