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ABSTRACTWe report the development of nucleic acid sequence-based amplification (NASBA) and quantitative real-time reverse transcription (RT)-PCR assays for the detection of La Crosse (LAC) virus in field-collected vector mosquito samples and human clinical samples. The sensitivities of these assays were compared to that of a standard plaque assay in Vero cells. The NASBA and quantitative real-time RT-PCR assays demonstrated sensitivities greater than that of the standard plaque assay. The specificities of these assays were determined by testing a battery of reference strain viruses, including representative strains of LAC virus and other arthropod-borne viruses. Additionally, these assays were used to detect LAC viral RNA in mosquito pool samples and human brain tissue samples and yielded results within less than 4 h. The NASBA and quantitative real-time RT-PCR assays detect LAC viral RNA in a sensitive, specific, and rapid manner; these findings support the use of these assays in surveillance and diagnostic laboratory systems.

ABSTRACTAn antigen capture immunoassay to detect West Nile (WN) virus antigen in infected mosquitoes and avian tissues has been developed. With this assay purified WN virus was detected at a concentration of 32 pg/0.1 ml, and antigen in infected suckling mouse brain and laboratory-infected mosquito pools could be detected when the WN virus titer was 102.1to 103.7PFU/0.1 ml. In a blindly coded set of field-collected mosquito pools (n= 100), this assay detected WN virus antigen in 12 of 18 (66.7%) TaqMan-positive pools, whereas traditional reverse transcriptase PCR detected 10 of 18 (55.5%) positive pools. A sample set of 73 organ homogenates from naturally infected American crows was also examined by WN virus antigen capture immunoassay and TaqMan for the presence of WN virus. The antigen capture assay detected antigen in 30 of 34 (88.2%) TaqMan-positive tissues. Based upon a TaqMan-generated standard curve of infectious WN virus, the limit of detection in the antigen capture assay for avian tissue homogenates was approximately 103PFU/0.1 ml. The recommended WN virus antigen capture protocol, which includes a capture assay followed by a confirmatory inhibition assay used to retest presumptive positive samples, could distinguish between the closely related WN and St. Louis encephalitis viruses in virus-infected mosquito pools and avian tissues. Therefore, this immunoassay demonstrates adequate sensitivity and specificity for surveillance of WN virus activity in mosquito vectors and avian hosts, and, in addition, it is easy to perform and relatively inexpensive compared with the TaqMan assay.

ABSTRACTThe authors report on the development and application of a rapid TaqMan assay for the detection of West Nile (WN) virus in a variety of human clinical specimens and field-collected specimens. Oligonucleotide primers and FAM- and TAMRA-labeled WN virus-specific probes were designed by using the nucleotide sequence of the New York 1999 WN virus isolate. The TaqMan assay was compared to a traditional reverse transcriptase (RT)-PCR assay and to virus isolation in Vero cells with a large number (˜500) of specimens obtained from humans (serum, cerebrospinal fluid, and brain tissue), field-collected mosquitoes, and avian tissue samples. The TaqMan assay was specific for WN virus and demonstrated a greater sensitivity than the traditional RT-PCR method and correctly identified WN virus in 100% of the culture-positive mosquito pools and 98% of the culture-positive avian tissue samples. The assay should be of utility in the diagnostic laboratory to complement existing human diagnostic testing and as a tool to conduct WN virus surveillance in the United States.

