Impact of variation in equine influenza A virus pathogenicity determinants on vaccine effectiveness and interspecies transmission

甲型流感病毒（IAV）被认为已经在马中传播了几个世纪，并且在过去已经跨越物种障碍感染人类。2005年，首次发现马甲型流感病毒（eqIAV）传播给犬的证据;该病毒随后在美国流行。我们回顾性地证明，2002年在英国，eqIAV也曾从马身上跳到猎狐犬身上，但没有得到证实。用2003年分离的eqIAV毒株感染小马的实验证实了比早期分离株更严重的临床症状（包括长时间咳嗽）。这与促炎性细胞因子的更大诱导相关。2003年毒株的基因组测序显示病毒NS 1蛋白的截短，其调节宿主细胞的细胞因子反应。使用反向遗传学转换NS基因片段，我们证实截短的NS 1在感染细胞中诱导更大的细胞因子应答。这表明，由于致病性增加的eqIAV毒株的出现，发生了物种跳跃。有趣的是，NS 1截短在适应狗时丢失。自20世纪60年代末以来，针对eqIAV的疫苗已经可用，自1982年以来，英国赛马的疫苗接种已成为强制性的。有一个更新疫苗株的过程，但马流感的周期性大流行仍然存在-这些流行发生在1963年，1979年，1989年，2003年和2019年。这些主要流行病通常与未接种疫苗的马的一些死亡有关。血凝素表面糖蛋白的变异作为疫苗分解的原因已被广泛研究。该项目的目的是研究决定IAV致病性的病毒蛋白变异在疫苗分解和种间传播中的作用。反向遗传学是一种技术，其中重组流感病毒可以通过将八个质粒（每个质粒编码八个病毒基因片段中的一个）体外转染到哺乳动物细胞中来产生。这允许单独研究病毒之间的特定变化，例如通过在“同基因”背景上仅交换编码NS 1的基因片段。该技术将通过生物信息学分析来补充，以比较流行性eqIAV毒株与流行性毒株之间决定IAV致病性的基因。将在细胞培养物中检测基因已交换或突变的病毒复制和诱导或抵抗促炎细胞因子应答的能力。使用已建立的技术和具有互补技能的合作者的参与应确保任何挑战都可以得到解决，并且这项工作的适当风险评估已经到位，识别与流行菌株出现相关的模式可以提高世界动物卫生组织（OIE）专家监测小组的能力，以预防下一次马流感流行。

Influenza A viruses (IAV) are believed to have circulated in horses for centuries and to have crossed species barriers to infect humans in the past. In 2005, the first evidence for transmission of equine influenza A virus (eqIAV) to dogs was reported; the virus subsequently became endemic in the USA. We retrospectively demonstrated that eqIAV had also jumped from horses to foxhounds in the UK in 2002, but did not become established. Experimental infection of ponies with an eqIAV strain isolated in 2003 confirmed the more severe clinical signs seen (including prolonged coughing) than with earlier isolates. This was associated with greater induction of pro-inflammatory cytokines. Genome sequencing of the 2003 strain revealed a truncation in the viral NS1 protein, which modulates the host cell cytokine response. Using reverse genetics to switch the NS gene segment, we confirmed that the truncated NS1 induced a greater cytokine response in infected cells. This suggests that the species jump occurred due to emergence of an eqIAV strain with increased pathogenicity. Interestingly, the NS1 truncation was lost on adaptation to dogs. Vaccines against eqIAV have been available since the late 1960s and vaccination of UK racehorses has been compulsory since 1982. There is a process for updating vaccine strains but there continue to be periodic major epidemics of equine influenza - these have occurred in 1963, 1979, 1989, 2003 and 2019. These major epidemics are usually associated with some deaths among unvaccinated horses. Variation in the haemagglutinin surface glycoprotein as a cause of vaccine breakdown has been extensively studied. The aim of this project is to investigate the role of variation in viral proteins that determine the pathogenicity of IAV in vaccine breakdown and interspecies transmission. Reverse genetics is a technique where a recombinant influenza virus can be generated by transfecting eight plasmids each encoding one of the eight viral gene segments into mammalian cells in vitro. This allows specific changes between viruses to be studied individually, for example by swapping only the gene segment encoding NS1 on an 'isogenic' background. This technique will be complemented by bioinformatics analysis to compare the genes that determine the pathogenicity of IAV in epidemic eqIAV strains with inter-epidemic strains. The ability of viruses in which genes have been swapped or mutated to replicate and induce or resist pro-inflammatory cytokine responses will be tested in cell culture. The use of established techniques and involvement of collaborators with complementary skills should ensure any challenges can be addressed and the appropriate risk assessment for this work is already in place.Identification of patterns associated with the emergence of an epidemic strain could improve the ability of the World Organization for Animal Health (OIE) Expert Surveillance Panel to pre-empt the next equine influenza epidemic.