Rapid acquisition of mammalian characteristics by avian influenza virus in single host infections.

甲型流感病毒的自然宿主是野生水禽，在那里大量不同亚型的病毒持续循环，几乎不会造成疾病。众所周知，野生鸟类将禽流感病毒(AIV)传播到养殖的家禽物种，导致具有重大生产和经济后果的疫情。近年来，在养殖家禽中爆发禽流感的报告有所增加，包括目前流行的H5N8和H7N9毒株。禽流感在家禽中的暴发增加了跨物种感染人类的机会，因为在养殖场或东南亚的活禽市场感染的禽类与人类非常接近。要使禽流感病毒引起持续的人类感染和继续传播，“关键的病毒特征”必须经过适应。其中一个特征是病毒聚合酶能够在人类上呼吸道的低温下在哺乳动物细胞中有效复制病毒基因组。在AIV聚合酶蛋白中可以发生几种明确的突变，这些突变可以增强哺乳动物细胞的活性。对数据库中的聚合酶蛋白序列的分析和我们的初步数据表明，对于某些禽流感病毒株，这些适应突变在来自禽类宿主的病毒分离株中出现的频率较低，而在从哺乳动物宿主分离的同一病毒株中，这些适应突变出现的频率较高。这表明，当某些AIV毒株感染哺乳动物宿主时，受感染的个体会发生快速适应，以增强病毒复制。在这个项目中，我们将利用重组AIV来衡量在病毒被免疫系统清除之前，感染个体是否可以快速适应哺乳动物宿主--小鼠。我们将用相关的AIV毒株感染小鼠，并在最初感染后的多个时间点对从感染小鼠中回收的病毒遗传物质进行测序，以表征病毒的任何变化并确定它们何时出现。H9N2型病毒在南亚和中亚大片地区的家禽中流行。H9N2病毒将内部基因片段捐赠给H7N9禽流感病毒株，该病毒目前在中国造成大量家禽和人感染。H7N9和H9N2都被报道在最初的哺乳动物宿主中快速适应聚合酶基因，因此可能存在内部H9N2基因的特征，使它们易于这种快速适应。我们将评估H9N2病毒聚合酶基因在哺乳动物细胞中的病毒聚合酶保真度和基线复制活性的病毒特征，并将其与其他重要的AIV亚型进行比较。我们将操纵病毒聚合酶的保真度，以询问这是否会改变快速适应的能力。宿主对流感病毒感染的免疫反应最终会解决感染问题，因此病毒抑制宿主免疫反应的能力可能会影响包含适应突变的病毒基因组的积累。因此，我们还将研究不同的AIV毒株抑制宿主免疫反应的能力，使用基因操作来产生这种特征发生改变的病毒。通过这种方式，我们将了解病毒基因组中编码的特定AIV特征是否可以改变哺乳动物宿主经历快速适应的倾向。我们的目标是提供关于哺乳动物对引起频繁家禽暴发的严重AIV毒株在单个宿主中的易应性的基本知识，并阐明促进这一现象的机制。希望这项研究将对家禽禽流感控制领域的政策制定者将作出的决定产生影响，这些决定往往具有深远的经济和社会代价。

The natural host for influenza A viruses are wild aquatic birds where large numbers of different subtypes continuously circulating causing little disease. Wild birds are known to spread avian influenza virus (AIV) to farmed poultry species causing outbreaks that have significant production and economic consequence. In recent years there have been increased reports of AIV outbreaks in farmed poultry, including the currently circulating strains H5N8 and H7N9. Outbreaks of AIV in poultry increases the opportunity of cross-species infections into humans because of close proximity in a farming situation or the live bird markets of South East Asia of infected birds with humans. For AIV to cause sustained human infections and onward transmission critical virus characteristics' must undergo adaptation. One characteristic is the ability of the viral polymerase to replicate the viral genome effectively in mammalian cells at the low temperatures found in the human upper respiratory tract. There are several well defined mutations that can occur in the AIV polymerase proteins that enhances activity in mammalian cells. Analysis of the polymerase protein sequence from databases and our preliminary data show that for some AIV strains there are low frequencies of these mutations in viral isolates from avian hosts but that in the same viral strains isolated from mammalian hosts these adapting mutations appear at high frequency. This suggests that upon infection of a mammalian host by certain AIV strains rapid adaptation to enhance virus replication occurs in the individual infected. In this project we will make use of recombinant AIVs to measure if rapid adaptation to a mammalian host, the mouse, can occur in the infected individual before the virus is cleared by the immune system. We will infect mice with pertinent AIV strains and sequence the viral genetic material recovered from the infected mice at multiple time-points after initial infection to characterise any changes to the virus and determine when they appear. H9N2 viruses are endemic in poultry in vast swathes of south and central Asia. H9N2 viruses donated the internal genetic segments to the H7N9 avian influenza strain that is currently causing numerous poultry and human infections in China. Both H7N9 and H9N2 have been reported to rapidly adapt polymerase genes in the initial mammalian host therefore there may be characteristics of the internal H9N2 genes that predispose them to this rapid adaption. We will assess the viral characteristics of polymerase fidelity and baseline replication activity in mammalian cells of the H9N2 viral polymerase genes and comparison these to other important AIV subtypes. We will manipulate the fidelity of the viral polymerase to ask if this alters the ability to rapidly adapt. The host immune response to influenza virus infection ultimately resolves infection, so the ability of a virus to suppress a host immune response may affect the accumulation of a viral genome containing adaptation mutations. We will therefore also investigate different AIV strain ability to suppress the host immune responses use genetic manipulation to generate viruses that have this characteristic altered. In this way we will understand if specific AIV characteristics that are encoded in the virus genome can change the propensity to undergo rapid adaptation in mammalian hosts. Our aim is to provide fundamental knowledge about the ease of mammalian adaptation in an individual host for serious AIV strains that cause frequent poultry outbreaks and shed some light on the mechanism that facilitate this phenomenon. It is hoped this research will have an impact on the decisions that policymakers in the area of avian influenza control in poultry will make which often have far-reaching economic and societal costs.