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Expanding testing capabilities is integral to managing the further spread of SARS-CoV-2 and developing reopening strategies, particularly in regards to identifying and isolating asymptomatic and pre-symptomatic individuals. Central to meeting testing demands are specimens that can be easily and reliably collected and laboratory capacity to rapidly ramp up to scale. We and others have demonstrated that high and consistent levels of SARS-CoV-2 RNA can be detected in saliva from COVID-19 inpatients, outpatients, and asymptomatic individuals. As saliva collection is non-invasive, extending this strategy to test pooled saliva samples from multiple individuals could thus provide a simple method to expand testing capacity. However, hesitation towards pooled sample testing arises due to the dilution of positive samples, potentially shifting weakly positive samples below the detection limit for SARS-CoV-2 and thereby decreasing the sensitivity. Here, we investigated the potential of pooling saliva samples by 5, 10, and 20 samples prior to RNA extraction and RT-qPCR detection of SARS-CoV-2. Based on samples tested, we conservatively estimated a reduction of 7.41%, 11.11%, and 14.81% sensitivity, for each of the pool sizes, respectively. Using these estimates we modeled anticipated changes in RT-qPCR cycle threshold to show the practical impact of pooling on results of SARS-CoV-2 testing. In tested populations with greater than 3% prevalence, testing samples in pools of 5 requires the least overall number of tests. Below 1% however, pools of 10 or 20 are more beneficial and likely more supportive of ongoing surveillance strategies.

The recent Zika virus (ZIKV) and chikungunya virus epidemics highlight the explosive nature of arthropod-borne viruses (arboviruses) transmitted by Aedes spp. mosquitoes1,2. Vector competence and the extrinsic incubation period (EIP) are two key entomological parameters used to assess the public health risk posed by arboviruses3. These are typically measured empirically by offering mosquitoes an infectious blood meal and temporally sampling mosquitoes to determine the infection and transmission status. This approach has been used for the better part of a century; however, it does not accurately capture the biology and behaviour of many mosquito vectors that refeed frequently (every 2–3 d)4. Here, we demonstrate that acquisition of a second non-infectious blood meal significantly shortens the EIP of ZIKV-infected Aedes aegypti by enhancing virus dissemination from the mosquito midgut. Similarly, a second blood meal increases the competence of this species for dengue virus and chikungunya virus as well as Aedes albopictus for ZIKV, suggesting that this phenomenon may be common among other virus–vector pairings and that A. albopictus might be a more important vector than once thought. Blood-meal-induced microperforations in the virus-impenetrable basal lamina that surrounds the midgut provide a mechanism for enhanced virus escape. Modelling of these findings reveals that a shortened EIP would result in a significant increase in the basic reproductive number, R0, estimated from experimental data. This helps to explain how A. aegypti can sustain explosive epidemics such as ZIKV despite relatively poor vector competence in single-feed laboratory trials. Together, these data demonstrate a direct and unrecognized link between mosquito feeding behaviour, EIP and vector competence. This study reports that flavivirus transmission by mosquitoes to mice is increased if mosquitoes feed subsequently on non-infectious blood, possibly because feeding causes microperforations in the gut. Modelling shows this could explain how A. aegypti can sustain an explosive epidemic such as Zika virus despite its perceived poor vector competence.

West Nile virus (WNV) is transmitted by mosquitoes and can cause severe disease, including meningoencephalitis. AgBR1 is a mosquito salivary protein that enhances Aedes aegypti mosquito-borne Zika virus pathogenesis in mice. Here, we show that AgBR1 antibodies reduce the initial West Nile viral load and delay lethal infection after feeding by an infected Aedes aegypti mosquito. Targeting AgBR1 may therefore be incorporated into strategies to prevent mosquito-transmitted West Nile virus infection.

The current quantitative reverse transcription PCR (RT-qPCR) assay recommended for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the United States requires analysis of 3 genomic targets per sample: 2 viral and 1 host. To simplify testing and reduce the volume of required reagents, we devised a multiplex RT-qPCR assay to detect SARS-CoV-2 in a single reaction. We used existing N1, N2, and RP primer and probe sets by the Centers for Disease Control and Prevention, but substituted fluorophores to allow multiplexing of the assay. The cycle threshold (Ct) values of our multiplex RT-qPCR were comparable to those obtained by the single assay adapted for research purposes. Low copy numbers (≥500 copies/reaction) of SARS-CoV-2 RNA were consistently detected by the multiplex RT-qPCR. Our novel multiplex RT-qPCR improves upon current single diagnostics by saving reagents, costs, time, and labor., The current RT-qPCR assay recommended for SARS-CoV-2 testing in the United States requires analysis of three genomic targets per sample: two viral and one host. This study describes a multiplex PCR that saves both reagents and labor while accurately detecting SARS-CoV-2 RNA.

