West Nile virus in mosquitoes in Greece

Epidemics of West Nile virus (WNV) occurred for two consecutive years in Greece (in 2010 and 2011). A total of 16,116 adult Culex pipiens mosquitoes collected in two peripheries, Central Macedonia and Thessaly, were tested for WNV infection. WNV lineage 2 was detected in 6/296 mosquito pools, three in each year. The H₂₄₉P substitution in the NS3 protein, previously associated with increased pathogenicity and thermotolerance, was detected in all six WNV-positive mosquito pools. When 21 individual C. pipiens mosquitoes were tested for the locus CQ11 to distinguish between the two C. pipiens forms, pipiens and molestus, 71.4 % were identified as pipiens, 4.7 % as molestus, and 19 % as hybrid pipiens/molestus, giving the first evidence that both C. pipiens biotypes are present in Greece, with a significant proportion being hybrids. The exact role of the C. pipiens forms and hybrids in the WNV epidemiology, in combination or not with the H₂₄₉P substitution in the virus genome, remains to be elucidated.     

Experimental studies on comparison of the potential vector competence of four species of Culex mosquitoes in China to transmit West Nile virus

To assess the risk that indigenous mosquitoes in China are capable of transmitting and sustaining West Nile virus (WNV), four important Culex mosquito species, Culex tritaeniorhynchus, Culex modestus, Culex pipiens pallens, and Culex pipiens quinquefasciatus, were allowed to feed on the artificial infectious blood meal with WNV dose of 10(6.8) plaque-forming unit/ml and tested approximately 2 wk later to determine infection and transmission rates. The results indicated that four Culex mosquitoes were competent laboratory vectors of WNV. The infection rates and transmission rates were statistical differences among different species of mosquito (chi2 = 20.620, P = 0.000; chi2 = 15.020, P = 0.005, respectively). The highest infection rate and transmission rate were obtained with Cx. tritaeniorhynchus (87.5 and 74.2%, respectively).     

Population variation of West Nile virus confers a host-specific fitness benefit in mosquitoes

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.     

West Nile virus infection in mosquitoes in the mid-south USA, 2002-2005

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was first detected in the Tennessee Valley and in Alabama in August 2001. In summer 2002, intensive viral activity was seen, but in subsequent years, viral activity settled into an enzootic pattern. Here, we report an analysis of viral activity in the mosquito fauna in the Mid-South from 2002 (the first year viral activity was detected in mosquitoes) through 2005. Eight mosquito species were infected with WNV during 2002. However, viral activity was only detected in four species--Culex salinarius Coquillett, Culex erraticus Dyar & Knab, Coquillettidia perturbans Walker, and Aedes vexans Meigen--in multiple years. The greatest number of positive pools was in Cx. erraticus and Cx. salinarius. Despite being specifically targeted for collection, Aedes albopictus Skuse was only found to be infected during the epiornitic year (2002), suggesting that under enzootic transmission conditions its role as a bridge vector in the region may not be significant. Virus-positive pools of Cx. erraticus were identified from winter-resting and early season dry ice-baited trap collections in 2005, implicating this species in WNV overwintering in Alabama. Molecular analysis of individuals initially identified as members of the Culex pipiens L. complex suggested that alleles characteristic of Cx. pipiens predominated in mosquitoes collected in Huntsville, AL, whereas alleles in the Auburn, AL, population were predominately characteristic of Culex quinquefaciatus Say. The southern boundary of the overlap zone of the two species seems to be located primarily between Huntsville and Auburn, a distance of 350 km.     

Serologic survey of potential vertebrate hosts for West Nile virus in Poland

A survey for antibodies to West Nile virus (WNV; genus ,Flavivirus) was carried out by plaque-re-duction neutralization microtesting in 78 horses, 20 domestic chickens, and 97 wild birds belonging to 10 species from different areas in Poland. Specific antibodies were detected in five juvenile (hatching-year) birds collected in 2006: three white storks (Ciconia ciconia) in a wildlife rehabilitation center (5.4% of all examined storks; the antibody titers in each bird were 1:320, 1:160, and 1:20), one free-living mute swan (Cygnus olor; the titer was 1:20), and one hooded crow (Corvus corone cornix; the titer 1:20) in a wildlife rehabilitation center; thus the overall seropositivity to WNV was 5.2% among all the birds sampled. These data do not rule out the presence of WNV activity in Poland with 100% certainty, but they indicate a significant trace that demands verification. In addition, one black-headed gull (Larus ridibundus) had neutralizing antibodies for the Usutu Flavivirus, the first case recorded in Poland.     

