We describe two new insect-specific flaviviruses (ISFs) isolated from mosquitoes in Australia, Binjari virus (BinJV) and Hidden Valley virus (HVV), that grow efficiently in mosquito cells but fail to replicate in a range of vertebrate cell lines. Phylogenetic analysis revealed that BinJV and HVV were closely related (90% amino acid sequence identity) and clustered with lineage II (dual-host affiliated) ISFs, including the Lammi and Nounané viruses. Using a panel of monoclonal antibodies prepared to BinJV viral proteins, we confirmed a close relationship between HVV and BinJV and revealed that they were antigenically quite divergent from other lineage II ISFs. We also constructed chimeric viruses between BinJV and the vertebrate-infecting West Nile virus (WNV) by swapping the structural genes (prM and E) to produce BinJ/WNV_KUN-prME and WNV_KUN/BinJV-prME. This allowed us to assess the role of different regions of the BinJV genome in vertebrate host restriction and revealed that while BinJV structural proteins facilitated entry to vertebrate cells, the process was inefficient. In contrast, the BinJV replicative components in wild-type BinJV and BinJ/WNV_KUN-prME failed to initiate replication in a wide range of vertebrate cell lines at 37°C, including cells lacking components of the innate immune response. However, trace levels of replication of BinJ/WNV_KUN-prME could be detected in some cultures of mouse embryo fibroblasts (MEFs) deficient in antiviral responses (IFNAR^−/− MEFs or RNase L^−/− MEFs) incubated at 34°C after inoculation. This suggests that BinJV replication in vertebrate cells is temperature sensitive and restricted at multiple stages of cellular infection, including inefficient cell entry and susceptibility to antiviral responses.

中文翻译：

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在澳大利亚蚊子中新的世系II昆虫特异性黄病毒的抗原表征和宿主限制因子的鉴定。

我们描述了两种新的昆虫特异性黄病毒（ISF），它们从澳大利亚的蚊子中分离出来，分别是Binjari病毒（BinJV）和Hidden Valley病毒（HVV），它们在蚊子细胞中有效生长，但无法在一系列脊椎动物细胞系中复制。系统发育分析表明，BinJV和HVV密切相关（90％的氨基酸序列同一性），并与沿袭II（与双宿主相关）的ISF聚集在一起，包括Lammi和Nounané病毒。使用一组针对BinJV病毒蛋白的单克隆抗体，我们确认了HVV和BinJV之间的密切关系，并揭示了它们与其他谱系II ISF的抗原性相当不同。我们还通过交换结构基因（prM和E）产生BinJ / WNV，在BinJV和感染脊椎动物的西尼罗河病毒（WNV）之间构建了嵌合病毒_KUN -prME和WNV _KUN / BinJV-prME。这使我们能够评估BinJV基因组的不同区域在脊椎动物宿主限制中的作用，并揭示了虽然BinJV结构蛋白有助于进入脊椎动物细胞，但该过程效率低下。相反，野生型BinJV和BinJ / WNV _KUN -prME中的BinJV复制成分未能在37°C的多种脊椎动物细胞系中启动复制，包括缺乏先天免疫应答成分的细胞。但是，在某些抗病毒应答不足的小鼠胚胎成纤维细胞（MEF）（IFNAR ^-/- MEFs或RNase L ^-/-）中，可以检测到BinJ / WNV _KUN -prME的痕量复制水平。接种后于34°C孵育。这表明脊椎动物细胞中的BinJV复制是温度敏感的，并且在细胞感染的多个阶段受到限制，包括无效的细胞进入和对抗病毒反应的敏感性。