We measured severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA concentrations in primary sewage sludge in the New Haven, Connecticut, USA, metropolitan area during the Coronavirus Disease 2019 (COVID-19) outbreak in Spring 2020. SARS-CoV-2 RNA was detected throughout the more than 10-week study and, when adjusted for time lags, tracked the rise and fall of cases seen in SARS-CoV-2 clinical test results and local COVID-19 hospital admissions. Relative to these indicators, SARS-CoV-2 RNA concentrations in sludge were 0–2 d ahead of SARS-CoV-2 positive test results by date of specimen collection, 0–2 d ahead of the percentage of positive tests by date of specimen collection, 1–4 d ahead of local hospital admissions and 6–8 d ahead of SARS-CoV-2 positive test results by reporting date. Our data show the utility of viral RNA monitoring in municipal wastewater for SARS-CoV-2 infection surveillance at a population-wide level. In communities facing a delay between specimen collection and the reporting of test results, immediate wastewater results can provide considerable advance notice of infection dynamics. Testing sewage for the novel coronavirus reveals epidemiological trends.

Macroautophagy is an evolutionarily conserved cellular process critical for maintaining cellular homeostasis. It can additionally function as an innate immune response to viral infection as has been demonstrated for a number of arthropod-borne (arbo-) viruses. Arboviruses are maintained in a transmission cycle between vertebrate hosts and invertebrate vectors yet the majority of studies assessing autophagy-arbovirus interactions have been limited to the mammalian host. Therefore we evaluated the role of autophagy during arbovirus infection of the invertebrate vector using the tractable Aag2 Aedes aegypti mosquito cell culture system. Our data demonstrates that autophagy is significantly induced in mosquito cells upon infection with two divergent arboviruses: dengue virus-2 (DENV-2; Flaviviridae, Flavivirus) and chikungunya virus (CHIKV; Togaviridae, Alphavirus). While assessing the role of autophagy during arbovirus infection, we observed a somewhat paradoxical outcome. Both induction and suppression of autophagy via torin-1 and spautin-1, respectively, resulted in increased viral titers for both viruses, yet suppression of autophagy-related genes had no effect. Interestingly, chemical modulators of autophagy had either no effect or opposite effects in another widely used mosquito cell line, C6/36 Aedes albopictus cells. Together, our data reveals a limited role for autophagy during arbovirus infection of mosquito cells. Further, our findings suggest that commonly used chemical modulators of autophagy alter mosquito cells in such a way as to promote viral replication; however, it is unclear if this occurs directly through autophagic manipulation or other means., Author summary Arthropod-borne (arbo) viruses, specifically those transmitted by Aedes aegypti mosquitoes, cause significant morbidity and mortality and pose a continued public health threat worldwide. Many of these viruses lack vaccines or therapeutics and current mosquito control strategies are underperforming. For these reasons, identifying vulnerabilities within the transmission cycle that can be targeted will be critical to the development of novel control interventions. Autophagy is a highly conserved cellular pathway and previous studies manipulating this pathway have shown promise in minimizing viral infections in mammalian hosts. In this study we examined arbovirus-autophagy interactions within mosquito cells. The goal was to elucidate the role of autophagy during infection of these cells in hopes of identifying critical interactions that can be targeted by novel approaches to block infection of and transmission by vector mosquitoes.