Insecticide resistance in malaria vector mosquitoes in a gold mining town in Ghana and implications for malaria control

Malaria control programmes in Africa, for the most part, address only treatment of the disease and supply of insecticide treated bed nets. The impact of these restricted programmes has been limited and new approaches are being advocated, including integrated vector management strategies and partnerships with industry. Mosquito surveys were carried out for AngloGold/Ashanti in preparation for their implementation of an integrated malaria control programme at the Obuasi gold mine in Ghana. Malaria vectors that were collected inside houses were identified to species and molecular forms by PCR, and tested for insecticide resistance using standard WHO bioassays and molecular target site insensitivity (kdr) assays. Species were identified as An. funestus s.s. and An. gambiae S and M forms. The An. gambiae S form samples showed resistance to DDT, pyrethroids and carbamates while An. funestus was resistant to DDT and carbamates. The An. gambiae M form occurred in very low numbers and could not be assessed reliably for resistance. The standard PCR assay for detection of the kdr mutation in An. gambiae S form showed little association with pyrethroid resistance. Subsequent sequencing of the II56 domain containing the kdr mutation from nine surviving mosquitoes showed that eight were homozygous resistant and one heterozygous. This correlated with the bioassay results and with previous studies on West African An. gambiae, but raised concerns about the reliability of the PCR assay for detection of the kdr mutation. As a result of these investigations AngloGold/Ashanti are implementing, in addition to treatment and case management, a vector control programme that includes insecticide resistance management by alternation of various classes of insecticides for house spraying, supply of ITNs, screening of houses and environmental management where appropriate, i.e. integrated vector management.     

Vector competence of North American mosquitoes (Diptera: Culicidae) for West Nile virus

We evaluated the potential for several North American mosquito species to transmit the newly introduced West Nile (WN) virus. Mosquitoes collected in the New York City metropolitan area during the recent WN virus outbreak, at the Assateague Island Wildlife Refuge, VA, or from established colonies were allowed to feed on chickens infected with WN virus isolated from a crow that died during the 1999 outbreak. These mosquitoes were tested approximately 2 wk later to determine infection, dissemination, and transmission rates. Aedes albopictus (Skuse), Aedes atropalpus (Coquillett), and Aedes japonicus (Theobald) were highly susceptible to infection, and nearly all individuals with a disseminated infection transmitted virus by bite. Culex pipiens L. and Aedes sollicitans (Walker) were moderately susceptible. In contrast, Aedes vexans (Meigen), Aedes aegypti (L.), and Aedes taeniorhynchus (Wiedemann) were relatively refractory to infection, but individual mosquitoes inoculated with WN virus did transmit virus by bite. Infected female Cx. pipiens transmitted WN virus to one of 1,618 F1 progeny, indicating the potential for vertical transmission of this virus. In addition to laboratory vector competence, host-feeding preferences, relative abundance, and season of activity also determine the role that these species could play in transmitting WN virus.     

Detection of West Nile virus lineage 2 in mosquitoes during a human outbreak in Greece

A human outbreak of West Nile virus (WNV) infections occurred in 2010 in central Macedonia, northern Greece. Most cases were observed close to four rivers forming a large Delta, a major Mediterranean wetland. WNV lineage 2 sequences were obtained from two pools of Culex pipiens mosquitoes trapped in sites where encephalitis cases occurred a few days before the trapping. The Greek strain showed the highest homology to Hungarian and South African strains, differing from the Russian WNV lineage 2 strain, which suggests that at least two lineage 2 strains have been introduced and established in Europe, causing severe disease to humans.     

Human-induced expanded distribution of Anopheles plumbeus, experimental vector of West Nile virus and a potential vector of human malaria in Belgium

For the majority of native species, human-created habitats provide a hostile environment that prevents their colonization. However, if the conditions encountered in this novel environment are part of the fundamental niche of a particular species, these low competitive environments may allow strong population expansion of even rare and stenotopic species. If these species are potentially harmful to humans, such anthropogenic habitat alterations may impose strong risks for human health. Here, we report on a recent and severe outbreak of the viciously biting and day-active mosquito Anopheles plumbeus Stephens, 1828, that is caused by a habitat shift toward human-created habitats. Although historic data indicate that the species was previously reported to be rare in Belgium and confined to natural forest habitats, more recent data indicate a strong population expansion all over Belgium and severe nuisance at a local scale. We show that these outbreaks can be explained by a recent larval habitat shift of this species from tree-holes in forests to large manure collecting pits of abandoned and uncleaned pig stables. Further surveys of the colonization and detection of other potential larval breeding places of this mosquito in this artificial environment are of particular importance for human health because the species is known as a experimental vector of West Nile virus and a potential vector of human malaria.     

