禽源H3N2亚型犬流感病毒跨越禽－犬宿主屏障的分子基础

Animal influenza is an important infectious disease caused by influenza A virus.The species specificity of influenza viruse is certain uasally, but interspecies transmission of avian influenza virus（AIV） across host barrier becomes onging.Since 2007, Asia's new hair H3N2 subtype canine influenza virus is widely prevalent in our country, causing an acute infectious disease in dogs-canine influenza that could be a potential threaten to human health. Sequence analysis demonstrated that this virus was avian orign.However,the mechanism of H3N2 subtype AIV crossing species barrier to infect dogs could not be expained by known theory . In the previous study, two viruses with different pathogenicities in dogs and ducks were selected.Their reverse genetic systems were established and a serious of one-gene substituted mutant viruses were rescued. In this project, we will use these mutant viruses to infect dogs.The crucial genes that determine the replication abilities of H3N2 avian-orign influenza viruses in dogs are expected to be found according to its replication and pathogenicity .In a further analysis, key amino acids of H3N2 canine influenza virus will be identified by site-direct methods and so on. The findings will provide a theroretical basis for an in-depth interpretation of the mechanism of H3N2 subtype canine influenza virus acorss host barrier to infect dogs.

动物流感是由A型流感病毒引起的一种重要传染病。A型流感病毒具有一定的宿主特异性，但近几年禽流感病毒跨越宿主屏障感染人类和哺乳动物的事件不断发生。2007年以来，亚洲新发H3N2亚型犬流感病毒在我国广泛流行，引起犬急性传染病-犬流感，同时给人类健康埋下了隐患。序列分析表明，新发犬流感病毒源于禽流感病毒，而用目前已知知识无法解释这类病毒跨越禽-犬种间屏障的分子机制。在早期研究中，我们筛选到两株高度同源的病毒，一株引起犬发病的犬流感病毒和一株不感染犬的鸭流感病毒，并建立它们的反向遗传操作系统，救获了一系列单基因替换的重组病毒。本项目拟利用前期救获的单基因替换重组病毒，人工感染实验犬，根据在犬体内的复制和对犬的致病能力来确定犬流感病毒跨越禽-犬宿主屏障的关键基因，进一步利用定点突变等方法，明确该类病毒在犬中流行的关键氨基酸位点，为深入解读H3N2亚型犬流感越禽-犬种间屏障的的分子机制研究奠定基础。

动物流感是由A型流感病毒引起的一种重要传染病。A型流感病毒具有一定的宿主特异性，但近几年禽流感病毒跨越宿主屏障感染人类和哺乳动物的事件不断发生。新发的犬流感病毒源于禽流感病毒，可以跨越禽-犬种间屏障感染犬，但却不感染禽。目前，该病毒的跨越禽-犬种间屏障的感染机制尚不清楚。前期研究中，我们筛选到两株高度同源的病毒，一株引起犬发病的H3N2犬流感病毒（C1）和一株不感染犬的H3N2鸭流感病毒（D6）,并建立了它们的反向遗传操作系统。本研究将拯救获得的rC1和rD6和它们的亲本毒分别感染鼠，结果发现拯救获得的rC1可在鼠中良好复制，而rD6却无法复制，这些致病性与亲本病毒C1和D6 完成一致。将一系列以C1为背景的单基因替换了D6的重组病毒感染鼠。除了C1+D6NP, C1+D6NA会引起鼠体重的正常增加，其余病毒包括C1+D6PB1, C1+D6PA, C1+D6HA, C1+D6M, C1+D6NS可导致鼠的体重在不同程度上减少。病毒的鸡胚滴定结果表明，C1+D6PB1, C1+D6PA, C1+D6HA, C1+D6NP, C1+D6M, C1+D6NS在鼠的肺组织上呈现不同程序的复制水平，而C1+D6NP, C1+D6NA在鼠的肺组织上不复制。此结果与荧光定量PCR的结果相同。C1+D6NP, C1+D6NA在鼠的肺组织皆无病理变化，而其它的重组病毒C1+D6PB1, C1+D6PA, C1+D6HA, C1+D6M, C1+D6NS与rC1相同，皆有不同程度的病理变化。这些结果表明，H3N2亚型犬流感病毒C1株的NA和NP基因是决定该病毒跨越禽-犬种间屏障的感染的关键基因。根据H3N2亚型犬流感和禽流感关键的差异氨基酸位点（前期研究基础），对C1毒株关键基因NA和NP相应的氨基酸位点进行了点突变，构建相应的重组质粒，并拯救相应的C1毒株点突变的重组病毒，包括rC1-NAL24M, rC1-NAK54E, rC1-NAS156P, rC1-NAG432R, rC1-NPV105M, rC1-NPK373T, rC1-NPT428A, rC1-NPK473N。rC1-NAK54E, rC1-NAS156P, rC1-NAG432R, rC1-NPV105M, rC1-NPK373T, rC1-NPT428A, rC1-NPK473N皆能在A549细胞上清中良好生长，