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Mechanical transmission is an understudied mode of arbovirus transmission that occurs when a biting insect transmits virus among hosts by the direct transfer of virus particles contaminating its mouthparts. Multiple arboviruses have been shown to be capable of utilizing this transmission route, but most studies were conducted 40 to 70 years ago using dated methodologies. To gain a better understanding of this phenomenon, we used molecular techniques to evaluate the efficiency of mechanical transmission by Aedes aegypti mosquitoes for two evolutionarily divergent arboviruses, chikungunya virus (CHIKV) and dengue virus (DENV). Viral RNA and/or infectious DENV could be detected on 13.8% of mosquito proboscises sampled immediately after an infectious bloodmeal, but positivity rates declined within hours. CHIKV RNA and/or infectious virus was detected on 38.8% of proboscises immediately after feeding but positivity rates dropped to 2.5% within 4 hours. RNA copy numbers were low for both viruses, and we were unable to demonstrate mechanical transmission of CHIKV using an established animal model, suggesting that this mode of transmission is unlikely under natural conditions.

Powassan virus is an emerging tick-borne virus of concern for public health, but very little is known about its transmission patterns and ecology. Here, we expanded the genomic dataset by sequencing 279 Powassan viruses isolated fromIxodes scapularisticks from the northeastern United States. Our phylogeographic reconstructions revealed that Powassan virus lineage II was likely introduced or emerged from a relict population in the Northeast between 1940-1975. Sequences strongly clustered by sampling location, suggesting a highly focal geographical distribution. Our analyses further indicated that Powassan virus lineage II emerged in the northeastern U.S. mostly following a south to north pattern, with a weighted lineage dispersal velocity of ~3 km/year. Since the emergence in the Northeast, we found an overall increase in the effective population size of Powassan virus lineage II, but with growth stagnating during recent years. The cascading effect of population expansion of white-tailed deer andI. scapularispopulations likely facilitated the emergence of Powassan virus in the northeastern U.S.Significance statementOur work provides important fundamental insights in the local transmission dynamics of an emerging tick-borne pathogen of public health concern. Without the availability of vaccines or specific treatments, prevention of Powassan virus infection is dependent on education and control. We identified that Powassan virus is maintained in highly localized transmission foci that have been maintained for several years, without introductions of new virus clades. This provides both opportunities for better education about high risk areas and effective targeted control in Powassan virus foci with a long lasting impact.

In this study we describe the generation of two new species of axenic mosquito,Aedes albopictusandAedes triseriatus.Along withAedes aegypti, axenic larvae of these three species were exposed to an environmental water source to document the assembly of the microbiome in a common garden experiment. Additionally, the larvae were reared either individually or combinatorially with the other species to characterize the effects of co-rearing on the composition of the microbiome. We found that the microbiome of the larvae was composed of a relatively low diversity collection of bacteria from the colonizing water. The abundance of bacteria in the water was a poor predictor for their abundance in the larvae, suggesting the larval microbiome is made up of a subset of relatively rare aquatic bacteria. We found eleven bacterial 16S rRNA gene amplicon sequence variants (ASVs) that were conserved amongst>90% of the mosquitoes sampled, including two found in 100% of the larvae, pointing to a conserved core of bacteria capable of colonizing all three species of mosquito. Yet, the abundance of these ASVs varied widely between larvae suggesting individuals harbored largely unique microbiome structures, even if they overlapped in membership. Finally, larvae reared in a tripartite mix of the host species consistently showed a convergence in the structure of their microbiome, indicating that multi-species interactions between hosts potentially lead to shifts in the composition of their respective microbiomes.IMPORTANCEThis study is the first report of the axenic (free of external microbes) rearing of two species of mosquito,Aedes albopictusandAedes triseriatus. With our previous report of axenicAedes aegypti, brings the number of axenic species to three. We designed a method to perform a common garden experiment to characterize the bacteria the three species of axenic larvae assemble from their surroundings. Furthermore, species could be reared in isolation or in multi-species combinations to assess how host species interactions influence the composition of the microbiome. We found all three species recruited a common core of bacteria from their rearing water, with a large contingent of rare and sporadically detected bacteria. Finally, we also show that co-rearing of mosquito larvae leads to a coalescence in the composition of their microbiome, indicating that host species interactions potentially influence the composition of the microbiome.

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.

The increasing availability of modern research tools has enabled a revolution in studies of non-model organisms. Yet, one aspect that remains difficult or impossible to control in many model and most non-model organisms is the presence and composition of the host-associated microbiota or the microbiome. In this review, we explore the development of axenic (microbe-free) mosquito models and what these systems reveal about the role of the microbiome in mosquito biology. Additionally, the axenic host is a blank template on which a microbiome of known composition can be introduced, also known as a gnotobiotic organism. Finally, we identify a “most wanted” list of common mosquito microbiome members that show the greatest potential to influence host phenotypes. We propose that these are high-value targets to be employed in future gnotobiotic studies. The use of axenic and gnotobiotic organisms will transition the microbiome into another experimental variable that can be manipulated and controlled. Through these efforts, the mosquito will be a true model for examining host microbiome interactions.

