HA和NA匹配度对H5亚型高致病性禽流感假病毒影响的研究

Impact of various HA and NA pair combinations from different H5 subtype highly pathogenic avian influenza virus on production of influenza pseudovirus

为研究不同H5亚型高致病性禽流感病毒(H5 HPAIV)的HA和NA匹配度对流感假病毒组装和功能的影响,本研究将5株H5 HPAIV的HA和NA基因节段分别克隆于pCMV/R载体,构建3个HA和5个NA重组质粒,两两随机组合后将HA、NA质粒与pCMV/RΔ8.9质粒、p HR-CMV-Luc质粒构成4质粒系统,利用磷酸钙法将4个质粒转染HEK293T细胞,检测各细胞培养上清液血凝效价,结果显示:有3个上清液无血凝效价,其余12个上清液的血凝效价在100 HAU/m L~400 HAU/m L,表明获得12株具有血凝效价的流感假病毒。超速离心后获得浓缩假病毒,通过western blot检测假病毒HA蛋白的表达,结果显示各假病毒均在75 ku、50 ku和25 ku有特异性条带。利用各假病毒分别感染MDCK.1细胞60 h后裂解细胞,采用荧光素酶试验检测假病毒的相对荧光素酶活性(RLA),结果显示各假病毒RLA在5.0×10~1~4.5×10~5不等。上述结果首次表明不同H5 HPAIV的HA和NA相互匹配会对假病毒的血凝效价和相对荧光酶活性(RLA)造成影响。本研究为后续流感病毒灭活疫苗制备及抗病毒药物的研发奠定基础。

This research aims to elucidate the impact of various HA and NA pair combinations from different H5 subtypes of the highly pathogenic avian influenza virus(H5 HPAIV)on the assembly and functionality of influenza pseudoviruses.Initially,the HA or NA gene segments from five H5 HPAIV strains were integrated into the pCMV/R vector,yielding three HA and five NA recombinant plasmids.These plasmids were then randomly paired,and in conjunction with pCMV/RΔ8.9 and pHR-CMV-Luc plasmids,a four-plasmid system was established.Subsequently,this system was introduced into HEK293T cells utilizing the calcium phosphate method to generate pseudoviruses.then the hemagglutination titers of each cell culture supernatant were tested.Our findings reveal that three of the supernatants had no hemagglutination titers,while the remaining twelve supernatants had hemagglutination titers between 100HAU/mL-400HAU/mL,indicating the acquisition of twelve influenza pseudoviruses with hemagglutin ationtiters.After ultracentrifugation,a concentrated pseudovirus was obtained.Further validation through western blot analysis identified accurate band formations at 75ku,50ku,and 25ku,and the relative luciferase activity assay displayed values spanning from 5.0×10~1 to 4.5×10~5.These findings strongly indicate that different combinations of HA and NA pairs extracted from various H5 HPAIV strains significantly influence the hemagglutination titer and relative luciferase activity of the synthesized pseudoviruses.This pioneering study thereby establishes a fundamental basis for the forthcoming developments in inactivated influenza virus vaccine production and pharmaceutical research and development.