Determinants of Arbovirus Vertical Transmission in Mosquitoes.

International audienceVertical transmission (VT) and horizontal transmission (HT) of pathogens refer to parental and non-parental chains of host-to-host transmission. Combining HT with VT enlarges considerably the range of ecological conditions in which a pathogen can persist, but the factors governing the relative frequency of each transmission mode are poorly understood for pathogens with mixed-mode transmission. Elucidating these factors is particularly important for understanding the epidemiology of arthropod-borne viruses (arboviruses) of public health significance. Arboviruses are primarily maintained by HT between arthropod vectors and vertebrate hosts in nature, but are occasionally transmitted vertically in the vector population from an infected female to her offspring, which is a proposed maintenance mechanism during adverse conditions for HT. Here, we review over a century of published primary literature on natural and experimental VT, which we previously assembled into large databases, to identify biological factors associated with the efficiency of arbovirus VT in mosquito vectors. Using a robust statistical framework, we highlight a suite of environmental, taxonomic, and physiological predictors of arbovirus VT. These novel insights contribute to refine our understanding of strategies employed by arboviruses to persist in the environment and cause substantial public health concern. They also provide hypotheses on the biological processes underlying the relative VT frequency for pathogens with mixed-mode transmission that can be tested empirically

Excretion of dengue virus RNA by Aedes aegypti allows non-destructive monitoring of viral dissemination in individual mosquitoes

International audienceSuccessful transmission of a vector-borne pathogen relies on a complex life cycle in the arthropod vector that requires initial infection of the digestive tract followed by systemic viral dissemination. The time interval between acquisition and subsequent transmission of the pathogen, called the extrinsic incubation period, is one of the most influential parameters of vector-borne pathogen transmission. However, the dynamic nature of this process is often ignored because vector competence assays are sacrificial and rely on end-point measurements. Here, we report that individual Aedes aegypti mosquitoes release large amounts of dengue virus (DENV) RNA in their excreta that can be non-sacrificially detected over time following oral virus exposure. Further, we demonstrate that detection of DENV RNA in excreta from individual mosquitoes is correlated to systemic viral dissemination with high specificity (0.9–1) albeit moderate sensitivity (0.64–0.89). Finally, we illustrate the potential of our finding to detect biological differences in the dynamics of DENV dissemination in a proof-of-concept experiment. Individual measurements of the time required for systemic viral dissemination, a prerequisite for transmission, will be valuable to monitor the dynamics of DENV vector competence, to carry out quantitative genetics studies, and to evaluate the risk of DENV transmission in field settings. Vector competence is the intrinsic ability of arthropods to acquire and subsequently transmit vector-borne pathogens , such as, malaria parasites or dengue viruses (DENV) 1. Experimental vector competence assessments of arthropod populations are an important component of assessing the risk of vector-borne disease. Vector competence is a quantitative trait that varies not only between arthropod species, but also within a vector species. For example, 24 populations of the mosquito Aedes aegypti sampled throughout Mexico and the United States displayed substantial variation in their vector competence for DENV

Genetic specificity and potential for local adaptation between dengue viruses and mosquito vectors

Background: Several observations support the hypothesis that vector-driven selection plays an important role in shaping dengue virus (DENV) genetic diversity. Clustering of DENV genetic diversity at a particular location may reflect underlying genetic structure of vector populations, which combined with specific vector genotype x virus genotype (G x G) interactions may promote adaptation of viral lineages to local mosquito vector genotypes. Although spatial structure of vector polymorphism at neutral genetic loci is well-documented, existence of G x G interactions between mosquito and virus genotypes has not been formally demonstrated in natural populations. Here we measure G x G interactions in a system representative of a natural situation in Thailand by challenging three isofemale families from field-derived Aedes aegypti with three contemporaneous low-passage isolates of DENV-1. Results: Among indices of vector competence examined, the proportion of mosquitoes with a midgut infection, viral RNA concentration in the body, and quantity of virus disseminated to the head/legs (but not the proportion of infected mosquitoes with a disseminated infection) strongly depended on the specific combinations of isofemale families and viral isolates, demonstrating significant G x G interactions. Conclusion: Evidence for genetic specificity of interactions in our simple experimental design indicates that vector competence of Ae. aegypti for DENV is likely governed to a large extent by G x G interactions in genetically diverse, natural populations. This result challenges the general relevance of conclusions from laboratory systems that consist of a single combination of mosquito and DENV genotypes. Combined with earlier evidence for fine-scale genetic structure of natural Ae. aegypti populations, our finding indicates that the necessary conditions for local DENV adaptation to mosquito vectors are met

