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

A Survey of Virus Recombination Uncovers Canonical Features of Artificial Chimeras Generated During Deep Sequencing Library Preparation

International audienceChimeric reads can be generated by in vitro recombination during the preparation of high-throughput sequencing libraries. Our attempt to detect biological recombination between the genomes of dengue virus (DENV; +ssRNA genome) and its mosquito host using the Illumina Nextera sequencing library preparation kit revealed that most, if not all, detected host–virus chimeras were artificial. Indeed, these chimeras were not more frequent than with control RNA from another species (a pillbug), which was never in contact with DENV RNA prior to the library preparation. The proportion of chimera types merely reflected those of the three species among sequencing reads. Chimeras were frequently characterized by the presence of 1-20 bp microhomology between recombining fragments. Within-species chimeras mostly involved fragments in opposite orientations and located less than 100 bp from each other in the parental genome. We found similar features in published datasets using two other viruses: Ebola virus (EBOV; -ssRNA genome) and a herpesvirus (dsDNA genome), both produced with the Illumina Nextera protocol. These canonical features suggest that artificial chimeras are generated by intra-molecular template switching of the DNA polymerase during the PCR step of the Nextera protocol. Finally, a published Illumina dataset using the Flock House virus (FHV; +ssRNA genome) generated with a protocol preventing artificial recombination revealed the presence of 1-10 bp microhomology motifs in FHV–FHV chimeras, but very few recombining fragments were in opposite orientations. Our analysis uncovered sequence features characterizing recombination breakpoints in short-read sequencing datasets, which can be helpful to evaluate the presence and extent of artificial recombination

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

Successful 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.status: publishe

Larval Exposure to the Bacterial Insecticide Bti Enhances Dengue Virus Susceptibility of Adult Aedes aegypti Mosquitoes

International audienceUnderstanding the interactions between pathogens sharing the same host can be complicated for holometabolous animals when larval and adult stages are exposed to distinct pathogens. In medically important insect vectors, the effect of pathogen exposure at the larval stage may influence susceptibility to human pathogens at the adult stage. We addressed this hypothesis in the mosquito Aedes aegypti, a major vector of arthropod-borne viruses (arboviruses), such as the dengue virus (DENV) and the chikungunya virus (CHIKV). We experimentally assessed the consequences of sub-lethal exposure to the bacterial pathogen Bacillus thuringiensis subsp. israelensis (Bti), during larval development, on arbovirus susceptibility at the adult stage in three Ae. aegypti strains that differ in their genetic resistance to Bti. We found that larval exposure to Bti significantly increased DENV susceptibility, but not CHIKV susceptibility, in the Bti-resistant strains. However, there was no major difference in the baseline arbovirus susceptibility between the Bti-resistant strains and their Bti-susceptible parental strain. Although the generality of our results remains to be tested with additional arbovirus strains, this study supports the idea that the outcome of an infection by a pathogen depends on other pathogens sharing the same host even when they do not affect the same life stage of the host. Our findings may also have implications for Bti as a mosquito biocontrol agent, indicating that the sub-optimal Bti efficacy may have counter-productive effects by increasing vector competence, at least for some combinations of arbovirus and mosquito strains

Epidemiological significance of dengue virus genetic variation in mosquito infection dynamics

The kinetics of arthropod-borne virus (arbovirus) transmission by their vectors have long been recognized as a powerful determinant of arbovirus epidemiology. The time interval between virus acquisition and transmission by the vector, termed extrinsic incubation period (EIP), combines with vector mortality rate and vector competence to determine the proportion of infected vectors that eventually become infectious. However, the dynamic nature of this process, and the amount of natural variation in transmission kinetics among arbovirus strains, are poorly documented empirically and are rarely considered in epidemiological models. Here, we combine newly generated empirical measurements in vivo and outbreak simulations in silico to assess the epidemiological significance of genetic variation in dengue virus (DENV) transmission kinetics by Aedes aegypti mosquitoes. We found significant variation in the dynamics of systemic mosquito infection, a proxy for EIP, among eight field-derived DENV isolates representing the worldwide diversity of recently circulating type 1 strains. Using a stochastic agent-based model to compute time-dependent individual transmission probabilities, we predict that the observed variation in systemic mosquito infection kinetics may drive significant differences in the probability of dengue outbreak and the number of human infections. Our results demonstrate that infection dynamics in mosquitoes vary among wild-type DENV isolates and that this variation potentially affects the risk and magnitude of dengue outbreaks. Our quantitative assessment of DENV genetic variation in transmission kinetics contributes to improve our understanding of heterogeneities in arbovirus epidemiological dynamics.status: publishe

Genetic Drift, Purifying Selection and Vector Genotype Shape Dengue Virus Intra-host Genetic Diversity in Mosquitoes

