Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics.

Arboviruses can emerge rapidly and cause explosive epidemics of severe disease. Some of the most epidemiologically important arboviruses, including dengue virus (DENV), Zika virus (ZIKV), Chikungunya (CHIKV) and yellow fever virus (YFV), are transmitted by Aedes mosquitoes, most notably Aedes aegypti and Aedes albopictus. After a mosquito blood feeds on an infected host, virus enters the midgut and infects the midgut epithelium. The virus must then overcome a series of barriers before reaching the mosquito saliva and being transmitted to a new host. The virus must escape from the midgut (known as the midgut escape barrier; MEB), which is thought to be mediated by transient changes in the permeability of the midgut-surrounding basal lamina layer (BL) following blood feeding. Here, we present a mathematical model of the within-mosquito population dynamics of DENV (as a model system for mosquito-borne viruses more generally) that includes the interaction of the midgut and BL which can account for the MEB. Our results indicate a dose-dependency of midgut establishment of infection as well as rate of escape from the midgut: collectively, these suggest that the extrinsic incubation period (EIP)-the time taken for DENV virus to be transmissible after infection-is shortened when mosquitoes imbibe more virus. Additionally, our experimental data indicate that multiple blood feeding events, which more closely mimic mosquito-feeding behavior in the wild, can hasten the course of infections, and our model predicts that this effect is sensitive to the amount of virus imbibed. Our model indicates that mutations to the virus which impact its replication rate in the midgut could lead to even shorter EIPs when double-feeding occurs. Mechanistic models of within-vector viral infection dynamics provide a quantitative understanding of infection dynamics and could be used to evaluate novel interventions that target the mosquito stages of the infection.

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.

Large-Scale Complete-Genome Sequencing and Phylodynamic Analysis of Eastern Equine Encephalitis Virus Reveals Source-Sink Transmission Dynamics in the United States.

Eastern equine encephalitis virus (EEEV) has a high case-fatality rate in horses and humans, and Florida has been hypothesized to be the source of EEEV epidemics for the northeastern United States. To test this hypothesis, we sequenced complete genomes of 433 EEEV strains collected within the United States from 1934 to 2014. Phylogenetic analysis suggested EEEV evolves relatively slowly and that transmission is enzootic in Florida, characterized by higher genetic diversity and long-term local persistence. In contrast, EEEV strains in New York and Massachusetts were characterized by lower genetic diversity, multiple introductions, and shorter local persistence. Our phylogeographic analysis supported a source-sink model in which Florida is the major source of EEEV compared to the other localities sampled. In sum, this study revealed the complex epidemiological dynamics of EEEV in different geographic regions in the United States and provided general insights into the evolution and transmission of other avian mosquito-borne viruses in this region.IMPORTANCE Eastern equine encephalitis virus (EEEV) infections are severe in horses and humans on the east coast of the United States with a >90% mortality rate in horses, an ∼33% mortality rate in humans, and significant brain damage in most human survivors. However, little is known about the evolutionary characteristics of EEEV due to the lack of genome sequences. By generating large collection of publicly available complete genome sequences, this study comprehensively determined the evolution of the virus, described the epidemiological dynamics of EEEV in different states in the United States, and identified Florida as one of the major sources. These results may have important implications for the control and prevention of other mosquito-borne viruses in the Americas.

Cache Valley Virus in Aedes japonicus japonicus Mosquitoes, Appalachian Region, United States.

We detected Cache Valley virus in Aedes japonicus, a widely distributed invasive mosquito species, in an Appalachian forest in the United States. The forest contained abundant white-tailed deer, a major host of the mosquito and virus. Vector competence trials indicated that Ae. j. japonicus mosquitoes can transmit this virus in this region.

West Nile Virus Seroprevalence, Connecticut, USA, 2000-2014.

West Nile virus (WNV) infection is mainly asymptomatic but can be severe in elderly persons. As part of studies on immunity and aging in Connecticut, USA, we detected WNV seroconversion in 8.5% of nonimmunosuppressed and 16.8% of immunosuppressed persons. Age was not a significant seroconversion factor. Our findings suggest that immune factors affect seroconversion.

Multiple bloodmeals enhance dissemination of arboviruses in three medically relevant mosquito genera.