Vector competence of Aedes vexans (Diptera: Culicidae) for West Nile virus and potential as an enzootic vector

Vector competence of Aedes vexans (Meigen) and Culex pipiens pipiens L. (Diptera: Culicidae) for West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was compared. Infection rates of both species were similar 14 d after feeding on chickens, with WNV titers ranging from 10(4.2) to 10(8.7) plaque-forming units (PFU)/ml. Median infectious doses and 95% confidence intervals (CI) were 10(6.0(5.8, 63)) and 10(5.7(5.4, 5.9)) PFU for Ae. vexans and Cx. p. pipiens, respectively. WNV transmission was not observed in Ae. vexans that fed on chickens with WNV titers < 10(5.0) PFU/ml, in contrast to a mean (95% CI) transmission rate of 7(2,18)% for Cx. p. pipiens. Mean WNV transmission rates for Ae. vexans and Cx. p. pipiens were 13(7,21)% and 10(5,19)%, respectively, after feeding on chickens with WNV titers of 10(5.3 +/- 0.1) and 10(5.7 +/- 0.1) PFU/ml, and 31(25,37)% and 41(30,53)% after feeding on chickens with WNV titers > or = 10(6.1 +/- 0.1) PFU/ml. Time postinfection (p.i.) significantly influenced WNV transmission by Ae. vexans as indicated by a nearly 10-fold increase in transmission rate between days 7 and 14 p.i. Mean WNV load expectorated with saliva ofAe. vexans was 10(2.4(2.1, 2.7)) PFU, and it was not significantly affected by the titer of chickens on which they originally fed or time p.i. These data indicate that vector competence of the primarily mammalophilic Ae. vexans, which also feeds on birds, approaches that of Cx. p. pipiens for WNV. Because peridomestic mammals, such as cottontail rabbits, squirrels, and chipmunks, develop WNV titers infective for Ae. vexans, this species may play a significant role in WNV enzootic cycles.     

The potential of Aedes triseriatus (Diptera: Culicidae) as an enzootic vector of West Nile virus

The susceptibility of Aedes triseriatus (Say) (Diptera: Culicidae) to low levels of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) was determined and compared with that of Culex pipiens L. to assess the likelihood of its participation in an enzootic cycle involving mammals. Ae. triseriatus and Cx. pipiens were exposed to WNV by feeding on baby chickens with WNV serum titers ranging from 10(4.1 +/- 0.1) to 10(8.6 +/- 0.1) plaque-forming units (PFU)/ml and from 10(4.1 +/- 0.1) to 10(7.0) PFU/ml, respectively. Infection rates and 95% confidence intervals (CIs) of 8% (4, 14) and 25% (15, 38) occurred in Ae. triseriatus and Cx. pipiens after feeding on chickens with WNV titers of 10(4.1 +/- 0.1) PFU/ml and increased to 65% (49, 79) and 100% (72, 100) in Ae. triseriatus and Cx. pipiens after feeding on chickens with titers of 10(7.1 +/- 0.1) PFU/ml. The mean infection rate of Ae. triseriatus ranged from 97% (84, 100) to 100% (79, 100) after feeding on chickens with WNV titers of > or = 10(8.2) PFU/ml. The infectious dose (ID)50 values for Ae. triseriatus and Cx. pipiens were 10(6.5) (6.4, 6.7) and 10(4.9) (4.6, 5.1) PFU/ml, respectively. The combined estimated transmission rate of Ae. triseriatus at 14 and 18 d after feeding on chickens with a mean WNV titer of 10(8.6 +/- 0.1) PFU/ml was 55%. Although Ae. triseriatus is significantly less susceptible to WNV than Cx. pipiens, the susceptibility of Ae. triseriatus to WNV titers < 10(5.0) PFU/ml and its ability to transmit WNV suggest that Ae. triseriatus has the potential to be an enzootic vector among mammalian populations.     

Immunity to West Nile virus

Over the past five years, West Nile (WN) virus has emerged as an important public health concern in the United States. Recent studies from experimental models of WN virus infection have increased our understanding of its pathogenesis and immunity. These include the demonstration that the gene encoding 2'-5'oligoadenylate synthetase is responsible for murine susceptibility to WN virus, the elucidation of the contributions of B, CD8(+) and gamma T cells in the control of murine WN virus infection, and the use of active immunization with envelope protein and passive transfer of immunoglobulin for immunotherapy. These efforts will facilitate the development of effective vaccines and therapies to combat WN virus.     

