Abstract：
Influenza remains one of the most prevalent viruses circulating amongst humans and has resulted in several pandemics. The prevention and control of H3N2 influenza is complicated by its propensity for evolution, which leads to vaccine mismatch and reduced vaccine efficacies. This study employed the strategy of serial passaging to compare the evolution of the human seasonal influenza strain A/Singapore/G2-31.1/2014(H3N2) in MDCK-SIAT1 versus primary chick embryo fibroblast (CEF) cells. Genetic analysis of the HA, NS1, NA, and PB1 gene segments by Sanger sequencing revealed the presence of specific mutations and a repertoire of viral quasispecies following serial passaging. Most quasispecies were also found in PB1, which exhibited consistently high transversion-to-transition ratios in all five MDCK-SIAT1 passages. Most notably, passage 5 virus harbored the D457G substitution in the HA2 subunit, while passage 3 virus acquired K53Q and Q69H mutations in PB1-F2. An A971 variant leading to a non-synonymous R316Q substitution in PB1 was also identified in MDCK-SIAT1 passages 2 and 4. With an increasing number of passages, the proportion of D457G mutations progressively increased and was associated with larger virus plaque sizes. However, microneutralization assays revealed no significant differences in the neutralizing antibody profiles of human-influenza-immune serum samples against pre-passaged virus and passage 5 virus. In contrast, viable virus was only detected in passage 1 of CEF cells, which gave rise to multiple viral RNA quasispecies. Our findings highlight that serial passaging is able to drive differential adaptation of H3N2 influenza in different host species and may alter viral virulence. More studies are warranted to elucidate the complex relationships between H3N2 virus evolution, viral virulence changes, and low vaccine efficacy.
摘要：
流感仍然是在人类中传播的最普遍的病毒之一，并导致了几次大流行。H3N2流感的预防和控制因其进化倾向而变得复杂，这导致疫苗不匹配和疫苗效力降低。这项研究采用了连续传代的策略，以比较人季节性流感株A/新加坡/G2-31.1/2014（H3N2）在MDCK-SIAT1与原代鸡胚成纤维细胞（CEF）细胞中的进化。HA的遗传分析，NS1,NA,通过Sanger测序和PB1基因片段显示，在连续传代后，存在特定的突变和病毒准种。在PB1中也发现了大多数准种，在所有五个MDCK-SIAT1传代中始终表现出较高的颠换过渡比。最值得注意的是,第5代病毒在HA2亚基中具有D457G取代，而第3代病毒在PB1-F2中获得K53Q和Q69H突变。在MDCK-SIAT1第2和第4代也鉴定了在PB1中导致非同义R316Q取代的A971变体。随着通道数量的增加，D457G突变的比例逐渐增加,并且与较大的病毒空斑大小相关.然而,微中和分析显示，人流感免疫血清样品针对传代前病毒和第5代病毒的中和抗体谱没有显着差异。相比之下,活病毒仅在CEF细胞的第1代检测到，从而产生了多种病毒RNA准种。我们的发现强调，连续传代能够驱动不同宿主物种对H3N2流感的差异适应，并可能改变病毒的毒力。有必要进行更多的研究来阐明H3N2病毒进化之间的复杂关系，病毒毒力变化，疫苗效力低。
