Bethany G. Bolling

Transmission dynamics of an insect-specific flavivirus in a naturally infected Culex pipiens laboratory colony and effects of co-infection on vector competence for West Nile virus.

We established a laboratory colony of Culex pipiens mosquitoes from eggs collected in Colorado and discovered that mosquitoes in the colony are naturally infected with Culex flavivirus (CxFV), an insect-specific flavivirus. In this study we examined transmission dynamics of CxFV and effects of persistent CxFV infection on vector competence for West Nile virus (WNV). We found that vertical transmission is the primary mechanism for persistence of CxFV in Cx. pipiens, with venereal transmission potentially playing a minor role. Vector competence experiments indicated possible early suppression of WNV replication by persistent CxFV infection in Cx. pipiens. This is the first description of insect-specific flavivirus transmission dynamics in a naturally infected mosquito colony and the observation of delayed dissemination of superinfecting WNV suggests that the presence of CxFV may impact the intensity of enzootic transmission of WNV and the risk of human exposure to this important pathogen.

Insect-Specific Flaviviruses from Culex Mosquitoes in Colorado, with Evidence of Vertical Transmission

Mosquitoes were collected in Colorado during 2006 and 2007 to examine spatial and seasonal patterns of risk for exposure to Culex vectors and West Nile virus. We used universal flavivirus primers to test pools of Culex mosquitoes for viral RNA. This led to the detection and subsequent isolation of two insect-specific flaviviruses: Culex flavivirus (CxFV), which was first described from Japan, and a novel insect flavivirus, designated Calbertado virus (CLBOV), which has also been detected in California and Canada. We recorded both viruses in Cx. tarsalis and Cx. pipiens from Colorado. Furthermore, quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed the presence of CxFV RNA in Cx. pipiens eggs and larvae from a laboratory colony established in 2005 and naturally infected with CxFV, suggesting vertical transmission as a means of viral maintenance in natural Culex populations. Finally, we present phylogenetic analyses of the relationships between insect-specific flaviviruses and other selected flaviviruses.

Seasonal Patterns for Entomological Measures of Risk for Exposure to Culex Vectors and West Nile Virus in Relation to Human Disease Cases in Northeastern Colorado

ABSTRACT We examined seasonal patterns for entomological measures of risk for exposure to Culex vectors and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in relation to human WNV disease cases in a five-county area of northeastern Colorado during 2006–2007. Studies along habitat/ elevation gradients in 2006 showed that the seasonal activity period is shortened and peak numbers occur later in the summer for Culex tarsalis Coquillett females in foothills-montane areas > 1,600 m compared with plains areas <1,600 m in Colorados Front Range. Studies in the plains of northeastern Colorado in 2007 showed that seasonal patterns of abundance for Cx. tarsalis and Culex pipiens L. females differed in that Cx. tarsalis reached peak abundance in early July (mean of 328.9 females per trap night for 18 plains sites), whereas the peak for Cx. pipiens did not occur until late August (mean of 16.4 females per trap night). During June-September in 2007, which was a year of intense WNV activity in Colorado with 578 reported WNV disease cases, we recorded WNV-infected Cx. tarsalis females from 16 of 18 sites in the plains. WNV infection rates in Cx. tarsalis females increased gradually from late June to peak in mid-August (overall maximum likelihood estimate for WNV infection rate of 8.29 per 1,000 females for the plains sites in mid-August). No WNV-infected Culex mosquitoes were recorded from sites > 1,600 m. The vector index for abundance of WNV-infected Cx. tarsalis females for the plains sites combined exceeded 0.50 from mid-July to mid-August, with at least one site exceeding 1.00 from early July to late August. Finally, we found that abundance of Cx. tarsalis females and the vector index for infected females were strongly associated with weekly numbers of WNV disease cases with onset 4–7 wk later (female abundance) or 1–2 wk later (vector index).

