The Neovolcanic Axis Is a Barrier to Gene Flow among Aedes aegypti Populations in Mexico That Differ in Vector Competence for Dengue 2 Virus

Background

Aedes aegypti is the main mosquito vector of the four serotypes of dengue virus (DENV). Previous population genetic and vector competence studies have demonstrated substantial genetic structure and major differences in the ability to transmit dengue viruses in Ae. aegypti populations in Mexico.

Methodology/Principal Findings

Population genetic studies revealed that the intersection of the Neovolcanic axis (NVA) with the Gulf of Mexico coast in the state of Veracruz acts as a discrete barrier to gene flow among Ae. aegypti populations north and south of the NVA. The mosquito populations north and south of the NVA also differed in their vector competence (VC) for dengue serotype 2 virus (DENV2). The average VC rate for Ae. aegypti mosquitoes from populations from north of the NVA was 0.55; in contrast the average VC rate for mosquitoes from populations from south of the NVA was 0.20. Most of this variation was attributable to a midgut infection and escape barriers. In Ae. aegypti north of the NVA 21.5% failed to develop midgut infections and 30.3% of those with an infected midgut failed to develop a disseminated infection. In contrast, south of the NVA 45.2% failed to develop midgut infections and 62.8% of those with an infected midgut failed to develop a disseminated infection.

Conclusions

Barriers to gene flow in vector populations may also impact the frequency of genes that condition continuous and epidemiologically relevant traits such as vector competence. Further studies are warranted to determine why the NVA is a barrier to gene flow and to determine whether the differences in vector competence seen north and south of the NVA are stable and epidemiologically significant.     

Vectorial Capacity of Aedes aegypti for Dengue Virus Type 2 Is Reduced with Co-infection of Metarhizium anisopliae

Background

Aedes aegypti, is the major dengue vector and a worldwide public health threat combated basically by chemical insecticides. In this study, the vectorial competence of Ae. aegypti co-infected with a mildly virulent Metarhizium anisopliae and fed with blood infected with the DENV-2 virus, was examined.

Methodology/Principal Findings

The study encompassed three bioassays (B). In B1 the median lethal time (LT₅₀) of Ae. aegypti exposed to M. anisopliae was determined in four treatments: co-infected (CI), single-fungus infection (SF), single-virus infection (SV) and control (C). In B2, the mortality and viral infection rate in midgut and in head were registered in fifty females of CI and in SV. In B3, the same treatments as in B1 but with females separated individually were tested to evaluate the effect on fecundity and gonotrophic cycle length. Survival in CI and SF females was 70% shorter than the one of those in SV and control. Overall viral infection rate in CI and SV were 76 and 84% but the mortality at day six post-infection was 78% (54% infected) and 6% respectively. Survivors with virus in head at day seven post-infection were 12 and 64% in both CI and SV mosquitoes. Fecundity and gonotrophic cycle length were reduced in 52 and 40% in CI compared to the ones in control.

Conclusion/Significance

Fungus-induced mortality for the CI group was 78%. Of the survivors, 12% (6/50) could potentially transmit DENV-2, as opposed to 64% (32/50) of the SV group, meaning a 5-fold reduction in the number of infective mosquitoes. This is the first report on a fungus that reduces the vectorial capacity of Ae. aegypti infected with the DENV-2 virus.     

Aedes (Stegomyia) albopictus (Skuse): A Potential Vector of Zika Virus in Singapore

Background

Zika virus (ZIKV) is a little known arbovirus until it caused a major outbreak in the Pacific Island of Yap in 2007. Although the virus has a wide geographic distribution, most of the known vectors are sylvatic Aedes mosquitoes from Africa where the virus was first isolated. Presently, Ae. aegypti is the only known vector to transmit the virus outside the African continent, though Ae. albopictus has long been a suspected vector. Currently, Ae. albopictus has been shown capable of transmitting more than 20 arboviruses and its notoriety as an important vector came to light during the recent chikungunya pandemic. The vulnerability of Singapore to emerging infectious arboviruses has stimulated our interest to determine the competence of local Ae. albopictus to transmit ZIKV.

