Mosquito immune cells enhance dengue and Zika virus dissemination in Aedes aegypti.

Mosquito-borne viruses cause more than 400 million annual infections and place over half of the world's population at risk. Despite this importance, the mechanisms by which arboviruses infect the mosquito host and disseminate to tissues required for transmission are not well understood. Here, we provide evidence that mosquito immune cells, known as hemocytes, play an integral role in the dissemination of dengue virus (DENV) and Zika virus (ZIKV) in the mosquito Aedes aegypti. We establish that phagocytic hemocytes are a focal point for virus infection and demonstrate that these immune cell populations facilitate virus dissemination to the ovaries and salivary glands. Additional transfer experiments confirm that virus-infected hemocytes confer a virus infection to non-infected mosquitoes more efficiently than free virus in acellular hemolymph, revealing that hemocytes are an important tropism to enhance virus dissemination in the mosquito host. These data support a "trojan horse" model of virus dissemination where infected hemocytes transport virus through the hemolymph to deliver virus to mosquito tissues required for transmission and parallels vertebrate systems where immune cell populations promote virus dissemination to secondary sites of infection. In summary, this study significantly advances our understanding of virus infection dynamics in mosquitoes and highlights conserved roles of immune cells in virus dissemination across vertebrate and invertebrate systems.

Field Isolation and Laboratory Vector-Host Studies of Brazoran Virus (Peribunyaviridae: Orthobunyavirus) from Florida.

Brazoran virus was first isolated from Culex mosquitoes in Texas in 2012, yet little is known about this virus. We report the isolation of this virus from Culex erraticus from southern Florida during 2016. The Florida strain had a nucleotide identity of 96.3% (S segment), 99.1% (M segment), and 95.8% (L segment) to the Texas isolate. Culex quinquefasciatus and Aedes aegypti colonies were subsequently fed virus blood meals to determine their vector competence for Brazoran virus. Culex quinquefasciatus was susceptible to midgut infection, but few mosquitoes developed disseminated infections. Aedes aegypti supported disseminated infection, but virus transmission could not be demonstrated. Suckling mice became infected by intradermal inoculation without visible disease signs. The virus was detected in multiple mouse tissues but rarely infected the brain. This study documents the first isolation of Brazoran virus outside of Texas. Although this virus infected Ae. aegypti and Cx. quinquefasciatus in laboratory trials, their vector competence could not be demonstrated, suggesting they are unlikely vectors of Brazoran virus.

Increased blood meal size and feeding frequency compromise Aedes aegypti midgut integrity and enhance dengue virus dissemination.

Aedes aegypti is a highly efficient vector for numerous pathogenic arboviruses including dengue virus (DENV), Zika virus, and yellow fever virus. This efficiency can in part be attributed to their frequent feeding behavior. We previously found that acquisition of a second, full, non-infectious blood meal could accelerate virus dissemination within the mosquito by temporarily compromising midgut basal lamina integrity; however, in the wild, mosquitoes are often interrupted during feeding and only acquire partial or minimal blood meals. To explore the impact of this feeding behavior further, we examined the effects of partial blood feeding on DENV dissemination rates and midgut basal lamina damage in Ae. aegypti. DENV-infected mosquitoes given a secondary partial blood meal had intermediate rates of dissemination and midgut basal lamina damage compared to single-fed and fully double-fed counterparts. Subsequently, we evaluated if basal lamina damage accumulated across feeding episodes. Interestingly, within 24 hours of feeding, damage was proportional to the number of blood meals imbibed; however, this additive effect returned to baseline levels by 96 hours. These data reveal that midgut basal lamina damage and rates of dissemination are proportional to feeding frequency and size, and further demonstrate the impact that mosquito feeding behavior has on vector competence and arbovirus epidemiology. This work has strong implications for our understanding of virus transmission in the field and will be useful when designing laboratory experiments and creating more accurate models of virus spread and maintenance.

Introducing an environmental microbiome to axenic Aedes aegypti mosquitoes documents bacterial responses to a blood meal.

IMPORTANCE: The blood meal of the female mosquito serves as a nutrition source to support egg development, so is an important aspect of its biology. Yet, the roles the microbiome may play in blood digestion are poorly characterized. We employed axenic mosquitoes to investigate how the microbiome differs between mosquitoes reared in the insectary versus mosquitoes that acquire their microbiome from the environment. Environmental microbiomes were more diverse and showed larger temporal shifts over the course of blood digestion. Importantly, only bacteria from the environmental microbiome performed hemolysis in culture, pointing to functional differences between bacterial populations. These data highlight that taxonomic differences between the microbiomes of insectary-reared and wild mosquitoes are potentially also related to their functional ecology. Thus, axenic mosquitoes colonized with environmental bacteria offer a way to investigate the role of bacteria from the wild in mosquito processes such as blood digestion, under controlled laboratory conditions.

The known unknowns of Powassan virus ecology.

Powassan virus (POWV; Family: Flaviviridae, Genus: Flavivirus) is the sole North American member of the tick-borne encephalitis sero-complex. While associated with high rates of morbidity and mortality, POWV has historically been of little public health concern due to low incidence rates. However, over the last 20 yr, incidence rates have increased highlighting the growing epidemiological threat. Currently, there are no vaccines or therapeutics with tick habitat reduction, acaricide application, and public awareness programs being our primary means of intervention. The effectiveness of these control strategies is dependent on having a sound understanding of the virus's ecology. In this Forum, we review what is currently known about POWV ecology, identify gaps in our knowledge, and discuss prevailing and alternative hypotheses about transmission dynamics, reservoir hosts, and spatial focality.