Mosquito Species Richness, Composition, and Abundance along Habitat-Climate-Elevation Gradients in the Northern Colorado Front Range

Abstract We exploited elevation gradients (1,500–2,400 m) ranging from plains to montane areas along the Poudre River and Big Thompson River in the northern Colorado Front Range to determine how mosquito species richness, composition, and abundance change along natural habitat-climate-elevation gradients. Mosquito collections in 26 sites in 2006 by using CO2-baited CDC light traps yielded a total of 7,136 identifiable mosquitoes of 27 species. Commonly collected species included Aedes vexans (Meigen) (n = 4,722), Culex tarsalis Coquillett (n = 825), Ochlerotatus increpitus (Dyar) (n = 546), Ochlerotatus trivittatus (Coquillett) (n = 303), Aedes cinereus Meigen (n = 280), Ochlerotatus melanimon (Dyar) (n = 146), Ochlerotatus dorsalis (Meigen) (n = 67), Culiseta inornata (Williston) (n = 52), Ochlerotatus pullatus (Coquillett) (n = 38), Ochlerotatus spencerii idahoensis (Theobald) (n = 37), and Culex pipiens L. (n = 29). Species richness was highest in plains habitats at elevations below 1,600 m. Numerous species were found exclusively or predominantly at low elevations below 1,700 m [Anopheles earlei Vargas, Anopheles freeborni Aitken, Coquilletidia perturbans (Walker), Culex erythrothorax (Dyar), Cx. pipiens, Culex territans Walker, Oc. dorsalis, Ochlerotatus hendersoni (Cockerell), Oc. melanimon, and Oc. trivittatus], whereas others occurred predominantly at high elevations above 2,300 m [Ae. cinereus, Culiseta incidens (Thomson), Culiseta morsitans (Theoblad), Ochlerotatus cataphylla (Dyar), Ochlerotatus intrudens (Dyar), Oc. pullatus, and Ochlerotatus punctor (Kirby)]. Ae. vexans and Cx. tarsalis were abundant in the plains (<1,600 m; mean June–August temperature >19.5°C), occurred at low abundances in foothills and low montane areas (1,610–1,730 m; 18.0–19.5°C), and they were collected only sporadically in montane areas above 1,750 m (mean June–August temperature <17.5°C). These findings suggest that future climate warming may lead to shifts in distribution patterns of West Nile virus vectors (e.g., Cx. tarsalis) toward higher elevations in Colorado.

Distribution and Phylogenetic Comparisons of a Novel Mosquito Flavivirus Sequence Present in Culex tarsalis Mosquitoes from Western Canada with Viruses Isolated in California and Colorado

In a previous study, a new flavivirus genome sequence was identified in Culex tarsalis mosquitoes obtained in Alberta, Canada and was shown to be genetically related to but distinct from members of the insect-specific flaviviruses. Nonstructural protein 5-encoding sequences amplified from Cx. tarsalis pools from western Canada have shown a high similarity to genome sequences of novel flaviviruses isolated from mosquitoes in California and Colorado. Despite wide distribution of this virus, designated Calbertado virus, strains demonstrate a high degree of nonstructural protein 5 nucleotide (> 90%) and amino acid (> 97%) identity. The ecology and geographic range of Calbertado virus warrants further study because it may potentially influence transmission of mosquito-borne flaviviruses, including important human pathogens such as West Nile and Saint Louis encephalitis viruses.

Mosquitoes and West Nile virus along a river corridor from prairie to montane habitats in eastern Colorado