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.

We assessed the relationship between municipality COVID-19 case rates and SARS-CoV-2 concentrations in the primary sludge of corresponding wastewater treatment facilities. Over 1,000 daily primary sludge samples were collected from six wastewater treatment facilities with catchments serving 18 cities and towns in the State of Connecticut, USA. Samples were analyzed for SARS-CoV-2 RNA concentrations during a six-month time period that overlapped with fall 2020 and winter 2021 COVID-19 outbreaks in each municipality. We fit a single regression model to estimate reported case rates in the six municipalities from SARS-CoV-2 RNA concentrations collected daily from corresponding wastewater treatment facilities. Results demonstrate the ability of SARS-CoV-2 RNA concentrations in primary sludge to estimate COVID-19 reported case rates across treatment facilities and wastewater catchments, with coverage probabilities ranging from 0.94 to 0.96. Leave-one-out cross validation suggests that the model can be broadly applied to wastewater catchments that range in more than one order of magnitude in population served. Estimation of case rates from wastewater data can be useful in locations with limited testing availability or testing disparities, or delays in individual COVID-19 testing programs.

SalivaDirect™ is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect™ means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets. Version 2 includes: Optimized thermocycler conditions Locally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclers Use of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates. Version 3 has been updated to remove steps for sample self-collection. Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx. Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers. Version 6 includes: Introducing multiple workflows for sample processing including heat Pre-treatment as an alternative to Proteinase K Updated list of RT-qPCR reagents, theromcyclers and protocol for 384 well format.

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.

SalivaDirect™ is an RNA-extraction free, dual-plexed RT-qPCR method for SARS-CoV-2 detection. It can be broadly implemented as it (1) does not require saliva collection tubes containing preservatives, (2) does not require specialized equipment for RNA extraction, and (3) is validated for use with products from multiple vendors. Thus, the simplicity and flexibility of SalivaDirect™ means that it is not as affected by supply chain bottlenecks as some other assays. Our method is RNA-extraction free which enables testing of low volume and minimally processed saliva in dual-plexed RT-qPCR for SARS-CoV-2 detection. Saliva will be treated with proteinase K followed by a heat inactivation step, and is then directly used as input in the dual-plexed RT-qPCR test. Our aim was not to design new primers and probes for RT-qPCR testing, but rather to use validated primer and probe sets (N1 and RP) developed by the US CDC. The human Ribonuclease P (RP) probe was modified with a different fluorophore so that the primer/probe set could be combined in a dualplex assay, reducing the number of tests to 1 assay with 2 sets. Version 2 includes: Optimized thermocycler conditions Locally validated alternative options for Proteinase K, RT-qPCR master mix, and thermocyclers Use of 8-strip tubes for sample processing step, due to contamination issues in 96-well plates. Version 3 has been updated to remove steps for sample self-collection. Version 4 has updated Ct thresholds for the ABI 7500 Fast Dx. Version 5 has an updated description for use, additional RP probe with ATTO647 fluorophore, and a detailed table with catalog numbers.

The microbiome is an assemblage of microorganisms living in association with a multicellular host. Numerous studies have identified a role for the microbiome in host physiology, development, immunity, and behaviour. The generation of axenic (germ-free) and gnotobiotic model systems has been vital to dissecting the role of the microbiome in host biology. We have previously reported the generation of axenic Aedes aegypti mosquitoes, the primary vector of several human pathogenic viruses, including dengue virus and Zika virus. In order to better understand the influence of the microbiome on mosquitoes, we examined the transcriptomes of axenic and conventionally reared Ae. aegypti before and after a blood meal. Our results suggest that the microbiome has a much lower effect on the mosquito’s gene expression than previously thought with only 170 genes influenced by the axenic state, while in contrast, blood meal status influenced 809 genes. The pattern of expression influenced by the microbiome is consistent with transient changes similar to infection rather than sweeping physiological changes. While the microbiome does seem to affect some pathways such as immune function and metabolism, our data suggest the microbiome is primarily serving a nutritional role in development with only minor effects in the adult.

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 report a time course of SARS-CoV-2 RNA concentrations in primary sewage sludge during the Spring COVID-19 outbreak in a northeastern U.S. metropolitan area. SARS-CoV-2 RNA was detected in all environmental samples and, when adjusted for the time lag, the virus RNA concentrations were highly correlated with the COVID-19 epidemiological curve (R 2 =0.99) and local hospital admissions (R 2 =0.99). SARS-CoV-2 RNA concentrations were a seven-day leading indicator ahead of compiled COVID-19 testing data and led local hospital admissions data by three days. Decisions to implement or relax public health measures and restrictions require timely information on outbreak dynamics in a community.