BACKGROUND: Mosquitoes in nature may acquire multiple bloodmeals (BMs) over the course of their lifetime; however, incorporation of frequent feeding behavior in laboratory vector competence studies is rarely done. We have previously shown that acquisition of a second non-infectious BM can enhance early dissemination of Zika virus (ZIKV), dengue virus, and chikungunya virus in Aedes aegypti and ZIKV in Aedes albopictus mosquitoes, yet it is unknown if other taxonomically-diverse virus-vector pairings show a similar trend under a sequential feeding regimen. METHODS: To test this, we evaluated the impact of a second noninfectious BM on the vector competence of Aedes aegypti and Anopheles quadrimaculatus for Mayaro virus, Culex quinquefasciatus for West Nile virus, Aedes triseriatus for La Crosse virus, and Aedes aegypti for Oropouche virus (OROV). Female mosquitoes were fed BMs containing these viruses and half of them were given a second noninfectious BM at 3 or 4-days post infection. Mosquitoes were harvested at various time points and assayed for virus infection in bodies and disseminated infection in legs by performing reverse transcription-quantitative polymerase chain reaction (RT-qPCR) assays. RESULTS: We found that a second noninfectious BM had no impact on midgut infection rates but increased virus dissemination for all but one of the virus-vector pairings- Ae. aegypti and OROV. Unlike the other arboviruses under consideration, which are strictly mosquito-borne, biting midges (Culicoides spp.) serve as the main vector of OROV and this virus rarely disseminated to the mosquito leg tissue in our study. CONCLUSIONS: Taken together, our findings show that sequential blood feeding enhances virus dissemination across diverse arbovirus-vector pairings, representing three mosquito genera and virus families, but a second BM was insufficient to overcome a strong midgut virus escape barrier in a nonnatural virus-vector pairing.

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.

Host-Feeding Behavior of Mosquitoes in the Florida Everglades.

Background: West Nile virus (WNV), Everglades virus (EVEV), and five species of Orthobunyavirus were isolated from mosquitoes collected in the Everglades in 2016-2017. Prior studies of blood meals of mosquitoes in southern Florida have related findings to acquisition and transmission of EVEV, St. Louis encephalitis virus, and WNV, but not the Orthobunyavirus viruses associated with the subgenus Melanoconion of the genus Culex. Materials and Methods: In the present study, blood-fed mosquitoes were collected in the Everglades in 2016, 2017, 2021, and 2022, and from an industrial site in Naples, FL in 2017. Blood meals were identified to host species by PCR assays using mitochondrial cytochrome b gene. Results: Blood meals were identified from Anopheles crucians complex and 11 mosquito species captured in the Florida Everglades and from 3 species collected from an industrial site. The largest numbers of blood-fed specimens were from Culex nigripalpus, Culex erraticus, Culex cedecei, and Aedes taeniorhynchus. Cx. erraticus fed on mammals, birds, and reptiles, particularly American alligator. This mosquito species could transmit WNV to American alligator in the wild. Cx. nigripalpus acquired blood meals primarily from birds and mammals and frequently fed on medium-sized mammals and white-tailed deer. Water and wading birds were the primary avian hosts for Cx. nigripalpus and Cx. erraticus in the Everglades. Wading birds are susceptible to WNV and could serve as reservoir hosts. Cx. cedecei fed on five species of rodents, particularly black and hispid cotton rats. EVEV and three different species of Orthobunyavirus have been isolated from the hispid cotton rat and Cx. cedecei in the Everglades. Cx. cedecei is likely acquiring and transmitting these viruses among hispid cotton rats and other rodents. The marsh rabbit was a frequent host for An. crucians complex. An. crucians complex, and other species could acquire Tensaw virus from rabbits. Conclusions: Our study contributes to a better understanding of the host and viral associations of mosquito species in southwestern Florida.

Isolations of Cache Valley virus from Aedes albopictus (Diptera: Culicidae) in New Jersey and evaluation of its role as a regional arbovirus vector.

The Asian tiger mosquito, Aedes albopictus (Skuse), is an invasive species and a major pest problem in urban and suburban locales in New Jersey. To assess its potential role as an arbovirus vector, we sampled Ae. albopictus from two New Jersey counties over a 3-yr period and estimated the prevalence of virus infection by Vero cell culture and reverse transcription-polymerase chain reaction assays. Three virus isolates were obtained from 34,567 field-collected Ae. albopictus, and all were identified as Cache Valley virus by molecular methods. Ae. albopictus (N = 3,138), collected in Mercer County from late July through early September 2011, also were retested for West Nile virus (WNV) by reverse transcription-polymerase chain reaction, and all were negative. These results corroborate previous findings showing that Ae. albopictus may occasionally acquire Cache Valley virus, a deer-associated arbovirus, in nature. In contrast, we did not detect WNV infection in Ae. albopictus despite concurrent WNV amplification in this region.

Jamestown Canyon virus comes into view: understanding the threat from an underrecognized arbovirus.