Methodology/Principal Findings

To determine the competence of Ae. albopictus to ZIKV, we orally infected local mosquito strains to a Ugandan strain virus. Fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 80–85%RH. Twelve mosquitoes were then sampled daily from day one to seven and on day 10 and 14 post infection (pi). Zika virus titre in the midgut and salivary glands of each mosquito were determined using tissue culture infectious dose₅₀ assay, while transmissibility of the virus was determined by detecting viral antigen in the mosquito saliva by qRT-PCR. High dissemination and transmission rate of ZIKV were observed. By day 7-pi, all mosquitoes have disseminated infection and 73% of these mosquitoes have ZIKV in their saliva. By day 10-pi, all mosquitoes were potentially infectious.

Conclusions/Significance

The study highlighted the potential of Ae. albopictus to transmit ZIKV and the possibility that the virus could be established locally. Nonetheless, the threat of ZIKV can be mitigated by existing dengue and chikungunya control program being implemented in Singapore.     

Oral Susceptibility of Singapore Aedes (Stegomyia) aegypti (Linnaeus) to Zika Virus

Background

Zika virus (ZIKV) is a little known flavivirus that caused a major outbreak in 2007, in the South-western Pacific Island of Yap. It causes dengue-like syndromes but with milder symptoms. In Africa, where it was first isolated, ZIKV is mainly transmitted by sylvatic Aedes mosquitoes. The virus has also been isolated from Ae. aegypti and it is considered to be the vector involved in the urban transmission of the virus. Transmission of the virus by an African strain of Ae. aegypti has also been demonstrated under laboratory conditions. The aim of the present study is to describe the oral susceptibility of a Singapore strain of Ae. aegypti to ZIKV, under conditions that simulate local climate.

Methodology/Principal Findings

To assess the receptivity of Singapore''s Ae. aegypti to the virus, we orally exposed a local mosquito strain to a Ugandan strain of ZIKV. Upon exposure, fully engorged mosquitoes were maintained in an environmental chamber set at 29°C and 70–75% RH. Eight mosquitoes were then sampled daily from day 1 to day 7, and subsequently on days 10 and 14 post exposure (pe). The virus titer of the midgut and salivary glands of each mosquito were determined using a tissue culture infectious dose₅₀ (TCID₅₀) assay. High midgut infection and salivary gland dissemination rates were observed. By day 5 after the infectious blood meal, ZIKV was found in the salivary glands of more than half of the mosquitoes tested (62%); and by day 10, all mosquitoes were potentially infective.

Conclusions/Significance

This study showed that Singapore''s urban Ae. aegypti are susceptible and are potentially capable of transmitting ZIKV. The virus could be established in Singapore should it be introduced. Nevertheless, Singapore''s current dengue control strategy is applicable to control ZIKV.     

Dual African Origins of Global Aedes aegypti s.l. Populations Revealed by Mitochondrial DNA

Background

Aedes aegypti is the primary global vector to humans of yellow fever and dengue flaviviruses. Over the past 50 years, many population genetic studies have documented large genetic differences among global populations of this species. These studies initially used morphological polymorphisms, followed later by allozymes, and most recently various molecular genetic markers including microsatellites and mitochondrial markers. In particular, since 2000, fourteen publications and four unpublished datasets have used sequence data from the NADH dehydrogenase subunit 4 mitochondrial gene to compare Ae. aegypti collections and collectively 95 unique mtDNA haplotypes have been found. Phylogenetic analyses in these many studies consistently resolved two clades but no comprehensive study of mtDNA haplotypes have been made in Africa, the continent in which the species originated.

Methods and Findings

ND4 haplotypes were sequenced in 426 Ae. aegypti s.l. from Senegal, West Africa and Kenya, East Africa. In Senegal 15 and in Kenya 7 new haplotypes were discovered. When added to the 95 published haplotypes and including 6 African Aedes species as outgroups, phylogenetic analyses showed that all but one Senegal haplotype occurred in a basal clade while most East African haplotypes occurred in a second clade arising from the basal clade. Globally distributed haplotypes occurred in both clades demonstrating that populations outside Africa consist of mixtures of mosquitoes from both clades.

