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.     

Avian defensive behavior and blood-feeding success of the West Nile vector mosquito, Culex pipiens

Avian defensive behavior against host-seeking arthropods influencestransmission of vector-borne pathogens by affecting mosquitobiting rate, either by preventing vector–host contactor by increasing the rate of multiple host feeding. We exposedhouse sparrows (Passer domesticus L.) and chickens (Gallus gallusdomesticus L.) to Culex pipiens pipiens L. overnight in a largeobservation cage and measured avian defensive behavior ratesand mosquito blood-feeding success. Both bird species exhibiteda range of defensive behaviors, 90% of which were foot stomps,head movements, and wing shakes. Total behavior rates increasedproportionately with mosquito density in both species, increasedafter the first hour of mosquito exposure, and decreased asindividual birds were exposed to mosquitoes multiple times.Mosquito blood-feeding success on house sparrows was high overall(82 ± 5%) and independent of behavior rates. Blood-feedingsuccess on chicks was lower (58 ± 5%) and negativelycorrelated with defensive behavior rate after the first hourof mosquito exposure. Results revealed a higher percentage ofpartial blood meals on chicks (18 ± 3% of all blood mealson chicks) than on house sparrows (4.9 ± 3%). Birds ofboth species ate an average of 9.4 ± 1.2% of mosquitoes,and this percentage was positively correlated with defensivebehavior. High mosquito feeding success on house sparrows supportsits role as a potential amplifying host of West Nile virus.     

West Nile virus genetic diversity is maintained during transmission by Culex pipiens quinquefasciatus mosquitoes

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.     

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.     

Temperature, viral genetics, and the transmission of West Nile virus by Culex pipiens mosquitoes

The distribution and intensity of transmission of vector-borne pathogens can be strongly influenced by the competence of vectors. Vector competence, in turn, can be influenced by temperature and viral genetics. West Nile virus (WNV) was introduced into the United States of America in 1999 and subsequently spread throughout much of the Americas. Previously, we have shown that a novel genotype of WNV, WN02, first detected in 2001, spread across the US and was more efficient than the introduced genotype, NY99, at infecting, disseminating, and being transmitted by Culex mosquitoes. In the current study, we determined the relationship between temperature and time since feeding on the probability of transmitting each genotype of WNV. We found that the advantage of the WN02 genotype increases with the product of time and temperature. Thus, warmer temperatures would have facilitated the invasion of the WN02 genotype. In addition, we found that transmission of WNV accelerated sharply with increasing temperature, T, (best fit by a function of T(4)) showing that traditional degree-day models underestimate the impact of temperature on WNV transmission. This laboratory study suggests that both viral evolution and temperature help shape the distribution and intensity of transmission of WNV, and provides a model for predicting the impact of temperature and global warming on WNV transmission.     

Infection and dissemination of West Nile virus in China by the potential vector,Culex pipiens pallens

The distribution of the West Nile virus (WNV) in the organs and tissues of the mosquito Culex pipiens pallens, a potential vector of WNV in China, was investigated up to 14 days after oral infection. The WNV antigen was detected in paraffin‐embedded mosquitoes using immunocytochemistry and viral titers of post‐infected mosquitoes determined by plaque assay. Viral titers sharply decreased 24 h post‐infection, were undetectable for the first few days, then rose over the course of infection. The first midgut infection appeared after one day, and the overall infection rate (based on midgut infection) was 43.9%. Other tissues, including hindgut, foregut, ovarian follicles, Malpighian tubules, and ommatidia, showed weak WNV antigens as early as three days post‐infection. Staining in the salivary glands first appeared after seven days, and the salivary gland infection rate on the 14th day was 37.5%. Specimens with no detectable WNV antigens in any tissues, and with positive results confined to the midgut, anterior midgut, and hindgut, were observed on the 14th day. The route of viral dissemination from the midgut, and the relative importance of amplifying tissues in mosquitoes' susceptibility to infection, were evaluated. The results indicate that Cx. p. pallens has the ability to harbor WNV throughout its alimentary system and that midgut epithelial cells may be the initial site of the replication of this virus in this species.     