This review examines the epidemiology, ecology, and evolution of Jamestown Canyon virus (JCV) and highlights new findings from the literature to better understand the virus, the vectors driving its transmission, and its emergence as an agent of arboviral disease. We also reanalyze data from the Connecticut Arbovirus Surveillance Program which represents the largest dataset on JCV infection in mosquitoes. JCV is a member of the California serogroup of the genus Orthobunyavirus, family Peribunyaviridae, and is found throughout much of temperate North America. This segmented, negative-sense RNA virus evolves predominately by genetic drift punctuated by infrequent episodes of genetic reassortment among novel strains. It frequently infects humans within affected communities and occasionally causes febrile illness and neuroinvasive disease in people. Reported human cases are relatively rare but are on the rise during the last 20 yr, particularly within the northcentral and northeastern United States. JCV appears to overwinter and reemerge each season by transovarial or vertical transmission involving univoltine Aedes (Diptera: Culicidae) species, specifically members of the Aedes communis (de Geer) and Ae. stimulans (Walker) Groups. The virus is further amplified in a mosquito-deer transmission cycle involving a diversity of mammalophilic mosquito species. Despite progress in our understanding of this virus, many aspects of the vector biology, virology, and human disease remain poorly understood. Remaining questions and future directions of research are discussed.

Ecological predictors of mosquito population and arbovirus transmission synchrony estimates.

Quantifying synchrony in species population fluctuations and determining its driving factors can inform multiple aspects of ecological and epidemiological research and policy decisions. We examined seasonal mosquito and arbovirus surveillance data collected in Connecticut, United States from 2001 to 2020 to quantify spatial relationships in 19 mosquito species and 7 arboviruses timeseries accounting for environmental factors such as climate and land cover characteristics. We determined that mosquito collections, on average, were significantly correlated up to 10 km though highly variable among the examined species. Few arboviruses displayed any synchrony and significant maximum correlated distances never exceeded 5 km. After accounting for distance, mixed effects models showed that mosquito or arbovirus identity explained more variance in synchrony estimates than climate or land cover factors. Correlated mosquito collections up to 10-20 km suggest that mosquito control operations for nuisance and disease vectors alike must expand treatment zones to regional scales for operations to have population-level impacts. Species identity matters as well, and some mosquito species will require much larger treatment zones than others. The much shorter correlated detection distances for arboviruses reinforce the notion that focal-level processes drive vector-borne pathogen transmission dynamics and risk of spillover into human populations.

Seasonality of Mosquitoes in North Dakota, 2003-2006, with Four New State Records.

Thirty-seven species and subspecies of mosquitoes were identified from 3,580,610 specimens collected in eastern (Cass, Nelson, and Richland counties) and western (Williams County) North Dakota in 2003-2006. Four species were new state records (Aedes schizopinax, Psorophora ciliata, Ps. ferox, and Ps. horrida). Aedes vexans was dominant (82.9%). Other relatively abundant species were Ae. trivittatus (7.7%), Ae. melanimon (2.7%), Culex tarsalis (2.6%), Ae. dorsalis (1.6%), Ae. sticticus (1.0), and Culiseta inornata (0.9%). The seasonality of the species is presented.

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.

Investigating the dose-dependency of the midgut escape barrier using a mechanistic model of within-mosquito dengue virus population dynamics.

Flaviviruses are arthropod-borne (arbo)viruses which can emerge rapidly and cause explosive epidemics of severe disease. Some of the most epidemiologically important flaviviruses, including dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV), are transmitted by Aedes mosquitoes, most notably Aedes aegypti and Aedes albopictus. After a mosquito blood feeds on an infected host, virus enters the midgut and infects the midgut epithelium. The virus must then overcome a series of barriers before reaching the mosquito saliva and being transmitted to a new host. The virus must escape from the midgut (known as the midgut escape barrier; MEB), which is thought to be mediated by transient changes in the permeability of the midgut-surrounding basal lamina layer (BL) following blood feeding. Here, we present a mathematical model of the within-mosquito population dynamics of flaviviruses that includes the interaction of the midgut and BL which can account for the MEB. Our results indicate a dose-dependency of midgut establishment of infection as well as rate of escape from the midgut: collectively, these suggest that the extrinsic incubation period (EIP) - the time taken for DENV virus to be transmissible after infection - is shortened when mosquitoes imbibe more virus. Additionally, our experimental data indicates that multiple blood feeding events, which more closely mimic mosquito-feeding behavior in the wild, can hasten the course of infections, and our model predicts that this effect is sensitive to the amount of virus imbibed. Our model indicates that mutations to the virus which impact its replication rate in the midgut could lead to even shorter EIPs when double-feeding occurs. Mechanistic models of within-vector viral infection dynamics provide a quantitative understanding of infection dynamics and could be used to evaluate novel interventions that target the mosquito stages of the infection. Author summary: Aedes mosquitoes are the main vectors of dengue virus (DENV), Zika virus (ZIKV) and yellow fever virus (YFV), all of which can cause severe disease in humans with dengue alone infecting an estimated 100-400 million people each year. Understanding the processes that affect whether, and at which rate, mosquitoes may transmit such viruses is, hence, paramount. Here, we present a mathematical model of virus dynamics within infected mosquitoes. By combining the model with novel experimental data, we show that the course of infection is sensitive to the initial dose of virus ingested by the mosquito. The data also indicates that mosquitoes which blood feed subsequent to becoming infected may be able to transmit infection earlier, which is reproduced in the model. This is important as many mosquito species feed multiple times during their lifespan and, any reduction in time to dissemination will increase the number of days that a mosquito is infectious and so enhance the risk of transmission. Our study highlights the key and complementary roles played by mathematical models and experimental data for understanding within-mosquito virus dynamics.

