Mosquitoes Transmit Unique West Nile Virus Populations during Each Feeding Episode.
ABSTRACT: Arthropod-borne viruses (arboviruses), such as Zika virus, chikungunya virus, and West Nile virus (WNV), pose continuous threats to emerge and cause large epidemics. Often, these events are associated with novel virus variants optimized for local transmission that first arise as minorities within a host. Thus, the conditions that regulate the frequency of intrahost variants are important determinants of emergence. Here, we describe the dynamics of WNV genetic diversity during its transmission cycle. By temporally sampling saliva from individual mosquitoes, we demonstrate that virus populations expectorated by mosquitoes are highly diverse and unique to each feeding episode. After transmission to birds, however, most genetic diversity is removed by strong purifying selection. Further, transmission of potentially mosquito-adaptive WNV variants is strongly influenced by genetic drift in mosquitoes. These results highlight the complex evolutionary forces a novel virus variant must overcome to alter infection phenotypes at the population level.
Project description:West Nile virus (WNV) exists in nature as a genetically diverse population of competing genomes. This high genetic diversity and concomitant adaptive plasticity has facilitated the rapid adaptation of WNV to North American transmission cycles and contributed to its explosive spread throughout the New World. WNV is maintained in nature in a transmission cycle between mosquitoes and birds, with intrahost genetic diversity highest in mosquitoes. The mechanistic basis for this increase in genetic diversity in mosquitoes is poorly understood. To determine whether the high mutational diversity of WNV in mosquitoes is driven by RNA interference (RNAi), we characterized the RNAi response to WNV in the midguts of orally exposed Culex pipiens quinquefasciatus using high-throughput, massively parallel sequencing and estimated viral genetic diversity. Our data demonstrate that WNV infection in orally exposed vector mosquitoes induces the RNAi pathway and that regions of the WNV genome that are more intensely targeted by RNAi are more likely to contain point mutations compared to weakly targeted regions. These results suggest that, under natural conditions, positive selection of WNV within mosquitoes is stronger in regions highly targeted by the host RNAi response. Further, they provide a mechanistic basis for the relative importance of mosquitoes in driving WNV diversification.
Project description:Arthropod-borne viruses are among the most genetically constrained RNA viruses, yet they have a remarkable propensity to adapt and emerge. We studied wild birds and mosquitoes naturally infected with West Nile virus (WNV) in a 'hot spot' of virus transmission in Chicago, IL, USA. We generated full coding WNV genome sequences from spatiotemporally matched bird and mosquito samples using high-throughput sequencing, allowing a molecular evolutionary assessment with deep coverage. Mean FST among samples was 0.66 (±0.02 SE) and was bimodal, with mean nucleotide diversity being higher between samples (interhost πN = 0.001; πS = 0.024) than within them (intrahost πN < 0.0001; πS < 0.001). Eight genomic sites with FST > 1.01 (in the PrM, NS2a, NS3, NS4b, and 5'-noncoding genomic regions) showed bird versus mosquito variant frequency differences of >30 per cent and/or polymorphisms fixed in ≥5 host or vector individuals, suggesting host tropism for these variants. However, phylogenetic analyses demonstrated a lack of grouping by bird or mosquito, most inter-sample differences were synonymous (mean interhost πN/πS = 0.04), and there was no significant difference between hosts and vectors in either their nucleotide diversities or levels of purifying selection (mean intrahost πN/πS = 0.28 in birds and πN/πS = 0.21 in mosquitoes). This finding contrasts with the 'trade-off' and 'selective sieve' hypotheses that have been proposed and tested in the laboratory, which predict strong host versus vector effects on WNV genetic variation, with heightened selective constraint in birds alternating with heightened viral diversity in mosquitoes. Overall, our data show WNV to be highly selectively constrained within and between both hosts and vectors but still able to vary at a limited number of sites across the genome. Such site-specific plasticity in the face of overall selective constraint may offer a mechanism whereby highly constrained viruses such as WNV and its relatives can still adapt and emerge.
