Abstract：
New World fruit bats were recently found to harbor two distinct and previously unknown influenza A viruses (IAVs) of the subtypes H17N10 and H18N11. Although viral genome sequences were detected in the liver, intestine, lung, and kidney of infected bats and the complete genome sequences have been isolated from their rectal swab samples, all attempts to isolate an infectious virus from bats in nature have failed. The lack of an infectious bat IAV isolate was overcome by reverse genetic approaches that led to the generation of an infectious virus in vitro. Using such synthetic bat IAVs enabled the identification of their unconventional cell entry via major histocompatibility complex II (MCH-II) molecules and their ability to replicate in mice, ferrets, and bats. Importantly, we also showed that these synthetic recombinant bat IAVs are not able to reassort with conventional IAVs, preventing the acquisition of enhanced transmission properties in non-bat species by reassortment with conventional IAVs. As authentic viruses are key for understanding the molecular biology of bat IAVs, in this chapter, we describe our recently established reverse genetics protocol for generating H17N10 and H18N11 in vitro. This step-by-step protocol starts with cloning of cDNA copies of the viral RNA segments into reverse genetics plasmids, followed by the generation of a highly concentrated stock and finally a method to determine viral titers.
摘要：
新世界水果蝙蝠最近被发现含有两种不同且以前未知的亚型H17N10和H18N11的甲型流感病毒（IAV）。尽管在肝脏中检测到病毒基因组序列，肠,肺，感染蝙蝠的肾脏和完整的基因组序列已经从他们的直肠拭子样本中分离出来，从自然界中的蝙蝠中分离出传染性病毒的所有尝试都失败了。通过反向遗传方法克服了缺乏感染性蝙蝠IAV分离株的缺陷，该方法导致在体外产生感染性病毒。使用这种合成蝙蝠IAV能够通过主要组织相容性复合物II（MCH-II）分子识别其非常规细胞进入及其在小鼠中复制的能力，雪貂,还有蝙蝠.重要的是,我们还表明，这些合成的重组蝙蝠IAV不能与传统的IAV重组，通过与常规IAVs重组来防止非蝙蝠物种获得增强的传播特性。由于真正的病毒是理解蝙蝠AAV分子生物学的关键，在这一章中,我们描述了我们最近建立的体外产生H17N10和H18N11的反向遗传学方案。这个一步一步的方案开始于将病毒RNA片段的cDNA拷贝克隆到反向遗传学质粒中，然后产生高度浓缩的原液，最后是确定病毒滴度的方法。
