Experimental vertical transmission of West Nile virus by Culex pipiens (Diptera: Culicidae)

Despite the detection of West Nile (WN) virus in overwintering Culex pipiens L. in New York in February 2000, the mechanism by which this virus persists throughout the winter to initiate infections in vertebrate hosts and vectors the following spring remains unknown. After a blood meal, parous mosquitoes generally do not survive until spring and gonotrophic dissociation occurs in only a small percentage of the population. To investigate vertical transmission as a means of viral survival during interepizootics, we intrathoracically inoculated Cx. pipiens and Aedes albopictus (Skuse) with WN virus and subsequently tested their F1 progeny for the presence of virus. Among the Cx. pipiens, we recovered virus from two of 1,417 adult progeny that had been reared at 18 degrees C for a minimal filial infection rate (MFIR) of approximately 1.4/1,000 and four of 1,873 adult progeny reared at 26 degrees C (MFIR = 2.1/1,000). The mean titer of the positive pools was 10(5.6) plaque-forming units (PFU)/ml (=10(5.9) PFU/mosquito for positive mosquitoes) of virus. Overall, the MFIR was approximately 1.8/1,000 for Cx. pipiens. Although reports indicate that Ae. albopictus vertically transmit various viruses in the Japanese encephalitis virus complex, we did not detect WN virus in any of > 13,000 F1 progeny of WN virus-inoculated specimens. Female Cx. pipiens that are vertically infected during the late summer season and then survive the winter could serve as a source of WN virus to initiate an infection cycle the following spring.     

Ultrastructural study of West Nile virus pathogenesis in Culex pipiens quinquefasciatus (Diptera: Culicidae)

The ultrastructural features of West Nile virus (WNV) replication and dissemination in orally infected Culex pipiens quinquefasciatus Say were analyzed over a 25-d infection period. To investigate the effects of virus replication on membrane induction, cellular organization, and cell viability in midgut and salivary gland tissues, midguts were dissected on days 3, 7, 14, and 21, and salivary glands were collected on days 7, 14, 21, and 25 postinfection (d.p.i.) for examination by transmission electron microscopy (TEM). Whole mosquito heads were embedded for TEM analysis 14 d.p.i. to localize WNV particles and to investigate the effects of replication on nervous tissues of the brain. Membrane proliferation was induced by WNV in the midgut epithelium, midgut muscles, and salivary glands, although extensive endoplasmic reticulum swelling was a unique feature of salivary gland infection. TEM revealed WNV-induced pathology in salivary glands at 14, 21, and 25 d.p.i., and we hypothesize that long-term virus infection of this tissue results in severe cellular degeneration and apoptotic-like cell death. This finding indicates that the efficiency of WNV transmission may decrease with mosquito age postinfection.     

Vertical transmission of West Nile Virus by three California Culex (Diptera: Culicidae) species

Three California Culex species previously identified as efficient laboratory vectors of West Nile (WN) virus were tested for their capability to vertically transmit WN virus. Wild-caught Culex pipiens pipiens L., Culex pipiens quinquefasciatus Say, and two populations of Culex tarsalis Coquillett females were inoculated intrathoracically with 10(2.7 +/- 0.1) plaque-forming units of WN virus. F1 progeny were reared at 18 degrees C and subsequently tested as adults for infectious virus on Vero cell culture. Virus was not detected in 197 pools comprising 4,884 Cx. p. pipiens. The minimum filial infection rate (MFIR) for Cx. p. quinquefasciatus was approximately 3.0/1,000 for 665 progeny tested in 28 pools. There was no virus detected in 102 pools of 2,453 progeny from Cx. tarsalis collected in Riverside County. The MFIR for Cx. tarsalis collected in Yolo County was approximately 6.9/1,000 for 2,165 progeny tested in 86 pools. Mosquito progeny infected vertically during the fall could potentially serve as a mechanism for WN virus to overwinter and initiate horizontal transmission the following spring.     

