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.

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.

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.

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.

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.

Experimental evolution of an RNA virus in wild birds: evidence for host-dependent impacts on population structure and competitive fitness.

Within hosts, RNA viruses form populations that are genetically and phenotypically complex. Heterogeneity in RNA virus genomes arises due to error-prone replication and is reduced by stochastic and selective mechanisms that are incompletely understood. Defining how natural selection shapes RNA virus populations is critical because it can inform treatment paradigms and enhance control efforts. We allowed West Nile virus (WNV) to replicate in wild-caught American crows, house sparrows and American robins to assess how natural selection shapes RNA virus populations in ecologically relevant hosts that differ in susceptibility to virus-induced mortality. After five sequential passages in each bird species, we examined the phenotype and population diversity of WNV through fitness competition assays and next generation sequencing. We demonstrate that fitness gains occur in a species-specific manner, with the greatest replicative fitness gains in robin-passaged WNV and the least in WNV passaged in crows. Sequencing data revealed that intrahost WNV populations were strongly influenced by purifying selection and the overall complexity of the viral populations was similar among passaged hosts. However, the selective pressures that control WNV populations seem to be bird species-dependent. Specifically, crow-passaged WNV populations contained the most unique mutations (~1.7× more than sparrows, ~3.4× more than robins) and defective genomes (~1.4× greater than sparrows, ~2.7× greater than robins), but the lowest average mutation frequency (about equal to sparrows, ~2.6× lower than robins). Therefore, our data suggest that WNV replication in the most disease-susceptible bird species is positively associated with virus mutational tolerance, likely via complementation, and negatively associated with the strength of selection. These differences in genetic composition most likely have distinct phenotypic consequences for the virus populations. Taken together, these results reveal important insights into how different hosts may contribute to the emergence of RNA viruses.

Modulation of flavivirus population diversity by RNA interference.

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.

Evaluation of ivermectin mass drug administration for malaria transmission control across different West African environments.

BACKGROUND: Mass drug administration (MDA) of ivermectin to humans for control and elimination of filarial parasites can kill biting malaria vectors and lead to Plasmodium transmission reduction. This study examines the degree and duration of mosquitocidal effects resulting from single MDAs conducted in three different West African countries, and the subsequent reductions in parity and Plasmodium sporozoite rates. METHODS: Indoor-resting, blood-fed and outdoor host-seeking Anopheles spp. were captured on days surrounding MDAs from 2008-2013 in Senegalese, Liberian and Burkinabé villages. Mortality was assessed on a portion of the indoor collection, and parity status was determined on host-seeking mosquitoes. The effect of MDA was then analysed against the time relative to the MDA, the distributed drugs and environmental variables. RESULTS: Anopheles gambiae survivorship was reduced by 33.9% for one week following MDA and parity rates were significantly reduced for more than two weeks after the MDAs. Sporozoite rates were significantly reduced by >77% for two weeks following the MDAs in treatment villages despite occurring in the middle of intense transmission seasons. These observed effects were consistent across three different West African transmission dynamics. CONCLUSIONS: These data provide a comprehensive and crucial evidence base for the significant reduction in malaria transmission following single ivermectin MDAs across diverse field sites. Despite the limited duration of transmission reduction, these results support the hypothesis that repeated MDAs with optimal timing could help sustainably control malaria as well as filarial transmission.

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.

Global genomics of Aedes aegypti unveils widespread and novel infectious viruses capable of triggering a small RNA response.

The mosquito Aedes aegypti is a prominent vector for arboviruses, but the breadth of mosquito viruses that infects this specie is not fully understood. In the broadest global survey to date of over 200 Ae. aegypti small RNA samples, we detected viral small interfering RNAs (siRNAs) and Piwi interacting RNAs (piRNAs) arising from mosquito viruses. We confirmed that most academic laboratory colonies of Ae. aegypti lack persisting viruses, yet two commercial strains were infected by a novel tombus-like virus. Ae. aegypti from North to South American locations were also teeming with multiple insect viruses, with Anphevirus and a bunyavirus displaying geographical boundaries from the viral small RNA patterns. Asian Ae. aegypti small RNA patterns indicate infections by similar mosquito viruses from the Americas and reveal the first wild example of dengue virus infection generating viral small RNAs. African Ae. aegypti also contained various viral small RNAs including novel viruses only found in these African substrains. Intriguingly, viral long RNA patterns can differ from small RNA patterns, indicative of viral transcripts evading the mosquitoes' RNA interference (RNAi) machinery. To determine whether the viruses we discovered via small RNA sequencing were replicating and transmissible, we infected C6/36 and Aag2 cells with Ae. aegypti homogenates. Through blind passaging, we generated cell lines stably infected by these mosquito viruses which then generated abundant viral siRNAs and piRNAs that resemble the native mosquito viral small RNA patterns. This mosquito small RNA genomics approach augments surveillance approaches for emerging infectious diseases.

Highly sensitive and specific detection of rare variants in mixed viral populations from massively parallel sequence data.

Viruses diversify over time within hosts, often undercutting the effectiveness of host defenses and therapeutic interventions. To design successful vaccines and therapeutics, it is critical to better understand viral diversification, including comprehensively characterizing the genetic variants in viral intra-host populations and modeling changes from transmission through the course of infection. Massively parallel sequencing technologies can overcome the cost constraints of older sequencing methods and obtain the high sequence coverage needed to detect rare genetic variants (< 1%) within an infected host, and to assay variants without prior knowledge. Critical to interpreting deep sequence data sets is the ability to distinguish biological variants from process errors with high sensitivity and specificity. To address this challenge, we describe V-Phaser, an algorithm able to recognize rare biological variants in mixed populations. V-Phaser uses covariation (i.e. phasing) between observed variants to increase sensitivity and an expectation maximization algorithm that iteratively recalibrates base quality scores to increase specificity. Overall, V-Phaser achieved > 97% sensitivity and > 97% specificity on control read sets. On data derived from a patient after four years of HIV-1 infection, V-Phaser detected 2,015 variants across the -10 kb genome, including 603 rare variants (< 1% frequency) detected only using phase information. V-Phaser identified variants at frequencies down to 0.2%, comparable to the detection threshold of allele-specific PCR, a method that requires prior knowledge of the variants. The high sensitivity and specificity of V-Phaser enables identifying and tracking changes in low frequency variants in mixed populations such as RNA viruses.

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.

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.

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.

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.

RNAi targeting of West Nile virus in mosquito midguts promotes virus diversification.

West Nile virus (WNV) exists in nature as a genetically diverse population of competing genomes. This high genetic diversity and concomitant adaptive plasticity has facilitated the rapid adaptation of WNV to North American transmission cycles and contributed to its explosive spread throughout the New World. WNV is maintained in nature in a transmission cycle between mosquitoes and birds, with intrahost genetic diversity highest in mosquitoes. The mechanistic basis for this increase in genetic diversity in mosquitoes is poorly understood. To determine whether the high mutational diversity of WNV in mosquitoes is driven by RNA interference (RNAi), we characterized the RNAi response to WNV in the midguts of orally exposed Culex pipiens quinquefasciatus using high-throughput, massively parallel sequencing and estimated viral genetic diversity. Our data demonstrate that WNV infection in orally exposed vector mosquitoes induces the RNAi pathway and that regions of the WNV genome that are more intensely targeted by RNAi are more likely to contain point mutations compared to weakly targeted regions. These results suggest that, under natural conditions, positive selection of WNV within mosquitoes is stronger in regions highly targeted by the host RNAi response. Further, they provide a mechanistic basis for the relative importance of mosquitoes in driving WNV diversification.

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.

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.