Long-term adaptation following virus host shifts results in higher replication rate, broader intra-host spread and lower pathogenicity

Influenza A viruses (IAVs) have their main natural reservoir in wild birds and have established in a limited number of mammals, sometimes with devastating consequences. This is illustrated by the four human pandemics that occurred in the last 100 years as well as the large number of outbreaks and epizooties in various species of domestic animals. Infection of a mammal by an avian-origin virus can cause variable outcomes ranging from lethal infections (such as those caused by H5N1 and H7N9 in humans) to outbreaks and pandemics that might lead to the establishment of new endemic viruses. H3N8 equine influenza virus (EIV) constitutes an example of an avian-origin IAV that jumped into a mammalian host -the horse- and established as an endemic lineage that has been circulating in many countries for over 50 years. Virus adaptation requires finely tuned interactions between virus and host proteins, as molecular incompatibilities could act as effective species barriers. We hypothesise that mammalian adaptation is evolutionarily driven and involves multiple changes in virus-host interactions that lead to higher fitness as well as in changes in virus pathogenesis. To test our hypothesis, we infected an interferon-competent equine cell line (E-Derm) with two evolutionarily distinct H3N8 IAVs (A/equine/Uruguay/1/1963 [EIV/63], and A/equine/Ohio/1/2003 [EIV/2003]) and quantified changes in gene expression levels by performing transcriptome analysis at different times post-infection. Cells infected with EIV/63 and EIV/2003 displayed significant differences in gene expression patterns, resulting in virus-specific changes on the regulation of intracellular pathways. Particularly, we observed that cells infected with EIV/2003 exhibit at early time-points a lower number of differentially expressed genes (DEGs) but associated with a high level of regulation. In addition, we observed significant differences between the two EIVs on the virus-induced lesions within the respiratory tract of the horse. In sum, our results provide an initial inventory of genes and intracellular pathways that are likely to be important for the adaptation of avian-origin IAVs to mammals, and also show that IAV evolution has a significant impact on host gene regulation and viral pathogenesis.

甲型流感病毒（Influenza A viruses, IAV）的主要自然储存宿主为野生鸟类，且已在有限的哺乳动物类群中定植，有时会引发灾难性后果。这一点可通过过去100年间发生的4次人类大流行，以及多种家畜暴发的大量疫情和兽疫得到佐证。禽源病毒感染哺乳动物后可引发多种结局：从致死性感染（如人类感染H5N1、H7N9病毒所致病例），到可能催生新型地方性流行病毒的暴发与大流行均有发生。

马H3N8亚型流感病毒（Equine Influenza Virus H3N8, EIV）便是禽源甲型流感病毒跨物种传播至马这一哺乳动物宿主，并形成地方性流行谱系的典型案例——该谱系已在全球多国传播超过50年。病毒的适应性定植依赖病毒与宿主蛋白间的精细互作，因为分子不相容性可构成有效的物种屏障。我们提出假说：哺乳动物适应性定植是进化驱动的过程，涉及病毒-宿主互作的多重改变，以提升病毒的增殖适合度，同时也会改变病毒的致病机制。

为验证该假说，我们使用两种进化谱系迥异的H3N8亚型马流感病毒（A/equine/Uruguay/1/1963 [EIV/63] 与A/equine/Ohio/1/2003 [EIV/2003]）感染具备干扰素应答能力的马源细胞系（E-Derm），并通过感染后不同时间点的转录组分析，量化细胞基因表达水平的变化。

感染EIV/63与EIV/2003的细胞展现出显著的基因表达模式差异，导致病毒特异性的细胞内通路调控改变。尤为值得注意的是，感染EIV/2003的细胞在感染早期的差异表达基因（Differentially Expressed Genes, DEGs）数量更少，但这些基因的调控幅度更高。此外，两种马流感病毒在诱发马呼吸道损伤方面也存在显著差异。

综上，本研究结果首次梳理了可能对禽源甲型流感病毒适应哺乳动物宿主至关重要的基因与细胞内通路，同时证实甲型流感病毒的进化过程会对宿主基因调控及病毒致病机制产生显著影响。