Evaluation of Vector-Enabled Xenosurveillance in Rural Guatemala.

Surveillance methods that permit rapid detection of circulating pathogens in low-resource settings are desperately needed. In this study, we evaluated a mosquito bloodmeal-based surveillance method ("xenosurveillance") in rural Guatemala. Twenty households from two villages (Los Encuentros and Chiquirines) in rural southwest Guatemala were enrolled and underwent weekly prospective surveillance from August 2019 to December 2019 (16 weeks). When febrile illness was reported in a household, recently blood-fed mosquitoes were collected from within dwellings and blood samples taken from each member of the household. Mosquitoes were identified to species and blood sources identified by sequencing. Shotgun metagenomic sequencing was used to identify circulating viruses. Culex pipiens (60.9%) and Aedes aegypti (18.6%) were the most abundant mosquitoes collected. Bloodmeal sources were most commonly human (32.6%) and chicken (31.6%), with various other mammal and avian hosts detected. Several mosquito-specific viruses were detected, including Culex orthophasma virus. Human pathogens were not detected. Therefore, xenosurveillance may require more intensive sampling to detect human pathogens in Guatemala and ecologically similar localities in Central America.

Elucidating the role of dsRNA sensing and Toll6 in antiviral responses of Culex quinquefasciatus cells.

The first step of any immune response is the recognition of foreign molecular structures inside the host organism. An important molecule that is generally foreign to eukaryotic cells is long double-stranded RNA (dsRNA), which can be generated during virus replication. The mechanisms of sensing viral dsRNA are well-studied in mammalian systems but are only poorly understood in insects, including disease vectors such as Culex quinquefasciatus mosquitoes. These mosquitoes are vectors for important arboviruses, such as West Nile virus, and Culex species mosquitoes are distributed across the globe in many temperate and tropical regions. The major antiviral response triggered by dsRNA in mosquitoes is RNA interference - a sequence-specific response which targets complementary viral RNA for degradation. However, here, we aimed to identify whether sequence-independent dsRNA sensing, mimicked by poly(I:C), can elicit an antiviral response. We observed a significant reduction in replication of La Crosse virus (LACV) in Cx. quinquefasciatus mosquito cells following poly(I:C) priming. We identified a number of antimicrobial peptides and Toll receptors that were upregulated at the transcript level by poly(I:C) stimulation. Notably, Toll6 was upregulated and we determined that a knockdown of Toll6 expression resulted also in increased LACV replication. Future efforts require genetic tools to validate whether the observed Toll6 antiviral activity is indeed linked to dsRNA sensing. However, large-scale functional genomic and proteomic approaches are also required to determine which downstream responses are part of the poly(I:C) elicited antiviral response.

Recognition of Arboviruses by the Mosquito Immune System.

Arthropod-borne viruses (arboviruses) pose a significant threat to both human and animal health worldwide. These viruses are transmitted through the bites of mosquitoes, ticks, sandflies, or biting midges to humans or animals. In humans, arbovirus infection often results in mild flu-like symptoms, but severe disease and death also occur. There are few vaccines available, so control efforts focus on the mosquito population and virus transmission control. One area of research that may enable the development of new strategies to control arbovirus transmission is the field of vector immunology. Arthropod vectors, such as mosquitoes, have coevolved with arboviruses, resulting in a balance of virus replication and vector immune responses. If this balance were disrupted, virus transmission would likely be reduced, either through reduced replication, or even through enhanced replication, resulting in mosquito mortality. The first step in mounting any immune response is to recognize the presence of an invading pathogen. Recent research advances have been made to tease apart the mechanisms of arbovirus detection by mosquitoes. Here, we summarize what is known about arbovirus recognition by the mosquito immune system, try to generate a comprehensive picture, and highlight where there are still gaps in our current understanding.

Intracellular Diversity of WNV within Circulating Avian Peripheral Blood Mononuclear Cells Reveals Host-Dependent Patterns of Polyinfection.

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.

The Incompetence of Mosquitoes-Can Zika Virus Be Adapted To Infect Culex tarsalis Cells?