CNAct-1 gene is differentially expressed in the subtropical mosquito Culex nigripalpus (Diptera: Culicidae), the primary West Nile Virus vector in Florida

Analysis of differentially expressed genes is a common molecular biological tool to investigate changes in mosquito genes after a bloodmeal or parasite exposure. We report here the characterization of a differentially expressed actin gene, CNAct-1, from the subtropical mosquito, Culex nigripalpus Theobald (Diptera: Culicidae). The CNAct-1 genomic clone is 1.525 kb, includes one 66-bp intron, and a 328-bp 3'-untranslated region. The 376-amino acid putative translation product shares high similarity with muscle-specific actin proteins from other insects, including Culex pipiens pipiens L., Aedes aegypti (L.), Anopheles gambiae Giles and Drosophila melanogaster (Meigen). CNAct-1 is expressed in second and third instars, late pupae, and adult females and males. Interestingly, Cx. nigripalpus actin was highly expressed in female mosquito midgut tissue isolated 6-12 h after ingestion of a bloodmeal. This expression profile indicates a unique function for CNAct-1 in midgut processes that are initiated after blood ingestion.     

Culex pipiens (Diptera: Culicidae): a bridge vector of West Nile virus to humans

Host-feeding patterns of Culex pipiens L. collected in southwest suburban Chicago in 2005 were studied using polymerase chain reaction (PCR) and DNA sequencing techniques. Culex spp. mosquitoes, most identified to Cx. pipiens and the remainder to Cx. restuans by PCR, had fed on 18 avian species, most commonly American robin (Turdus migratorious), house sparrow (Passer domesticus), and mourning dove (Zenaida macroura). Additional blood meals were derived from four mammal species, primarily humans and raccoons (Procyon lotor). During a West Nile virus (WNV) epidemic in 2005, West Nile virus (WNV) RNA was detected in heads and thoraces of five Cx. pipiens (n = 335, 1.5%) using quantitative PCR. The hosts of these virus-infected, blood-fed mosquitoes included two American robins, one house sparrow, and one human. This is the first report of a WNV-infected Cx. pipiens mosquito collected during an epidemic of WNV that was found to have bitten a human. These results fulfill a criterion for incrimination of Cx. pipiens as a bridge vector.     

Extrinsic incubation periods for horizontal and vertical transmission of West Nile virus by Culex pipiens pipiens (Diptera: Culicidae)

Culex pipiens pipiens L. (Diptera: Culicidae), infected per os from a membrane feeder, transmitted West Nile virus (family Flaviviridae, genus Flavivirus, WNV) at 26 degrees C horizontally during feeding to hamsters and suckling mice and vertically to F1 progeny during egg deposition. Horizontal transmission rates increased with extrinsic incubation, with 75-100% of the females transmitting on days 16 through 25 postinfection (pi). No females deposited eggs infected with WNV after the first bloodmeal on 3-8 d pi. Females vertically transmitted WNV during egg laying after their second, third, and fourth bloodmeals on days 13-33 pi. The vertical transmission rate was 4.7%. The vertical minimal infection rate was 0.52 infected F1 specimens/1,000 specimens tested from females feeding during their second and later bloodmeals on hamsters or suckling mice. The sequence of horizontal and vertical transmission is reported. A female may transmit WNV 1) horizontally to a host during feeding and subsequently vertically to her offspring during egg laying, 2) vertically to her offspring during oviposition without prior horizontal transmission to a host, and 3) horizontally to a host without vertically transmitting the virus. These two means of transmission by Cx. p. pipiens contribute to the relatively high minimum infection rates that are reached in late summer and to the survival of virus during winter and initiation of amplification in the spring in the northeastern United States.     

Rainfall-directed oviposition behavior of Culex nigripalpus (Diptera: Culicidae) and its influence on St. Louis encephalitis virus transmission in Indian River County, Florida

Resting Culex nigripalpus Theobald were collected with a ground aspirator three times a week in Indian River County, Fla., from January 1985 through December 1987. The number of green (newly emerged), empty, blood-fed, and gravid females in each collection was counted; up to 100 empty females were dissected to estimate the abundance of empty, parous females in the population. In general, this species was uncommon during the dry season from January through June but increased during the wet season in July and remained abundant through early winter. During the wet season, green, parous, and gravid females were periodically abundant. Time series analysis demonstrated that green and parous mosquitoes were positively associated through time and that gravid females were associated negatively with daily rainfall. The strongest correlations were recorded in 1985, a year during which St. Louis encephalitis virus was detected with increased frequency in Indian River County. Laboratory experiments in a large outdoor wind tunnel confirmed that high relative humidity was important for the initiation and successful completion of oviposition by gravid, field-collected Cx. nigripalpus mosquitoes. Thus, intermittent rainfall during autumn may serve to delay oviposition, increase longevity, synchronize blood feeding, and enhance transmission of St. Louis encephalitis virus by infected mosquitoes in the field.     