Backward Bifurcation and Optimal Control in Transmission Dynamics of West Nile Virus

The paper considers a deterministic model for the transmission dynamics of West Nile virus (WNV) in the mosquito-bird-human zoonotic cycle. The model, which incorporates density-dependent contact rates between the mosquito population and the hosts (birds and humans), is rigorously analyzed using dynamical systems techniques and theories. These analyses reveal the existence of the phenomenon of backward bifurcation (where the stable disease-free equilibrium of the model co-exists with a stable endemic equilibrium when the reproduction number of the disease is less than unity) in WNV transmission dynamics. The epidemiological consequence of backward bifurcation is that the classical requirement of having the reproduction number less than unity, while necessary, is no longer sufficient for WNV elimination from the population. It is further shown that the model with constant contact rates can also exhibit this phenomenon if the WNV-induced mortality in the avian population is high enough. The model is extended to assess the impact of some anti-WNV control measures, by re-formulating the model as an optimal control problem with density-dependent demographic parameters. This entails the use of two control functions, one for mosquito-reduction strategies and the other for personal (human) protection, and redefining the demographic parameters as density-dependent rates. Appropriate optimal control methods are used to characterize the optimal levels of the two controls. Numerical simulations of the optimal control problem, using a set of reasonable parameter values, suggest that mosquito reduction controls should be emphasized ahead of personal protection measures.     

New evidence for the potential role of Culex pipiens mosquitoes in the transmission cycle of West Nile virus in Tunisia

Physiological and molecular characteristics of natural populations of Culex pipiens Linnaeus, 1758 (Diptera: Culicidae) were investigated to elucidate how this species is potentially involved in the transmission of West Nile virus in Tunisia. A total of 215 Cx. pipiens females from 11 breeding habitats were analysed in the laboratory to estimate autogeny and stenogamy rates. They were tested individually for the locus CQ11 to distinguish between the two Cx. pipiens forms, pipiens and molestus. All tested Cx. pipiens populations were stenogamous. Females from underground breeding sites were all autogeneous, whereas females from above‐ground habitats were mostly anautogeneous. Of all the females tested, 59.7% were identified as pipiens, 22.4% as molestus, and 17.9% as hybrid pipiens/molestus. Furthermore, both Cx. pipiens forms and their hybrids were found to co‐occur in sympatry in all sites. The results of this study represent the first evidence that both Cx. pipiens forms and their hybrids are present in Tunisia. Because hybrids able to act as bridge vectors are present in all studied habitats, Tunisia can be considered to have a high degree of receptivity for the establishment of West Nile virus zoonotic cycles.     

Culex pipiens, an experimental efficient vector of West Nile and Rift Valley fever viruses in the Maghreb region

West Nile fever (WNF) and Rift Valley fever (RVF) are emerging diseases causing epidemics outside their natural range of distribution. West Nile virus (WNV) circulates widely and harmlessly in the old world among birds as amplifying hosts, and horses and humans as accidental dead-end hosts. Rift Valley fever virus (RVFV) re-emerges periodically in Africa causing massive outbreaks. In the Maghreb, eco-climatic and entomologic conditions are favourable for WNV and RVFV emergence. Both viruses are transmitted by mosquitoes belonging to the Culex pipiens complex. We evaluated the ability of different populations of Cx. pipiens from North Africa to transmit WNV and the avirulent RVFV Clone 13 strain. Mosquitoes collected in Algeria, Morocco, and Tunisia during the summer 2010 were experimentally infected with WNV and RVFV Clone 13 strain at titers of 10(7.8) and 10(8.5) plaque forming units/mL, respectively. Disseminated infection and transmission rates were estimated 14-21 days following the exposure to the infectious blood-meal. We show that 14 days after exposure to WNV, all mosquito st developed a high disseminated infection and were able to excrete infectious saliva. However, only 69.2% of mosquito strains developed a disseminated infection with RVFV Clone 13 strain, and among them, 77.8% were able to deliver virus through saliva. Thus, Cx. pipiens from the Maghreb are efficient experimental vectors to transmit WNV and to a lesser extent, RVFV Clone 13 strain. The epidemiologic importance of our findings should be considered in the light of other parameters related to mosquito ecology and biology.     

Characterization of Culex pipiens Complex (Diptera: Culicidae) Populations in Colorado,USA Using Microsatellites

Mosquitoes such as those in the Culex pipiens complex are important vectors of disease. This study was conducted to genetically characterize Cx. pipiens complex populations in the state of Colorado, USA, and to determine the number of genetic clusters represented by the data. Thirteen populations located among four major river basins were sampled (n = 597 individuals) using a panel of 14 microsatellites. The lowest-elevation sites had the highest Expected Heterozygosity (H_E) values (range 0.54–0.65). AMOVA results indicated the presence of statistically significant amounts of variation within each level when populations were analyzed as one group or when they were grouped either by river basin or by their position on the east or west side of the Rocky Mountains. Most pairwise F_ST values were significant via permutation test (range 0–0.10), with the highest values from comparisons with Lamar, in southeast CO. A neighbor joining tree based on Cavalli–Sforza and Edwards’s chord distances was consistent with the geographic locations of populations, as well as with the AMOVA results. There was a significant isolation by distance effect, and the cluster analysis resolved five groups. Individuals were also assayed with an additional microsatellite marker, Cxpq78, proposed to be monomorphic in Cx. pipiens but polymorphic in the closely related but biologically distinct species Cx. quinquefasciatus. Low frequencies (≤3%) of Cx. quinquefasciatus alleles for this marker were noted, and mostly confined to populations along the Interstate 25 corridor. Pueblo was distinct in that it had 10% Cx. quinquefasciatus alleles, mostly of one allele size. The degree of population genetic structure observed in this study is in contrast with that of Cx. tarsalis, the other major vector of WNV in the western U.S., and likely reflects the two species’ different dispersal strategies.     