Conclusions

Populations of Ae. aegypti outside Africa consist of mosquitoes arising from one of two ancestral clades. One clade is basal and primarily associated with West Africa while the second arises from the first and contains primarily mosquitoes from East Africa     

Age-Dependent Effects of Oral Infection with Dengue Virus on Aedes aegypti (Diptera: Culicidae) Feeding Behavior,Survival, Oviposition Success and Fecundity

Background

Aedes aegypti is the main vector of dengue, a disease that is increasing its geographical range as well as incidence rates. Despite its public health importance, the effect of dengue virus (DENV) on some mosquito traits remains unknown. Here, we investigated the impact of DENV-2 infection on the feeding behavior, survival, oviposition success and fecundity of Ae. aegypti females.

Methods/Principal Findings

After orally-challenging Ae. aegypti females with a DENV-2 strain using a membrane feeder, we monitored the feeding behavior, survival, oviposition success and fecundity throughout the mosquito lifespan. We observed an age-dependent cost of DENV infection on mosquito feeding behavior and fecundity. Infected individuals took more time to ingest blood from anesthetized mice in the 2^nd and 3^rd weeks post-infection, and also longer overall blood-feeding times in the 3^rd week post-infection, when females were around 20 days old. Often, infected Ae. aegypti females did not lay eggs and when they were laid, smaller number of eggs were laid compared to uninfected controls. A reduction in the number of eggs laid per female was evident starting on the 3^rd week post-infection. DENV-2 negatively affected mosquito lifespan, since overall the longevity of infected females was halved compared to that of the uninfected control group.

Conclusions

The DENV-2 strain tested significantly affected Ae. aegypti traits directly correlated with vectorial capacity or mosquito population density, such as feeding behavior, survival, fecundity and oviposition success. Infected mosquitoes spent more time ingesting blood, had reduced lifespan, laid eggs less frequently, and when they did lay eggs, the clutches were smaller than uninfected mosquitoes.     

Fluctuations at a Low Mean Temperature Accelerate Dengue Virus Transmission by Aedes aegypti

Background

Environmental factors such as temperature can alter mosquito vector competence for arboviruses. Results from recent studies indicate that daily fluctuations around an intermediate mean temperature (26°C) reduce vector competence of Aedes aeygpti for dengue viruses (DENV). Theoretical predictions suggest that the mean temperature in combination with the magnitude of the diurnal temperature range (DTR) mediate the direction of these effects.

Methodology/Principal Findings

We tested the effect of temperature fluctuations on Ae. aegypti vector competence for DENV serotype-1 at high and low mean temperatures, and confirmed this theoretical prediction. A small DTR had no effect on vector competence around a high (30°C) mean, but a large DTR at low temperature (20°C) increased the proportion of infected mosquitoes with a disseminated infection by 60% at 21 and 28 days post-exposure compared to a constant 20°C. This effect resulted from a marked shortening of DENV extrinsic incubation period (EIP) in its mosquito vector; i.e., a decrease from 29.6 to 18.9 days under the fluctuating vs. constant temperature treatment.

Conclusions

Our results indicate that Ae. aegypti exposed to large fluctuations at low temperatures have a significantly shorter virus EIP than under constant temperature conditions at the same mean, leading to a considerably greater potential for DENV transmission. These results emphasize the value of accounting for daily temperature variation in an effort to more accurately understand and predict the risk of mosquito-borne pathogen transmission, provide a mechanism for sustained DENV transmission in endemic areas during cooler times of the year, and indicate that DENV transmission could be more efficient in temperate regions than previously anticipated.     

Variation in vector competence for dengue viruses does not depend on mosquito midgut binding affinity

Background

Dengue virus genotypes of Southeast Asian origin have been associated with higher virulence and transmission compared to other genotypes of serotype 2 (DEN-2). We tested the hypothesis that genetic differences in dengue viruses may result in differential binding to the midgut of the primary vector, Aedes aegypti, resulting in increased transmission or vectorial capacity.