Due to their error-prone replication, RNA viruses typically exist as a diverse population of closely related genomes, which is considered critical for their fitness and adaptive potential. Intra-host demographic fluctuations that stochastically reduce the effective size of viral populations are a challenge to maintaining genetic diversity during systemic host infection. Arthropod-borne viruses (arboviruses) traverse several anatomical barriers during infection of their arthropod vectors that are believed to impose population bottlenecks. These anatomical barriers have been associated with both maintenance of arboviral genetic diversity and alteration of the variant repertoire. Whether these patterns result from stochastic sampling (genetic drift) rather than natural selection, and/or from the influence of vector genetic heterogeneity has not been elucidated. Here, we used deep sequencing of full-length viral genomes to monitor the intra-host evolution of a wild-type dengue virus isolate during infection of several mosquito genetic backgrounds. We estimated a bottleneck size ranging from 5 to 42 founding viral genomes at initial midgut infection, irrespective of mosquito genotype, resulting in stochastic reshuffling of the variant repertoire. The observed level of genetic diversity increased following initial midgut infection but significantly differed between mosquito genetic backgrounds despite a similar initial bottleneck size. Natural selection was predominantly negative (purifying) during viral population expansion. Taken together, our results indicate that dengue virus intra-host genetic diversity in the mosquito vector is shaped by genetic drift and purifying selection, and point to a novel role for vector genetic factors in the genetic breadth of virus populations during infection. Identifying the evolutionary forces acting on arboviral populations within their arthropod vector provides novel insights into arbovirus evolution.status: publishe

Epidemiological significance of dengue virus genetic variation in mosquito infection dynamics.

The kinetics of arthropod-borne virus (arbovirus) transmission by their vectors have long been recognized as a powerful determinant of arbovirus epidemiology. The time interval between virus acquisition and transmission by the vector, termed extrinsic incubation period (EIP), combines with vector mortality rate and vector competence to determine the proportion of infected vectors that eventually become infectious. However, the dynamic nature of this process, and the amount of natural variation in transmission kinetics among arbovirus strains, are poorly documented empirically and are rarely considered in epidemiological models. Here, we combine newly generated empirical measurements in vivo and outbreak simulations in silico to assess the epidemiological significance of genetic variation in dengue virus (DENV) transmission kinetics by Aedes aegypti mosquitoes. We found significant variation in the dynamics of systemic mosquito infection, a proxy for EIP, among eight field-derived DENV isolates representing the worldwide diversity of recently circulating type 1 strains. Using a stochastic agent-based model to compute time-dependent individual transmission probabilities, we predict that the observed variation in systemic mosquito infection kinetics may drive significant differences in the probability of dengue outbreak and the number of human infections. Our results demonstrate that infection dynamics in mosquitoes vary among wild-type DENV isolates and that this variation potentially affects the risk and magnitude of dengue outbreaks. Our quantitative assessment of DENV genetic variation in transmission kinetics contributes to improve our understanding of heterogeneities in arbovirus epidemiological dynamics

Genetic Drift, Purifying Selection and Vector Genotype Shape Dengue Virus Intra-host Genetic Diversity in Mosquitoes

International audienceDue to their error-prone replication, RNA viruses typically exist as a diverse population of closely related genomes, which is considered critical for their fitness and adaptive potential. Intra-host demographic fluctuations that stochastically reduce the effective size of viral populations are a challenge to maintaining genetic diversity during systemic host infection. Arthropod-borne viruses (arboviruses) traverse several anatomical barriers during infection of their arthropod vectors that are believed to impose population bottlenecks. These anatomical barriers have been associated with both maintenance of arboviral genetic diversity and alteration of the variant repertoire. Whether these patterns result from stochastic sampling (genetic drift) rather than natural selection, and/or from the influence of vector genetic heterogeneity has not been elucidated. Here, we used deep sequencing of full-length viral genomes to monitor the intra-host evolution of a wild-type dengue virus isolate during infection of several mosquito genetic backgrounds. We estimated a bottleneck size ranging from 5 to 42 founding viral genomes at initial midgut infection, irrespective of mosquito genotype, resulting in stochastic reshuffling of the variant repertoire. The observed level of genetic diversity increased following initial midgut infection but significantly differed between mosquito genetic backgrounds despite a similar initial bottleneck size. Natural selection was predominantly negative (purifying) during viral population expansion. Taken together, our results indicate that dengue virus intra-host genetic diversity in the mosquito vector is shaped by genetic drift and purifying selection, and point to a novel role for vector genetic factors in the genetic breadth of virus populations during infection. Identifying the evolutionary forces acting on arboviral populations within their arthropod vector provides novel insights into arbovirus evolution

Time course of DENV prevalence and viral load.

<p>(A) Bar graphs show the percentage of DENV-infected samples stratified by time point, tissue and isofemale line. Relative numbers of positive samples are indicated above the bars. (B) Box plots show the number of DENV genome copies per infected sample stratified by time point, tissue and isofemale line. Solid dots represent individual samples selected for deep sequencing and open dots represent samples that were not sequenced. dpe = days post exposure. * <i>p</i><0.05.</p

SNV markers used for bottleneck size estimation.

<p>SNV markers used for bottleneck size estimation.</p