Nocturnal activity of mosquitoes (Diptera: Culicidae) in a West Nile virus focus in Connecticut

Six species of mosquitoes (Diptera: Culicidae) were collected in sufficient numbers for analysis in segregating traps set at 2-h intervals by using CO2 and light as attractants in a West Nile virus (family Flaviviridae, genus Flavivirus, WNV) focus in Stratford, CT. The Kolmogorov-Smirnov one-sided test for two samples was used to analyze the data. Mosquito activity began shortly before sunset and continued until shortly after sunrise the next morning. All species had geometric means that were significantly higher during the 2-h period shortly after sunset compared with the 2-h collection before sunset. Species, known to be naturally infected with WNV, were often attracted to these traps in about equal numbers at 2-h intervals during an 8- to 10-h period commencing shortly after sunset. Differences of geometric means were not significant among the four or five 2-h collection periods commencing at sunset for Aedes vexans (Meigen), Culex salinarius Coquillett, and Aedes cinereus Meigen. Aedes cantator (Coquillett) had a significantly higher geometric mean for the 2-h period commencing at sunset, and Coquillettidia perturbans (Walker) was captured in significantly greater numbers during the 2-h period starting at sunset compared with periods commencing 6 h after sunset. Culex pipiens L. tended to have an activity pattern that was primarily nocturnal. Time of night, not meteorological conditions, was the most important factor in determining the nightly variation in the number of trapped mosquitoes. Parity rates of Cx. pipiens collected during specific periods of the night were not significant. In total, 39 isolations of WNV were made from seven species collected primarily during periods of total darkness. Humans are at risk of being bitten by infected mosquitoes throughout the night.     

Aedes japonicus japonicus (Diptera: Culicidae) from Germany have vector competence for Japan encephalitis virus but are refractory to infection with West Nile virus

The interplay between arthropod-borne (arbo) viruses and their vectors is usually complex and often exert unique relationships. Aedes japonicus japonicus (Hulecoeteomyia japonica or Ochlerotatus japonicus japonicus), an invasive mosquito species with laboratory proven vector competence for a number of emerging viruses has been newly introduced to Germany and is currently expanding its range throughout the country. On the other hand, West Nile virus (WNV), an emerging arbovirus originating from Africa, is already circulating in several European countries and might soon be introduced to Germany. Because newly introduced and rapidly expanding vector species pose a potential risk for public health in Germany, we assessed the vectorial capacity of German Ae. j. japonicus populations for WNV and Japanese encephalitis virus (JEV). The results indicate that German Ae. j. japonicus are susceptible for JEV but are refractory to infection with WNV. Of 67 Ae. j. japonicus females challenged by feeding of WNV-containing blood, none had measurable amounts of WNV-RNA (0 % infection rate) on day 14 post-infection. In contrast, all females challenged with JEV were positive for JEV-RNA (100 % infection rate) on day 14 post-infection. The reason for WNV resistance remains to be determined but is independent from co-infection with other flaviviruses or the presence of endosymbiotic Wolbachia, since we found no evidence for other flavivirus infections within 1,033 tested A. j. japonicus females from the sampling region, nor detectable Wolbachia infection within 30 randomly selected individuals.     

A live attenuated West Nile virus strain as a potential veterinary vaccine

This article reviews the development of two attenuated West Nile virus (WNV) variants, WNI-25 and WNI-25A. These variants have lost the neuroinvasion trait of the parental virus. Attenuation was achieved through serial passages in mosquito cells and neutralization escape from WNV-specific monoclonal antibody. Genetic analysis reveals amino acid changes between the parental and each of the variants. The attenuated variants preserve the ability to replicate in mice and geese and to induce a protective immune response. WNI-25A was found to be a genetically stable virus. This variant was successfully used as a live vaccine to protect geese against a wild-type virulent WNV field isolate that closely resembles the WNV isolated during the 1999 New York epidemic.     

Sentinel birds in wild-bird resting sites as potential indicators for West Nile virus infections in Germany

West Nile virus (WNV) is a mosquito-transmitted flavivirus with wild birds as its natural hosts. Ravens, falcons and jays are highly susceptible for WNV and develop deadly encephalitis, while other bird species undergo only subclinical infections. Migratory birds are efficient vectors for geographic spreading of WNV. Until now, WNV infections have not been diagnosed in Germany, but infections in humans and horses have occurred recently in Austria, Hungary and Italy. To investigate potential WNV introduction by infected wild birds, we have monitored the serological status of ducks in three national sentinel stations. No WNV-positive reactions were found, whereas sera from coots from northern Iran were positive.