Alexander T. Ciota

West Nile virus infection of Drosophila melanogaster induces a protective RNAi response

To determine if West Nile virus (WNV) infection of insect cells induces a protective RNAi response, Drosophila melanogaster S2 and Aedes albopictus C6/36 cells were infected with WNV, and the production of WNV-homologous small RNAs was assayed as an indicator of RNAi induction. A distinct population of approximately 25 nt WNV-homologous small RNAs was detected in infected S2 cells but not C6/36 cells. RNAi knockdown of Argonaute 2 in S2 cells resulted in slightly increased susceptibility to WNV infection, suggesting that some WNV-homologous small RNAs produced in infected S2 cells are functional small interfering RNAs. WNV was shown to infect adult D. melanogaster, and adult flies containing mutations in each of four different RNAi genes (Argonaute 2, spindle-E, piwi, and Dicer-2) were significantly more susceptible to WNV infection than wildtype flies. These results combined with the analysis of WNV infection of S2 and C6/36 cells support the conclusion that WNV infection of D. melanogaster, but perhaps not Ae. albopictus, induces a protective RNAi response.

The Effect of Temperature on Life History Traits of Culex Mosquitoes

ABSTRACT Climatic changes forecasted in the coming years are likely to result in substantial alterations to the distributions and populations of vectors of arthropod-borne pathogens. Characterization of the effect of temperature shifts on the life history traits of specific vectors is needed to more accurately define how such changes could impact the epidemiological patterns of vector-borne disease. Here, we determined the effect of temperatures including 16, 20, 24, 28, and 32°C on development time, immature survival, adult survival, mosquito size, blood feeding, and fecundity of both field and colonized populations of the Culex mosquitoes Culex pipiens L., Culex quinquefasciatus Say, and Culex restuans Theobald. Our results demonstrate that temperature significantly affects all of these traits, yet also that the extent of this effect is at times incongruent among temperatures, as well as being population and species-specific. Comparisons of colonized mosquitoes with field populations generally demonstrate decreased adult and immature survival, increased blood feeding and egg production, and significant variation in the effects of temperature, indicating that such colonies are not fully representative of natural populations. Results with field populations in general indicate that increases in temperature are likely to accelerate mosquito development, and that this effect is greater at temperatures below 24°C, but also that temperature significantly increases mortality. Among field populations, Cx. restuans were most affected by temperature increases, with decreased longevity relative to other species and significant increases in adult and immature mortality measured with each incremental temperature increase. Despite the unique climates characteristic of the geographic ranges of Cx. quinquefasciatus and Cx. pipiens, evidence of significant species-specific adaptation to temperature ranges was not seen. Taken together, these results indicate that geographic region, as well as species and population differences, must be considered when measuring the effect of temperature on vector populations.

Characterization of mosquito-adapted West Nile virus

West Nile virus (WNV), a mosquito-borne flavivirus, has significantly expanded its geographical and host range since its 1999 introduction into North America. The underlying mechanisms of evolution of WNV and other arboviruses are still poorly understood. Studies evaluating virus adaptation and fitness in relevant in vivo systems are largely lacking. In order to evaluate the capacity for host-specific adaptation and the genetic correlates of adaptation in vivo, this study measured phenotypic and genotypic changes in WNV resulting from passage in Culex pipiens mosquitoes. An increase in replicative ability of WNV in C. pipiens was attained for the two lineages of WNV tested. This adaptation for replication in mosquitoes did not result in a replicative cost in chickens, but did decrease cell-to-cell spread of virus in vertebrate cell culture. Genetic analyses of one mosquito-adapted lineage revealed a total of nine consensus nucleotide substitutions with no accumulation of a significant mutant spectrum. These results differed significantly from previous in vitro studies. When St Louis encephalitis virus (SLEV), a closely related flavivirus, was passaged in C. pipiens, moderately attenuated growth in C. pipiens was observed for two lineages tested. These results suggest that significant differences in the capacity for mosquito adaptation may exist between WNV and SLEV, and demonstrate that further comparative studies in relevant in vivo systems will help elucidate the still largely unknown mechanisms of arboviral adaptation in ecologically relevant hosts.