Methodology/Principal Finding

Two strains of each of the four DEN-2 genotypes (Southeast Asian, American, Indian, and West African) were tested to determine their binding affinity for mosquito midguts from two distinct populations (Tapachula, Chiapas, Mexico and McAllen, Texas, USA). Our previous studies demonstrated that Southeast Asian viruses disseminated up to 65-fold more rapidly in Ae. aegypti from Texas and were therefore more likely to be transmitted to humans. Results shown here demonstrate that viruses from all four genotypes bind to midguts at the same rate, in a titer-dependent manner. In addition, we show population differences when comparing binding affinity for DEN-2 between the Tapachula and McAllen mosquito colonies.

Conclusions

If midgut binding potential is the same for all DEN-2 viruses, then viral replication differences in these tissues and throughout the mosquito can thus probably explain the significant differences in dissemination and vector competence. These conclusions differ from the established paradigms to explain mosquito barriers to infection, dissemination, and transmission.     

High Efficiency of Temperate Aedes albopictus to Transmit Chikungunya and Dengue Viruses in the Southeast of France

Background

Since 2005, cases of chikungunya (CHIK) were caused by an unusual vector, Aedes albopictus. This mosquito, present in Europe since 1979, has gained importance since its involvement in the first CHIK outbreak in Italy in 2007. The species is capable of transmitting experimentally 26 arboviruses. However, the vectorial status of its temperate populations has remained little investigated. In 2010, autochthonous cases of CHIK and dengue (DEN) were reported in southeastern France. We evaluated the potential of a French population of Ae. albopictus in the transmission of both viruses.

Methodology and Principal Findings

We used two strains of each virus, CHIK and DEN: one strain was isolated from an imported case, and one from an autochthonous case. We used as controls Aedes aegypti from India and Martinique, the source of the imported cases of CHIK and DEN, respectively. We showed that Ae. albopictus from Cagnes-sur-Mer (AL-CSM) was as efficient as the typical tropical vector Ae. aegypti from India to experimentally transmit both CHIK strains isolated from patients in Fréjus, with around 35–67% of mosquitoes delivering up to 14 viral particles at day 3 post-infection (pi). The unexpected finding came from the high efficiency of AL-CSM to transmit both strains of DENV-1 isolated from patients in Nice. Almost 67% of Ae. albopictus AL-CSM which have ensured viral dissemination were able to transmit at day 9 pi when less than 21% of the typical DEN vector Ae. aegypti from Martinique could achieve transmission.

Conclusions/Significance

Temperate Ae. albopictus behaves differently compared to its counterpart from tropical regions, where recurrent epidemic outbreaks occur. Its potential responsibility for outbreaks in Europe should not be minimized.     

Chikungunya Virus and Aedes Mosquitoes: Saliva Is Infectious as soon as Two Days after Oral Infection

Background

Aedes aegypti and Aedes albopictus are potential vectors of chikungunya virus (CHIKV). The recent CHIKV outbreaks were caused by a new variant characterized by a mutation in the E1 glycoprotein gene (E1-226V) which has favored a better transmissibility by Ae. albopictus. As Ae. albopictus tends to replace Ae. aegypti in many regions, one question remained: is Ae. albopictus as efficient as Ae. aegypti to transmit the variant E1-226V of CHIKV?

Methodology and Findings

We infected orally both species with the variant E1-226V and estimated the infection, the viral dissemination, and the transmission rate by real time RT-PCR. Additionally, we used an in vitro assay to determine the amount of virus delivered by mosquitoes in their saliva. We found that Ae. aegypti as well as Ae. albopictus ensured a high replication of the virus which underwent an efficient dissemination as detectable in the salivary glands at day 2 post-infection (pi). Infectious CHIKV particles were delivered by salivary glands from day 2 with a maximum at day 6 pi for Ae. albopictus (10^3.3 PFU) and day 7 pi for Ae. aegypti (10^2.5 PFU).

Conclusions

Ae. albopictus is slightly more efficient than Ae. aegypti to transmit the variant E1-226V of CHIKV. These results will help to design an efficient vector control to limit transmission as soon as the first human cases are diagnosed.     

Patterns of Geographic Expansion of Aedes aegypti in the Peruvian Amazon

Background and Objectives

In the Peruvian Amazon, the dengue vector Aedes aegypti is abundant in large urban centers such as Iquitos. In recent years, it has also been found in a number of neighboring rural communities with similar climatic and socioeconomic conditions. To better understand Ae. aegypti spread, we compared characteristics of communities, houses, and containers in infested and uninfested communities.