Mosquito cell lines have been used extensively in research to isolate and propagate arthropod-borne viruses and understand virus-vector interactions. Despite their utility as an in vitro tool, these cell lines are poorly defined and may harbor insect-specific viruses. Accordingly, we screened four commonly-used mosquito cell lines, C6/36 and U4.4 cells from Aedes albopictus, Aag2 cells from Aedes aegypti, and Hsu cells from Culex quinquefasciatus, for the presence of adventitious (i.e. exogenous) viruses. All four cell lines stained positive for double-stranded RNA, indicative of RNA virus replication. We subsequently identified viruses infecting Aag2, U4.4 and Hsu cell lines using untargeted next-generation sequencing, but not C6/36 cells. PCR confirmation revealed that these sequences stem from active viral replication and/or integration into the cellular genome. Our results show that these commonly-used mosquito cell lines are persistently-infected with several viruses. This finding may be critical to interpreting data generated in these systems. Centers for Disease Control and Prevention [U50/CCU116806-01, U01/CK000509-01]; US Department of Agriculture Hatch Funds and Multistate Research Project [CONH00773, NE1443]; National Institute of Health, National Institute of Allergy and Infectious Diseases [AI067380, AI099042]; NIH/NCATS [UL1 TR001082] This work was supported in part by grants from the Centers for Disease Control and Prevention (U50/CCU116806-01 and U01/CK000509-01), the US Department of Agriculture Hatch Funds and Multistate Research Project (CONH00773 and NE1443), and the National Institute of Health, National Institute of Allergy and Infectious Diseases (AI067380, AI099042), and NIH/NCATS (UL1 TR001082).

Arthropod-borne (arbo-) viruses, like all viruses, are obligate intracellular parasites that have evolved mechanisms to subvert cellular processes and evade anti-viral defenses to replicate and persist. An increasing body of research is beginning to recognize the intimate relationship between arboviruses and the cellular autophagy pathway. As a result, new therapeutic approaches that modify the autophagic response to viral infection have shown great promise. The preponderance of work thus far, however, has originated from vertebrate systems. Efforts to elucidate the role of autophagy during arbovirus infection of invertebrates have emerged, providing new insights into arbovirus-vector interactions; interactions that could be exploited for novel control strategies.

Arthropod-borne viruses (arboviruses), such as Zika virus, chikungunya virus, and West Nile virus (WNV), pose continuous threats to emerge and cause large epidemics. Often, these events are associated with novel virus variants optimized for local transmission that first arise as minorities within a host. Thus, the conditions that regulate the frequency of intrahost variants are important determinants of emergence. Here, we describe the dynamics of WNV genetic diversity during its transmission cycle. By temporally sampling saliva from individual mosquitoes, we demonstrate that virus populations expectorated by mosquitoes are highly diverse and unique to each feeding episode. After transmission to birds, however, most genetic diversity is removed by strong purifying selection. Further, transmission of potentially mosquito-adaptive WNV variants is strongly influenced by genetic drift in mosquitoes. These results highlight the complex evolutionary forces a novel virus variant must overcome to alter infection phenotypes at the population level.

Ticks transmit a diverse array of viruses such as tick-borne encephalitis virus, Powassan virus, and Crimean-Congo hemorrhagic fever virus that are reemerging in many parts of the world. Most tick-borne viruses (TBVs) are RNA viruses that replicate using error-prone polymerases and produce genetically diverse viral populations that facilitate their rapid evolution and adaptation to novel environments. This article reviews the mechanisms of virus transmission by tick vectors, the molecular evolution of TBVs circulating in nature, and the processes shaping viral diversity within hosts to better understand how these viruses may become public health threats. In addition, remaining questions and future directions for research are discussed.

Macroautophagy is an evolutionarily conserved cellular process critical for maintaining cellular homeostasis. It can additionally function as an innate immune response to viral infection as has been demonstrated for a number of arthropod-borne (arbo-) viruses. Arboviruses are maintained in a transmission cycle between vertebrate hosts and invertebrate vectors yet the majority of studies assessing autophagy-arbovirus interactions have been limited to the mammalian host. Therefore we evaluated the role of autophagy during arbovirus infection of the invertebrate vector using the tractable Aag2Aedes aegyptimosquito cell culture system. Our data demonstrates that autophagy is significantly induced in mosquito cells upon infection with two flaviviruses, dengue virus (DENV) and Zika virus (ZIKV), as well as an unrelated mosquito-borne virus, chikungunya virus (CHIKV;Togaviridae). While assessing the role of autophagy during arbovirus infection, we observed a somewhat paradoxical outcome. Both induction and suppression of autophagy via torin 1 and spautin-1, respectively, resulted in increased viral titers for all three viruses, yet suppression of autophagy-related genes had no effect. Interestingly, chemical modulators of autophagy had either no effect or opposite effects in another widely used mosquito cell line, C6/36Aedes albopictuscells. Together, our data reveals a limited role for autophagy during arbovirus infection of mosquito cells. Further, our findings suggest that commonly used chemical modulators of autophagy alter mosquito cells in such a way as to promote viral replication; however, it is unclear if this occurs directly through autophagic manipulation or other means.Author SummaryArthropod-borne (arbo) viruses, specifically those transmitted by Aedes aegypti mosquitoes, cause significant morbidity and mortality and pose a continued public health threat worldwide. Many of these viruses lack vaccines or therapeutics and current mosquito control strategies are underperforming. For these reasons, identifying vulnerabilities within the transmission cycle that can be targeted will be critical to the development of novel control interventions. Autophagy is a highly conserved cellular pathway and previous studies manipulating this pathway have shown promise in minimizing viral infections in mammalian hosts. In this study we examined arbovirus-autophagy interactions within vector mosquitoes. The goal was to elucidate the role of autophagy during infection of mosquitoes in hopes of identifying critical interactions that can be targeted by novel approaches to block infection of and transmission by vector mosquitoes.

