Ecological Distribution and CQ11 Genetic Structure of Culex pipiens Complex (Diptera: Culicidae) in Italy

Mosquitoes in the Culex pipiens complex are considered to be involved in the transmission of a range of pathogens, including West Nile virus (WNV). Although its taxonomic status is still debated, the complex includes species, both globally distributed or with a more limited distribution, morphologically similar and characterised by different physiological and behavioural traits, which affect their ability as vectors. In many European countries, Cx. pipiens and its sibling species Culex torrentium occur in sympatry, exhibiting similar bionomic and morphological characters, but only Cx. pipiens appears to play a vector role in WNV transmission. This species consists of two biotypes, pipiens and molestus, which can interbreed when in sympatry, and their hybrids can act as WNV-bridge vectors, due to intermediate ecological features. Considering the yearly WNV outbreaks since 2008 and given the morphological difficulties in recognising species and biotypes, our aim was to molecularly identify and characterised Cx. pipiens and Cx. torrentium in Italy, using recently developed molecular assays. Culex torrentium was not detected; as in other European countries, the pipiens and molestus biotypes were widely found in sympatry with hybrids in most environments. The UPGMA cluster analysis applied to CQ11 genotypic frequencies mainly revealed two groups of Cx. pipiens populations that differed in ecological features. The high propensity of the molestus biotype to exist in hypogean environments, where the habitat’s physical characteristics hinder and preclude the gene flow, was shown. These results confirmed the CQ11 assay as a reliable diagnostic method, consistent with the ecological and physiological aspects of the populations analysed. Since the assessment of the actual role of three biotypes in the WNV circulation remains a crucial point to be elucidated, this extensive molecular screening of Cx. pipiens populations can provide new insights into the ecology of the species and may give useful indications to plan and implement WNV surveillance activities in Italy.     

Comparing Competitive Fitness of West Nile Virus Strains in Avian and Mosquito Hosts

Enzootic transmission of West Nile virus (WNV; Flaviviridae, Flavivirus) involves various species of birds and ornithophilic mosquitoes. Single nucleotide substitutions in the WNV genome may impact viral fitness necessary for WNV adaptation and evolution as previously shown for the WN02 genotype. In an effort to study phenotypic change, we developed an in vivo fitness competition model in two biologically relevant hosts for WNV. The House Finch (HOFI; Haemorhous mexicanus) and Culex tarsalis mosquitoes represent moderately susceptible hosts for WNV, are highly abundant in Western North America and frequently are infected with WNV in nature. Herein, we inoculated HOFIs and Cx. tarsalis competitively (dually) and singly with infectious-clone derived viruses of the founding California isolate COAV997-2003 (COAV997-IC), the founding North American isolate NY99 (NY99-IC), and a 2004 field isolate from California (CA-04), and compared the replicative capacities (fitness) of these viruses to a genetically marked virus of COAV997 (COAV997-5nt) by measuring RNA copy numbers. COAV997 and COAV997-5nt exhibited neutral fitness in HOFIs and Cx. tarsalis, and the temperature-sensitive phenotype of COAV997 did not affect replication in HOFIs as none of the infected birds became febrile. The NY99 and CA-04 isolates demonstrated elevated fitness in HOFIs compared to COAV997-5nt, whereas all viruses replicated to similar titers and RNA copies in Cx. tarsalis, and the only fitness differences were related to infection rates. Our data demonstrated that competitive replication allows for the sensitive comparison of fitness differences among two genetically closely related viruses using relevant hosts of WNV while eliminating host-to-host differences. In conclusion, our approach may be helpful in understanding the extent of phenotypic change in fitness associated with genetic changes in WNV.     