Threshold Conditions for West Nile Virus Outbreaks

In this paper, we study the stability and saddle-node bifurcation of a model for the West Nile virus transmission dynamics. The existence and classification of the equilibria are presented. By the theory of K-competitive dynamical systems and index theory of dynamical systems on a surface, sufficient and necessary conditions for local stability of equilibria are obtained. We also study the saddle-node bifurcation of the system. Explicit subthreshold conditions in terms of parameters are obtained beyond the basic reproduction number which provides further guidelines for accessing control of the spread of the West Nile virus. Our results suggest that the basic reproductive number itself is not enough to describe whether West Nile virus will prevail or not and suggest that we should pay more attention to the initial state of West Nile virus. The results also partially explained the mechanism of the recurrence of the small scale endemic of the virus in North America. Supported by the Chinese NSF grants 10531030 and 10671143. Supported by the Chinese NSF grants 10801074. Supported by Canada Research Chairs Program, Mathematics for Information Technology and Complex Systems (MITACS), National Microbiology Laboratory, Natural Sciences and Engineering Research Council (NSERC), Canadian Foundation of Innovation (CFI) and Ontario Innovation Trust (OIT), Ontario Ministry of Health and Long-term Care, Peel, Toronto, Chat-Kent Health Units, and Public Health Agency of Canada (PHAC). Supported by NSERC, MITACS, CFI/OIT a new opportunity fund, Early Research Award of Ministry of Research and Innovation (ERA) of Ontario, Infectious Diseases Branch of Ministry of Health and Long Term Care (MOH) of Ontario and PHAC.     

Odorant-Binding Protein from Culex tarsalis,the Most Competent Vector of West Nile Virus in California

We have identified and cloned an odorantbinding protein from the female mosquito, Culex tarsalis (CtarOBP). As expected for an olfactory protein, CtarOBP was detected by gel electrophoresis analysis in antennae but not in control tissues (legs). The isolated protein was identified by in-gel digestion and subsequent analysis of internal fragments by tandem mass spectrometry (MS-MS). Based on the amino acid sequences of two peptides generated by enzymatic digestion, degenerate primers were designed for cDNA cloning. The complete cDNA (cloned by RACE) encoded a protein with a signal peptide (24 residues) and a mature protein of 125 amino acid residues. The calculated molecular mass and isoelectric point of the mature protein were 14,515 Da and pl 5.5, respectively. CtarOBP showed the hallmark of odorant-binding proteins, 6 cysteine residues, and high sequence homology (61–96%) to previously characterized mosquito OBPs.     

Dynamics of West Nile Virus Persistence in House Sparrows (Passer domesticus)

West Nile Virus (WNV) is now endemic throughout North America, with annual recurrence dependent upon successful overwintering when cold temperatures drive mosquito vectors into inactivity and halt transmission. To investigate whether avian hosts may serve as an overwintering mechanism, groups of eight to ten House Sparrows were experimentally infected with a WN02 genotype of WNV and then held until necropsy at 3, 5, 7, 9, 12, 15, or 18 weeks post-infection (pi) when they were assessed for the presence of persistent infection. Blood was collected from all remaining birds every two weeks pi, and sera tested for WNV RNA and WNV neutralizing antibodies. West Nile virus RNA was present in the sera of some birds up to 7 weeks pi and all birds retained neutralizing antibodies throughout the experiment. The detection of persistently infected birds decreased with time, from 100% (n = 13) positive at 3 weeks post-infection (pi) to 12.5% (n = 8) at 18 weeks pi. Infectious virus was isolated from the spleens of birds necropsied at 3, 5, 7 and 12 weeks pi. The current study confirmed previous reports of infectious WNV persistence in avian hosts, and further characterized the temporal nature of these infections. Although these persistent infections supported the hypothesis that infected birds may serve as an overwintering mechanism, mosquito-infectious recrudescent viremias have yet to be demonstrated thereby providing proof of principle.     

Transmission Dynamics of the West Nile Virus in Mosquito Vector Populations under the Influence of Weather Factors in the Danube Delta,Romania

EcoHealth - Mosquitoes were collected in the Danube Delta during the active seasons of 2011–2013. For Culex spp. mosquitoes, the abundance was calculated. Culex pipiens (sensu lato), (s.l.)...     

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.