How viruses evolves within hosts can dictate infection outcomes; however, accurately reconstructing this process is challenging from samples with low virus concentrations. We evaluated our highly multiplexed amplicon approach - PrimalSeq - to enrich for viral RNA and demonstrate how virus concentration, sequencing coverage depth, primer mismatches, and technical replicates influence the accuracy of measuring intrahost virus diversity. Using this data, we developed an open source experimental protocol and computational tool (iVar; github.com/andersen-lab/ivar) for using PrimalSeq to measure intrahost virus diversity using Illumina and compared the results to Oxford Nanopore sequencing. Furthermore, we demonstrate the utility of our approach by measuring Zika and West Nile virus diversity from varied sample types, and show that the accumulation of genetic diversity is heavily influenced by experimental and biological systems.

Productive arbovirus infections require mechanisms to suppress or circumvent the cellular RNA interference (RNAi) pathway, a major antiviral response in mosquitoes. In this study, we demonstrate that two flaviviruses, Dengue virus and Kunjin virus, significantly repress siRNA-mediated RNAi in infected human cells as well as during infection of the mosquito vector Culex quinquefasciatus. Arthropod-borne flaviviruses generate a small structured non-coding RNA from the viral 3′ UTR referred to as sfRNA. Analysis of infections with a mutant Kunjin virus that is unable to generate appreciable amounts of the major sfRNA species indicated that RNAi suppression was associated with the generation of the non-coding sfRNA. Co-immunoprecipitation of sfRNA with RNAi mediators Dicer and Ago2 suggest a model for RNAi suppression. Collectively, these data help to establish a clear role for sfRNA in RNAi suppression and adds to the emerging impact of viral long non-coding RNAs in modulating aspects of anti-viral immune processes.

To test the hypothesis that RNA interference (RNAi) imposes diversifying selection on RNA virus genomes, we quantified West Nile virus (WNV) quasispecies diversity after passage in Drosophila cells in which RNAi was left intact, depleted, or stimulated against WNV. As predicted, WNV diversity was significantly lower in RNAi-depleted cells and significantly greater in RNAi-stimulated cells relative to that in controls. These findings reveal that an innate immune defense can shape viral population structure.

Arthropod-borne viruses (arboviruses) represent an emerging threat to human and livestock health globally. In particular, those transmitted by mosquitoes present the greatest challenges to disease control efforts. An understanding of the molecular basis for mosquito innate immunity to arbovirus infection is therefore critical to investigations regarding arbovirus evolution, virus-vector ecology, and mosquito vector competence. In this review, we discuss the current state of understanding regarding mosquito innate immunity to West Nile virus. We draw from the literature with respect to other virus-vector pairings to attempt to draw inferences to gaps in our knowledge about West Nile virus and relevant vectors.

Arthropod-borne RNA viruses exist within hosts as heterogeneous populations of viral variants and, as a result, possess great genetic plasticity. Understanding the micro-evolutionary forces shaping these viruses can provide insights into how they emerge, adapt, and persist in new and changing ecological niches. While considerable attention has been directed toward studying the population dynamics of mosquito-borne viruses, little is known about tick-borne virus populations. Therefore, using a mouse and Ixodes scapularis tick transmission model, we examined Powassan virus (POWV; Flaviviridae, Flavivirus) populations in and between both the vertebrate host and arthropod vector. We found that genetic bottlenecks, RNAi-mediated diversification, and selective constraints collectively influence POWV evolution. Together, our data provide a mechanistic explanation for the slow, long-term evolutionary trends of POWV, and suggest that all arthropod-borne viruses encounter similar selective pressures at the molecular level (i.e. RNAi), yet evolve much differently due to their unique rates and modes of transmission.

