Identification of the molecular basis of differential host responses to rapidly evolving Avian Influenza viruses in different avian species

禽流感是由高致病性禽流感病毒(HPAIV)H5N1引起的，自2006年首次爆发以来，正在给印度家禽业造成巨大损失。尽管根除并确认了无病状态，但重新出现的情况仍在继续。由于每次暴发都需要根除方圆3公里范围内的所有家禽，因此每一次暴发都会对印度农村地区的小规模和边缘家禽养殖户产生重大的经济和社会影响。这在印度尤为严重，那里的后院家禽很受欢迎，人口稠密的村庄通常位于根除区域内。作为一种应通报的疾病，禽流感可能会对国际贸易产生严重影响，并进一步损害印度的家禽业。由于HPAI H5N1具有潜在的人畜共患病的潜在危险，并且由于家禽和人类与活禽市场文化共存，人们总是担心出现大流行流感。HPAI H5N1影响几种禽类，包括家禽、火鸡、鹌鹑、珍珠鸡和野生鸟类；然而，对感染的反应差异很大。鸭子和水禽往往具有抵抗力，即它们被感染，但能够清除病毒或无症状地携带病毒，并充当宿主。相比之下，家禽高度敏感，即它们受到感染，无法清除病毒，从而导致高死亡率。自2006年以来，印度的禽流感疫情主要影响到家禽。令人惊讶的是，在2011年的疫情中，乌鸦和鸭子的死亡率也很高，此前人们认为它们对H5N1感染具有抵抗力。这些新的禽流感病毒分支在正常抵抗物种中促进高致病性的机制尚不完全清楚，但可能是由促进病毒复制和宿主免疫反应失调的病毒突变引起的。虽然禽流感病毒在鸡中的致病机制在一定程度上是已知的，但对其他禽类的耐药性机制了解很少，这些禽流感病毒可以作为禽类的感染库。在分子水平上，耐药物种可能具有能够阻止疾病进展和/或改善免疫病理学的途径。随着许多禽类基因组的测序和尖端工具的可用，如RNAseq，现在有可能检查任何鸟类对病毒感染的全球反应。我们提出了一种比较转录学的方法来识别导致不同鸟类对H5N1病毒宿主易感性差异的分子特征。我们提出了一种综合的方法，结合病毒病理学、转录组分析、生物信息学和体外和体内基因功能测试，并辅之以对赋予耐药物种更高毒力的病毒基因差异的相应分析，以探讨导致这些物种差异的分子机制。六个禽类物种，鸡/火鸡(高度易感，严重死亡)，鹅/鸽子(携带零星死亡)和鸭子/乌鸦(对大多数禽流感病毒感染具有抵抗力，但对不同分支的病毒有不同的反应)，将感染来自不同分支的H5N1病毒，以及LPAIV H9N2。全球对感染的反应将通过RNAseq、生物信息学和关键组织的细胞组织化学来研究。通过比较敏感/抗性物种和不同禽流感病毒分支中参与宿主反应的途径将被用来识别可能的宿主抗性基因。它们的作用将在基因敲除和过度表达实验中得到测试。将通过反向遗传学和其他功能分析来探索病毒分离株之间的序列差异在正常抗病禽类中表现出不同致病性的作用。从这些比较中获得的知识可用于制定可持续战略，以控制家禽中的禽流感感染。

Avian Influenza, caused by the highly pathogenic avian influenza virus (HPAIV) H5N1, is taking a huge toll on the Indian poultry industry since the first outbreak in 2006. Despite eradication and confirmation of disease free status, re-emergence continues. Since each outbreak needs eradication of all poultry within a 3km radius, every episode has a major economic and social impact on small and marginal poultry farmers of rural India. This is particularly acute in India where backyard poultry is popular and thickly populated villages usually lie within the eradication zone. As a notifiable disease, Avian Influenza can have serious implications for international trade and further harm the poultry industry of India. Due to its zoonotic potential it has public health concerns and owing to the co-habitation of poultry and humans with a culture of live bird markets, there is always a fear of the emergence of pandemic flu.HPAI H5N1 affects several avian species, including domesticated chickens, turkeys, quails, guinea fowl as well as wild birds; however the response to infection varies widely. Ducks and waterfowl are often resistant i.e. they become infected but are capable of clearing the virus or carry the virus without symptoms, and act as reservoirs. In contrast, poultry are highly susceptible i.e. they become infected and are not able to clear the virus, which results in high mortality. Since 2006 Avian Influenza outbreaks in India have mainly affected domestic chickens. Strikingly in the 2011 outbreak, high mortality was also observed in crows and ducks, previously assumed to be resistant to H5N1 infection. The mechanisms that promote high pathogenicity of these recent clades of AIV in normally resistant species are not completely understood, but may be caused by viral mutations that facilitate virus replication and dysregulation of the host immune response.Though the pathogenesis of AIV in chickens is known to some extent, little is understood about mechanisms of resistance in other birds, which can act as reservoirs of infection to poultry. At the molecular level, resistant species may have pathways able to check disease progression and/or ameliorate immunopathology. With many avian genomes sequenced and with the availability of cutting edge tools, like RNAseq, it is now possible to examine global responses to viral infection in any bird. We propose a comparative transcriptomic approach to identify molecular signatures responsible for the differences in host susceptibility to H5N1 seen between avian species. We propose an integrated approach, combining viral pathology, transcriptome analysis, bioinformatics and functional tests of genes in vitro and in vivo, complemented by corresponding analyses of the differences in viral genes that confer higher virulence in the resistant species to investigate the molecular mechanisms underlying these species differences.Six avian species, chickens/turkeys (highly susceptible with heavy mortality), geese/pigeons (carriers with sporadic mortality) and ducks/crows (resistant to most AIV infections but having differential response to virus of different clades), will be infected with H5N1 from different clades, as well as LPAIV H9N2. Global responses to infection will be studied by RNAseq, bioinformatics and cytohistochemistry of critical tissues. Comparison of pathways involved in host responses in susceptible/resistant species and different AIV clades will be used to identify putative host resistant genes. Their roles will be tested in knockdown and over-expression experiments. The roles of sequence differences between virus isolates that show differential pathogenicity in normally resistant birds will be probed by reverse genetics and other functional assays. The knowledge gained from these comparisons can be used to develop sustainable strategies to control Avian Influenza infections in domestic poultry.