The molecular evolutionary mechanisms underpinning virus-host interactions are increasingly recognized as key drivers of virus emergence, host specificity, and the likelihood that viruses can undergo a host shift that alters epidemiology and transmission biology. Zika virus (ZIKV) is mainly transmitted between humans by Aedes aegypti mosquitoes. However, the 2015 to 2017 outbreak stimulated discussion regarding the role of Culex spp. mosquitoes in transmission. Reports of ZIKV-infected Culex mosquitoes, in nature and under laboratory conditions, resulted in public and scientific confusion. We previously found that Puerto Rican ZIKV does not infect colonized Culex quinquefasciatus, Culex pipiens, or Culex tarsalis, but some studies suggest they may be competent ZIKV vectors. Therefore, we attempted to adapt ZIKV to Cx. tarsalis by serially passaging virus on cocultured Ae. aegypti (Aag2) and Cx. tarsalis (CT) cells to identify viral determinants of species specificity. Increasing fractions of CT cells resulted in decreased overall virus titer and no enhancement of Culex cell or mosquito infection. Next-generation sequencing of cocultured virus passages revealed synonymous and nonsynonymous variants throughout the genome that arose as CT cell fractions increased. We generated nine recombinant ZIKVs containing combinations of the variants of interest. None of these viruses showed increased infection of Culex cells or mosquitoes, demonstrating that variants associated with passaging were not specific to increased Culex infection. These results reveal the challenge of a virus adapting to a new host, even when pushed to adapt artificially. Importantly, they also demonstrate that while ZIKV may occasionally infect Culex mosquitoes, Aedes mosquitoes likely drive transmission and human risk. IMPORTANCE ZIKV is mainly transmitted between humans by Aedes mosquitoes. In nature, ZIKV-infected Culex mosquitoes have been found, and ZIKV infrequently infects Culex mosquitoes under laboratory conditions. Yet, most studies show that Culex mosquitoes are not competent vectors for ZIKV. We attempted to adapt ZIKV to Culex cells to identify viral determinants of species specificity. We sequenced ZIKV after it was passaged on a mixture of Aedes and Culex cells and found that it acquired many variants. We generated recombinant viruses containing combinations of the variants of interest to determine if any of these changes enhance infection in Culex cells or mosquitoes. Recombinant viruses did not show increased infection in Culex cells or mosquitoes, but some variants increased infection in Aedes cells, suggesting adaptation to those cells instead. These results reveal that arbovirus species specificity is complex, and that virus adaptation to a new genus of mosquito vectors likely requires multiple genetic changes.

Intracellular diversity of WNV within circulating avian peripheral blood mononuclear cells reveals host-dependent patterns of polyinfection.

Error-prone replication of RNA viruses generates the genetic diversity required for adaptation within rapidly changing environments. Thus, arthropod-borne virus (arbovirus) populations exist in nature as mutant swarms that are maintained between arthropods and vertebrates. Previous studies have demonstrated that West Nile virus (WNV) population dynamics are host dependent: In American crows, which experience extremely high viremia, purifying selection is weak and population diversity is high compared to American robins, which have 100 to 1000-fold lower viremia. WNV passed in robins experiences fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows higher genetic diversity within individual avian peripheral-blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a novel, barcoded version of WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes that each contained. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation. These studies further document the role of particular, ecologically relevant hosts in shaping virus population structure. Author Summary: WNV mutational diversity in vertebrates is species-dependent. In crows, low frequency variants are common, and viral populations are more diverse. In robins, fewer mutations become permanent fixtures of the overall viral population. We infected crows, robins and a chicken cell line with a genetically marked (barcoded) WNV. Higher levels of virus led to multiple unique WNV genomes infecting individual cells, even when a genotype was present at low levels in the input viral stock. Our findings suggest that higher levels of circulating virus in natural hosts allow less fit viruses to survive in RNA virus populations through complementation by more fit viruses. This is significant as it allows less represented and less fit viruses to be maintained at low levels until they potentially emerge when virus environments change. Overall our data reveal new insights on the relationships between host susceptibility to high viremia and virus evolution.

Identification of MicroRNAs in the West Nile Virus Vector Culex tarsalis (Diptera: Culicidae).

