Highly conserved motifs in NS1 and PB1-F2 proteins of highly pathogenic avian influenza viruses and their functionalities in virulence, immune responses and pathological alterations like acute lung injury (ALI)

据世界卫生组织估计，呼吸道感染造成的死亡占全球死亡总数的6%以上。重要的是，在所有能够感染呼吸道的病原体中，甲型流感病毒（IAV）是最常见和最具破坏性的。除了数百万人住院治疗和数千人死亡之外，这些病原体每年在季节性流感期间造成巨大的经济损失。更令人担忧的是，IAV有可能导致大流行爆发，造成数百万人死亡，例如1918年的西班牙流感爆发，IAV被认为是即将到来的危及生命的大流行的潜在病原体。禽流感病毒的人畜共患潜力是自1997年以来在人间发生大流行疫情和H5N1亚型高致病性禽流感病毒（HPAI）向人间持续散发传播的根本原因。60%的高致死率强调需要了解人类感染高致病性禽流感的生物学原理。为了了解这种高病死率，已经开展了针对不同病毒蛋白的多种方法。除了高致病性禽流感的血凝素蛋白对其毒力和最终传播的贡献外，聚合酶蛋白在提高H5N1禽流感病毒复制效率方面的作用也得到了研究。目前对非结构蛋白NS1和PB1-F2的研究较少。这两种蛋白都携带免疫调节和干扰素拮抗特性，并被描述为毒力决定因素。一般来说，氨基酸基序在季节性人类和高致病性禽流感中存在差异，这成为了解高致病性禽流感感染严重程度的关注焦点。在NS1蛋白方面，几乎所有的禽流感病毒分离株中都存在一个SH3结合基序（aa212-217）， H5N1分离株中存在一个特殊的位置，而季节性的人禽流感病毒则缺乏这个基序。在PB1-F2的背景下，四种氨基酸（促炎基序；L62, R75， R79和L82）已被确定为细胞因子释放和炎症反应的贡献者，并且在禽类IAV中高度存在，包括在人类H5N1分离株中高度流行。在本研究中，将广泛研究NS1和PB1-F2中所描述的基序对不同宿主（鸟类和哺乳动物）的高致病性禽流感毒力、免疫反应调节以及最终免疫病理和急性肺损伤的影响。了解高致病性禽流感人毒力的基本机制和这些基序的影响可能会加强更有指导的监测能力，并为高致病性禽流感感染制定更多的治疗方案。

As estimated by the World Health Organization, infections of the respiratory tract are responsible for more than 6 percent of all deaths worldwide. Importantly, among all pathogens having the capability to infect the respiratory tract, influenza A viruses (IAV) are among the most common and devastating. In addition to millions of hospitalizations and thousands of deaths, these pathogens lead to enormous economic losses during the seasonal influenza each year. Even more worrisome, IAV have the potential to cause pandemic outbreaks with several million victims, such as the Spanish flu outbreak in 1918 and IAV are discussed as potential causative agent for upcoming life-threatening pandemics. The zoonotic potential of IAV is fundamental for the occurrence of pandemic outbreaks in humans and the ongoing, sporadic transmission of highly pathogenic avian IAV (HPAI) of the subtype H5N1 to humans since 1997. The high case fatality rate of 60%, emphasizes the need to understand the biology of HPAI infection of humans. To understand this high case fatality rate, multiple approaches focusing on different viral proteins have been conducted. Beside the contribution of the hemagglutinin protein of HPAI to their virulence and finally transmission, the polymerase proteins have been studied for their role in increasing replication efficiency of H5N1 IAV. Less effort has been put into study of the non-structural proteins NS1 and PB1-F2 to this point. Both proteins carry immune modulatory and interferon antagonistic properties and are described as virulence determinants. In general, amino-acid motifs, which are differentially present in seasonal human and HPAI came into focus of interest for understanding the severeness of HPAI infections. Regarding the NS1 protein, a SH3 binding motif (aa212-217) is present in almost all avian IAV isolates with a special position of H5N1 isolates in contrast to seasonal human IAV lacking this motif. In the context of PB1-F2, four amino acids (pro-inflammatory motif; L62, R75, R79 and L82) have been identified as contributor to cytokine release and inflammatory responses and are highly present in avian IAV, including a high prevalence in human H5N1 isolates.In this study, the impact of the depicted motifs in NS1 and PB1-F2 on virulence of HPAI, modulation of immune responses and finally immune pathology and acute lung injury in different hosts (avian and mammalian) will be broadly investigated. Understanding the basal mechanisms of HPAI virulence in humans and the impact of these motifs potentially strengthen the ability of more guided surveillance and set up of additional treatment options for HPAI infections.