Methods

We conducted pupal-demographic surveys and deployed ovitraps in 34 communities surrounding the city of Iquitos. Communities surveyed were located along two transects: the Amazon River and a 95km highway. We calculated entomological indices, mapped Ae. aegypti presence, and developed univariable and multivariable logistic regression models to predict Ae. aegypti presence at the community, household, or container level.

Results

Large communities closer to Iquitos were more likely to be infested with Ae. aegypti. Within infested communities, houses with Ae. aegypti had more passively-filled containers and were more often infested with other mosquito genera than houses without Ae. aegypti. For containers, large water tanks/drums and containers with solar exposure were more likely to be infested with Ae. aegypti. Maps of Ae. aegypti presence revealed a linear pattern of infestation along the highway, and a scattered pattern along the Amazon River. We also identified the geographical limit of Ae. aegypti expansion along the highway at 19.3 km south of Iquitos.

Conclusion

In the Peruvian Amazon, Ae. aegypti geographic spread is driven by human transportation networks along rivers and highways. Our results suggest that urban development and oviposition site availability drive Ae. aegypti colonization along roads. Along rivers, boat traffic is likely to drive long-distance dispersal via unintentional transport of mosquitoes on boats.     

Dengue infection increases the locomotor activity of Aedes aegypti females

Background

Aedes aegypti is the main vector of the virus causing Dengue fever, a disease that has increased dramatically in importance in recent decades, affecting many tropical and sub-tropical areas of the globe. It is known that viruses and other parasites can potentially alter vector behavior. We investigated whether infection with Dengue virus modifies the behavior of Aedes aegypti females with respect to their activity level.

Methods/Principal Findings

We carried out intrathoracic Dengue 2 virus (DENV-2) infections in Aedes aegypti females and recorded their locomotor activity behavior. We observed an increase of up to ∼50% in the activity of infected mosquitoes compared to the uninfected controls.

Conclusions

Dengue infection alters mosquito locomotor activity behavior. We speculate that the higher levels of activity observed in infected Aedes aegypti females might involve the circadian clock. Further studies are needed to assess whether this behavioral change could have implications for the dynamics of Dengue virus transmission.     

Imaginal discs--a new source of chromosomes for genome mapping of the yellow fever mosquito Aedes aegypti

Background

The mosquito Aedes aegypti is the primary global vector for dengue and yellow fever viruses. Sequencing of the Ae. aegypti genome has stimulated research in vector biology and insect genomics. However, the current genome assembly is highly fragmented with only ∼31% of the genome being assigned to chromosomes. A lack of a reliable source of chromosomes for physical mapping has been a major impediment to improving the genome assembly of Ae. aegypti.

Methodology/Principal Findings

In this study we demonstrate the utility of mitotic chromosomes from imaginal discs of 4^th instar larva for cytogenetic studies of Ae. aegypti. High numbers of mitotic divisions on each slide preparation, large sizes, and reproducible banding patterns of the individual chromosomes simplify cytogenetic procedures. Based on the banding structure of the chromosomes, we have developed idiograms for each of the three Ae. aegypti chromosomes and placed 10 BAC clones and a 18S rDNA probe to precise chromosomal positions.

Conclusion

The study identified imaginal discs of 4^th instar larva as a superior source of mitotic chromosomes for Ae. aegypti. The proposed approach allows precise mapping of DNA probes to the chromosomal positions and can be utilized for obtaining a high-quality genome assembly of the yellow fever mosquito.     

Ecological modeling of Aedes aegypti (L.) pupal production in rural Kamphaeng Phet, Thailand

Background

Aedes aegypti (L.) is the primary vector of dengue, the most important arboviral infection globally. Until an effective vaccine is licensed and rigorously administered, Ae. aegypti control remains the principal tool in preventing and curtailing dengue transmission. Accurate predictions of vector populations are required to assess control methods and develop effective population reduction strategies. Ae. aegypti develops primarily in artificial water holding containers. Release recapture studies indicate that most adult Ae. aegypti do not disperse over long distances. We expect, therefore, that containers in an area of high development site density are more likely to be oviposition sites and to be more frequently used as oviposition sites than containers that are relatively isolated from other development sites. After accounting for individual container characteristics, containers more frequently used as oviposition sites are likely to produce adult mosquitoes consistently and at a higher rate. To this point, most studies of Ae. aegypti populations ignore the spatial density of larval development sites.