ABSTRACT: We conducted studies on mosquitoes and West Nile virus (WNV) along a riparian corridor following the South Platte River and Big Thompson River in northeastern Colorado and extending from an elevation of 1,215 m in the prairie landscape of the eastern Colorado plains to 1,840 m in low montane areas at the eastern edge of the Rocky Mountains in the central part of the state. Mosquito collection during June–September 2007 in 20 sites along this riparian corridor yielded a total of 199,833 identifiable mosquitoes of 17 species. The most commonly collected mosquitoes were, in descending order: Aedes vexans, Culex tarsalis, Ae. dorsalis, Ae. trivittatus, Ae. melanimon, Cx. pipiens, and Culiseta inornata. Species richness was higher in the plains than in foothills-montane areas, and abundances of several individual species, including the WNV vectors Cx. tarsalis and Cx. pipiens and the nuisance-biter and potential secondary WNV vector Ae. vexans, decreased dramatically from the plains (1,215–1,487 m) to foothills-montane areas (1,524–1,840 m). Ae. vexans and Cx. tarsalis had a striking pattern of uniformly high abundances between 1,200–1,450 m followed by a gradual decrease in abundance above 1,450 m to reach very low numbers above 1,550 m. Culex species were commonly infected with WNV in the plains portion of the riparian corridor in 2007, with 14 of 16 sites yielding WNV-infected Cx. tarsalis and infection rates for Cx. tarsalis females exceeding 2.0 per 1,000 individuals in ten of the sites. The Vector Index for abundance of WNV-infected Cx. tarsalis females during June–September exceeded 0.5 in six plains sites along the South Platte River but was uniformly low (0–0.1) in plains, foothills and montane sites above 1,500 m along the Big Thompson River. A population genetic analysis of Cx. tarsalis revealed that all collections from the ∼190 km riparian transect in northeastern Colorado were genetically uniform but that these collections were genetically distinct from collections from Delta County on the western slope of the Continental Divide. This suggests that major waterways in the Great Plains serve as important dispersal corridors for Cx. tarsalis but that the Continental Divide is a formidable barrier to this WNV vector.

The distribution of potential West Nile virus vectors, Culex pipiens pipiens and Culex pipiens quinquefasciatus (Diptera: Culicidae), in Mexico City.

BackgroundCulex spp. mosquitoes are considered to be the most important vectors of West Nile virus (WNV) detected in at least 34 species of mosquitoes in the United States. In North America, Culex pipiens pipiens, Culex pipiens quinquefasciatus, and Culex tarsalis are all competent vectors of WNV, which is considered to be enzootic in the United States and has also been detected in equines and birds in many states of Mexico and in humans in Nuevo Leon. There is potential for WNV to be introduced into Mexico City by various means including infected mosquitoes on airplanes, migrating birds, ground transportation and infected humans. Little is known of the geographic distribution of Culex pipiens complex mosquitoes and hybrids in Mexico City. Culex pipiens pipiens preferentially feed on avian hosts; Culex pipiens quinquefasciatus have historically been considered to prefer mammalian hosts; and hybrids of these two species could theoretically serve as bridge vectors to transmit WNV from avian hosts to humans and other mammalian hosts. In order to address the potential of WNV being introduced into Mexico City, we have determined the identity and spatial distribution of Culex pipiens complex mosquitoes and their hybrids.ResultsMosquito larvae collected from 103 sites throughout Mexico City during 2004-2005 were identified as Culex, Culiseta or Ochlerotatus by morphological analysis. Within the genus Culex, specimens were further identified as Culex tarsalis or as belonging to the Culex pipiens complex. Members of the Culex pipiens complex were separated by measuring the ratio of the dorsal and ventral arms (DV/D ratio) of the male genitalia and also by using diagnostic primers designed for the Ace.2 gene. Culex pipiens quinquefasciatus was the most abundant form collected.ConclusionsImportant WNV vectors species, Cx. p. pipiens, Cx. p. quinquefasciatus and Cx. tarsalis, are all present in Mexico City. Hybrids of Cx. p. pipiens and Cx. p. quinquefasciatus were also collected and identified. The presence and abundance of these WNV competent vectors is a cause for concern. Understanding the distribution of these vectors can help improve viral surveillance activities and mosquito control efforts in Mexico City.