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).

This protocol describes the generation of axenic Aedes aegypti, which can be raised from larvae to adults in the absence of a microflora. The total protocol including the time taken to grow E. coli (used in one of the diets) takes two days to set up. The mosquitoes will develop normally with a slight developmental lag compared to conventionally raised mosquitoes (~1 week). Two larval diets are described, a liver:yeast (LY) extract diet and an LY diet supplemented with heat-inactivated E. coli. Axenic mosquitoes raised on food supplemented with heat-inactivated E. coli develop slightly faster than axenic mosquitoes raised without killed bacteria. This axenic system allows the manipulation of the mosquito microbiome and transitions the microbiome into a variable that can be controlled and manipulated in a mechanistic manner.

Infectious disease surveillance is hindered by several factors, including limited infrastructure and geographic isolation of many resource-poor regions. In addition, the complexities of sample acquisition, processing, and analysis, even in developed regions, can be rate limiting. Therefore, new strategies to survey human populations for emerging pathogens are necessary. Xenosurveillance is a method that utilizes mosquitoes as sampling devices to search for genetic signatures of pathogens in vertebrates. Previously we demonstrated that xenosurveillance can detect viral RNA in both laboratory and field settings. However, its ability to detect bacteria and parasites remains to be assessed. Accordingly, we fed Anopheles gambiae mosquitoes blood that contained Trypanosoma brucei gambiense and Bacillus anthracis. In addition, we determined whether two additional emerging viruses, Middle East Respiratory Syndrome Coronavirus and Zika virus could be detected by this method. Pathogen-specific real-time reverse transcription polymerase chain reaction was used to evaluate the sensitivity of xenosurveillance across multiple pathogen taxa and over time. We detected RNA from all pathogens at clinically relevant concentrations from mosquitoes processed up to 1 day postbloodfeeding. These results demonstrate that xenosurveillance may be used as a tool to expand surveillance for viral, parasitic, and bacterial pathogens in resource-limited areas.

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.

The emerging and increasing prevalence of bacterial antibiotic resistance is a significant public health challenge. To begin to tackle this problem, it will be critical to not only understand the origins of this resistance but also document environmental reservoirs of antibiotic resistance. In this study we investigated the possibility that both colony and field caught mosquitoes could harbor antibiotic resistant bacteria. Specifically, we characterized the antibiotic resistant bacterial populations from colony-reared Aedes aegypti larvae and adults and two field caught mosquito species Coquillettidia perturbans and Ochlerotatus canadensis. The cultured bacterial populations were dominated by isolates belonging to the class Gammaproteobacteria. Among the antibiotic resistant populations, we found bacteria resistant to carbenicillin, kanamycin, and tetracycline, including bacteria resistant to a cocktail of all three antibiotics in combination. The antibiotic resistant bacteria were numerically rare, at most 5% of total cell counts. Isolates were characterized by 16S rRNA gene sequencing, and clustering into Operational Taxonomic Units (OTUs; 99% sequence identity). 27 antibiotic resistant OTUs were identified, although members of an OTU did not always share the same resistance profile. This suggests the clustering was either not sensitive enough to distinguish different bacteria taxa or different antibiotic resistant sub-populations exist within an OTU. Finally, the antibiotic selection opened up a niche to culture mosquito-associated fungi, and 10 fungal OTUs (28S rRNA gene sequencing) were identified. Two fungal OTUs both classified to the class Microbotryomycetes were commonly identified in the field-caught mosquitoes. Thus, in this study we demonstrate that antibiotic resistant bacteria and certain fungi are common and conserved mosquito microbiome members. These observations highlight the potential of invertebrates to serve as vehicles for the spread of antibiotic resistance throughout the environment.