West Nile virus is similar to most other RNA viruses in that it exists in nature as a genetically diverse population. However, the role of this genetic diversity within natural transmission cycles and its importance to virus perpetuation remain poorly understood. Therefore, we determined whether highly genetically diverse populations are more fit compared to less genetically diverse WNV populations. Specifically, we generated three WNV populations that varied in their genetic diversity and evaluated their fitness relative to genetically marked control WNV in vivo in Culex quinquefasciatus mosquitoes and chickens. Our results demonstrate that high genetic diversity leads to fitness gains in vector mosquitoes, but not chickens.

Background Novel surveillance strategies are needed to detect the rapid and continuous emergence of infectious disease agents. Ideally, new sampling strategies should be simple to implement, technologically uncomplicated, and applicable to areas where emergence events are known to occur. To this end, xenosurveillance is a technique that makes use of blood collected by hematophagous arthropods to monitor and identify vertebrate pathogens. Mosquitoes are largely ubiquitous animals that often exist in sizable populations. As well, many domestic or peridomestic species of mosquitoes will preferentially take blood-meals from humans, making them a unique and largely untapped reservoir to collect human blood. Methodology/Principal findings We sought to take advantage of this phenomenon by systematically collecting blood-fed mosquitoes during a field trail in Northern Liberia to determine whether pathogen sequences from blood engorged mosquitoes accurately mirror those obtained directly from humans. Specifically, blood was collected from humans via finger-stick and by aspirating bloodfed mosquitoes from the inside of houses. Shotgun metagenomic sequencing of RNA and DNA derived from these specimens was performed to detect pathogen sequences. Samples obtained from xenosurveillance and from finger-stick blood collection produced a similar number and quality of reads aligning to two human viruses, GB virus C and hepatitis B virus. Conclusions/Significance This study represents the first systematic comparison between xenosurveillance and more traditional sampling methodologies, while also demonstrating the viability of xenosurveillance as a tool to sample human blood for circulating pathogens., Author summary Infectious diseases continue to be a burden on mankind, particularly in the developing countries of the tropics. Recognition of pathogen transmission in humans is a crucial step to thwarting epidemics of these pathogens. However, sampling human blood or tissue is invasive and logistically difficult. Xenosurveillance takes advantage of the blood-feeding behavior of mosquitoes to sample human blood for the presence of infectious disease agents. In this study, we aimed to compare xenosurveillance to a more traditional sampling method to assess the usefulness of this technique in field settings where it could potentially be beneficial. DNA and RNA next generation sequencing followed by an in-house bioinformatic pipeline identified viruses and parasites of human origin in blood collected by either mosquitoes or finger-stick. Xenosurveillance produces samples of comparable quality to finger-stick blood collections while alleviating many of the difficulties of direct human sampling. This study suggests xenosurveillance can be a complimentary strategy for infectious disease surveillance in low-resource areas.

Dengue viruses (DENV) cause significant morbidity and mortality worldwide and are transmitted by the mosquito Aedes aegypti. Mosquitoes become infected after ingesting a viremic bloodmeal, and molecular mechanisms involved in bloodmeal digestion may affect the ability of DENV to infect the midgut. We used RNA interference (RNAi) to silence expression of four midgut serine proteases and assessed the effect of each RNAi phenotype on DENV-2 infectivity of Aedes aegypti. Silencing resulted in significant reductions in protease mRNA levels and correlated with a reduction in activity except in the case of late trypsin. RNA silencing of chymotrypsin, early and late trypsin had no effect on DENV-2 infectivity. However, silencing of 5G1 or the addition of soybean trypsin inhibitor to the infectious bloodmeals significantly increased midgut infection rates. These results suggest that some midgut serine proteases may actually limit DENV-2 infectivity of Ae. aegypti.

Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes.