Culex restuans (Diptera: Culicidae) relative abundance and vector competence for West Nile Virus

The abundance and vector competence of Culex restuans Theobald and Culex pipiens L. were compared to determine the relative importance of these species as West Nile virus (WNV) vectors in the northeastern United States. Abundance was estimated from egg raft collections at 12 sites in Albany, Suffolk, and Richmond counties, New York, during July, August, and September 2002 and 2003. Cx. restuans was more abundant than Cx. pipiens in both urban and rural areas, comprising 86% of 1,623 egg rafts collected. Vector competence for WNV was estimated after feeding on an artificial bloodmeal and in vitro transmission assays. The vector competence of the two species for WNV was similar, but the dynamics of infection seems to be mosquito species dependent. These findings suggest an important role for Cx. restuans in WNV transmission cycles in New York.     

West Nile virus-infected dead corvids increase the risk of infection in Culex mosquitoes (Diptera: Culicidae) in domestic landscapes

A comparative study of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) infection rates in Culex mosquitoes collected at 13 sites, seven reporting WNV-positive dead corvids (case sites) and six without reported dead birds (control sites) was conducted in Davis, CA, from 14 to 21 July at the beginning of the 2006 WNV outbreak. In total, 3051 Culex mosquitoes were collected using gravid traps and CO2-baited traps; WNV-infected mosquitoes were only collected with CO2-baited traps. WNV-infected Culex pipiens L. were collected at one of the seven case sites. Six of seven case sites yielded WNV-infected Culex tarsalis Coquillett, whereas only one of six control sites had WNV-infected Cx. tarsalis. Overall, the odds of finding WNV-positive mosquitoes were 19.75 times greater at sites reporting a WNV-infected dead corvid than sites without a WNV-infected dead corvid. Maximum likelihood estimates of the overall infection rates at the case sites were 3.48/1000 for Cx. tarsalis and 8.69/1000 for Cx. pipiens compared with 1.02/1000 in Cx. tarsalis collected at the control sites. Results indicate that Cx. tarsalis was important in early season enzootic transmission within Davis and that sites reporting WNV-infected dead corvids are areas to focus control and surveillance efforts.     

Effect of environmental temperature on the ability of Culex pipiens (Diptera: Culicidae) to transmit West Nile virus

Environmental temperature can affect the ability of mosquitoes to transmit an arbovirus. However, results of various studies indicate that these effects are not consistent among viruses or mosquito species, and there is no information available on the effect of environmental temperature on the ability of North American mosquito species to transmit West Nile (WN) virus. We evaluated the effect of incubation temperature (18, 20, 26, or 30 degrees C) on the ability of Culex pipiens L. derived from specimens collected during the outbreak in New York in 1999 to transmit a strain of WN virus obtained from a crow that died during this outbreak. Although mosquitoes fed on the same viremic chickens, infection rates were directly related to subsequent incubation temperatures. In mosquitoes held at 30 degrees C, virus was recovered from nearly all mosquitoes tested, disseminated infections were detected as early as 4 d after the infectious blood meal, and >90% of all mosquitoes had a disseminated infection 12 or more days after the infectious blood meal. In contrast, for mosquitoes held at 18 degrees C, disseminated infections were not detected until 25 d after the infectious blood meal, and even after 28 d, <30% contained a disseminated infection. Results for mosquitoes held at 20 and 26 degrees C were intermediate for both infection and dissemination rates. The effect of environmental temperature should to be considered when evaluating the vector competence of these mosquitoes and modeling risk of WN virus transmission in nature.     