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

In 2006–2007, stable flies, Stomoxys calcitrans (L.) (Diptera: Muscidae), were suspected of being enzootic vectors of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) during a die-off of American white pelicans (Pelecanus erythrorhynchos Gmelin) (Pelecanidae) in Montana, USA. WNV-positive stable flies were observed feeding en masse on incapacitated, WNV-positive pelicans, arousing suspicions that the flies could have been involved in WNV transmission among pelicans, and perhaps to livestock and humans. We assessed biological transmission by infecting stable flies intrathoracically with WNV and testing them at 2-d intervals over 20 d. Infectious WNV was detected in fly bodies in decreasing amounts over time for only the first 6 d postinfection, an indication that WNV did not replicate within fly tissues and that stable flies cannot biologically transmit WNV. We assessed mechanical transmission using a novel technique. Specifically, we fed WNV-infected blood to individual flies by using a cotton swab (i.e., artificial donor), and at intervals of 1 min–24 h, we allowed flies to refeed on a different swab saturated with WNV-negative blood (i.e., artificial recipient). Flies mechanically transmitted viable WNV from donor to recipient swabs for up to 6 h postinfection, with the majority of the transmission events occurring within the first hour. Flies mechanically transmitted WNV RNA to recipient swabs for up to 24 h, mostly within the first 6 h. Given its predilection to feed multiple times when disturbed, these findings support the possibility that the stable fly could mechanically transmit WNV.

In total, 1,324 Culex pipiens pipiens L. female mosquitoes were collected at Ft. Hancock, Monmouth County, New Jersey, from January to March 2001–2003. Mosquitoes were held in an insectary at 27°C and a photoperiod of 16:8 (L:D) h for 6 to 21 d after which they were tested in 34 pools. West Nile viral RNA was detected in one pool by a TaqMan reverse transcription-polymerase chain reaction assay; however, infectious virus could not be isolated using either Vero cell plaque assay or C6/36 mosquito cells. Twenty females dissected in January and March 2003 confirmed ovarian diapause status. We suggest that the mode of infection in this pool of overwintering females may have been due to vertical (transgenerational) transmission.

At adult emergence, the midgut is distended with gas and the diverticula are empty. Gas then disappears from the midgut and, by 24 h the diverticula are inflated to a volume which remains unchanged for at least 48 h. By 24 h after ingestion of blood, the diverticula are nearly devoid of gas and are then re-inflated. Diverticular gas volume decreases immediately following a sugar meal in specimens starved for 24 h and then increases at a constant rate. Diverticular gas volume does not decrease immediately following ingestion of sugar in specimens starved for 72 h. We suggest the following: (1) diverticular gas decreases specific gravity when an increase in the efficiency of utilization of energy reserves would be advantageous, i.e., when reserves are depleted and when the female is ready to fly to an oviposition site; and (2) the proportion of a sugar meal which goes to the diverticula and to the midgut is a function of physiological state.

Studies were conducted on the functions of the ventral air space (VAS) and 1st abdominal spiracles in Ae. aegypti pupae. The VAS is filled with gas from the tracheal system during larval-pupal ecdysis. Gas appears to be forced into the VAS by hemostatic pressure caused by contraction of body musculature. Mean VAS gas volume (0.8000 ± 0.0339 mm3) does not change between larval-pupal and pupal-adult ecdysis. There is a moderate positive correlation between size and VAS gas volume. Although gas can be withdrawn from the VAS via the 1st abdominal spiracles, pupae do not replace lost VAS gas. Therefore, VAS gas volume probably is not regulated. The VAS cuticle is hydrophobic, and the remaining pupal cuticle is hydrophilic. Due to the hydrophobic property, VAS gas is held in place during ecdysis and throughout the pupal-pharate adult period. The mechanisms of both temporary and permanent buoyancy changes in pupae are discussed.

Biting persistence was measured for Aedes aegypti (L.) mosquitoes given different levels of larval and adult nutrition. Persistence was measured as the total number of times during a 60-min trial that a mosquito returned to land on a defensive host after being disturbed and dislocated from the host, and as the rate of decline of landings over the 60 min test (pattern of persistence). Large females were significantly more persistent than smaller females, regardless of whether the females were provided water or 5% sucrose for 3 d before the test. Access to sucrose for 3 or 7 d did not influence the total number of landings of large females. Large females starved for 7 d exhibited significantly reduced persistence compared with large females starved for only 3 d. Water-fed specimens displayed a more rapid decline in the pattern of persistence than sucrose-fed specimens. Field-collected females, intermediate in size between the large and small laboratory females, had dry weights indicating they were relatively starved. They had a total number of landings similar to the small laboratory females and a persistence pattern similar to sucrose-fed specimens. Sizerelated differences in persistence may be one factor causing differences in parity rates and vector potential observed between large and small females in Ae. aegypti field populations.