Effects of ivermectin treatment of backyard chickens on mosquito dynamics and West Nile virus transmission

BackgroundVector control strategies typically rely on pesticides to target mosquitoes involved in enzootic and zoonotic transmission of West Nile virus (WNV). Nevertheless, increasing insecticide resistance and a desire to reduce pesticide usage provide the impetus for developing alternative strategies. Ivermectin (IVM), an antiparasitic drug which is widely used in human and veterinary medicine, is a potential alternative for targeted control because Culex mosquitoes experience increased mortality following ingestion of IVM in bloodmeals.Methodology/Principal findingsWe conducted a randomized field trial to investigate the impact of treating backyard chicken flocks with IVM in urban neighborhoods across Davis, California on mosquito populations and WNV transmission dynamics. We observed a significant reduction in WNV seroconversions in treated vs. untreated chickens, suggesting a reduction in WNV transmission intensity around treated flocks. We also detected a reduction in parity rates of Cx. tarsalis near treated vs. untreated flocks and increased mortality in wild mosquitoes following a bloodmeal on treated chickens (IVM serum concentration > 5ng/mL) vs. chickens with IVM serum concentrations < 5 ng/mL. However, we did not find a significant difference in abundance or infection prevalence in mosquitoes between treatment groups associated with the reductions in seroconversions. Mosquito immigration from surrounding larval habitat, relatively low WNV activity in the study area, and variable IVM serum concentrations likely contributed to uncertainty about the impact.Conclusions/SignificanceTaken together, our results point to a reduction in WNV transmission due to the impact of IVM on Culex mosquito populations and support the ongoing investigation of oral administration of IVM to wild birds for local control of WNV transmission, although further work is needed to optimize dosing and understand effects on entomological endpoints.     

Mosquito population structure,pathogen surveillance and insecticide resistance monitoring in urban regions of Crete,Greece

BackgroundIn Greece vector borne diseases (VBD) and foremost West Nile virus (WNV) pose an important threat to public health and the tourist industry, the primary sector of contribution to the national economy. The island of Crete, is one of Greece’s major tourist destinations receiving annually over 5 million tourists making regional VBD control both a public health and economic priority.MethodologyUnder the auspices of the Region of Crete, a systematic integrative surveillance network targeting mosquitoes and associated pathogens was established in Crete for the years 2018–2020. Using conventional and molecular diagnostic tools we investigated the mosquito species composition and population dynamics, pathogen infection occurrences in vector populations and in sentinel chickens, and the insecticide resistance status of the major vector species.Principal findingsImportant disease vectors were recorded across the island including Culex pipiens, Aedes albopictus, and Anopheles superpictus. Over 75% of the sampled specimens were collected in the western prefectures potentially attributed to the local precipitation patterns, with Cx. pipiens being the most dominant species. Although no pathogens (flaviviruses) were detected in the analysed mosquito specimens, chicken blood serum analyses recorded a 1.7% WNV antibody detection rate in the 2018 samples. Notably detection of the first WNV positive chicken preceded human WNV occurrence in the same region by approximately two weeks. The chitin synthase mutation I1043F (associated with high diflubenzuron resistance) was recorded at an 8% allelic frequency in Lasithi prefecture Cx. pipiens mosquitoes (sampled in 2020) for the first time in Greece. Markedly, Cx. pipiens populations in all four prefectures were found harboring the kdr mutations L1014F/C/S (associated with pyrethroid resistance) at a close to fixation rate, with mutation L1014C being the most commonly found allele (≥74% representation). Voltage gated sodium channel analyses in Ae. albopictus revealed the presence of the kdr mutations F1534C and I1532T (associated with putative mild pyrethroid resistance phenotypes) yet absence of V1016G. Allele F1534C was recorded in all prefectures (at an allelic frequency range of 25–46.6%) while I1532T was detected in populations from Chania, Rethymnon and Heraklion (at frequencies below 7.1%). Finally, no kdr mutations were detected in the Anopheles specimens included in the analyses.Conclusions/SignificanceThe findings of our study are of major concern for VBD control in Crete, highlighting (i) the necessity for establishing seasonal integrated entomological/pathogen surveillance programs, supporting the design of targeted vector control responses and; ii) the need for establishing appropriate insecticide resistance management programs ensuring the efficacy and sustainable use of DFB and pyrethroid based products in vector control.     