Arbovirus transmission by Culex nigripalpus in Florida, 2005

Understanding the transmission patterns of West Nile and St. Louis encephalitis viruses (family Flaviviridae, genus Flavivirus, WNV and SLEV) could result in an increased ability to predict transmission risk to humans. To examine transmission patterns between vector and host, we trapped mosquitoes in three Florida counties from June to November 2005 by using chicken-baited lard can mosquito traps. These traps were used to monitor for presence of WNV and SLEV in mosquitoes and subsequent transmission of these viruses to chickens. In total, 166,615 female mosquitoes were sorted into 4,009 pools based on species and bloodfed status, and they were tested for presence of WNV and SLEV. Sera from 209 chickens were tested for WNV and SLEV antibodies. We detected eight WNV-positive Culex nigripalpus Theobald mosquito pools; SLEV was not detected in any pools. Six positive pools were collected in August and September from Duval County, one pool in September from Manatee County, and one pool in November from Indian River County. Of the eight chickens potentially exposed to WNV, antibodies were detected in only one chicken, indicating a low rate of transmission relative to the observed mosquito infection rates. Low virus transmission rates relative to infection rates would suggest that using sentinel chicken seroconversion data as a means of arbovirus surveillance may underestimate the prevalence of WNV in the mosquito population. However, using mosquito infection rates may overestimate the risk of arboviral transmission. A variety of factors might account for the observed low level of transmission including a lack of viral dissemination in mosquito vectors.     

Mosquito (Diptera: Culicidae) bloodmeal sources during a period of West Nile virus transmission in Puerto Rico

Host bloodmeals of indigenous Caribbean mosquitoes have not been studied previously. We identified vertebrate DNA in 90 blood-engorged mosquitoes belonging to four genera (Aedes, Culex, Deinocerites, and Uranotaenia) and 12 species that were collected in Puerto Rico within a geographic and temporal focus of West Nile virus transmission in 2007. It was found that 62 (68.8%) bloodmeals were from reptiles, 18 (20.0%) from birds, and 10 (11.1%) from mammals. Only one bloodmeal of 18 derived from Culex (Culex) species was passerine, suggesting a preference for nonpasserine birds and other vertebrates (i.e., reptiles) among the candidate WNV vectors. We interpret the results with respect to vectorial capacity for West Nile virus, an emerging arbovirus throughout the Caribbean Basin.     

Temporal and geographic genetic variation in Culex nigripalpus theobald (Culicidae: Diptera), a vector of St. Louis encephalitis virus,from Florida

A field population of Culex (Culex) nigripalpus Theobald from Vero Beach, FL sampled monthly over a period of 24 mo, a colony sample and 10 geographic samples were analyzed for genetic variation at 14 enzyme loci using polyacrylamide gel electrophoresis. The Cx. nigripalpus colony sample showed significantly lower genetic variation than the field-collected samples, measured by mean number of alleles per locus (colony 1.4 +/- 0.1 versus field 2.1 +/- 0.22), percentage of polymorphic loci (colony 35.7% versus field 54.8 +/- 7.7%), but mean observed heterozygosity (Ho = colony 0.16 +/- 0.07 versus field 0.17 +/- 0.03) and mean Hardy-Weinberg expected heterozygosity (He = colony 0.14 +/- 0.06 versus field 0.18 +/- 0.02) did not differ significantly. Three of the 14 loci (Aldox, Gpd, and Gpi) from the Vero Beach field samples showed distinct temporal patterns in the frequency of the most common allele. Higher mean observed heterozygosity (Ho) occurred during months following high rainfall in the Vero Beach field samples than during months following low rainfall. The average Nm value of 3.6 indicated high gene flow among the temporally distributed samples of the Vero Beach population. Genetic variability values between geographic samples from Panhandle, FL and south Florida were not significant. Gene flow estimates based on F(ST) = 0.039 provided a Nm of 6.2 indicating high levels of gene flow among the geographic samples of Cx. nigripalpus. The average Nei's and modified Rogers' genetic distances among the 10 populations were 0.009 +/- 0.001 and 0.081 +/- 0.004, respectively. The cluster analysis did not suggest geographic clustering, Because Cx. nigripalpus is the vector of St. Louis encephalitis (SLE) in Florida, temporal and geographic genetic variation in this species is discussed in relation to the seasonal and geographic SLE virus activity in Florida.     