MicroRNAs (miRNAs) are a group of small noncoding RNAs that regulate gene expression during important biological processes including development and pathogen defense in most living organisms. Presently, no miRNAs have been identified in the mosquito Culex tarsalis (Diptera: Culicidae), one of the most important vectors of West Nile virus (WNV) in North America. We used small RNA sequencing data and in vitro and in vivo experiments to identify and validate a repertoire of miRNAs in Cx. tarsalis mosquitoes. Using bioinformatic approaches we analyzed small RNA sequences from the Cx. tarsalis CT embryonic cell line to discover orthologs for 86 miRNAs. Consistent with other mosquitoes such as Aedes albopictus and Culex quinquefasciatus, miR-184 was found to be the most abundant miRNA in Cx. tarsalis. We also identified 20 novel miRNAs from the recently sequenced Cx. tarsalis genome, for a total of 106 miRNAs identified in this study. The presence of selected miRNAs was biologically validated in both the CT cell line and in adult Cx. tarsalis mosquitoes using RT-qPCR and sequencing. These results will open new avenues of research into the role of miRNAs in Cx. tarsalis biology, including development, metabolism, immunity, and pathogen infection.

Culex Mosquito Piwi4 Is Antiviral against Two Negative-Sense RNA Viruses.

Culex spp. mosquitoes transmit several pathogens concerning public health, including West Nile virus and Saint Louis encephalitis virus. Understanding the antiviral immune system of Culex spp. mosquitoes is important for reducing the transmission of these viruses. Mosquitoes rely on RNA interference (RNAi) to control viral replication. While the siRNA pathway in mosquitoes is heavily studied, less is known about the piRNA pathway. The piRNA pathway in mosquitoes has recently been connected to mosquito antiviral immunity. In Aedes aegypti, Piwi4 has been implicated in antiviral responses. The antiviral role of the piRNA pathway in Culex spp. mosquitoes is understudied compared to Ae. aegypti. Here, we aimed to identify the role of PIWI genes and piRNAs in Culex quinquefasciatus and Culex tarsalis cells during virus infection. We examined the effect of PIWI gene silencing on virus replication of two arboviruses and three insect-specific viruses in Cx. quinquefasciatus derived cells (Hsu) and Cx. tarsalis derived (CT) cells. We show that Piwi4 is antiviral against the La Crosse orthobunyavirus (LACV) in Hsu and CT cells, and the insect-specific rhabdovirus Merida virus (MERDV) in Hsu cells. None of the silenced PIWI genes impacted replication of the two flaviviruses Usutu virus (USUV) and Calbertado virus, or the phasivirus Phasi-Charoen-like virus. We further used small RNA sequencing to determine that LACV-derived piRNAs, but not USUV-derived piRNAs were generated in Hsu cells and that PIWI gene silencing resulted in a small reduction in vpiRNAs. Finally, we determined that LACV-derived DNA was produced in Hsu cells during infection, but whether this viral DNA is required for vpiRNA production remains unclear. Overall, we expanded our knowledge on the piRNA pathway and how it relates to the antiviral response in Culex spp mosquitoes.

Optimized In Vitro CRISPR/Cas9 Gene Editing Tool in the West Nile Virus Mosquito Vector, Culex quinquefasciatus.

Culex quinquefasciatus mosquitoes are a globally widespread vector of multiple human and animal pathogens, including West Nile virus, Saint Louis encephalitis virus, and lymphatic filariasis. Since the introduction of West Nile virus to the United States in 1999, a cumulative 52,532 cases have been reported to the CDC, including 25,849 (49.2%) neuroinvasive cases and 2456 (5%) deaths. Viral infections elicit immune responses in their mosquito vectors, including the RNA interference (RNAi) pathway considered to be the cornerstone antiviral response in insects. To investigate mosquito host genes involved in pathogen interactions, CRISPR/Cas9-mediated gene-editing can be used for functional studies of mosquito-derived cell lines. Yet, the tools available for the study of Cx. quinquefasciatus-derived (Hsu) cell lines remain largely underdeveloped compared to other mosquito species. In this study, we constructed and characterized a Culex-optimized CRISPR/Cas9 plasmid for use in Hsu cell cultures. By comparing it to the original Drosophila melanogaster CRISPR/Cas9 plasmid, we showed that the Culex-optimized plasmid demonstrated highly efficient editing of the genomic loci of the RNAi proteins Dicer-2 and PIWI4 in Hsu cells. These new tools support our ability to investigate gene targets involved in mosquito antiviral response, and thus the future development of gene-based vector control strategies.