Resting and host-seeking adult female Psorophora columbiae (Dyar & Knab), Aedes atlanticus Dyar & Knab, Aedes vexans (Meigen), and Culex salinarius Coquillett were collected. Wing length and parity of each individual were determined. Results showed that average wing lengths within the four species did not differ between the resting and host-seeking populations. Parous C. salinarius and A. vexans had significantly longer wing lengths than did nulliparous females of the same species. Within each species, individuals were grouped in size classes of 1, 2, and 3 SD above and below the mean. In all four species, the percent parous in the larger size classes was higher than in the smaller size classes. Results indicate that large-bodied mosquitoes are more successful at blood-feeding than are smaller individuals.

During August and September of 1991, an epizootic of eastern equine encephalitis (EEE) virus in horses occurred in Wayne and Holmes counties, OH. This was the first recorded epizootic of EEE virus in the state. Twelve horses were confirmed positive for EEE virus through virus isolation or seroconversion, and seven additional horses with compatible symptoms were in close spatial and temporal proximity to the confirmed cases and were presumed to have died from EEE virus. The outbreak was centered around the Killbuck Wildlife Area, a 2,147-ha tract maintained by the state, half of which consists of wooded swamp and marsh. Mosquitoes were collected in upland areas before the epizootic and in the swamp basin at the end of the epizootic to identify the mosquito species involved in EEE virus transmission. We collected and tested 22,095 specimens for the presence of virus. EEE virus was isolated from one pool of the most likely epizootic vector, Coquillettidia perturbans (Walker). The minimum infection rate for EEE virus in this species was 0.1/1,000. Dense populations of Aedes vexans (Meigen) and Culex salinarius Coquillett occurred in the area, but their densities peaked after the epizootic. It is unlikely that these species were involved in epizootic transmission. IgM antibody to EEE virus was detected in three bird species collected in the swamp.

Aedes albopictus (Skuse) mosquitoes were fed on snowy egrets, Egretta thula (Thayer and Bangs), that had been infected by subcutaneous inoculation of eastern equine encephalitis (EEE) virus. Freshly fed mosquitoes were frozen and tested to determine how much virus they had ingested. Other fed mosquitoes from the same lots were incubated for 7 d at 27°C before testing. Seven lots of Ae. albopictus fed on viremic birds. Based on average amounts of virus ingested and day 7 virus infection rates in mosquitoes from the same lots, the amount of virus required to infect 50% of the mosquitoes was calculated to be 102.8 Vero cell plaque-forming units (PFU). The infection threshold (i.e., the amount of virus required to infect from 1 to 5% of mosquitoes) was determined to be =10 PFU per blood meal. These parameters indicate that Ae. albopictus is sufficiently susceptible to infection with EEE virus to enable it to acquire infectious doses from a wide variety of viremic birds and possibly from equines.

The blood-feeding patterns of Aedes vexans and Aedes trivittatus were studied in 3 habitat types in northern Indiana, USA. Both species fed primarily on mammals, and both exhibited a high degree of variability in the type of hosts used. Daily variation in blood feeding was detected among and within locations. Aedes vexans fed mainly on large mammals (deer, horses, and cows). However, the blood-feeding pattern varied depending upon which large mammal was most abundant in the area studied. Aedes trivittatus was more opportunistic and fed extensively on a wider variety of hosts than Ae. vexans. The results are discussed in terms of the “host image” and host-seeking behavior of the mosquito species, the effect of environmental variables on blood feeding, and the importance of local variation in blood feeding to studies of the ecology of mosquitoborne pathogens.

