Mimicking evolution to define mechanisms of airborne transmission of H7N9 viruses

亚洲谱系H7N9禽流感病毒（AIV）已导致超过1500例人畜共患感染，其中615例死亡。这些病毒尚未在人类中传播;然而，这些病毒很有可能演变为通过空气传播途径传播并导致大流行。我们之前使用雪貂评估了原型亚洲谱系病毒A/Anhui/1/2013（H7N9）进行两轮连续空气传播的能力。在这些研究中，我们发现病毒在两轮传播中能够传播给50-66%的呼吸道接触雪貂。在随后的深度序列分析中，我们在90-99%的所有变异病毒中发现了2-5个突变。这些突变发生在血凝素（HA）、神经氨酸酶（NA）和病毒聚合酶基因中。由于空气传播与上呼吸道细胞中增强的结合和复制有关，我们假设所鉴定的突变将改变病毒的分子特性，以增强在原代人鼻和气管上皮细胞中的复制。我们的目标是：目标1。确定先前鉴定的HA和NA突变在具有A/PR/8疫苗骨架的H7N9病毒中的作用。将产生在A/PR 8疫苗骨架上携带H7N9 HA和NA的病毒。 突变将被引入HA和NA基因片段，并将评价几种性质，包括受体结合偏好、融合pH、热稳定性、NA活性和通过免疫血清的抗体识别变化。 目标二。评价先前确定的突变对病毒聚合酶的作用。 为了评估病毒聚合酶突变的影响，将进行体外聚合酶复溶试验。具体地，将评估具有和不具有鉴定的突变的野生型H7N9聚合酶的活性。目标3.确定先前鉴定的突变的引入是否改变了原代人气道上皮细胞中的病毒复制。 为了确定鉴定的突变是否影响病毒复制，我们将评估重组H7N9-A/PR 8病毒在原代人气道上皮细胞中生长的复制动力学。原代人细胞将包括鼻、气管、支气管和小气道上皮细胞。总的来说，这些研究将确定所鉴定的突变对病毒不同分子特性的影响，同时还确定突变是否改变了人类细胞中的病毒嗜性。 我们的研究结果将产生关于AIV如何演变为通过空气传播途径传播的新见解，并将产生解释H7N9病毒演变和评估其大流行潜力所需的关键知识。

The Asian lineage H7N9 avian influenza viruses (AIV) have caused >1500 human zoonotic infections with 615 deaths. These viruses have not spread in humans; however, there is a high potential for these viruses to evolve to transmit via the airborne route and cause a pandemic. Using ferrets, we previously evaluated the ability of the prototypic Asian lineage virus, A/Anhui/1/2013 (H7N9), to undergo two continuous rounds of airborne transmission. In these studies, we found that the virus was able to transmit to 50-66% of respiratory contact ferrets during both rounds of transmission. In a subsequent deep sequence analysis, we identified 2-5 mutations in 90-99% of all variant viruses that transmitted. These mutations were in the hemagglutinin (HA), neuraminidase (NA), and viral polymerase genes. As airborne transmission is associated with enhanced binding and replication in cells of the upper airways, we hypothesize that the identified mutations will alter the molecular properties of the virus to enhance replication in primary human nasal and tracheal epithelial cells. Our aims are: Aim 1. Determine the role of previously identified HA and NA mutations in an H7N9 virus with the A/PR/8 vaccine backbone. Viruses carrying the H7N9 HA and NA on the A/PR8 vaccine backbone will generated. Mutations will be introduced into the HA and NA gene segments and several properties including receptor-binding preference, pH of fusion, thermostability, NA activity, and changes in antibody recognition via immune serum will be evaluated. Aim 2. Evaluate the role of previously identified mutations on the viral polymerase. To assess the impact of mutations in the viral polymerase, in vitro polymerase reconstitution assays will be performed. Specifically, the activity of the wild-type H7N9 polymerase with and without the identified mutations will be assessed. Aim 3. Determine if the introduction of previously identified mutations alters viral replication in primary human airway epithelial cells. To determine if the identified mutations impact viral replication, we will evaluate the replication kinetics of recombinant H7N9-A/PR8 viruses for their growth in primary human airway epithelial cells. Primary human cells will include nasal, tracheal, bronchial, and small airway epithelial cells. Collectively, these studies will determine the effect of the identified mutations on different molecular properties of the virus, while also determining if the mutations alter the viral tropism in human cells. Our findings will generate new insight on how AIV evolve to transmit via the airborne route and will yield critical knowledge required to interpret the evolution and assess the pandemic potential of H7N9 viruses.