The effect of hybridization of Culex pipiens complex mosquitoes on transmission of West Nile virus

BackgroundCulex pipiens L. complex mosquitoes have a global distribution and are primary vectors of pathogens of public health significance. In the U.S., Cx. pipiens bioformes, Cx. pipiens form pipiens and Cx. pipiens form molestus, as well as Cx. quinquefasciatus, are primary vectors of West Nile virus (WNV; Flaviviridae, Flavivirus). These mosquitoes reside in distinct but overlapping ecological niches and readily hybridize in areas where they coexist. Although species and population-specific differences in vector competence of Culex mosquitoes for WNV have been identified, the extent to which hybridization within this complex alters WNV transmission potential has not been well characterized.FindingsWNV vector competence of laboratory colonies of Cx. p. f. pipiens, Cx. p. f. molestus, and Cx. quinquefasciatus was assessed and compared to hybrid populations created from reciprocal mating of these lines. The results demonstrate that hybridization has a significant effect on WNV infection, dissemination, and, particularly, transmission in Culex pipiens L. complex mosquitoes. Specifically, enhanced transmission of WNV was measured in all hybrid populations relative to one or both parental stains.ConclusionThese findings demonstrate that environmental or anthropogenic changes resulting in fluctuations in the distribution and extent of hybrid populations of Culex mosquitoes could have a significant impact on transmission patterns of WNV in nature.

Cooperative interactions in the West Nile virus mutant swarm.

BackgroundRNA viruses including arthropod-borne viruses (arboviruses) exist as highly genetically diverse mutant swarms within individual hosts. A more complete understanding of the phenotypic correlates of these diverse swarms is needed in order to equate RNA swarm breadth and composition to specific adaptive and evolutionary outcomes.ResultsHere, we determined clonal fitness landscapes of mosquito cell-adapted West Nile virus (WNV) and assessed how altering the capacity for interactions among variants affects mutant swarm dynamics and swarm fitness. Our results demonstrate that although there is significant mutational robustness in the WNV swarm, genetic diversity also corresponds to substantial phenotypic diversity in terms of relative fitness in vitro. In addition, our data demonstrate that increasing levels of co-infection can lead to widespread strain complementation, which acts to maintain high levels of phenotypic and genetic diversity and potentially slow selection for individual variants. Lastly, we show that cooperative interactions may lead to swarm fitness levels which exceed the relative fitness levels of any individual genotype.ConclusionsThese studies demonstrate the profound effects variant interactions can have on arbovirus evolution and adaptation, and provide a baseline by which to study the impact of this phenomenon in natural systems.

Experimental passage of St. Louis encephalitis virus in vivo in mosquitoes and chickens reveals evolutionarily significant virus characteristics.

St. Louis encephalitis virus (SLEV; Flaviviridae, flavivirus) was the major cause of epidemic flaviviral encephalitis in the U.S. prior to the introduction of West Nile virus (WNV) in 1999. However, outbreaks of SLEV have been significantly more limited then WNV in terms of levels of activity and geographic dispersal. One possible explanation for these variable levels of activity is that differences in the potential for each virus to adapt to its host cycle exist. The need for arboviruses to replicate in disparate hosts is thought to result in constraints on both evolution and host-specific adaptation. If cycling is the cause of genetic stability observed in nature and arboviruses lack host specialization, then sequential passage should result in both the accumulation of mutations and specialized viruses better suited for replication in that host. Previous studies suggest that WNV and SLEV differ in capacity for both genetic change and host specialization, and in the costs each accrues from specializing. In an attempt to clarify how selective pressures contribute to epidemiological patterns of WNV and SLEV, we evaluated mutant spectra size, consensus genetic change, and phenotypic changes for SLEV in vivo following 20 sequential passages via inoculation in either Culex pipiens mosquitoes or chickens. Results demonstrate that the capacity for genetic change is large for SLEV and that the size of the mutant spectrum is host-dependent using our passage methodology. Despite this, a general lack of consensus change resulted from passage in either host, a result that contrasts with the idea that constraints on evolution in nature result from host cycling alone. Results also suggest that a high level of adaptation to both hosts already exists, despite host cycling. A strain significantly more infectious in chickens did emerge from one lineage of chicken passage, yet other lineages and all mosquito passage strains did not display measurable host-specific fitness gains. In addition, increased infectivity in chickens did not decrease infectivity in mosquitoes, which further contrasts the concept of fitness trade-offs for arboviruses.