Methodology

Pupal surveys were carried out from 2004 to 2007 in rural Kamphaeng Phet, Thailand. In total, 84,840 samples of water holding containers were used to estimate model parameters. Regression modeling was used to assess the effect of larval development site density, access to piped water, and seasonal variation on container productivity. A varying-coefficients model was employed to account for the large differences in productivity between container types. A two-part modeling structure, called a hurdle model, accounts for the large number of zeroes and overdispersion present in pupal population counts.

Findings

The number of suitable larval development sites and their density in the environment were the primary determinants of the distribution and abundance of Ae. aegypti pupae. The productivity of most container types increased significantly as habitat density increased. An ecological approach, accounting for development site density, is appropriate for predicting Ae. aegypti population levels and developing efficient vector control programs.     

Comparison of Vector Competence of Aedes mediovittatus and Aedes aegypti for Dengue Virus: Implications for Dengue Control in the Caribbean

Background

Aedes mediovittatus mosquitoes are found throughout the Greater Antilles in the Caribbean and often share the same larval habitats with Ae. Aegypti, the primary vector for dengue virus (DENV). Implementation of vector control measures to control dengue that specifically target Ae. Aegypti may not control DENV transmission in Puerto Rico (PR). Even if Ae. Aegypti is eliminated or DENV refractory mosquitoes are released, DENV transmission may not cease when other competent mosquito species like Ae. Mediovittatus are present. To compare vector competence of Ae. Mediovittatus and Ae. Aegypti mosquitoes, we studied relative infection and transmission rates for all four DENV serotypes.

Methods

To compare the vector competence of Ae. Mediovittatus and Ae. Aegypti, mosquitoes were exposed to DENV 1–4 per os at viral titers of 5–6 logs plaque-forming unit (pfu) equivalents. At 14 days post infectious bloodmeal, viral RNA was extracted and tested by qRT-PCR to determine infection and transmission rates. Infection and transmission rates were analyzed with a generalized linear model assuming a binomial distribution.

Results

Ae. Aegypti had significantly higher DENV-4 infection and transmission rates than Ae. mediovittatus.

Conclusions

This study determined that Ae. Mediovittatus is a competent DENV vector. Therefore dengue prevention programs in PR and the Caribbean should consider both Ae. Mediovittatus and Ae. Aegypti mosquitoes in their vector control programs.     

Towards a semen proteome of the dengue vector mosquito: protein identification and potential functions

Background

No commercially licensed vaccine or treatment is available for dengue fever, a potentially lethal infection that impacts millions of lives annually. New tools that target mosquito control may reduce vector populations and break the cycle of dengue transmission. Male mosquito seminal fluid proteins (Sfps) are one such target since these proteins, in aggregate, modulate the reproduction and feeding patterns of the dengue vector, Aedes aegypti. As an initial step in identifying new targets for dengue vector control, we sought to identify the suite of proteins that comprise the Ae. aegypti ejaculate and determine which are transferred to females during mating.

Methodology and Principal Findings

Using a stable-isotope labeling method coupled with proteomics to distinguish male- and female-derived proteins, we identified Sfps and sperm proteins transferred from males to females. Sfps were distinguished from sperm proteins by comparing the transferred proteins to sperm-enriched samples derived from testes and seminal vesicles. We identified 93 male-derived Sfps and 52 predicted sperm proteins that are transferred to females during mating. The Sfp protein classes we detected suggest roles in protein activation/inactivation, sperm utilization, and ecdysteroidogenesis. We also discovered that several predicted membrane-bound and intracellular proteins are transferred to females in the seminal fluids, supporting the hypothesis that Ae. aegypti Sfps are released from the accessory gland cells through apocrine secretion, as occurs in mammals. Many of the Ae. aegypti predicted sperm proteins were homologous to Drosophila melanogaster sperm proteins, suggesting conservation of their sperm-related function across Diptera.

