SummaryAedes aegypti mosquitoes are responsible for transmitting many medically important viruses such as those that cause Zika and dengue. The inoculation of viruses into mosquito bite sites is an important and common stage of all mosquito-borne virus infections. We show, using Semliki Forest virus and Bunyamwera virus, that these viruses use this inflammatory niche to aid their replication and dissemination in vivo. Mosquito bites were characterized by an edema that retained virus at the inoculation site and an inflammatory influx of neutrophils that coordinated a localized innate immune program that inadvertently facilitated virus infection by encouraging the entry and infection of virus-permissive myeloid cells. Neutrophil depletion and therapeutic blockade of inflammasome activity suppressed inflammation and abrogated the ability of the bite to promote infection. This study identifies facets of mosquito bite inflammation that are important determinants of the subsequent systemic course and clinical outcome of virus infection.

The exogenous siRNA pathway is important in restricting arbovirus infection in mosquitoes. Less is known about the role of the PIWI-interacting RNA pathway, or piRNA pathway, in antiviral responses. Viral piRNA-like molecules have recently been described following infection of mosquitoes and derived cell lines with several arboviruses. The piRNA pathway has thus been suggested to function as an additional small RNA-mediated antiviral response to the known infection-induced siRNA response. Here we show that piRNA-like molecules are produced following infection with the naturally mosquito-borne Semliki Forest virus in mosquito cell lines. We show that knockdown of piRNA pathway proteins enhances the replication of this arbovirus and defines the contribution of piRNA pathway effectors, thus characterizing the antiviral properties of the piRNA pathway. In conclusion, arbovirus infection can trigger the piRNA pathway in mosquito cells, and knockdown of piRNA proteins enhances virus production.

Bunyamwera virus (family Bunyaviridae, genus Bunyavirus) contains a tripartite negative-sense RNA genome. The smallest RNA segment, S, encodes the nucleocapsid protein N and a nonstructural protein, NSs, in overlapping reading frames. We have generated a mutant virus lacking NSs, called BUNdelNSs, by reverse genetics. Compared with the wild-type (wt) virus, BUNdelNSs exhibited a smaller plaque size and generated titers of virus approximately 1 log lower. In mammalian cells, the mutant expressed greatly increased levels of N protein; significantly, the marked inhibition of host cell protein synthesis shown by wt virus was considerably impaired by BUNdelNSs. When inoculated by the intracerebral route BUNdelNSs killed BALB͞c mice with a slower time course than wt and exhibited a reduced cell-to-cell spread, and titers of virus in the brain were lower. In addition, the abrogation of NSs expression changed Bunyamwera virus from a noninducer to an inducer of an interferon-␤ promoter. These results suggest that, although not essential for growth in tissue culture or in mice, the bunyavirus NSs protein has several functions in the virus life cycle and contributes to viral pathogenesis.reverse genetics ͉ host cell shutoff ͉ interferon antagonist

Production of alpha/beta interferons (IFN-␣/␤) in response to viral infection is one of the main defense mechanisms of the innate immune system. Many viruses therefore encode factors that subvert the IFN system to enhance their virulence. Bunyamwera virus (BUN) is the prototype of the Bunyaviridae family. By using reverse genetics, we previously produced a recombinant virus lacking the nonstructural protein NSs (BUNdelNSs) and showed that NSs is a nonessential gene product that contributes to viral pathogenesis. Here we demonstrate that BUNdelNSs is a strong inducer of IFN-␣/␤, whereas in cells infected with the wild-type counterpart expressing NSs (wild-type BUN), neither IFN nor IFN mRNA could be detected. IFN induction by BUNdelNSs correlated with activation of NF-B and was dependent on virally produced double-stranded RNA and on the IFN transcription factor IRF-3. Furthermore, both in cultured cells and in mice lacking a functional IFN-␣/␤ system, BUNdelNSs replicated to wild-type BUN levels, whereas in IFN-competent systems, wild-type BUN grew more efficiently. These results suggest that BUN NSs is an IFN induction antagonist that blocks the transcriptional activation of IFN-␣/␤ in order to increase the virulence of Bunyamwera virus.