Relationship Between Distance From Major Larval Habitats and Abundance of Adult Mosquitoes in Semiarid Plains Landscapes in Colorado

ABSTRACT We examined the relationship between distance from major larval habitats and abundance of adult mosquitoes in the semiarid plains landscape characteristic of eastern Colorado. Mosquito collection was conducted from late June to early August 2007 and included trap locations at distances ranging from <10 m up to 20–150 m and 160–373 m from three major larval habitats: one area along a river corridor and two small reservoirs. The study yielded 65,140 mosquitoes of 14 species, and five species were sufficiently abundant to be included in statistical analyses: Aedes vexans (Meigen), Culex tarsalis Coquillett, Ochlerotatus dorsalis (Meigen) (=Ae. dorsalis), Ochlerotatus melanimon (Dyar) (=Ae. melanimon), and Culex pipiens L. Distance to nearest major larval habitat was not strongly related to Culex abundance within the ≈ 400-m range from larval habitats examined in this study. Abundance of Ae. vexans declined significantly with distance from the larval habitat, whereas abundance was significantly higher in the 20–150- and 160–373-m classes compared with areas within 10 m of the larval habitat for both Ochlerotatus species. Except for Ae. vexans, however, we did not find monotonic increasing or decreasing abundance trends associated with distance from larval habitats for the 400-m range examined. This, combined with a finding that fine-scale habitat heterogeneity influenced abundance for most of the mosquitoes examined, underscores the importance of considering not only distance from larval habitat but also fine-scale habitat heterogeneity to understand how important nuisance-biters and West Nile virus (family Flaviviridae, genus Flavivirus, WNV) vectors use the landscape. We also discuss how these results relate to previous studies from western North America and explore their relevance to operational implementation of adulticides to suppress mosquito vectors during WNV disease outbreaks in the Great Plains.

Genetic Determinants of Differential Oral Infection Phenotypes of West Nile and St. Louis Encephalitis Viruses in Culex spp. Mosquitoes

St. Louis encephalitis virus (SLEV) has shown greater susceptibility to oral infectivity than West Nile virus (WNV) in Culex mosquitoes. To identify the viral genetic elements that modulate these disparate phenotypes, structural chimeras (WNV-pre-membrane [prM] and envelope [E] proteins [prME]/SLEV.IC (infectious clone) and SLEV-prME/WNV.IC) were constructed in which two of the structural proteins, the prM and E, were interchanged between viruses. Oral dose-response assessment with the chimeric/parental WNV and SLEV was performed to characterize the infection phenotypes in Culex mosquitoes by artificial blood meals. The median infectious dose required to infect 50% of Cx. quinquefasciatus with WNV was indistinguishable from that of the SLEV-prME/WNV.IC chimeric virus. Similarly, SLEV and WNV-prME/SLEV.IC virus exhibited an indistinguishable oral dose-response relationship in Cx. quinquefasciatus. Infection rates for WNV.IC and SLEV-prME/WNV.IC were significantly lower than SLEV.IC and WNV-prME/SLEV.IC infection rates. These results indicated that WNV and SLEV oral infectivities are not mediated by genetic differences within the prM and E proteins.

Temporal and Spatial Variability of Entomological Risk Indices for West Nile Virus Infection in Northern Colorado: 2006–2013

Abstract West Nile virus (WNV) is enzootic in northern Colorado. Annual surveillance activities in Fort Collins, CO, include collecting female Culex mosquitoes and testing them for the presence of WNV RNA in order to calculate 1) Culex female abundance, 2) WNV infection rate, and 3) the vector index (VI). These entomological risk indices inform public policy regarding the need for emergency adulticiding. Currently, these are calculated on a citywide basis. In this study, we present descriptive data from historical surveillance records spanning 2006–2013 to discern seasonal and yearly patterns of entomological risk for WNV infection. Also, we retrospectively test the hypothesis that entomological risk is correlated with human transmission risk and is heterogeneous within the City of Fort Collins. Four logistically relevant zones within the city were established and used to test this hypothesis. Zones in the eastern portion of the city consistently had significantly higher Culex abundance and VI compared with zones in the west, leading to higher entomological risk indicators for human WNV infection in the east. Moreover, the relative risk of a reported human case of WNV infection was significantly higher in the eastern zones of the city. Our results suggest that a more spatially targeted WNV management program may better mitigate human risk for WNV infection in Fort Collins, and possibly other cities where transmission is enzootic, while at the same time reducing pesticide use.