Conclusion and Significance

This is the first study to directly identify Sfps transferred from male Ae. aegypti to females. Our data lay the groundwork for future functional analyses to identify individual seminal proteins that may trigger female post-mating changes (e.g., in feeding patterns and egg production). Therefore, identification of these proteins may lead to new approaches for manipulating the reproductive output and vectorial capacity of Ae. aegypti.     

Role of Bunyamwera Orthobunyavirus NSs Protein in Infection of Mosquito Cells

Background

Bunyamwera orthobunyavirus is both the prototype and study model of the Bunyaviridae family. The viral NSs protein seems to contribute to the different outcomes of infection in mammalian and mosquito cell lines. However, only limited information is available on the growth of Bunyamwera virus in cultured mosquito cells other than the Aedes albopictus C6/36 line.

Methodology and Principal Findings

To determine potential functions of the NSs protein in mosquito cells, replication of wild-type virus and a recombinant NSs deletion mutant was compared in Ae. albopictus C6/36, C7-10 and U4.4 cells, and in Ae. aegypti Ae cells by monitoring N protein production and virus yields at various times post infection. Both viruses established persistent infections, with the exception of NSs deletion mutant in U4.4 cells. The NSs protein was nonessential for growth in C6/36 and C7-10 cells, but was important for productive replication in U4.4 and Ae cells. Fluorescence microscopy studies using recombinant viruses expressing green fluorescent protein allowed observation of three stages of infection, early, acute and late, during which infected cells underwent morphological changes. In the absence of NSs, these changes were less pronounced. An RNAi response efficiently reduced virus replication in U4.4 cells transfected with virus specific dsRNA, but not in C6/36 or C7/10 cells. Lastly, Ae. aegypti mosquitoes were exposed to blood-meal containing either wild-type or NSs deletion virus, and at various times post-feeding, infection and disseminated infection rates were measured. Compared to wild-type virus, infection rates by the mutant virus were lower and more variable. If the NSs deletion virus was able to establish infection, it was detected in salivary glands at 6 days post-infection, 3 days later than wild-type virus.

Conclusions/Significance

Bunyamwera virus NSs is required for efficient replication in certain mosquito cell lines and in Ae. aegypti mosquitoes.     

Deletion of the NSm Virulence Gene of Rift Valley Fever Virus Inhibits Virus Replication in and Dissemination from the Midgut of Aedes aegypti Mosquitoes

Background

Previously, we investigated the role of the Rift Valley fever virus (RVFV) virulence genes NSs and NSm in mosquitoes and demonstrated that deletion of NSm significantly reduced the infection, dissemination, and transmission rates of RVFV in Aedes aegypti mosquitoes. The specific aim of this study was to further characterize midgut infection and escape barriers of RVFV in Ae. aegypti infected with reverse genetics-generated wild type RVFV (rRVF-wt) or RVFV lacking the NSm virulence gene (rRVF-ΔNSm) by examining sagittal sections of infected mosquitoes for viral antigen at various time points post-infection.

Methodology and Principal Findings

Ae. aegypti mosquitoes were fed an infectious blood meal containing either rRVF-wt or rRVF-ΔNSm. On days 0, 1, 2, 3, 4, 6, 8, 10, 12, and 14 post-infection, mosquitoes from each experimental group were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and examined for RVFV antigen by immunofluorescence assay. Remaining mosquitoes at day 14 were assayed for infection, dissemination, and transmission. Disseminated infections were observed in mosquitoes as early as three days post infection for both virus strains. However, infection rates for rRVF-ΔNSm were statistically significantly less than for rRVF-wt. Posterior midgut infections in mosquitoes infected with rRVF-wt were extensive, whereas midgut infections of mosquitoes infected with rRVF-ΔNSm were confined to one or a few small foci.

Conclusions/Significance

Deletion of NSm resulted in the reduced ability of RVFV to enter, replicate, and disseminate from the midgut epithelial cells. NSm appears to have a functional role in the vector competence of mosquitoes for RVFV at the level of the midgut barrier.