Project description:Intrahost genetic diversity was analysed in naturally infected mosquitoes and birds to determine whether West Nile virus (WNV) exists in nature as a quasispecies and to quantify selective pressures within and between hosts. WNV was sampled from ten infected birds and ten infected mosquito pools collected on Long Island, NY, USA, during the peak of the 2003 WNV transmission season. A 1938 nt fragment comprising the 3' 1159 nt of the WNV envelope (E) coding region and the 5' 779 nt of the non-structural protein 1 (NS1) coding region was amplified and cloned and 20 clones per specimen were sequenced. Results from this analysis demonstrate that WNV infections are derived from a genetically diverse population of genomes in nature. The mean nucleotide diversity was 0.016 % within individual specimens and the mean percentage of clones that differed from the consensus sequence was 19.5 %. WNV sequences in mosquitoes were significantly more genetically diverse than WNV in birds. No host-dependent bias for particular types of mutations was observed and estimates of genetic diversity did not differ significantly between E and NS1 coding sequences. Non-consensus clones obtained from two avian specimens had highly similar genetic signatures, providing preliminary evidence that WNV genetic diversity may be maintained throughout the enzootic transmission cycle, rather than arising independently during each infection. Evidence of purifying selection was obtained from both intra- and interhost WNV populations. Combined, these data support the observation that WNV populations may be structured as a quasispecies and document strong purifying natural selection in WNV populations.
Project description:The emergence of mosquito-borne RNA viruses, such as West Nile virus (WNV), is facilitated by genetically complex virus populations within hosts. Here, we determine whether WNV enzootic (Culex tarsalis, Cx. quinquefasciatus, and Cx. pipiens) and bridge vectors (Aedes aegypti) have differential impacts on viral mutational diversity and fitness. During systemic mosquito infection, WNV faced stochastic reductions in genetic diversity that rapidly was recovered during intra-tissue population expansions. Interestingly, this intrahost selection and diversification was mosquito species dependent with Cx. tarsalis and Cx. quinquefasciatus exhibiting greater WNV divergence. However, recovered viral populations contained a preponderance of potentially deleterious mutations (i.e., high mutational load) and had lower relative fitness in avian cells compared to input virus. These findings demonstrate that the adaptive potential associated with mosquito transmission varies depending on the mosquito species and carries a significant fitness cost in vertebrates.
Project description:Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes.
Project description:West Nile virus is similar to most other RNA viruses in that it exists in nature as a genetically diverse population. However, the role of this genetic diversity within natural transmission cycles and its importance to virus perpetuation remain poorly understood. Therefore, we determined whether highly genetically diverse populations are more fit compared to less genetically diverse WNV populations. Specifically, we generated three WNV populations that varied in their genetic diversity and evaluated their fitness relative to genetically marked control WNV in vivo in Culex quinquefasciatus mosquitoes and chickens. Our results demonstrate that high genetic diversity leads to fitness gains in vector mosquitoes, but not chickens.
Project description:West Nile virus (WNV) is now the predominant circulating arthropod-borne virus in the United States with >15,000 human cases and >600 fatalities since 1999. Conventionally, mosquitoes become infected when feeding on viremic birds and subsequently transmit the virus to susceptible hosts. Here, we demonstrate nonviremic transmission of WNV between cofeeding mosquitoes. Donor, Culex pipiens quinquefasciatus mosquitoes infected with WNV were fed simultaneously with uninfected "recipient" mosquitoes on naïve mice. At all times, donor and recipient mosquitoes were housed in separate sealed containers, precluding the possibility of mixing. Recipients became infected in all five trials, with infection rates as high as 5.8% and no detectable viremia in the hosts. Remarkably, a 2.3% infection rate was observed when 87 uninfected mosquitoes fed adjacent to a single infected mosquito. This phenomenon could potentially enhance virus survival, transmission, and dispersion and obviate the requirement for viremia. All vertebrates, including immune and insusceptible animals, might therefore facilitate mosquito infection. Our findings question the status of dead-end hosts in the WNV transmission cycle and may partly explain the success with which WNV established and rapidly dispersed throughout North America.