The Role of Climatic and Density Dependent Factors in Shaping Mosquito Population Dynamics: The Case of Culex pipiens in Northwestern Italy

Culex pipiens mosquito is a species widely spread across Europe and represents a competent vector for many arboviruses such as West Nile virus (WNV), which has been recently circulating in many European countries, causing hundreds of human cases. In order to identify the main determinants of the high heterogeneity in Cx. pipiens abundance observed in Piedmont region (Northwestern Italy) among different seasons, we developed a density-dependent stochastic model that takes explicitly into account the role played by temperature, which affects both developmental and mortality rates of different life stages. The model was calibrated with a Markov chain Monte Carlo approach exploring the likelihood of recorded capture data gathered in the study area from 2000 to 2011; in this way, we disentangled the role played by different seasonal eco-climatic factors in shaping the vector abundance. Illustrative simulations have been performed to forecast likely changes if temperature or density–dependent inputs would change. Our analysis suggests that inter-seasonal differences in the mosquito dynamics are largely driven by different temporal patterns of temperature and seasonal-specific larval carrying capacities. Specifically, high temperatures during early spring hasten the onset of the breeding season and increase population abundance in that period, while, high temperatures during the summer can decrease population size by increasing adult mortality. Higher densities of adult mosquitoes are associated with higher larval carrying capacities, which are positively correlated with spring precipitations. Finally, an increase in larval carrying capacity is expected to proportionally increase adult mosquito abundance.     

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

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.     

Wolbachia Enhances West Nile Virus (WNV) Infection in the Mosquito Culex tarsalis

Novel strategies are required to control mosquitoes and the pathogens they transmit. One attractive approach involves maternally inherited endosymbiotic Wolbachia bacteria. After artificial infection with Wolbachia, many mosquitoes become refractory to infection and transmission of diverse pathogens. We evaluated the effects of Wolbachia (wAlbB strain) on infection, dissemination and transmission of West Nile virus (WNV) in the naturally uninfected mosquito Culex tarsalis, which is an important WNV vector in North America. After inoculation into adult female mosquitoes, Wolbachia reached high titers and disseminated widely to numerous tissues including the head, thoracic flight muscles, fat body and ovarian follicles. Contrary to other systems, Wolbachia did not inhibit WNV in this mosquito. Rather, WNV infection rate was significantly higher in Wolbachia-infected mosquitoes compared to controls. Quantitative PCR of selected innate immune genes indicated that REL1 (the activator of the antiviral Toll immune pathway) was down regulated in Wolbachia-infected relative to control mosquitoes. This is the first observation of Wolbachia-induced enhancement of a human pathogen in mosquitoes, suggesting that caution should be applied before releasing Wolbachia-infected insects as part of a vector-borne disease control program.     

Ecological Niche Modelling of Potential West Nile Virus Vector Mosquito Species and Their Geographical Association with Equine Epizootics in Italy

In Italy, West Nile virus (WNV) equine outbreaks have occurred annually since 2008. Characterizing WNV vector habitat requirements allows for the identification of areas at risk of viral amplification and transmission. Maxent-based ecological niche models were developed using literature records of 13 potential WNV Italian vector mosquito species to predict their habitat suitability range and to investigate possible geographical associations with WNV equine outbreak occurrence in Italy from 2008 to 2010. The contribution of different environmental variables to the niche models was also assessed. Suitable habitats for Culex pipiens, Aedes albopictus, and Anopheles maculipennis were widely distributed; Culex modestus, Ochlerotatus geniculatus, Ochlerotatus caspius, Coquillettidia richiardii, Aedes vexans, and Anopheles plumbeus were concentrated in north-central Italy; Aedes cinereus, Culex theileri, Ochlerotatus dorsalis, and Culiseta longiareolata were restricted to coastal/southern areas. Elevation, temperature, and precipitation variables showed the highest predictive power. Host population and landscape variables provided minor contributions. WNV equine outbreaks had a significantly higher probability to occur in habitats suitable for Cx. modestus and Cx. pipiens, providing circumstantial evidence that the potential distribution of these two species coincides geographically with the observed distribution of the disease in equines.     

