Aedes aegypti anti-salivary gland antibody concentration and dengue virus exposure history in healthy individuals living in an endemic area in Colombia.

INTRODUCTION: Mosquito salivary proteins are able to induce an antibody response that reflects the level of human-vector contact. IgG antibodies against dengue virus (DENV-IgG) are indicators of previous exposure. The risk of DENV transmission is not only associated to mosquito or dengue factors, but also to socioeconomic factors that may play an important role in the disease epidemiology. OBJECTIVE: To determine the effect of the presence of Aedes aegypti mosquitos in different stages in households and the history of dengue exposure on vector-human contact determined by the level of anti-salivary protein antibodies in people living in a Colombian endemic area. MATERIALS AND METHODS: A pilot study of 58 households and 55 human subjects was conducted in Norte de Santander, Colombia. A questionnaire for socioeconomic factors was administered and houses were examined for the presence of Ae. aegypti specimens in the aquatic stages. The level of DENV-IgG antibodies (DENV-IgG), in addition to IgG and IgM anti- Ae. aegypti salivary gland extract (SGE) antibodies (SGE-IgG, SGE-IgM) were evaluated by ELISA using blood collected in filter paper. RESULTS: We found a significant higher level of SGE-IgG antibodies in subjects living in houses with Ae. aegypti in aquatic stages. We also found a higher concentration of SGE-IgG antibodies in people exposed to DENV, a positive correlation between IgM-SGE and IgG-DENV and a negative correlation with IgG-SGE. CONCLUSION: Anti-salivary proteins antibodies are consistent with the presence of Ae. aegypti aquatic stages inside houses and DENV-IgG antibodies concentrations.

Chikungunya viral fitness measures within the vector and subsequent transmission potential.

Given the recent emergence of chikungunya in the Americas, the accuracy of forecasting and prediction of chikungunya transmission potential in the U.S. requires urgent assessment. The La Reunion-associated sub-lineage of chikungunya (with a valine substitution in the envelope protein) was shown to increase viral fitness in the secondary vector, Ae. albopictus. Subsequently, a majority of experimental and modeling efforts focused on this combination of a sub-lineage of the East-Central-South African genotype (ECSA-V)-Ae. albopictus, despite the Asian genotype being the etiologic agent of recent chikungunya outbreaks world-wide. We explore a collection of data to investigate relative transmission efficiencies of the three major genotypes/sub-lineages of chikungunya and found difference in the extrinsic incubation periods to be largely overstated. However, there is strong evidence supporting the role of Ae. albopictus in the expansion of chikungunya that our R0 calculations cannot attribute to fitness increases in one vector over another. This suggests other ecological factors associated with the Ae. albopictus-ECSA-V cycle may drive transmission intensity differences. With the apparent bias in literature, however, we are less prepared to evaluate transmission where Ae. aegypti plays a significant role. Holistic investigations of CHIKV transmission cycle(s) will allow for more complete assessment of transmission risk in areas affected by either or both competent vectors.

Infection with dengue-2 virus alters proteins in naturally expectorated saliva of Aedes aegypti mosquitoes.

BACKGROUND: Dengue virus (DENV) is responsible for up to approximately 300 million infections and an increasing number of deaths related to severe manifestations each year in affected countries throughout the tropics. It is critical to understand the drivers of this emergence, including the role of vector-virus interactions. When a DENV-infected Aedes aegypti mosquito bites a vertebrate, the virus is deposited along with a complex mixture of salivary proteins. However, the influence of a DENV infection upon the expectorated salivary proteome of its vector has yet to be determined. METHODS: Therefore, we conducted a proteomic analysis using 2-D gel electrophoresis coupled with mass spectrometry based protein identification comparing the naturally expectorated saliva of Aedes aegypti infected with DENV-2 relative to that of uninfected Aedes aegypti. RESULTS: Several proteins were found to be differentially expressed in the saliva of DENV-2 infected mosquitoes, in particular proteins with anti-hemostatic and pain inhibitory functions were significantly reduced. Hypothetical consequences of these particular protein reductions include increased biting rates and transmission success, and lead to alteration of transmission potential as calculated in our vectorial capacity model. CONCLUSIONS: We present our characterizations of these changes with regards to viral transmission and mosquito blood-feeding success. Further, we conclude that our proteomic analysis of Aedes aegypti saliva altered by DENV infection provides a unique opportunity to identify pro-viral impacts key to virus transmission.

Effect of dengue-2 virus infection on protein expression in the salivary glands of Aedes aegypti mosquitoes.

Dengue virus (DENV) is the most important mosquito-transmitted flavivirus that is transmitted throughout the tropical and subtropical regions of the world. The primary mosquito vector of DENV in urban locations is Aedes aegypti. Key to understanding the transmission of DENV is the relationship between pathogen and vector. Accordingly, we report our preliminary characterization of the differentially expressed proteins from Ae. aegypti mosquitoes after DENV infection. We investigated the virus-vector interaction through changes in the proteome of the salivary glands of mosquitoes with disseminated DENV serotype 2 (DENV-2) infections using two-dimensional gel electrophoresis and identification by mass spectrometry. Our findings indicate that DENV-2 infection in the Ae. aegypti salivary gland alters the expression of structural, secreted, and metabolic proteins. These changes in the salivary gland proteome highlight the virally influenced environment caused by a DENV-2 infection and warrant additional investigation to determine if these differences extend to the expectorated saliva.

Use of anti-Aedes aegypti salivary extract antibody concentration to correlate risk of vector exposure and dengue transmission risk in Colombia.