Project description:St. Louis encephalitis virus (SLEV; Flaviviridae; Flavivirus) is a member of the Japanese encephalitis serocomplex and a close relative of West Nile virus (WNV). Although SLEV remains endemic to the US, both levels of activity and geographical dispersal are relatively constrained when compared to the widespread distribution of WNV. In recent years, WNV appears to have displaced SLEV in California, yet both viruses currently coexist in Texas and several other states. It has become clear that viral swarm characterization is required if we are to fully evaluate the relationship between viral genomes, viral evolution, and epidemiology. Mutant swarm size and composition may be particularly important for arboviruses, which require replication not only in diverse tissues but also divergent hosts. In order to evaluate temporal, spatial, and host-specific patterns in the SLEV mutant swarm, we determined the size, composition, and phylogeny of the intrahost swarm within primary mosquito isolates from both Texas and California. Results indicate a general trend of decreasing intrahost diversity over time in both locations, with recent isolates being highly genetically homogeneous. Additionally, phylogenic analyses provide detailed information on the relatedness of minority variants both within and among strains and demonstrate how both geographic isolation and seasonal maintenance have shaped the viral swarm. Overall, these data generally provide insight into how time, space, and unique transmission cycles influence the SLEV mutant swarm and how understanding these processes can ultimately lead to a better understanding of arbovirus evolution and epidemiology.
Project description:BACKGROUND: The natural history and potential impact of mosquito-specific flaviviruses on the transmission efficiency of West Nile virus (WNV) is unknown. The objective of this study was to determine whether or not prior infection with Culex flavivirus (CxFV) Izabal altered the vector competence of Cx. quinquefasciatus Say for transmission of a co-circulating strain of West Nile virus (WNV) from Guatemala. METHODS AND FINDINGS: CxFV-negative Culex quinquefasciatus and those infected with CxFV Izabal by intrathoracic inoculation were administered WNV-infectious blood meals. Infection, dissemination, and transmission of WNV were measured by plaque titration on Vero cells of individual mosquito bodies, legs, or saliva, respectively, two weeks following WNV exposure. Additional groups of Cx. quinquefasciatus were intrathoracically inoculated with WNV alone or WNV+CxFV Izabal simultaneously, and saliva collected nine days post inoculation. Growth of WNV in Aedes albopictus C6/36 cells or Cx. quinquefasciatus was not inhibited by prior infection with CxFV Izabal. There was no significant difference in the vector competence of Cx. quinquefasciatus for WNV between mosquitoes uninfected or infected with CxFV Izabal across multiple WNV blood meal titers and two colonies of Cx. quinquefasciatus (p>0.05). However, significantly more Cx. quinquefasciatus from Honduras that were co-inoculated simultaneously with both viruses transmitted WNV than those inoculated with WNV alone (p = 0.0014). Co-inoculated mosquitoes that transmitted WNV also contained CxFV in their saliva, whereas mosquitoes inoculated with CxFV alone did not contain virus in their saliva. CONCLUSIONS: In the sequential infection experiments, prior infection with CxFV Izabal had no significant impact on WNV replication, infection, dissemination, or transmission by Cx. quinquefasciatus, however WNV transmission was enhanced in the Honduras colony when mosquitoes were inoculated simultaneously with both viruses.
Project description:Laboratory and field research was conducted to determine if Culex tarsalis Coquillett expectorated West Nile virus (WNV) during sugar feeding and if a lure or bait station could be developed to exploit this behavior for WNV surveillance. Experimentally infected Cx. tarsalis repeatedly expectorated WNV onto filter paper strips and into vials with wicks containing sucrose that was readily detectable by a quantitative reverse transcriptase-polymerase chain reaction assay. Few females (33%, n = 27) became infected by imbibing sugar solutions spiked with high concentrations (10(7) plaque forming units/ml) of WNV, indicating sugar feeding stations probably would not be a source of WNV infection. In nature, sugar bait stations scented with the floral attractant phenyl acetaldehyde tracked WNV transmission activity in desert but not urban or agricultural landscapes in California. When deployed in areas of the Coachella Valley with WNV activity during the summer of 2011, 27 of 400 weekly sugar samples (6.8%) tested positive for WNV RNA by reverse transcriptase-polymerase chain reaction. Prevalence of positives varied spatially, but positive sugar stations were detected before concurrent surveillance measures of infection (mosquito pools) or transmission (sentinel chicken seroconversions). In contrast, sugar bait stations deployed in urban settings in Los Angeles or agricultural habits near Bakersfield in Kern County supporting WNV activity produced 1 of 90 and 0 of 60 positive weekly sugar samples, respectively. These results with sugar bait stations will require additional research to enhance bait attractancy and to understand the relationship between positive sugar stations and standard metrics of arbovirus surveillance.