Dengue-1 Virus Clade Replacement in Thailand Associated with Enhanced Mosquito Transmission

International audienceDengue viruses (DENV) are characterized by extensive genetic diversity and can be organized in multiple, genetically distinct lineages that arise and die out on a regular basis in regions where dengue is endemic. A fundamental question for understanding DENV evolution is the relative extent to which stochastic processes (genetic drift) and natural selection acting on fitness differences among lineages contribute to lineage diversity and turnover. Here, we used a set of recently collected and archived low-passage DENV-1 isolates from Thailand to examine the role of mosquito vector-virus interactions in DENV evolution. By comparing the ability of 23 viruses isolated on different dates between 1985 and 2009 to be transmitted by a present-day Aedes aegypti population from Thailand, we found that a major clade replacement event in the mid-1990s was associated with virus isolates exhibiting increased titers in the vector's hemocoel, which is predicted to result in a higher probability of transmission. This finding is consistent with the hypothesis that selection for enhanced transmission by mosquitoes is a possible mechanism underlying major DENV clade replacement events. There was significant variation in transmission potential among isolates within each clade, indicating that in addition to vector-driven selection, other evolutionary forces act to maintain viral genetic diversity. We conclude that occasional adaptive processes involving the mosquito vector can drive major DENV lineage replacement events

A major genetic locus controlling natural Plasmodium falciparum infection is shared by East and West African Anopheles gambiae

Background: Genetic linkage mapping identified a region of chromosome 2L in the Anopheles gambiae genome that exerts major control over natural infection by Plasmodium falciparum. This 2L Plasmodium-resistance interval was mapped in mosquitoes from a natural population in Mali, West Africa, and controls the numbers of P. falciparum oocysts that develop on the vector midgut. An important question is whether genetic variation with respect to Plasmodium-resistance exists across Africa, and if so whether the same or multiple geographically distinct resistance mechanisms are responsible for the trait. Methods: To identify P falciparum resistance loci in pedigrees generated and infected in Kenya, East Africa, 28 microsatellite loci were typed across the mosquito genome. Genetic linkage mapping was used to detect significant linkage between genotype and numbers of midgut oocysts surviving to 7–8 days post-infection. Results: A major malaria-control locus was identified on chromosome 2L in East African mosquitoes, in the same apparent position originally identified from the West African population. Presence of this resistance locus explains 75% of parasite free mosquitoes. The Kenyan resistance locus is named EA_Pfin1 (East Africa_ Plasmodium falciparum Infection Intensity). Conclusion: Detection of a malaria-control locus at the same chromosomal location in both East and West African mosquitoes indicates that, to the level of genetic resolution of the analysis, the same mechanism of Plasmodium-resistance, or a mechanism controlled by the same genomic region, is found across Africa, and thus probably operates in A. gambiae throughout its entire range

Detection of each DENV serotype by RT-LAMP

Background 4 one-step, real-time, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays were developed for the detection of dengue virus (DENV) serotypes by considering 2,056 full genome DENV sequences. DENV1 and DENV2 RT-LAMP assays were validated with 31 blood and 11 serum samples from Tanzania, Senegal, Sudan and Mauritania. DENV3 and DENV4 RT-LAMP assays were validated with 25 serum samples from Cambodia Methodology/Principal findings 4 final reaction primer mixes were obtained by using a combination of Principal Component Analysis of the full DENV genome sequences, and LAMP primer design based on sequence alignments using the LAVA software. These mixes contained 14 (DENV1), 12 (DENV2), 8 (DENV3) and 3 (DENV4) LAMP primer sets. The assays were evaluated with an External Quality Assessment panel from Quality Control for Molecular Diagnostics. The assays were serotype-specific and did not cross-detect with other flaviviruses. The limits of detection, with 95% probability, were 22 (DENV1), 542 (DENV2), 197 (DENV3) and 641 (DENV4) RNA molecules, and 100% reproducibility in the assays was obtained with up to 102 (DENV1) and 103 RNA molecules (DENV2, DENV3 and DENV4). Validation of the DENV2 assay with blood samples from Tanzania resulted in 23 samples detected by RT-LAMP, demonstrating that the assay is 100% specific and 95.8% sensitive (positive predictive value of 100% and a negative predictive value of 85.7%). All serum samples from Senegal, Sudan and Mauritania were detected and 3 untyped as DENV1. The sensitivity of RT-LAMP for DENV4 samples from Cambodia did not quite match qRT-PCR. Conclusions/Significance We have shown a novel approach to design LAMP primers that makes use of fast growing sequence databases. The DENV1 and DENV2 assays were validated with viral RNA extracted clinical samples, showing very good performance parameters