Resting adult Aedes triseriatus and Ae. hendersoni females were collected in a woodlot enzootic for La Crosse encephalitis (LAC) virus (Cook Co., Illinois) and in an area without LAC virus activity (St. Joseph Co., Indiana). Multiple tests were performed on each specimen to identify the species (cellulose acetate electrophoresis of the thorax), to detect disseminated LAC infection (fluorescent-antibody-stained head squash), to identify the blood meal host of engorged females (precipitin test), and to detect presence of LAC virus in the blood meal (Vero cell plaque assay). No virus was detected in any of the field-collected specimens. However, the techniques performed on specimens in the laboratory were highly effective, indicating their utility in areas with a higher field-infection rate. In the nonenzootic area, Ae. triseriatus fed predominantly on Eastern Chipmunks (61%) and Ae. hendersoni fed predominantly on Red Squirrels and Fox Squirrels (85%). In the enzootic area, both Ae. triseriatus and Ae. hendersoni fed extensively on Gray Squirrels (65 and 95%, respectively). The relative density of the 2 species in resting populations differed between areas. In the nonenzootic area, the ratio of Ae. triseriatus:Ae. hendersoni was 1.89:1, while in the enzootic area, the ratio was 0.43:1. The percentage of the resting Ae. hendersoni population that was blood engorged in the 2 areas ($\bar x$ = 47.5%) was much higher than that of Ae. triseriatus ($\bar x$ = 16.5%), suggesting that Ae. hendersoni seeks hosts in the forest canopy and, once blood fed, rests in the forest understory. Results are discussed with reference to the potential influence of local variations in host use on the dynamics of the La Crosse virus cycle.

The population structure and nectar feeding patterns of the mosquito Culiseta melanura were studied in and around a freshwater swamp in the EEE virus enzootic areas of southeastern Massachusetts, USA. During the summer of 1978, resting boxes were used to collect 12,994 female Cs. melanura, 1747 of which were dissected for parity and tested for the presence of plant nectar by the anthrone test for fructose. Large numbers of parous and nulliparous females were present at the perimeter of the swamp habitats and in the upland areas up to 4.8 km from the swamps. Population density fluctuations and seasonal parity rates suggested 4 generations or emergences over the course of the season. Population age structures varied in the 2 habitat types. Blood-engorged and gravid females were more prevalent at the swamp perimeter and nulliparous females were more prevalent outside the swamp. Parous females were equally prevalent in both habitats. Parity rate averaged 23.7% for the season, peaked in mid-summer at 40–45%, and was used to calculate a daily survival probability ranging from 0.749 to 0.814 during the study. Nectar feeding at the swamp perimeter differed between population components; parous females were positive for nectar more frequently than nulliparous females, and both were positive more frequently than gravid females. Nectar feeding outside the swamp did not vary between population components. Overall, the percentage of mosquitoes positive for nectar was higher at the perimeter than outside the swamp. Gravid females contained nectar throughout the egg maturation process. Results are discussed with reference to population dispersal, behavior of mosquitoes in various physiological states, and the EEE virus cycle.

The percentage of resting mosquitoes that had fed on blood or were gravidparous was determined in two pastures associated with the riceland agroecosystem in southwestern Louisiana. In one pasture, all of the cattle were treated with permethrin every 14 d over a 22-wk period, whereas cattle in the other pasture were untreated. The percentage of blood-engorged Psorophora columbiae (Dyar & Knab) was significantly lower in the vicinity of the treated herd. The effectiveness of the permethrin treatment in reducing blood feeding decreased over the interval between applications and as a result of increased rainfall. The proportion of Ps. columbiae that was gravid or parous was not significantly affected by the treatment. Anopheles quadrimaculatus Say engorgement rates also were significantly reduced by the treatment; however, Anopheles crucians Wiedemann and Culex salinarius Coquillett engorgement rates did not differ between the two areas. The results indicate that host management by permethrin treatment has potential for controlling Ps. columbiae and An. quadrimaculatus populations in the Louisiana riceland-pasture agroecosystem.

