West Nile virus (WNV) is an enveloped virus with a single-stranded positive-sense RNA genome from the Flaviviridae family. WNV is spread by mosquitoes and able to infect humans, causing encephalitis and meningitis that can be fatal; it therefore presents a significant risk for human health. In insects, innate response to RNA virus infection mostly relies on RNA interference and JAK/SAT pathways; however, some evidence indicates that it can also involve microRNAs (miRNAs). miRNAs are small noncoding RNAs that regulate gene expression at posttranscriptional level and play an important role in a number of processes, including immunity and antiviral response. In this study, we focus on the miRNA-mediated response to WNV in mosquito cells. We demonstrate that in response to WNV infection the expression of a mosquito-specific miRNA, aae-miR-2940, is selectively downregulated in Aedes albopictus cells. This miRNA is known to upregulate the metalloprotease m41 FtsH gene, which we have also shown to be required for efficient WNV replication. Correspondingly, downregulation of aae-miR-2940 reduced the metalloprotease level and restricted WNV replication. Thus, we have identified a novel miRNA-dependent mechanism of antiviral response to WNV in mosquitoes. IMPORTANCE A detailed understanding of vector-pathogen interactions is essential to address the problems posed by vector-borne diseases. Host and viral miRNAs play an important role in regulating expression of viral and host genes involved in endogenous processes, including antiviral response. There has been no evidence to date for the role of mosquito miRNAs in response to flaviviruses. In this study, we show that downregulation of aae-miR-2940 in mosquito cells acts as a potential antiviral mechanism in the mosquito host to inhibit WNV replication by repressing the expression of the metalloprotease m41 FtsH gene, which is required for efficient WNV replication. This is the first identification of an miRNA-dependent antiviral mechanism in mosquitoes, which inhibits replication of WNV. Our findings should facilitate identification of targets in the mosquito genome that can be utilized to suppress vector population and/or limit WNV replication.

Flaviviruses, including Zika virus (ZIKV), utilise host mRNA degradation machinery to produce subgenomic flaviviral RNA (sfRNA). In mammalian hosts, this noncoding RNA facilitates replication and pathogenesis of flaviviruses by inhibiting IFN-signalling, whereas the function of sfRNA in mosquitoes remains largely elusive. Herein, we conduct a series of in vitro and in vivo experiments to define the role of ZIKV sfRNA in infected Aedes aegypti employing viruses deficient in production of sfRNA. We show that sfRNA-deficient viruses have reduced ability to disseminate and reach saliva, thus implicating the role for sfRNA in productive infection and transmission. We also demonstrate that production of sfRNA alters the expression of mosquito genes related to cell death pathways, and prevents apoptosis in mosquito tissues. Inhibition of apoptosis restored replication and transmission of sfRNA-deficient mutants. Hence, we propose anti-apoptotic activity of sfRNA as the mechanism defining its role in ZIKV transmission.

West Nile virus (WNV) is a mosquito-transmitted flavivirus that naturally circulates between mosquitos and birds but can also infect humans, causing severe neurological disease. The early host response to WNV infection in vertebrates primarily relies on the type I interferon pathway; however, recent studies suggest that microRNAs (miRNAs) may also play a notable role. In this study, we assessed the role of host miRNAs in response to WNV infection in human cells. We employed small RNA sequencing (RNA-seq) analysis to determine changes in the expression of host miRNAs in HEK293 cells infected with an Australian strain of WNV, Kunjin (WNV _KUN ), and identified a number of host miRNAs differentially expressed in response to infection. Three of these miRNAs were confirmed to be significantly upregulated in infected cells by quantitative reverse transcription (qRT)-PCR and Northern blot analyses, and one of them, miR-532-5p, exhibited a significant antiviral effect against WNV _KUN infection. We have demonstrated that miR-532-5p targets and downregulates expression of the host genes SESTD1 and TAB3 in human cells. Small interfering RNA (siRNA) depletion studies showed that both SESTD1 and TAB3 were required for efficient WNV _KUN replication. We also demonstrated upregulation of mir-532-5p expression and a corresponding decrease in the expression of its targets, SESTD1 and TAB3, in the brains of WNV _KUN -infected mice. Our results show that upregulation of miR-532-5p and subsequent suppression of the SESTD1 and TAB3 genes represent a host antiviral response aimed at limiting WNV _KUN infection and highlight the important role of miRNAs in controlling RNA virus infections in mammalian hosts. IMPORTANCE West Nile virus (WNV) is a significant viral pathogen that poses a considerable threat to human health across the globe. There is no specific treatment or licensed vaccine available for WNV, and deeper insight into how the virus interacts with the host is required to facilitate their development. In this study, we addressed the role of host microRNAs (miRNAs) in antiviral response to WNV in human cells. We identified miR-532-5p as a novel antiviral miRNA and showed that it is upregulated in response to WNV infection and suppresses the expression of the host genes TAB3 and SESTD1 required for WNV replication. Our results show that upregulation of miR-532-5p and subsequent suppression of the SESTD1 and TAB3 genes represent an antiviral response aimed at limiting WNV infection and highlight the important role of miRNAs in controlling virus infections in mammalian hosts.