Dynamics of Eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans, and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, like previous years, cases were driven by frequent short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Dynamics of eastern equine encephalitis virus during the 2019 outbreak in the Northeast United States.

Eastern equine encephalitis virus (EEEV) causes a rare but severe disease in horses and humans and is maintained in an enzootic transmission cycle between songbirds and Culiseta melanura mosquitoes. In 2019, the largest EEEV outbreak in the United States for more than 50 years occurred, centered in the Northeast. To explore the dynamics of the outbreak, we sequenced 80 isolates of EEEV and combined them with existing genomic data. We found that, similar to previous years, cases were driven by multiple independent but short-lived virus introductions into the Northeast from Florida. Once in the Northeast, we found that Massachusetts was important for regional spread. We found no evidence of any changes in viral, human, or bird factors which would explain the increase in cases in 2019, although the ecology of EEEV is complex and further data is required to explore these in more detail. By using detailed mosquito surveillance data collected by Massachusetts and Connecticut, however, we found that the abundance of Cs. melanura was exceptionally high in 2019, as was the EEEV infection rate. We employed these mosquito data to build a negative binomial regression model and applied it to estimate early season risks of human or horse cases. We found that the month of first detection of EEEV in mosquito surveillance data and vector index (abundance multiplied by infection rate) were predictive of cases later in the season. We therefore highlight the importance of mosquito surveillance programs as an integral part of public health and disease control.

Ecology and Epidemiology of Eastern Equine Encephalitis Virus in the Northeastern United States: An Historical Perspective.

In the current review, we examine the regional history, ecology, and epidemiology of eastern equine encephalitis virus (EEEV) to investigate the major drivers of disease outbreaks in the northeastern United States. EEEV was first recognized as a public health threat during an outbreak in eastern Massachusetts in 1938, but historical evidence for equine epizootics date back to the 1800s. Since then, sporadic disease outbreaks have reoccurred in the Northeast with increasing frequency and northward expansion of human cases during the last 20 yr. Culiseta melanura (Coquillett) (Diptera: Culicidae) serves as the main enzootic vector that drives EEEV transmission among wild birds, but this mosquito species will occasionally feed on mammals. Several species have been implicated as bridge vectors to horses and humans, with Coquilletstidia perturbans (Walker) as a leading suspect based on its opportunistic feeding behavior, vector competence, and high infection rates during recent disease outbreaks. A diversity of bird species are reservoir competent, exposed to EEEV, and serve as hosts for Cs. melanura, with a few species, including the wood thrush (Hlocichia mustelina) and the American robin (Turdus migratorius), contributing disproportionately to virus transmission based on available evidence. The major factors responsible for the sustained resurgence of EEEV are considered and may be linked to regional landscape and climate changes that support higher mosquito densities and more intense virus transmission.

Impacts of Lysinibacillus sphaericus on mosquito larval community composition and larval competition between Culex pipiens and Aedes albopictus.

Effectiveness of mosquito larvicide active ingredients (AI), such as Lysinibacillus sphaericus, varies between species, yet little is known regarding how differential effectiveness manifests in larval communities in applied settings. To examine how differential effectiveness of L. sphaericus influences larval community dynamics, we performed two experiments. We performed a field experiment in which containers were seeded with a standardized nutrient treatment, mosquitoes colonized the containers, and then containers received one of three L. sphaericus applications. We then performed competition assays between Culex pipiens and Aedes albopictus in low nutrient environments using multiple interspecific ratios and the presence/absence of a low dose of L. sphaericus. Field results demonstrated elimination of Culex spp. from treated containers while container breeding Aedes spp. proliferated across all treatments. Lysinibacillus sphaericus did not influence competition between Cx. pipiens and Ae. albopictus, and the L. sphaericus application eliminated Cx. pipiens in all treatment replicates while survival of Ae. albopictus was similar between treated and untreated containers across interspecific ratios. Lysinibacillus sphaericus is an effective AI for control of Culex spp. However, different AIs should be utilized in habitats containing non-Culex genera while a mix of AIs should be utilized where coexistence of multiple genera is expected or confirmed.

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.