Norte de Santander is a region in Colombia with a high incidence of dengue virus (DENV). In this study, we examined the serum concentration of anti-Aedes salivary gland extract (SGE) antibodies as a biomarker of DENV infection and transmission, and assessed the duration of anti-SGE antibody concentration after exposure to the vector ceased. We also determined whether SGE antibody concentration could differentiate between positive and negative DENV infected individuals and whether there are differences in exposure for each DENV serotype. We observed a significant decrease in the concentration of IgG antibodies at least 40 days after returning to an "Ae. aegypti-free" area. In addition, we found significantly higher anti-SGE IgG concentrations in DENV positive patients with some difference in exposure to mosquito bites among DENV serotypes. We conclude that the concentration of IgG antibodies against SGE is an accurate indicator of risk of dengue virus transmission and disease presence.

Development of a transmission model for dengue virus.

BACKGROUND: Dengue virus (DENV) research has historically been hampered by the lack of a susceptible vertebrate transmission model. Recently, there has been progress towards such models using several varieties of knockout mice, particularly those deficient in type I and II interferon receptors. Based on the critical nature of the type I interferon response in limiting DENV infection establishment, we assessed the permissiveness of a mouse strain with a blunted type I interferon response via gene deficiencies in interferon regulatory factors 3 and 7 (IRF3/7 -/- -/-) with regards to DENV transmission success. We investigated the possibility of transmission to the mouse by needle and infectious mosquito, and subsequent transmission back to mosquito from an infected animal during its viremic period. METHODS: Mice were inoculated subcutaneously with non-mouse adapted DENV-2 strain 1232 and serum was tested for viral load and cytokine production each day. Additionally, mosquitoes were orally challenged with the same DENV-2 strain via artificial membrane feeder, and then allowed to forage or naïve mice. Subsequently, we determined acquisition potential by allowing naïve mosquitoes on forage on exposed mice during their viremic period. RESULTS: Both needle inoculation and infectious mosquito bite(s) resulted in 100% infection. Significant differences between these groups in viremia on the two days leading to peak viremia were observed, though no significant difference in cytokine production was seen. Through our determination of transmission and acquisition potentials, the transmission cycle (mouse-to mosquito-to mouse) was completed. We confirmed that the IRF3/7 -/- -/- mouse supports DENV replication and is competent for transmission experiments, with the ability to use a non-mouse adapted DENV-2 strain. A significant finding of this study was that this IRF3/7 -/- -/- mouse strain was able to be infected by and transmit virus to mosquitoes, thus providing means to replicate the natural transmission cycle of DENV. CONCLUSION: As there is currently no approved vaccine for DENV, public health monitoring and a greater understanding of transmission dynamics leading to outbreak events are critical. The further characterization of DENV using this model will expand knowledge of key entomological, virological and immunological components of infection establishment and transmission events.

Diversification of West Nile virus in a subtropical region.

BACKGROUND: West Nile virus (WNV) has spread across North, Central, and South America since its introduction in 1999. At the start of this spread, Florida was considered a potentially important area with regards to transmission due to its geographic, climatological, and demographic conditions. Curiously, the anticipated high levels of transmission or disease outbreaks have not been observed. As other studies have predicted that the lack of intense WNV transmission is not due to vector incompetence, we sought to evaluate the role of viral strain diversity in WNV transmission in Florida. Therefore, a phylogentic analysis was carried out on several isolates collected from three distinct locations in Florida. RESULTS: Contrasting with a positive control collected in Indian River County, Florida during 2003 that contains the original NY99 genotype with valanine at amino acid 159 of the envelope region, all of the isolates collected in 2005 contain the WN02 genotype composed of a substation with alanine at that position indicating the window of introduction of the WN02 genotype occurred between 2003 and 2005. From the eight isolates collected in Duval, Indian River, and Manatee Counties; there is also a silent nucleotide substitution that differentiates the isolates collected on the Atlantic side of the state compared to the isolate collected on the Gulf side, which groups closer to isolates from other locations near the Gulf. CONCLUSION: As a whole, the Florida isolates contained numerous variable nucleotide and amino acid sites from the reference sequences, as well as each other; indicating greater nucleotide diversity within the Florida 2005 isolates than within other regions. Finally, a series of three amino acid substitutions surrounding a set of histidines located in the envelope coding region that hypothesized to play a role in conformational changes was found in the isolate collected in Indian River County, perhaps changing the antigenicity of the homodimer. Taken together, these findings expand our understanding of the temporal and spatial compartmentalization of West Nile virus subtypes within North America.

A method to increase efficiency in testing pooled field-collected mosquitoes.

Testing field-caught mosquito collections can result in thousands of pools, and testing pools of 50 mosquitoes each can be both time consuming and cost prohibitive. Consequently, we have developed an alternative approach to testing mosquito pools for arboviruses, utilizing a superpool strategy. When mosquito samples are processed for extraction of viral RNA and subsequent virus testing via quantitative real-time polymerase chain reaction, each pool is tested individually. Using the method described here, 0.025 ml from each of 10 pools is combined into a superpool for RNA extraction and testing. When a virus-positive superpool sample is found, each of the original 10 pools that constitute this sample is tested individually in order to find the specific positive sample. By retesting the original samples after the initial superpool screen, we are still able to obtain reliable estimates for minimum infection rates or maximum likelihood estimations. To test this principle, we created controlled mosquito pools of known titer and subjected them to our superpool process. We were able to detect our entire range of laboratory-created pools as being West Nile virus (WNV) positive. In 2005, field surveillance efforts from our laboratory resulted in over 4,000 mosquito pools tested, with 8 resulting WNV-positive samples. We found that all of these field samples were detected as WNV positive using the superpool method and contained calculated virus titers from < 0.1 to 4.1 log10 plaque-forming units/ml WNV, indicating that the limit of superpool detection of WNV is below this point. These results reveal that the superpool method could be accurately used to detect WNV in field-collected specimens.