The small non-coding RNA response to virus infection in the <i>Leishmania</i> vector <i>Lutzomyia longipalpis</i>

Sandflies are well known vectors for Leishmania but also transmit a number of arthropod-borne viruses (arboviruses). Few studies have addressed the interaction between sandflies and arboviruses. RNA interference (RNAi) mechanisms utilize small non-coding RNAs to regulate different aspects of host-pathogen interactions. The small interfering RNA (siRNA) pathway is a broad antiviral mechanism in insects. In addition, at least in mosquitoes, another RNAi mechanism mediated by PIWI interacting RNAs (piRNAs) is activated by viral infection. Finally, endogenous microRNAs (miRNA) may also regulate host immune responses. Here, we analyzed the small non-coding RNA response to Vesicular stomatitis virus (VSV) infection in the sandfly Lutzoymia longipalpis. We detected abundant production of virus-derived siRNAs after VSV infection in adult sandflies. However, there was no production of virus-derived piRNAs and only mild changes in the expression of vector miRNAs in response to infection. We also observed abundant production of virus-derived siRNAs against two other viruses in Lutzomyia Lulo cells. Together, our results suggest that the siRNA but not the piRNA pathway mediates an antiviral response in sandflies. In agreement with this hypothesis, pre-treatment of cells with dsRNA against VSV was able to inhibit viral replication while knock-down of the central siRNA component, Argonaute-2, led to increased virus levels. Our work begins to elucidate the role of RNAi mechanisms in the interaction between L. longipalpis and viruses and should also open the way for studies with other sandfly-borne pathogens.白蛉（sandflies）是利什曼原虫（Leishmania）的公认传播媒介，同时还可传播多种节肢动物传播病毒（arboviruses，虫媒病毒）。目前针对白蛉与虫媒病毒之间互作的研究仍较为匮乏。RNA干扰（RNA interference, RNAi）机制通过小型非编码RNA调控宿主-病原体互作的多个环节。小干扰RNA（small interfering RNA, siRNA）通路是昆虫中广泛存在的抗病毒免疫机制。此外，至少在蚊类中，由PIWI互作RNA（PIWI interacting RNAs, piRNAs）介导的另一RNAi通路可被病毒感染激活。最后，内源性微RNA（microRNAs, miRNAs）亦可调控宿主免疫应答。本研究针对长须罗蛉（Lutzomyia longipalpis）感染水疱性口炎病毒（Vesicular stomatitis virus, VSV）后的小型非编码RNA应答展开分析。我们在成年白蛉感染VSV后检测到大量病毒来源的小干扰RNA生成，但未检测到病毒来源的piRNAs，且感染仅会引起该媒介昆虫miRNAs的表达出现轻微变化。我们还在罗蛉属Lulo细胞中检测到针对另外两种病毒的大量病毒来源siRNA生成。综上，我们的研究结果表明，白蛉中介导抗病毒应答的是siRNA通路而非piRNA通路。与此假说一致，用针对VSV的双链RNA（dsRNA）预处理细胞可抑制病毒复制，而敲低siRNA通路核心组分Argonaute-2则会导致病毒载量升高。本研究初步阐明了RNAi机制在长须罗蛉与病毒互作中的作用，同时也为开展其他白蛉传播病原体的相关研究开辟了新的思路。