West Nile Virus: High Transmission Rate in North-Western European Mosquitoes Indicates Its Epidemic Potential and Warrants Increased Surveillance

Background

West Nile virus (WNV) is a highly pathogenic flavivirus transmitted by Culex spp. mosquitoes. In North America (NA), lineage 1 WNV caused the largest outbreak of neuroinvasive disease to date, while a novel pathogenic lineage 2 strain circulates in southern Europe. To estimate WNV lineage 2 epidemic potential it is paramount to know if mosquitoes from currently WNV-free areas can support further spread of this epidemic.

Methodology/Principal Findings

We assessed WNV vector competence of Culex pipiens mosquitoes originating from north-western Europe (NWE) in direct comparison with those from NA. We exposed mosquitoes to infectious blood meals of lineage 1 or 2 WNV and determined the infection and transmission rates. We explored reasons for vector competence differences by comparing intrathoracic injection versus blood meal infection, and we investigated the influence of temperature. We found that NWE mosquitoes are highly competent for both WNV lineages, with transmission rates up to 25%. Compared to NA mosquitoes, transmission rates for lineage 2 WNV were significantly elevated in NWE mosquitoes due to better virus dissemination from the midgut and a shorter extrinsic incubation time. WNV infection rates further increased with temperature increase.

Conclusions/Significance

Our study provides experimental evidence to indicate markedly different risk levels between both continents for lineage 2 WNV transmission and suggests a degree of genotype-genotype specificity in the interaction between virus and vector. Our experiments with varying temperatures explain the current localized WNV activity in southern Europe, yet imply further epidemic spread throughout NWE during periods with favourable climatic conditions. This emphasizes the need for intensified surveillance of virus activity in current WNV disease-free regions and warrants increased awareness in clinics throughout Europe.     

Culex pipiens and Stegomyia albopicta (= Aedes albopictus) populations as vectors for lineage 1 and 2 West Nile virus in Europe

The emerging disease West Nile fever is caused by West Nile virus (WNV), one of the most widespread arboviruses. This study represents the first test of the vectorial competence of European Culex pipiens Linnaeus 1758 and Stegomyia albopicta (= Aedes albopictus) (both: Diptera: Culicidae) populations for lineage 1 and 2 WNV isolated in Europe. Culex pipiens and S. albopicta populations were susceptible to WNV infection, had disseminated infection, and were capable of transmitting both WNV lineages. This is the first WNV competence assay to maintain mosquito specimens under environmental conditions mimicking the field (day/night) conditions associated with the period of maximum expected WNV activity. The importance of environmental conditions is discussed and the issue of how previous experiments conducted in fixed high temperatures may have overestimated WNV vector competence results with respect to natural environmental conditions is analysed. The information presented should be useful to policymakers and public health authorities for establishing effective WNV surveillance and vector control programmes. This would improve preparedness to prevent future outbreaks.     

Molecular evidence for trypanosomatids in Culex mosquitoes collected during a West Nile virus survey

Adult mosquitoes were previously collected and tested for West Nile virus during an intense WNV outbreak in 2003-2004 along the Cache la Poudre River in Colorado, USA. A subset of these mosquitoes was also tested for infection with trypanosomatids using nested PCR to amplify 18S rRNA. Of the 69 pools of Culex pipiens that were screened for both pathogens, 4.3% were positive for WNV and 11.6% tested positive for trypanosomes; no pools were found to be co-infected with both pathogens. One hundred and forty-three pools of Culex tarsalis, considered to be the principal WNV vector in this area, were tested in the same manner. 7.7% were positive for WNV and 20.3% of these pools tested positive for trypanosomes. Five pools of C. tarsalis were found to be co-infected with both pathogens, which was approximately 2.2 times more frequent than would be expected if these pathogens are independent of each other. Sequencing and maximum parsimony analysis of 18S rRNA revealed that four of the isolates arise in or near clades of described avian trypanosomes, likely indicating that these are vectored pathogens between birds and mosquitoes. Unexpectedly, the majority (24/28, 86%) of our positive samples form their own separate clade within the order Trypanosomatida with 100% bootstrap support. We have identified a potential new clade of trypanosomatids that exist within important mosquito vectors and discuss the potential ecological connections between these trypanosomes, arboviruses and mosquitoes.     