The relationship between dry weight at adult emergence and wing length cubed was compared by regression analysis for three mosquito species reared in the laboratory and five species collected from the field. The regression equations were compared by ANCOVA between sexes and sources within species, and among species. Results indicated that the proportional relationship of wing length to teneral adult dry weight is highly variable and that unique regression formulae are required for each mosquito species and sex. Also, the relationship between wing length and dry weight differed within species among specimens reared in the lab versus those collected from the field.

The relationship of larval nutrition and adult body size to the susceptibility of Aedes aegypti (L.) to Ross River virus infection was examined. Large adult mosquitoes produced by feeding larvae a high-level diet consumed significantly more virus particles than did smaller mosquitoes. However, when a correction for body size was made, smaller mosquitoes were found to consume significantly more virus per unit of body weight. A host viremia of 2.4 log10 PFU/ml failed to infect mosquitoes of any size. Large Ae. aegypti mosquitoes were significantly more susceptible than small mosquitoes when fed on hosts with viremias of 4.8, 5.7, 6.4, and 7.5 log10 PFU/ml, but differences in susceptibility were less apparent at higher viremias.

Pupal density, wing length at emergence, host-seeking female abundance, and host-seeking female wing length and parity were determined monthly for a population of Aedes albopictus (Skuse) in a southwestern Louisiana tire dump from January 1988 through January 1989. Pupae and host-seeking females were first collected in April. Pupal densities were consistently high from June through September. Host-seeking female abundance was highest during July and August. Average female wing length was longest during spring, declined in midsummer, and increased slightly during fall. Average wing length was negatively correlated with average pupal density during the month of collection and with average air temperature recorded the previous month. Host-seeking females had consistently longer wings than emerging females, and parous host-seeking females had longer wings than nulliparous host-seeking females. Average wing length of the parous host-seeking females was consistently long throughout the season. Parity of host-seeking females parity was highest during July when ≈21 parous host-seeking females were collected per hour. These results indicate that seasonal patterns in immature population density influence average body size and parity structure in Ae. albopictus.

In October 1995, epidemic “hemorrhagic fever,” without jaundice or renal manifestations was reported in rural Nicaragua following heavy flooding; 2259 residents were evaluated for nonmalarial febrile illnesses (cumulative incidence, 6.1%) and 15 (0.7%) died with pulmonary hemorrhage. A case-control study found that case-patients were more likely than controls to have ever walked in creeks (matched odds ratio [MOR], 15.0; 95% confidence interval [CI], 1.7–132.3), have household rodents (MOR, 10.4; 95% CI, 1.1 -97.1), or own dogs with titers ≥400 to Leptospira species (MOR, 23.4; 95% CI, 3.6–∞). Twenty-six of 51 case-patients had serologic or postmortem evidence of acute leptospirosis. Leptospira species were isolated from case-patients and potential animal reservoirs. This leptospirosis epidemic likely resulted from exposure to flood waters contaminated by urine from infected animals, particularly dogs. Leptospirosis should be included in the differential diagnosis for nonmalarial febrile illness, particularly during periods of flooding or when pulmonary hemorrhage occurs.

Aedes triseriatus (Say) pupae and host-seeking females were collected over 19 wk in a tire dump located in southwestern Louisiana. Tire location influenced production of mosquitoes, sex ratio, and body size of adults. Tires located in a wooded area of the dump produced 3.6 times more adult Ae. triseriatus than did tires in the sun. However, tires in the sun produced mosquitoes with significantly longer wings than did tires in shade. Also, average wing length of females in shaded tires increased significantly later in the summer. Average wing length of host-seeking parous females was significantly greater than nulliparous host-seeking females, and average wing lengths of both host-seeking populations were significantly greater than that of emerging females. Thus, body size was related to survival and blood-feeding success in Ae. triseriatus.