Impact of extrinsic incubation temperature on natural selection during Zika virus infection of Aedes aegypti and Aedes albopictus.

Arthropod-borne viruses (arboviruses) require replication across a wide range of temperatures to perpetuate. While vertebrate hosts tend to maintain temperatures of approximately 37°C-40°C, arthropods are subject to ambient temperatures which can have a daily fluctuation of > 10°C. Temperatures impact vector competence, extrinsic incubation period, and mosquito survival unimodally, with optimal conditions occurring at some intermediate temperature. In addition, the mean and range of daily temperature fluctuations influence arbovirus perpetuation and vector competence. The impact of temperature on arbovirus genetic diversity during systemic mosquito infection, however, is poorly understood. Therefore, we determined how constant extrinsic incubation temperatures of 25°C, 28°C, 32°C, and 35°C control Zika virus (ZIKV) vector competence and population dynamics within Aedes aegypti and Aedes albopictus mosquitoes. We also examined fluctuating temperatures which better mimic field conditions in the tropics. We found that vector competence varied in a unimodal manner for constant temperatures peaking between 28°C and 32°C for both Aedes species. Transmission peaked at 10 days post-infection for Aedes aegypti and 14 days for Aedes albopictus. Conversely, fluctuating temperature decreased vector competence. Using RNA-seq to characterize ZIKV population structure, we identified that temperature alters the selective environment in unexpected ways. During mosquito infection, constant temperatures more often elicited positive selection whereas fluctuating temperatures led to strong purifying selection in both Aedes species. These findings demonstrate that temperature has multiple impacts on ZIKV biology, including major effects on the selective environment within mosquitoes.

Comparison of Chikungunya Virus and Zika Virus Replication and Transmission Dynamics in Aedes aegypti Mosquitoes.

Chikungunya virus (CHIKV) and Zika virus (ZIKV) are arthropod-borne viruses transmitted mainly by Aedes aegypti mosquitoes. These viruses have become endemic in large parts of North, Central, and South America. Arboviruses persistently infect mosquitoes throughout their life span and become infectious (i.e., expectorate infectious virus in saliva) after a period of time called the extrinsic incubation period (EIP). The duration of this infectiousness, however, is not well characterized. This is an important shortcoming because many epidemiological models assume that mosquitoes continue to be infectious for the duration of their life span. To define the duration of infectiousness for CHIKV and ZIKV, mosquitoes were infected orally with these viruses. Every 2 days, legs/wings, midguts, salivary glands, and saliva were collected from 30 to 60 mosquitoes and viral load measured. In CHIKV-infected mosquitoes, infectious virus in saliva peaked early (2-4 dpi), and then decreased rapidly and was rarely observed after 10 dpi. Viral RNA in infected tissues also decreased after the initial peak (4-8 dpi) but did so much less drastically. In ZIKV-infected mosquitoes, the infectious virus in saliva peaked at 12-14 dpi and dropped off only slightly after 14 dpi. In infected tissues, viral RNA increased early during infection, and then plateaued after 6-10 days. Our findings suggest that significant variation exists in the duration of the infectious period for arboviruses that is in part influenced by virus clearance from expectorated saliva.

Discrete viral E2 lysine residues and scavenger receptor MARCO are required for clearance of circulating alphaviruses.