Population genetic data suggest a role for mosquito‐mediated dispersal of West Nile virus across the western United States

After introduction, West Nile virus (WNV) spread rapidly across the western United States between the years 2001 and 2004. This westward movement is thought to have been mediated by random dispersive movements of resident birds. Little attention has been placed on the role of mosquito vectors in virus dispersal across North America. The mosquito vector largely responsible for WNV amplification and transmission of WNV in the western USA is Culex tarsalis. Here we present population genetic data that suggest a potential role for C. tarsalis in the dispersal of WNV across the western USA. Population genetic structure across the species range of C. tarsalis in the USA was characterized in 16 states using 12 microsatellite loci. structure and geneland analyses indicated the presence of three broad population clusters. Barriers to gene flow were resolved near the Sonoran desert in southern Arizona and between the eastern Rocky Mountains and High Plains plateau. Small genetic distances among populations within clusters indicated that gene flow was not obstructed over large portions of the West Coast and within the Great Plains region. Overall, gene flow in C. tarsalis appears to be extensive, potentially mediated by movement of mosquitoes among neighbouring populations and hindered in geographically limited parts of its range. The pattern of genetic clustering in C. tarsalis is congruent with the pattern of invasion of WNV across the western United States, raising the possibility that movement of this important vector may be involved in viral dispersal.     

Polymorphic microsatellite loci from the West Nile virus vector Culex tarsalis

Since its introduction in 1999, West Nile virus (WNV) has spread across North America. Culex tarsalis is a highly efficient WNV vector species. Many traits such as virus susceptibility, autogeny and host preference vary geographically and temporally in C. tarsalis. Culex tarsalis genomic libraries were developed and were highly enriched for microsatellite inserts (42–96%). We identified 12 loci that were polymorphic in wild C. tarsalis populations. These microsatellites are the first DNA‐based genetic markers developed for C. tarsalis and will be useful for investigating population structure and constructing genetic maps in this mosquito.     

Vector competence of northern and southern European Culex pipiens pipiens mosquitoes for West Nile virus across a gradient of temperatures

In Europe, West Nile virus (WNV) outbreaks have been limited to southern and central European countries. However, competent mosquito vectors and susceptible bird hosts are present in northern Europe. Differences in temperature and vector competence of mosquito populations may explain the absence of WNV outbreaks in northern Europe. The aim of the present study was to directly compare vector competence of northern and southern European Culex pipiens (Cx. p.) pipiens mosquitoes for WNV across a gradient of temperatures. WNV infection and transmission rates were determined for two Cx. p. pipiens populations originating from The Netherlands and Italy, respectively. Mosquitoes were orally exposed by providing an infectious bloodmeal, or by injecting WNV (lineage 2) in the thorax, followed by 14‐day incubation at 18, 23, or 28 °C. No differences in infection or transmission rates were found between the Cx. p. pipiens populations with both infection methods, but WNV transmission rates were significantly higher at temperatures above 18 °C. The absence of WNV outbreaks in northern Europe cannot be explained by differences in vector competence between Cx. p. pipiens populations originating from northern and southern Europe. This study suggests that low temperature is a key limiting factor for WNV transmission.     

Evaluation of vector competence for West Nile virus in Italian Stegomyia albopicta (=Aedes albopictus) mosquitoes

West Nile virus (WNV) is a zoonotic arboviral pathogen transmitted by mosquitoes in a cycle that involves wild birds as reservoir hosts. The virus is responsible for outbreaks of viral encephalitis in humans and horses. In Europe, Culex pipiens (Diptera: Culicidae) is considered to be the main vector of WNV, but other species such as Stegomyia albopicta (=Aedes albopictus) (Diptera: Culicidae) may also act as competent vectors of this virus. Since 2008 human cases of WNV disease have been reported in northeast Italy. In 2011, new areas of southern Italy became involved and a first outbreak of WNV lineage 1 occurred on the island of Sardinia. On the assumption that a potential involvement of St. albopicta in WNV transmission cannot be excluded, and in order to evaluate the competence of this species for the virus, an experimental infection of an St. albopicta laboratory colony, established from mosquitoes collected in Sardinia, was carried out. The results were compared with those obtained in a colony of the main vector Cx. pipiens. The study showed St. albopicta collected on Sardinia to be susceptible to WNV infection, which suggests this Italian mosquito species is able to act as a possible secondary vector, particularly in urban areas where the species reaches high levels of seasonal abundance.     