Mosquitoes rely on RNA interference (RNAi) as their primary defense against viral infections. To this end, the combination of RNAi and invertebrate cell culture systems has become an invaluable tool in studying virus-vector interactions. Nevertheless, a recent study failed to detect an active RNAi response to West Nile virus (WNV) infection in C6/36 (Aedes albopictus) cells, a mosquito cell line frequently used to study arthropod-borne viruses (arboviruses). Therefore, we sought to determine if WNV actively evades the host's RNAi response or if C6/36 cells have a dysfunctional RNAi pathway. C6/36 and Drosophila melanogaster S2 cells were infected with WNV (Flaviviridae), Sindbis virus (SINV, Togaviridae) and La Crosse virus (LACV, Bunyaviridae) and total RNA recovered from cell lysates. Small RNA (sRNA) libraries were constructed and subjected to high-throughput sequencing. In S2 cells, virus-derived small interfering RNAs (viRNAs) from all three viruses were predominantly 21 nt in length, a hallmark of the RNAi pathway. However, in C6/36 cells, viRNAs were primarily 17 nt in length from WNV infected cells and 26–27 nt in length in SINV and LACV infected cells. Furthermore, the origin (positive or negative viral strand) and distribution (position along viral genome) of S2 cell generated viRNA populations was consistent with previously published studies, but the profile of sRNAs isolated from C6/36 cells was altered. In total, these results suggest that C6/36 cells lack a functional antiviral RNAi response. These findings are analogous to the type-I interferon deficiency described in Vero (African green monkey kidney) cells and suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions at the molecular level., Author Summary Cell culture systems are invaluable tools for studying virus-host interactions. These systems are typically easy to maintain and manipulate; however, they can fail to accurately mimic the host environment encountered by viruses. Therefore, defining the limitations of each system is critical to properly interpreting the results. C6/36 Aedes albopictus cells are commonly used to study arthropod-borne viruses (arboviruses), such as West Nile virus (WNV). Recent evidence suggests that the RNA interference (RNAi) pathway, a critical aspect of the cellular innate antiviral immune response in invertebrates, may not actively target WNV in C6/36 cells. However, it is unknown whether this observation is limited to WNV. Therefore, we examined small RNA populations from C6/36 and Drosophila melanogastor S2 cells infected with WNV, Sindbis virus and La Crosse virus by high-throughput sequencing. We demonstrate that the RNAi pathway actively targets each of the three viruses in S2 cells, but does not in C6/36 cells. These findings suggest that C6/36 cells may fail to accurately model mosquito-arbovirus interactions.

West Nile virus (WNV) exists in nature as a genetically diverse population of competing genomes. This high genetic diversity and concomitant adaptive plasticity has facilitated the rapid adaptation of WNV to North American transmission cycles and contributed to its explosive spread throughout the New World. WNV is maintained in nature in a transmission cycle between mosquitoes and birds, with intrahost genetic diversity highest in mosquitoes. The mechanistic basis for this increase in genetic diversity in mosquitoes is poorly understood. To determine whether the high mutational diversity of WNV in mosquitoes is driven by RNA interference (RNAi), we characterized the RNAi response to WNV in the midguts of orally exposed Culex pipiens quinquefasciatus using high-throughput, massively parallel sequencing and estimated viral genetic diversity. Our data demonstrate that WNV infection in orally exposed vector mosquitoes induces the RNAi pathway and that regions of the WNV genome that are more intensely targeted by RNAi are more likely to contain point mutations compared to weakly targeted regions. These results suggest that, under natural conditions, positive selection of WNV within mosquitoes is stronger in regions highly targeted by the host RNAi response. Further, they provide a mechanistic basis for the relative importance of mosquitoes in driving WNV diversification., Author Summary West Nile virus (WNV) was introduced into New York state in 1999 and has since spread across the Americas. It is transmitted in nature between adult female mosquitoes and birds and occasionally infects humans and horses. Within the host, WNV exists as a diverse assortment of closely related mutants. WNV populations within mosquitoes are more complex genetically than are those within birds. The reasons for this discrepancy are unknown, but may be related to the host's innate antivirus response. We demonstrate that WNV is targeted by RNA interference, a highly sequence-specific pathway in the mosquito. Further, we present data that correlates the intensity of this targeting with virus mutation under natural conditions. These results provide a mechanistic explanation for the increasead complexity of WNV populations in mosquitoes: the RNAi response creates an intracellular environment where rare genotypes are favored. In addition, our results suggest that genetically diverse WNV populations may have an advantage over less diverse populations because they present a more complex target for the RNAi response. Finally, these data suggest that WNV, and possibly other viruses with high mutation rates, may escape an engineered antivirus intervention that is highly sequence-specific.

