Elucidation of virulence determinants of highly pathogenic non-H5, non-H7 avian influenza viruses

禽流感病毒（AIV）在鸟类中的16种血凝素（HA）亚型分为低致病性（LP）和高致病性（HP）。到目前为止，HP禽流感（禽鼠疫）仅由在HA蛋白内具有多碱基切割位点（CS）的H5和H7亚型病毒引起。有趣的是，很少有非H5/H7毒株符合HPAIV的官方定义。这些病毒的毒力标记是未知的日期，但可以使用反向遗传学系统的AIV，已经建立在我们的实验室进行调查。2012年，从鹌鹑中分离出一种独特的H4 N2病毒，其HA为多碱基CS。然而，该病毒在鸡中显示出低毒力，并且在不添加外源胰蛋白酶的情况下不能在细胞培养物中生长。研究了H4 N2转变为HP表型的可能性：（1）在含胚鸡蛋或鸡中连续传代后，（2）通过增加HA CS处的碱性氨基酸数量，（3）去除靠近CS的高度保守的糖基化位点，以及（4）通过与HPAI H5/H7病毒重配。此外，2014年欧洲港海豹的死亡归因于H10 N7病毒，该病毒与野生鸟类中的AIV密切相关。与来自鸟类的H10 Nx相比，在海豹H10 N7病毒中观察到了几个遗传标记。在这个建议中，我们将研究这些突变对毒力，发病机制和适应在体内和体外的影响。总之，低毒力，最近的H4 N2病毒是第一个天然存在的非H5/H7 AIV与多元HA CS。该病毒与H5/H7亚型禽流感病毒相似，但其生物学功能尚待进一步研究。同样，将研究海豹H10 N7病毒特有的突变对哺乳动物适应的影响。

The 16 hemagglutinin (HA) subtypes of avian influenza viruses (AIV) in birds are classified into low pathogenic (LP) or highly pathogenic (HP). Up to now, HP avian influenza (fowl plague) was only caused by viruses specifying subtypes H5 and H7 with a polybasic cleavage site (CS) within the HA protein. Interestingly, there are few non-H5/H7 strains complying with the official definition of HPAIV. Virulence markers of those viruses are not known to date but can be investigated using reverse genetics systems for AIV that have been established in our laboratory. A unique H4N2 virus with a polybasic CS at the HA was isolated from quails in 2012. However, the virus displayed low virulence in chickens and did not grow in cell culture without the addition of exogenous trypsin. The potential of H4N2 to shift to an HP phenotype was investigated (1) after serial passages in embryonated chicken eggs or chickens, (2) by increasing the number of basic amino acids at the HA CS, (3) removal of high conserved glycosylation sites close to the CS and (4) by reassortment with HPAI H5/H7 viruses. Moreover, mortalities in harbor seals in Europe in 2014 was attributed to H10N7 virus, which was closely related to AIV in wild birds. Several genetic markers were observed in the seal-H10N7 virus compared to H10Nx from birds. In this proposal, we will study the impact of these mutations on virulence, pathogenesis and adaptation in vivo and in vitro. In summary, the low virulent, recent H4N2 virus is the first naturally occurring non-H5/H7 AIV with polybasic HA CS. Mutations and gene segments conferred high virulence of this virus in chickens resembling H5/H7 HPAIV were determined, however, their biological functions remain to be studied. Likewise, the impact of mutations peculiar to seal-H10N7 virus on adaptation in mammals will be investigated.