Susceptibility and barriers to infection of Colorado mosquitoes with Rift Valley fever virus

Rift Valley fever virus (RVFV) causes morbidity and mortality in humans and domestic ungulates in sub-Saharan Africa, Egypt, and the Arabian Peninsula. Mosquito vectors transmit RVFV between vertebrates by bite, and also vertically to produce infectious progeny. Arrival of RVFV into the United States by infected mosquitoes or humans could result in significant impacts on food security, human health, and wildlife health. Elucidation of the vectors involved in the post-introduction RVFV ecology is paramount to rapid implementation of vector control. We performed vector competence experiments in which field-collected mosquitoes were orally exposed to an epidemic strain of RVFV via infectious blood meals. We targeted floodwater Aedes species known to feed on cattle, and/or deer species (Aedes melanimon Dyar, Aedes increpitus Dyar, Aedes vexans [Meigen]). Two permanent-water-breeding species were targeted as well: Culiseta inornata (Williston) of unknown competence considering United States populations, and Culex tarsalis Coquillett as a control species for which transmission efficiency is known. We tested the potential for midgut infection, midgut escape (dissemination), ovarian infection (vertical transmission), and transmission by bite (infectious saliva). Tissues were assayed by plaque assay and RT-qPCR, to quantify infectious virus and confirm virus identity. Tissue infection data were analyzed using a within-host model under a Bayesian framework to determine the probabilities of infection outcomes (midgut-limited infection, disseminated infection, etc.) while estimating barriers to infection between tissues. Permanent-water-breeding mosquitoes (Cx. tarsalis and Cs. inornata) exhibited more efficient horizontal transmission, as well as potential for vertical transmission, which is contrary to the current assumptions of RVFV ecology. Barrier estimates trended higher for Aedes spp., suggesting systemic factors in the differences between these species and Cx. tarsalis and Cs. inornata. These data indicate higher potential for vertical transmission than previously appreciated, and support the consensus of RVFV transmission including a broad range of potential vectors.     

Testing of UK Populations of Culex pipiens L. for Schmallenberg Virus Vector Competence and Their Colonization

Background

Schmallenberg virus (SBV), an arboviral pathogen of ruminants, emerged in northern Europe during 2011 and has subsequently spread across a vast geographic area. While Culicoides biting midges (Diptera: Ceratopogonidae) have been identified as a biological transmission agent of SBV, the role of mosquitoes (Diptera: Culicidae) as potential vectors has not been defined beyond small-scale field collections in affected areas. Culex pipiens L. are one of the most widespread mosquitoes in northern Europe; they are present on farms across the region and have previously been implicated as vectors of several other arboviruses. We assessed the ability of three colony lines of Cx. pipiens, originating from geographically diverse field populations, to become fully infected by SBV using semi-quantitative real-time RT-PCR (sqPCR).

Findings

Two colony lines of Cx. pipiens were created in the UK (‘Brookwood’ and ‘Caldbeck’) from field collections of larvae and pupae and characterised using genetic markers. A third strain of Cx. pipiens from CVI Wageningen, The Netherlands, was also screened during experiments. Intrathoracic inoculation of the Brookwood line resulted in infections after 14 days that were characterised by high levels of RNA throughout individuals, but which demonstrated indirect evidence of salivary gland barriers. Feeding of 322 individuals across the three colony lines on a membrane based infection system resulted in no evidence of full dissemination of SBV, although infections did occur in a small proportion of Cx. pipiens from each line.

Conclusions/Significance

This study established two novel lines of Cx. pipiens mosquitoes of UK origin in the laboratory and subsequently tested their competence for SBV. Schmallenberg virus replication and dissemination was restricted, demonstrating that Cx. pipiens is unlikely to be an epidemiologically important vector of the virus in northern Europe.     

