Egypt is a hotspot for H5- and H9-subtype avian influenza A virus (AIV) infections and co-infections in poultry by both subtypes have been frequently reported. However, natural genetic reassortment of these subtypes has not been reported yet. Here, we evaluated the genetic compatibility and replication efficiency of reassortants between recent isolates of an Egyptian H5N1 and a H9N2 AIV (H5N1_EGY and H9N2_EGY). All internal viral proteins-encoding segments of the contemporaneous G1-like H9N2_EGY, expressed individually and in combination in the genetic background of H5N1_EGY, were genetically compatible with the other H5N1_EGY segments. At 37 °C the replication efficiencies of H5N1_EGY reassortants expressing the H9N2_EGY polymerase subunits PB2 and PA (H5N1_PB2-H9N2EGY, H5N1_PA-H9N2EGY) were higher than the wild-type H5N1_EGY in Madin-Darby canine kidney (MDCK-II) cells. This could not be correlated to viral polymerase activity as this was found to be improved for H5N1_PB2-H9N2EGY, but reduced for H5N1_PA-H9N2EGY. At 33 °C and 39 °C, H5N1_PB2-H9N2EGY and H5N1_PA-H9N2EGY replicated to higher levels than the wild-type H5N1_EGY in human Calu-3 and A549 cell lines. Nevertheless, in BALB/c mice both reassortants caused reduced mortality compared to the wild-type H5N1_EGY. Genetic analysis of the polymerase-encoding segments revealed that the PA_H9N2EGY and PB2_H9N2EGY encode for a distinct uncharacterized mammalian-like variation (367K) and a well-known mammalian signature (591K), respectively. Introducing the single substitution 367K into the PA of H5N1_EGY enabled the mutant virus H5N1_PA-R367K to replicate more efficiently at 37 °C in primary human bronchial epithelial (NHBE) cells and also in A549 and Calu-3 cells at 33 °C and 39 °C. Furthermore, H5N1_PA-R367K caused higher mortality in BALB/c mice. These findings demonstrate that H5N1 (Clade 2.2.1.2) reassortants carrying internal proteins-encoding segments of G1-like H9N2 viruses can emerge and may gain improved replication fitness. Thereby such H5N1/H9N2 reassortants could augment the zoonotic potential of H5N1 viruses, especially by acquiring unique mammalian-like aa signatures.

中文翻译：

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来自最近携带H9N2（G1）禽流感病毒（AIV）菌株的赖氨酸367的PA赋予哺乳动物系统中同期H5N1改变的复制效率和致病性。

埃及是H5和H9亚型禽流感A病毒（AIV）感染和家禽两种亚型共感染的热点地区。但是，尚未报道这些亚型的自然遗传重配。在这里，我们评估了埃及H5N1和H9N2 AIV（H5N1 _EGY和H9N2 _EGY）最近分离株之间重配子的遗传相容性和复制效率。在H5N1 _EGY的遗传背景下单独或组合表达的同时G1样H9N2 _EGY的所有内部病毒蛋白编码片段与其他H5N1 _EGY片段在遗传上相容。在37°C下H5N1 _EGY的复制效率在Madin-Darby犬肾（MDCK-II）细胞中，表达H9N2 _EGY聚合酶亚基PB2和PA（H5N1 _{PB2-H9N2EGY，} H5N1 _PA-H9N2EGY）_的重组子高于野生型H5N1 _EGY。这不能与病毒聚合酶活性相关，因为发现对于H5N1 _PB2-H9N2EGY可以提高_，但是对于H5N1 _{PA-H9N2EGY可以降低}。在33°C和39°C下，在人Calu-3和A549细胞系中，H5N1 _PB2-H9N2EGY和H5N1 _{PA-H9N2EGY的}复制水平高于野生型H5N1 _EGY。然而，与野生型H5N1 _EGY相比，BALB / c小鼠中的两种重配体均可降低死亡率。聚合酶编码片段的遗传分析表明，PA _H9N2EGY和PB2 _{H9N2EGY分别}编码一个明显的未表征的哺乳动物样变异（367K）和一个众所周知的哺乳动物特征码（591K）。在H5N1 _EGY的PA中引入单取代367K可使突变病毒H5N1 _PA-R367K在37°C的条件下在原代人支气管上皮（NHBE）细胞以及在33°C的A549和Calu-3细胞中更有效地复制39°C。此外，H5N1 _PA-R367K引起BALB / c小鼠更高的死亡率。这些发现表明，带有G1样H9N2病毒内部蛋白编码片段的H5N1（进化枝2.2.1.2）重配子可以出现，并且可以提高复制适应性。因此，这种H5N1 / H9N2重配子可以增强H5N1病毒的人畜共患病潜力，尤其是通过获得独特的哺乳动物样氨基酸特征。