Understanding the Emergence of Highly Pathogenic Avian Influenza Viruses

(A)实验实验室部分： 利用反向遗传学，将产生具有最小多碱基裂解位点(MBCS)基序的低致病性禽流感病毒(H5N1和H6N1)，并将测定鸡的静脉致病指数(IVPI)和50%禽类感染剂量(BID50)，并与LPAI H5N1和H6N1进行比较。接下来，这些病毒将在鸡中连续传代(10倍)，看看最小的MBCS是否会演变为MBCS和高致病性禽流感(HPAI)表型。将使用深度测序(RML研究技术科，基因组学单位)来分析每一代鸡病毒的切割位点基序。 由于H5暂停的影响和NIAID，NIH随后的限制，该项目的这一部分仍然被搁置。2012/13财政年度没有就这一项目开展任何工作(Fuchier等人，《科学》2012；Fuchier等人，《Science》2012；Fuchier等人，《Science》2013)。 (B)实地调查部分： 从历史上看，所有甲型流感病毒的天然宿主都被认为是野鸟。然而，最近在危地马拉黄肩蝙蝠身上发现了一种新型H17甲型流感病毒，这使人们除了野生鸟类外，还将重点放在了新的宿主上。特别是某些甲型流感病毒对动物和公众健康都是直接威胁。特别是，高致病性的甲型禽流感病毒(HPAI H5和H7亚型)和多种猪流感病毒(H1、H2和H3)因跨越动物和人类的物种屏障而臭名昭著。刚果共和国和更广泛的刚果盆地地区的公共卫生、动物卫生、农业生产力和粮食安全直接受到甲型流感病毒暴发的威胁。此外，流感病毒可能以人畜共患病的方式传播给人类，可能会引发疫情和出现新的大流行。 到目前为止，关于动物流感病毒，特别是禽流感病毒在RC的流行、发病率和特性的信息很少或根本没有发表，目前没有禽流感病毒监测方案。因此，确实需要参考禽流感病毒的传播情况来制定基线信息。 2012/13财政年度，在布拉柴维尔(加拿大首都)的国家圣地实验室建立了实验室基础设施，并开发和实施了诊断/监测分析(Grolla等人，J Clin Virol 2012；Mombouli等人，Emerg Infec Dis 2013；Nisii等人，PLoS Path 2013)。监测研究已于2012/13财政年度开始，目前正在分析第一批结果，并准备出版。 作为现场项目，该装置被用来调查在RC出现的Chikunyunga病毒。此次疫情与埃及伊蚊和白纹伊蚊有关，而邻国加蓬的疫情主要与白纹伊蚊有关。我们的研究证实了白纹伊蚊在RC中的存在及其在该地区基孔肯雅病毒传播中的有效媒介作用(Mombouli等人，Emerg Infec Dis 2013)。 该项目将由NIH，LV，NIAID病毒生态单位的文森特·蒙斯特博士继续进行。费尔德曼博士将不再担任该项目的首席研究员。

(A) Experimental Laboratory Component: Using reverse genetics, low pathogenic avian influenza (LPAI) viruses (H5N1 and H6N1) with the minimal multibasic cleavage site (MBCS) motif will be generated and the intravenous pathogenicity index (IVPI) and 50% bird infectious dose (BID50) will be determined in chicken and compared to LPAI H5N1 and H6N1. Next, these viruses will be serially passaged (10x) in chicken to see whether the minimal MBCS will evolve into a MBCS and the highly pathogenic avian influenza (HPAI) phenotype. Deep sequencing (RML Research Technologies Section, Genomics Unit) will be used to analyze the cleavage site motif of viruses from chicken in each passage. This part of the project is still on hold due to the effects of the H5 moratorium and subsequent restrictions by NIAID, NIH. No work has been done on this project in fiscal year 2012/13 (Fouchier et al., Science 2012; Fouchier et al., Science 2012; Fouchier et al., Science 2013) . (B) Field Survey Component: Historically, the natural reservoir for all influenza A viruses were considered wild birds. However, the recent identification of a novel H17 influenza A virus in yellow-shouldered bats in Guatemala has put the focus on novel reservoirs besides wild birds. Certain influenza A viruses in particular are a direct threat to both animal and public health. In particular, Highly Pathogenic Avian influenza A viruses (HPAI H5 and H7 subtypes) and a wide variety of swine influenza viruses (H1, H2 and H3) are notorious for crossing the species barrier from animals to humans. Public health, animal health, agricultural productivity and food security in the Republic of Congo (RC) and the wider Congo basin region are directly threatened by outbreaks of influenza A viruses. In addition, the potential zoonotic transmission of influenza viruses to the human population could spark outbreaks and the emergence of a new pandemic. To date little or no information has been published on the prevalence, incidence and identity of animal influenza viruses, in particular avian influenza viruses, in RC and there are currently no surveillance programs for avian influenza viruses in place. Therefore, there is a real need to develop baseline information with reference to the circulation of avian influenza viruses. During the fiscal year 2012/13, the laboratory infrastructure has been established at the Laboratoire National de Sant Publique in Brazzaville (RC capital) and diagnostic/surveillance assays have been developed and implemented (Grolla et al., J Clin Virol 2012; Mombouli et al., Emerg Infec Dis 2013; Nisii et al., PLoS Path 2013). Surveillance studies have started in fiscal year 2012/13 and first results are currently analyzed and prepared for publication. As a site project, the set up was used to investigate the emergence of Chikunyunga virus in RC. The outbreak was associated with both Aedes aegypti and Aedes albopictus mosquitoes, whereas outbreaks in neighboring Gabon are predominantly associated with Aedes albopictus. Our study confirms the presence of Aedes albopictus in RC and its role as a potent vector in Chikungunya virus transmission in the region (Mombouli et al., Emerg Infec Dis 2013). This project will be continued by Dr. Vincent Munster, Viral Ecology Unit, LV, NIAID, NIH. Dr. Feldmann will no longer serve as the principal investigator for this project.