Background Scaling SARS-CoV-2 testing to meet demands of safe reopenings continues to be plagued by assay costs and supply chain shortages. In response, we developed SalivaDirect, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). Methods We simplified our saliva-based diagnostic test by (1) not requiring collection tubes with preservatives, (2) replacing nucleic acid extraction with a simple enzymatic and heating step, and (3) testing specimens with a dualplex qRT-PCR assay. Moreover, we validated SalivaDirect with reagents and instruments from multiple vendors to minimize supply chain issues. Findings From our hospital cohort, we show a high positive agreement (94%) between saliva tested with SalivaDirect and nasopharyngeal swabs tested with a commercial qRT-PCR kit. In partnership with the National Basketball Association (NBA) and National Basketball Players Association (NBPA), we tested 3,779 saliva specimens from healthy individuals and detected low rates of invalid (0.3%) and false-positive (, Context and Significance Frequent testing is critical to limit SARS-CoV-2 transmission. In response to this need, we developed SalivaDirect, a sensitive, simplified, and flexible testing framework, which received Emergency Use Authorization (EUA) from the U.S. Food and Drug Administration (FDA). We tested saliva collected from a hospital cohort and showed a high positive agreement (94%) as compared to paired nasopharyngeal swabs tested with a commercial diagnostic kit. Then, we partnered with the National Basketball Association (NBA) to test a large cohort of mostly healthy individuals, and we detected low rates of invalid (0.3%) and false-positive (0.03%–0.05%) results. Our study shows that SalivaDirect can help to increase testing capacity by providing access to an affordable framework that is less prone to supply chain shortages., Graphical Abstract, Highlights SalivaDirect is a simplified saliva-based test for detection of SARS-CoV-2 The testing framework is flexible to minimize the risk of supply chain issues SalivaDirect is sensitive, with low rates of invalid and false-positive results Laboratories can be designated to use SalivaDirect to increase testing capacity, SalivaDirect is a sensitive saliva-based COVID-19 diagnostic test, which received Emergency Use Authorization from the U.S. FDA. With the ability to designate other laboratories, the flexible and simplified framework helps to increase the capacity of existing laboratory infrastructure for SARS-CoV-2 testing.

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.

The mosquito gut microbiome plays an important role in mosquito development and fitness, providing a promising avenue for novel mosquito control strategies. Here we present a method for rearing axenic (bacteria free) Aedes aegypti mosquitoes, consisting of feeding sterilized larvae on agar plugs containing a high concentration of liver and yeast extract. This approach allows for the complete development to adulthood while maintaining sterility; however, axenic mosquito’s exhibit delayed development time and stunted growth in comparison to their bacterially colonized cohorts. These data challenge the notion that live microorganisms are required for mosquito development, and suggest that the microbiota’s main role is nutritional. Furthermore, we colonize axenic mosquitoes with simplified microbial communities ranging from a single bacterial species to a three-member community, demonstrating the ability to control the composition of the microbiota. This axenic system will allow the systematic manipulation of the mosquito microbiome for a deeper understanding of microbiota-host interactions., The mosquito gut microbiota is important for the insect’s development and fitness. Here Correa et al. present a method for rearing microbial-free Aedes aegypti mosquitoes, showing that the microbiota is not essential for mosquito development, and providing a method for manipulation of the mosquito microbiome.

SUMMARY PARAGRAPHThe recent Zika virus (ZIKV) and chikungunya virus (CHIKV) epidemics highlight the explosive nature of arthropod-borne (arbo) viruses transmitted by Aedes aegypti 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 bloodmeal and temporally sampling mosquitoes to determine 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 behavior of many mosquito vectors which refeed frequently (every 2-3 days)4. Here we demonstrate that administration of a second non-infectious bloodmeal significantly shortens the EIP of ZIKV-infected Ae. aegypti by enhancing virus escape from the mosquito midgut. Similarly, a second bloodmeal increased the competence of this species for dengue virus and CHIKV. This effect was also observed for ZIKV in Aedes albopictus, suggesting that this species might be a more important vector than once thought and that this phenomenon may be common among other virus-vector pairings. Modeling of these findings reveals that a shortened EIP would result in a significant increase in the basic reproductive number, R0. This increase helps explain how Ae.aegypti can sustain an explosive epidemic like ZIKV despite its relatively poor vector competence in single-feed laboratory trials. Together, these data demonstrate a direct and unrecognized link between mosquito feeding behavior, EIP, and vector competence.

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.

Aedes aegypti utilizes blood for energy production, egg maturation and replenishment of maternal reserves. The principle midgut enzymes responsible for bloodmeal digestion are endoproteolytic serine-type proteases within the S1.A subfamily. While there are hundreds of serine protease-like genes in the A. aegypti genome, only five are known to be expressed in the midgut. We describe the cloning, sequencing and expression profiling of seven additional serine proteases and provide a genomic and phylogenetic assessment of these findings. Of the seven genes, four are constitutively expressed and three are transcriptionally induced upon blood feeding. The amount of transcriptional induction is strongly correlated among these genes. Alignments reveal that, in general, the conserved catalytic triad, active site and accessory catalytic residues are maintained in these genes and phylogenetic analysis shows that these genes fall within three distinct clades; trypsins, chymotrypsins and serine collagenases. Interestingly, a previously described trypsin consistently arose with other serine collagenases in phylogenetic analyses. These results suggest that multiple gene duplications have arisen within the S1.A subfamily of midgut serine proteases and/or that A. aegypti has evolved an array of proteases with a broad range of substrate specificities for rapid, efficient digestion of bloodmeals.

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.