替换基因片段对流感病毒生长的影响

Effects of different gene segments of influenza virus on the growth of recombinants

摘要:

摘要:【目的】探索流感病毒内部基因对病毒滴度的影响，构建高产流感疫苗种子病毒。【方法】将A/chicken/ZJ/China/2013(H5N1) (ZJ)病毒的6个内部基因或点突变体或聚合酶复合物基因逐个替换鸡胚高度适应的A/Puerto Rico/8/1934(H1N1)病毒(PR8)的相同基因，构建重组病毒，通过血凝试验比较重组病毒在鸡胚上的增殖滴度。【结果】PB2基因影响最大，替换后未能产生重组病毒;PB1、PA、M基因替换后，重组病毒滴度分别下降了3.7、3.4、3.0个滴度(log2);NS基因替换后基本没有影响;聚合酶复合体基因替换后，病毒滴度稍有下降(7.6 log2)，没有提供像完全来自PR8的聚合酶复合体相同的生长特性(8.4 log2);PR8 PB2基因627位点替换成谷氨酸(E)后，病毒滴度从8.4 log2上升到8.7 log2。【结论】合适优化的基因组合可以通过病毒RNA与蛋白之间、蛋白与蛋白之间的相互作用促进病毒复制，从而筛选出能在鸡胚中高效复制的重组体，为高产流感疫苗的生产奠定基础。

Abstract:

Abstract:［Objective］We explored which internal genes of influenza virus that affect the titer of recombinant viruses and contribute to the high yield of Influenza A seed virus in ovo.［Methods］ Six internal genes or mutant or polymerase complex of A/Puerto Rico/8/1934 (H1N1) (PR8) virus genes were replaced individually by corresponding gene of A/chicken/ZJ/China/2013 (H5N1) virus，and the hemagglutination titers of recombinant viruses were compared by HA assay.［Results］PB2 gene had the greatest influence，its replace failed to generate recombinant virus． When PB1，PA，or M gene was replaced，the titers of recombinant viruses dropped by 3.7，3.4，3.0 (log2)，respectively．NS gene had little influence upon HA titer． When polymerase complex genes were replaced，virus titer dropped slightly to 7.6 log2，and it did not confer the same growth characteristics (8.4 log2) found when a complete polymerase complex was of PR8 origin．When amino acids of position 627 of PR8 PB2 gene were mutated to glutamic acid，virus titer rose from 8.4 log2 to 8.7 log2.［Conclusion］ The optimal gene combinations may facilitate replication through viral RNA and protein interaction with cellular components as well as interaction of viral RNA and protein or protein-protein interactions within the virus．These multi-factorial contributions resulted in selection of a high replication competent reassortant in embryonated chicken eggs in comparison to the respective low yield wild type viruses，and laid the foundation for high yield of influenza vaccine production.