Arboviruses are transmitted by distantly related arthropod vectors such as mosquitoes (class Insecta) and ticks (class Arachnida). RNA interference (RNAi) is the major antiviral mechanism in arthropods against arboviruses. Unlike in mosquitoes, tick antiviral RNAi is not understood, although this information is important to compare arbovirus/host interactions in different classes of arbovirus vectos. Using an Ixodes scapularis-derived cell line, key Argonaute proteins involved in RNAi and the response against tick-borne Langat virus (Flaviviridae) replication were identified and phylogenetic relationships characterized. Analysis of small RNAs in infected cells showed the production of virus-derived small interfering RNAs (viRNAs), which are key molecules of the antiviral RNAi response. Importantly, viRNAs were longer (22 nucleotides) than those from other arbovirus vectors and mapped at highest frequency to the termini of the viral genome, as opposed to mosquito-borne flaviviruses. Moreover, tick-borne flaviviruses expressed subgenomic flavivirus RNAs that interfere with tick RNAi. Our results characterize the antiviral RNAi response in tick cells including phylogenetic analysis of genes encoding antiviral proteins, and viral interference with this pathway. This shows important differences in antiviral RNAi between the two major classes of arbovirus vectors, and our data broadens our understanding of arthropod antiviral RNAi.

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RNA interference (RNAi) is an important mosquito defense mechanism against arbovirus infection. In this paper we study the processes underlying antiviral RNAi in Aedes albopictus-derived U4.4 mosquito cells infected with Semliki Forest virus (SFV) (Togaviridae; Alphavirus). The production of virus-derived small interfering RNAs (viRNAs) from viral double-stranded RNA (dsRNA) is a key event in this host response. dsRNA could be formed by RNA replication intermediates, by secondary structures in RNA genomes or antigenomes, or by both. Which of these dsRNAs is the substrate for the generation of viRNAs is a fundamental question. Here we used deep sequencing of viRNAs and bioinformatic analysis of RNA secondary structures to gain insights into the characteristics and origins of viRNAs. An asymmetric distribution of SFV-derived viRNAs with notable areas of high-level viRNA production (hot spots) and no or a low frequency of viRNA production (cold spots) along the length of the viral genome with a slight bias toward the production of genome-derived viRNAs over antigenome-derived viRNAs was observed. Bioinformatic analysis suggests that hot spots of viRNA production are rarely but not generally associated with putative secondary structures in the SFV genome, suggesting that most viRNAs are derived from replicative dsRNA. A pattern of viRNAs almost identical to those of A. albopictus cells was observed for Aedes aegypti-derived Aag2 cells, suggesting common mechanisms that lead to viRNA production. Hot-spot viRNAs were found to be significantly less efficient at mediating antiviral RNAi than cold-spot viRNAs, pointing toward a nucleic acid-based viral decoy mechanism to evade the RNAi response.

Chikungunya virus (CHIKV) is a reemerging mosquito-borne alphavirus that has caused severe epidemics in Africa and Asia and occasionally in Europe. As of today, there is no licensed vaccine available to prevent CHIKV infection. Here we describe the development and evaluation of novel CHIKV vaccine candidates that were attenuated by deleting a large part of the gene encoding nsP3 or the entire gene encoding 6K and were administered as viral particles or infectious genomes launched by DNA. The resulting attenuated mutants were genetically stable and elicited high magnitudes of binding and neutralizing antibodies as well as strong T cell responses after a single immunization in C57BL/6 mice. Subsequent challenge with a high dose of CHIKV demonstrated that the induced antibody responses protected the animals from viremia and joint swelling. The protective antibody response was long-lived, and a second homologous immunization further enhanced immune responses. In summary, this report demonstrates a straightforward means of constructing stable and efficient attenuated CHIKV vaccine candidates that can be administered either as viral particles or as infectious genomes launched by DNA. IMPORTANCESimilar to other infectious diseases, the best means of preventing CHIKV infection would be by vaccination using an attenuated vaccine platform which preferably raises protective immunity after a single immunization. However, the attenuated CHIKV vaccine candidates developed to date rely on a small number of attenuating point mutations and are at risk of being unstable or even sensitive to reversion. We report here the construction and preclinical evaluation of novel CHIKV vaccine candidates that have been attenuated by introducing large deletions. The resulting mutants proved to be genetically stable, attenuated, highly immunogenic, and able to confer durable immunity after a single immunization. Moreover, these mutants can be administered either as viral particles or as DNA-launched infectious genomes, enabling evaluation of the most feasible vaccine modality for a certain setting. These CHIKV mutants could represent stable and efficient vaccine candidates against CHIKV.