West Nile virus epidemics in North America are driven by shifts in mosquito feeding behavior

West Nile virus (WNV) has caused repeated large-scale human epidemics in North America since it was first detected in 1999 and is now the dominant vector-borne disease in this continent. Understanding the factors that determine the intensity of the spillover of this zoonotic pathogen from birds to humans (via mosquitoes) is a prerequisite for predicting and preventing human epidemics. We integrated mosquito feeding behavior with data on the population dynamics and WNV epidemiology of mosquitoes, birds, and humans. We show that Culex pipiens, the dominant enzootic (bird-to-bird) and bridge (bird-to-human) vector of WNV in urbanized areas in the northeast and north-central United States, shifted its feeding preferences from birds to humans by 7-fold during late summer and early fall, coinciding with the dispersal of its preferred host (American robins, Turdus migratorius) and the rise in human WNV infections. We also show that feeding shifts in Cx. tarsalis amplify human WNV epidemics in Colorado and California and occur during periods of robin dispersal and migration. Our results provide a direct explanation for the timing and intensity of human WNV epidemics. Shifts in feeding from competent avian hosts early in an epidemic to incompetent humans after mosquito infection prevalences are high result in synergistic effects that greatly amplify the number of human infections of this and other pathogens. Our results underscore the dramatic effects of vector behavior in driving the transmission of zoonotic pathogens to humans.     

Evidence of two inapparent nonoccluded viral infections of Culex tarsalis

Culex tarsalis from a mosquito colony maintained at Bakersfield, California, were infected with two noninclusion cytoplasmic viruses. Both viruses were polyhedral with average diameters of ca. 56 nm and 33 nm for C. tarsalis virus 1 and 2 (CTV1 and CTV2), respectively. CTV1 particles had an average prevalence in the colony of 24% over a 2-year period and CTV2 had a prevalence of 5%.The viruses were first observed in negatively stained preparations of crushed heads using sodium phosphotungstate. Both viruses were seen in thin sectioned material from the salivary glands, fat body, and nervous tissue of infected mosquitoes.CTV1 was retained through six serial passages by needle inoculations of infected mosquito suspensions into virus-free C. tarsalis adults. An estimated total dilution of >;10¹⁸ indicated CTV1 had multiplied in C. tarsalis. The coincident appearance of CTV2 in some of the inoculated mosquitoes through the 5th passage suggested that this virus also multiplied on serial passage.CTV1 had a median incubation period of ca. 6.6 days in adult C. tarsalis kept at 25°C. Preliminary data indicated that the median survival period of male and female C. tarsalis injected with CTV1 was reduced 2 or 3 days over that of inoculated control insects when incubated at 25°C. Infection may cause a degeneration of salivary glands in adult females.CTV1 did not infect Culex pipiens and did not produce plaques in Vero or duck embryo cell cultures.     

West Nile virus cluster analysis and vertical transmission in Culex pipiens complex mosquitoes in Sacramento and Yolo Counties,California, 2011

West Nile virus (WNV) is now endemic in California, with annual transmission documented by the statewide surveillance system. Although much is known about the horizontal avian‐mosquito transmission cycle, less is known about vertical transmission under field conditions, which may supplement virus amplification during summer and provide a mechanism to infect overwintering female mosquitoes during fall. The current study identified clusters of WNV‐infected mosquitoes in Sacramento and Yolo Counties, CA, during late summer 2011 and tested field‐captured ovipositing female mosquitoes and their progeny for WNV RNA to estimate the frequency of vertical transmission. Space‐time clustering of WNV‐positive Culex pipiens complex pools was detected in the northern Elk Grove area of Sacramento County between July 18 and September 18, 2011 (5.22 km radius; p<0.001 and RR=7.80). Vertical transmission by WNV‐infected females to egg rafts was 50% and to larvae was 40%. The estimated minimal filial infection rate from WNV‐positive, ovipositing females was 2.0 infected females/1,000. The potential contribution of vertical transmission to WNV maintenance and amplification are discussed.