The magnitude and duration of vertebrate viremia is a critical determinant of arbovirus transmission, geographic spread, and disease severity. We find that multiple alphaviruses, including chikungunya (CHIKV), Ross River (RRV), and o'nyong 'nyong (ONNV) viruses, are cleared from the circulation of mice by liver Kupffer cells, impeding viral dissemination. Clearance from the circulation was independent of natural antibodies or complement factor C3, and instead relied on scavenger receptor SR-A6 (MARCO). Remarkably, lysine to arginine substitutions at distinct residues within the E2 glycoproteins of CHIKV and ONNV (E2 K200R) as well as RRV (E2 K251R) allowed for escape from clearance and enhanced viremia and dissemination. Mutational analysis revealed that viral clearance from the circulation is strictly dependent on the presence of lysine at these positions. These findings reveal a previously unrecognized innate immune pathway that controls alphavirus viremia and dissemination in vertebrate hosts, ultimately influencing disease severity and likely transmission efficiency.

Small RNA responses of Culex mosquitoes and cell lines during acute and persistent virus infection.

RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication of different viruses.

Evaluation of a novel West Nile virus transmission control strategy that targets Culex tarsalis with endectocide-containing blood meals.

Control of arbovirus transmission remains focused on vector control through application of insecticides directly to the environment. However, these insecticide applications are often reactive interventions that can be poorly-targeted, inadequate for localized control during outbreaks, and opposed due to environmental and toxicity concerns. In this study, we developed endectocide-treated feed as a systemic endectocide for birds to target blood feeding Culex tarsalis, the primary West Nile virus (WNV) bridge vector in the western United States, and conducted preliminary tests on the effects of deploying this feed in the field. In lab tests, ivermectin (IVM) was the most effective endectocide tested against Cx. tarsalis and WNV-infection did not influence mosquito mortality from IVM. Chickens and wild Eurasian collared doves exhibited no signs of toxicity when fed solely on bird feed treated with concentrations up to 200 mg IVM/kg of diet, and significantly more Cx. tarsalis that blood fed on these birds died (greater than 80% mortality) compared to controls (less than 25% mortality). Mosquito mortality following blood feeding correlated with IVM serum concentrations at the time of blood feeding, which dropped rapidly after the withdrawal of treated feed. Preliminary field testing over one WNV season in Fort Collins, Colorado demonstrated that nearly all birds captured around treated bird feeders had detectable levels of IVM in their blood. However, entomological data showed that WNV transmission was non-significantly reduced around treated bird feeders. With further development, deployment of ivermectin-treated bird feed might be an effective, localized WNV transmission control tool.

Arbovirus coinfection and co-transmission: A neglected public health concern?

Epidemiological synergy between outbreaks of viruses transmitted by Aedes aegypti mosquitoes, such as chikungunya, dengue, and Zika viruses, has resulted in coinfection of humans with multiple viruses. Despite the potential impact on public health, we know only little about the occurrence and consequences of such coinfections. Here, we review the impact of coinfection on clinical disease in humans, discuss the possibility for co-transmission from mosquito to human, and describe a role for modeling transmission dynamics at various levels of co-transmission. Solving the mystery of virus coinfections will reveal whether they should be viewed as a serious concern for public health.

Dengue type 1 viruses circulating in humans are highly infectious and poorly neutralized by human antibodies.

The four dengue virus (DENV) serotypes are mosquito-borne flaviviruses of humans. The interactions between DENVs and the human host that lead to asymptomatic, mild, or severe disease are poorly understood, in part, because laboratory models are poor surrogates for human DENV disease. Virologists are interested in how the properties of DENVs replicating in people compare with virions propagated on laboratory cell lines, which are widely used for research and vaccine development. Using clinical samples from a DENV type 1 epidemic in Sri Lanka and new ultrasensitive assays, we compared the properties of DENVs in human plasma and after one passage on laboratory cell lines. DENVs in plasma were 50- to 700-fold more infectious than cell culture-grown viruses. DENVs produced by laboratory cell lines were structurally immature and hypersensitive to neutralization by human antibodies compared with DENVs circulating in people. Human plasma and cell culture-derived virions had identical genome sequences, indicating that these phenotypic differences were due to the mature state of plasma virions. Several dengue vaccines are under development. Recent studies indicate that vaccine-induced antibodies that neutralized DENVs in cell culture assays were not sufficient for protecting people from DENV infections. Our results about structural differences between DENVs produced in humans versus cell lines may be key to understanding vaccine failure and developing better models for vaccine evaluation.