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses., Author Summary Viruses are constantly emerging into new areas and pose significant challenges to public health. Chikungunya and West Nile viruses (WNV), both mosquito-borne RNA viruses, are quintessential examples of how increased globalization has facilitated the expansion of viruses into new territories. Rapid evolution of both of these agents has contributed to their rapid spread and health burden. Thus, characterizing how selection shapes zoonotic RNA viruses in their natural hosts is important to understand their emergence. As an ecological generalist able to infect hundreds of bird species, WNV is an excellent tool to study how different animal hosts can differentially drive virus evolution. We examined the genetic composition and fitness of WNV produced during replication in wild-caught American crows, house sparrows and American robins, species that range in mortality following WNV infection (crows the highest, robins the lowest). We demonstrate host-dependent effects on WNV population structure and fitness. Our study provides insights on how different virus-animal interactions can influence the success of a virus in the next host and ultimately the success of virus emergence into new host systems.

The exogenous RNA interference (RNAi) pathway is an important antiviral defense against arboviruses in mosquitoes, and virus-specific small interfering (si)RNAs are key components of this pathway. Understanding the biogenesis of siRNAs in mosquitoes could have important ramifications in using RNAi to control arbovirus transmission. Using deep sequencing technology, we characterized dengue virus type 2 (DENV2)-specific small RNAs produced during infection of Aedes aegypti mosquitoes and A. aegypti Aag2 cell cultures and compared them to those produced in the C6/36 Aedes albopictus cell line. We show that the size and mixed polarity of virus-specific small RNAs from DENV-infected A. aegypti cells indicate that they are products of Dicer-2 (Dcr2) cleavage of long dsRNA, whereas C6/36 cells generate DENV2-specific small RNAs that are longer and predominantly positive polarity, suggesting that they originate from a different small RNA pathway. Examination of virus-specific small RNAs after infection of the two mosquito cell lines with the insect-only flavivirus cell fusing agent virus (CFAV) corroborated these findings. An in vitro assay also showed that Aag2 A. aegypti cells are capable of siRNA production, while C6/36 A. albopictus cells exhibit inefficient Dcr2 cleavage of long dsRNA. Defective expression or function of Dcr2, the key initiator of the RNAi pathway, might explain the comparatively robust growth of arthropod-borne viruses in the C6/36 cell line, which has been used frequently as a surrogate for studying molecular interactions between arboviruses and cells of their mosquito hosts., Author Summary Understanding how arthropod-borne viruses (arboviruses) establish persistent infections in mosquitoes will help us to find ways to prevent viral disease transmission by these insects. RNA silencing pathways in mosquitoes and other insects, particularly RNA interference (RNAi), have been shown to be important in antiviral defense. In this study we describe small RNAs involved in RNA silencing that are derived from the genome of the arbovirus dengue virus type-2 (DENV2) in infected Aedes aegypti mosquito cell lines and mosquitoes. We also show that C6/36, a mosquito cell line from A. albopictus, appears to process DENV2 RNA for silencing differently from A. aegypti mosquitoes, revealing that other small RNA pathways in mosquito cells might have a role in antiviral immunity in this cell line and provide insight into using mosquito cell cultures to study the antiviral response to arboviruses in mosquitoes.

Anopheles gambiae are a major vector of malaria in sub-Saharan Africa. Viruses that naturally infect these mosquitoes may impact their physiology and ability to transmit pathogens. We therefore used metagenomics sequencing to search for viruses in adult Anopheles mosquitoes collected from Liberia, Senegal, and Burkina Faso. We identified a number of virus and virus-like sequences from mosquito midgut contents, including 14 coding-complete genome segments and 26 partial sequences. The coding-complete sequences define new viruses in the order Mononegavirales, and the families Flaviviridae, and Totiviridae. The identification of a flavivirus infecting Anopheles mosquitoes broadens our understanding of the evolution and host range of this virus family. This study increases our understanding of virus diversity in general, begins to define the virome of a medically important vector in its natural setting, and lays groundwork for future studies examining the potential impact of these viruses on anopheles biology and disease transmission.