Quantification of intrahost bottlenecks of West Nile virus in Culex pipiens mosquitoes using an artificial mutant swarm

Mosquito-borne viruses are predominantly RNA viruses which exist within hosts as diverse mutant swarms. Defining the way in which stochastic forces within mosquito vectors shape these swarms is critical to advancing our understanding of the evolutionary and adaptive potential of these pathogens. There are multiple barriers within a mosquito which a viral swarm must traverse in order to ultimately be transmitted. Here, using artificial mutant swarms composed of neutral variants of West Nile virus (WNV), we tracked changes to swarm breadth over time and space in Culex pipiens mosquitoes. Our results demonstrate that all variants have the potential to survive intrahost bottlenecks, yet mean swarm breadth decreases during both midgut infection and transmission when starting populations contain higher levels of minority variants. In addition, WNV swarms are subject to temporal sweeps which act to significantly decrease intrahost diversity over time. Taken together, these data demonstrate the profound effects that stochastic forces can have in shaping arboviral mutant swarms.

Effects of Zika Virus Strain and Aedes Mosquito Species on Vector Competence

In the Western Hemisphere, Zika virus is thought to be transmitted primarily by Aedes aegypti mosquitoes. To determine the extent to which Ae. albopictus mosquitoes from the United States are capable of transmitting Zika virus and the influence of virus dose, virus strain, and mosquito species on vector competence, we evaluated multiple doses of representative Zika virus strains in Ae. aegypti and Ae. albopictus mosquitoes. Virus preparation (fresh vs. frozen) significantly affected virus infectivity in mosquitoes. We calculated 50% infectious doses to be 6.1–7.5 log10 PFU/mL; minimum infective dose was 4.2 log10 PFU/mL. Ae. albopictus mosquitoes were more susceptible to infection than Ae. aegypti mosquitoes, but transmission efficiency was higher for Ae. aegypti mosquitoes, indicating a transmission barrier in Ae. albopictus mosquitoes. Results suggest that, although Zika virus transmission is relatively inefficient overall and dependent on virus strain and mosquito species, Ae. albopictus mosquitoes could become major vectors in the Americas.

Sequence-Specific Fidelity Alterations Associated with West Nile Virus Attenuation in Mosquitoes.

High rates of error-prone replication result in the rapid accumulation of genetic diversity of RNA viruses. Recent studies suggest that mutation rates are selected for optimal viral fitness and that modest variations in replicase fidelity may be associated with viral attenuation. Arthropod-borne viruses (arboviruses) are unique in their requirement for host cycling and may necessitate substantial genetic and phenotypic plasticity. In order to more thoroughly investigate the correlates, mechanisms and consequences of arbovirus fidelity, we selected fidelity variants of West Nile virus (WNV; Flaviviridae, Flavivirus) utilizing selection in the presence of a mutagen. We identified two mutations in the WNV RNA-dependent RNA polymerase associated with increased fidelity, V793I and G806R, and a single mutation in the WNV methyltransferase, T248I, associated with decreased fidelity. Both deep-sequencing and in vitro biochemical assays confirmed strain-specific differences in both fidelity and mutational bias. WNV fidelity variants demonstrated host-specific alterations to replicative fitness in vitro, with modest attenuation in mosquito but not vertebrate cell culture. Experimental infections of colonized and field populations of Cx. quinquefaciatus demonstrated that WNV fidelity alterations are associated with a significantly impaired capacity to establish viable infections in mosquitoes. Taken together, these studies (i) demonstrate the importance of allosteric interactions in regulating mutation rates, (ii) establish that mutational spectra can be both sequence and strain-dependent, and (iii) display the profound phenotypic consequences associated with altered replication complex function of flaviviruses.

Characterization of Rabensburg Virus, a Flavivirus Closely Related to West Nile Virus of the Japanese Encephalitis Antigenic Group

Rabensburg virus (RABV), a Flavivirus with ∼76% nucleotide and 90% amino acid identity with representative members of lineage one and two West Nile virus (WNV), previously was isolated from Culex pipiens and Aedes rossicus mosquitoes in the Czech Republic, and phylogenetic and serologic analyses demonstrated that it was likely a new lineage of WNV. However, no direct link between RABV and human disease has been definitively established and the extent to which RABV utilizes the typical WNV transmission cycle is unknown. Herein, we evaluated vector competence and capacity for vertical transmission (VT) in Cx. pipiens; in vitro growth on avian, mammalian, and mosquito cells; and infectivity and viremia production in birds. RABV infection and replication only were detected on mosquito cells. Experimentally inoculated birds did not become infected. Cx. pipiens had poor peroral vector competence and a higher VT rate as compared to US-WNV in Cx. pipiens. As a result, we postulate that RABV is an intermediate between the mosquito-specific and horizontally transmitted flaviviruses.