Sequential Infection of Aedes aegypti Mosquitoes with Chikungunya Virus and Zika Virus Enhances Early Zika Virus Transmission.

In urban settings, chikungunya, Zika, and dengue viruses are transmitted by Aedes aegypti mosquitoes. Since these viruses co-circulate in several regions, coinfection in humans and vectors may occur, and human coinfections have been frequently reported. Yet, little is known about the molecular aspects of virus interactions within hosts and how they contribute to arbovirus transmission dynamics. We have previously shown that Aedes aegypti exposed to chikungunya and Zika viruses in the same blood meal can become coinfected and transmit both viruses simultaneously. However, mosquitoes may also become coinfected by multiple, sequential feeds on single infected hosts. Therefore, we tested whether sequential infection with chikungunya and Zika viruses impacts mosquito vector competence. We exposed Ae. aegypti mosquitoes first to one virus and 7 days later to the other virus and compared infection, dissemination, and transmission rates between sequentially and single infected groups. We found that coinfection rates were high after sequential exposure and that mosquitoes were able to co-transmit both viruses. Surprisingly, chikungunya virus coinfection enhanced Zika virus transmission 7 days after the second blood meal. Our data demonstrate heterologous arbovirus synergism within mosquitoes, by unknown mechanisms, leading to enhancement of transmission under certain conditions.

Variation in competence for ZIKV transmission by Aedes aegypti and Aedes albopictus in Mexico.

BACKGROUND: ZIKV is a new addition to the arboviruses circulating in the New World, with more than 1 million cases since its introduction in 2015. A growing number of studies have reported vector competence (VC) of Aedes mosquitoes from several areas of the world for ZIKV transmission. Some studies have used New World mosquitoes from disparate regions and concluded that these have a variable but relatively low competence for the Asian lineage of ZIKV. METHODOLOGY/PRINCIPAL FINDINGS: Ten Aedes aegypti (L) and three Ae. albopictus (Skuse) collections made in 2016 from throughout Mexico were analyzed for ZIKV (PRVABC59-Asian lineage) VC. Mexican Ae. aegypti had high rates of midgut infection (MIR), dissemination (DIR) and salivary gland infection (SGIR) but low to moderate transmission rates (TR). It is unclear whether this low TR was due to heritable salivary gland escape barriers or to underestimating the amount of virus in saliva due to the loss of virus during filtering and random losses on surfaces when working with small volumes. VC varied among collections, geographic regions and whether the collection was made north or south of the Neovolcanic axis (NVA). The four rates were consistently lower in northeastern Mexico, highest in collections along the Pacific coast and intermediate in the Yucatan. All rates were lowest north of the NVA. It was difficult to assess VC in Ae. albopictus because rates varied depending upon the number of generations in the laboratory. CONCLUSIONS/SIGNIFICANCE: Mexican Ae. aegypti and Ae. albopictus are competent vectors of ZIKV. There is however large variance in vector competence among geographic sites and regions. At 14 days post infection, TR varied from 8-51% in Ae. aegypti and from 2-26% in Ae. albopictus.

Adventitious viruses persistently infect three commonly used mosquito cell lines.

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.

Using barcoded Zika virus to assess virus population structure in vitro and in Aedes aegypti mosquitoes.

Arboviruses such as Zika virus (ZIKV, Flaviviridae; Flavivirus) must replicate in both mammalian and insect hosts possessing strong immune defenses. Accordingly, transmission between and replication within hosts involves genetic bottlenecks, during which viral population size and genetic diversity may be significantly reduced. To help quantify these bottlenecks and their effects, we constructed 4 "barcoded" ZIKV populations that theoretically contain thousands of barcodes each. After identifying the most diverse barcoded virus, we passaged this virus 3 times in 2 mammalian and mosquito cell lines and characterized the population using deep sequencing of the barcoded region of the genome. C6/36 maintain higher barcode diversity, even after 3 passages, than Vero. Additionally, field-caught mosquitoes exposed to the virus to assess bottlenecks in a natural host. A progressive reduction in barcode diversity occurred throughout systemic infection of these mosquitoes. Differences in bottlenecks during systemic spread were observed between different populations of Aedes aegypti.