Three species of axenic mosquito larvae recruit a shared core of bacteria in a common garden experiment.

In this study, we describe the generation of two new species of axenic mosquito, Aedes albopictus and Aedes triseriatus. Along with Aedes 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 of their abundance in the larvae, suggesting the larval microbiome is made up of a subset of relatively rare aquatic bacteria. We found 11 bacterial 16S rRNA gene amplicon sequence variants (ASVs) that were conserved among ≥90% of the mosquitoes sampled, including 2 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. IMPORTANCE This study is the first report of the axenic (free of external microbes) rearing of two species of mosquito, Aedes albopictus and Aedes triseriatus. Our previous report of axenic Aedes 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.

Comparison of acarological risk metrics derived from active and passive surveillance and their concordance with tick-borne disease incidence.

Tick-borne diseases continue to threaten human health across the United States. Both active and passive tick surveillance can complement human case surveillance, providing spatio-temporal information on when and where humans are at risk for encounters with ticks and tick-borne pathogens. However, little work has been done to assess the concordance of the acarological risk metrics from each surveillance method. We used data on Ixodes scapularis and its associated human pathogens from Connecticut (2019-2021) collected through active collections (drag sampling) or passive submissions from the public to compare county estimates of tick and pathogen presence, infection prevalence, and tick abundance by life stage. Between the surveillance strategies, we found complete agreement in estimates of tick and pathogen presence, high concordance in infection prevalence estimates for Anaplasma phagocytophilum, Borrelia burgdorferi sensu stricto, and Babesia microti, but no consistent relationships between actively and passively derived estimates of tick abundance or abundance of infected ticks by life stage. We also compared nymphal metrics (i.e., pathogen prevalence in nymphs, nymphal abundance, and abundance of infected nymphs) with reported incidence of Lyme disease, anaplasmosis, and babesiosis, but did not find any consistent relationships with any of these metrics. The small spatial and temporal scale for which we had consistently collected active and passive data limited our ability to find significant relationships. Findings are likely to differ if examined across a broader spatial or temporal coverage with greater variation in acarological and epidemiological outcomes. Our results indicate similar outcomes between some actively and passively derived tick surveillance metrics (tick and pathogen presence, pathogen prevalence), but comparisons were variable for abundance estimates.

Phylogeographic reconstruction of the emergence and spread of Powassan virus in the northeastern United States.

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 from Ixodes scapularis ticks 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 and 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 United States mostly following a south-to-north pattern, with a weighted lineage dispersal velocity of ~3 km/y. 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 and I. scapularis populations likely facilitated the emergence of Powassan virus in the northeastern United States.

Limited Capacity for Aedes aegypti to Mechanically Transmit Chikungunya Virus and Dengue Virus.

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.

The Axenic and Gnotobiotic Mosquito: Emerging Models for Microbiome Host Interactions.

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.

Increased SARS-CoV-2 Testing Capacity with Pooled Saliva Samples.

We analyzed feasibility of pooling saliva samples for severe acute respiratory syndrome coronavirus 2 testing and found that sensitivity decreased according to pool size: 5 samples/pool, 7.4% reduction; 10 samples/pool, 11.1%; and 20 samples/pool, 14.8%. When virus prevalence is >2.6%, pools of 5 require fewer tests; when <0.6%, pools of 20 support screening strategies.

SalivaDirect: A simplified and flexible platform to enhance SARS-CoV-2 testing capacity.

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 (<0.05%) results. CONCLUSIONS: We demonstrate that saliva is a valid alternative to swabs for SARS-CoV-2 screening and that SalivaDirect can make large-scale testing more accessible and affordable. Uniquely, we can designate other laboratories to use our sensitive, flexible, and simplified platform under our EUA (https://publichealth.yale.edu/salivadirect/). FUNDING: This study was funded by the NBA and NBPA (N.D.G.), the Huffman Family Donor Advised Fund (N.D.G.), a Fast Grant from Emergent Ventures at the Mercatus Center at George Mason University (N.D.G.), the Yale Institute for Global Health (N.D.G.), and the Beatrice Kleinberg Neuwirth Fund (A.I.K.). C.B.F.V. is supported by NWO Rubicon 019.181EN.004.

Predicting daily COVID-19 case rates from SARS-CoV-2 RNA concentrations across a diversity of wastewater catchments.

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 1700 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 10 month time period that overlapped with October 2020 and winter/spring 2021 COVID-19 outbreaks in each municipality. We fit lagged regression models 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. Lags of 0 to 1 days resulted in the greatest predictive power for the model. 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. The close relationship between case rates and SARS-CoV-2 concentrations demonstrates the utility of using primary sludge samples for monitoring COVID-19 outbreak dynamics. Estimating case rates from wastewater data can be useful in locations with limited testing availability, testing disparities, or delays in individual COVID-19 testing programs.

Detection of SARS-CoV-2 RNA by multiplex RT-qPCR.

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.

Pooling saliva to increase SARS-CoV-2 testing capacity.

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.

Measurement of SARS-CoV-2 RNA in wastewater tracks community infection dynamics.

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.

Limited influence of the microbiome on the transcriptional profile of female Aedes aegypti mosquitoes.

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 impact of autophagy on arbovirus infection of mosquito cells.

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.

Successive blood meals enhance virus dissemination within mosquitoes and increase transmission potential.

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.