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.     

Early evening questing and oviposition activity by the Culex (Diptera: Culicidae) vectors of West Nile virus in northeastern North America

To determine whether the Culex (Diptera: Culicidae) mosquitoes that transmit West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in the northeastern United States seek hosts and oviposit contemporaneously, we recorded when these mosquitoes attacked caged birds and when they deposited eggs. They traversed oviposition sites most frequently approximately 2 h after astronomical sunset, and eggs generally were deposited at that time. Although they most frequently approached avian hosts approximately 2 h after sunset during midsummer, they are more opportunistic during mid- to late fall. Because the Culex mosquitoes that serve as the main vectors of West Nile virus in the northeastern United States quest for hosts and seek to oviposit well after sunset, insecticidal aerosols would be most effective when applied at that time.     

Host-feeding patterns of Culex pipiens and other potential mosquito vectors (Diptera: Culicidae) of West Nile virus (Flaviviridae) collected in Portugal

The host blood-feeding patterns of mosquito vectors affects the likelihood of human exposure to zoonotic pathogens, including West Nile Virus (family Flaviviridae, genus Flavivirus, WNV). In Portugal, data are unavailable regarding the blood-feeding habits of common mosquito species, including Culex pipiens L., considered the primary vector of WNV to humans. The sources of bloodmeals in 203 blood-fed mosquitoes of nine species collected from June 2007 to November 2010 in 34 Portuguese counties were analyzed by sequencing cytochrome-b partial fragments. Cx. pipiens was the most common species collected and successfully analyzed (n = 135/78). In addition, blood-fed females of the following species were analyzed: Ochlerotatus caspius Pallas (n = 20), Culex theileri Theobald (n = 16), Anopheles maculipennis s.l. Meigen (n = 10), Culiseta longiareolata Macquart (n = 7), Aedes aegypti L. (n = 6), Culex perexiguus Theobald (n = 3), Culiseta annulata Schrank (n = 3), and Ochlerotatus detritus Haliday (n = 3). The Cx. pipiens mosquitoes fed predominantly on birds (n = 55/78, 70.5%), with a high diversity of avian species used as hosts, although human blood was identified in 18 specimens (18/78, 23.1%). No significant differences were found between the host-feeding patterns of blood-fed Cx. pipiens collected in residential and nonresidential habitats. The occurrence of human derived blood meals and the presence of a mix avian-human bloodmeal accordingly suggest this species as a potential vector of WNV. Therefore, in Portugal, Cx. pipiens may play a role both in the avian-to-avian enzootic WNV cycle and in the avian-to-mammal transmission. In this context, the identity of Cx. pipiens (considering the forms molestus and pipiens) and the potential consequence on feeding behavior and WNV transmission are discussed.     

Blood feeding and oviposition by Culex nigripalpus (Diptera: Culicidae) before, during, and after a widespread St. Louis encephalitis virus epidemic in Florida

A widespread epidemic of St. Louis encephalitis (SLE) virus was reported from central Florida from late July through December 1990 with 226 clinical cases and 11 deaths in humans. The abundance of blood-fed and gravid Culex nigripalpus Theobald females, the vector of SLE in Florida, was monitored at a resting site in Indian River County, FL, for the 3 yr before the epidemic, the epidemic year, and for 1 yr following the epidemic. Each study year was divided into four 3-mo periods that depict the subtropical SLE transmission cycle; a January-March maintenance phase, an April-June amplification phase, a July-September early transmission phase, and an October-December late transmission phase. The abundance of blood-fed and gravid females was significantly greater during the amplification, early transmission, and late transmission phases of the 1990 SLE epidemic when compared with the 3 pre-epidemic years. Throughout the study, Cx. nigripalpus blood feeding and oviposition was driven by epic (> 50 mm) rainfall events. The abundance of gravid females decreased, whereas the number of blood-fed females increased following heavy rainfalls. Epizootic and epidemic transmission of SLE to sentinel chickens and humans in Indian River County was greatest immediately following heavy rainfalls that synchronized Cx. nigripalpus oviposition and blood feeding.