Female Culex tarsalis fed heparinized chicken blood-western equine encephalomyelitis virus (WEEV) mixtures through a biomembrane feeder were compared with females fed sweetened blood-virus mixtures presented in pledgets or as hanging drops or to restrained chickens with natural or artificial viremias. Results indicated that sodium heparin did not adversely affect the infection of Culex tarsalis with WEEV. Overall advantages of the biomembrane system included 1) increased blood feeding frequency, 2) control of the infectious virus dose, and 3) greater or similar infection rates and body titers to females taking blood meals from viremic chickens. Anesthetizing females with triethylamine for in vitro transmission assessment using the capillary tube method produced results similar to immobilization using cold or CO2 + cold. Our research provided insight into tools useful to investigate the infection and transmission of WEEV by Cx. tarsalis.

Abstract Nestling mourning doves and house finches produced elevated viremias after inoculation with 2–3 log10 plaque-forming units (PFU) of St Louis encephalitis (SLE) virus and infected 67 and 70% of Culex tarsalis Coquillett that engorged upon them, respectively. Mosquito infection rates as well as the quantity of virus produced after extrinsic incubation increased as a function of the quantity of virus ingested and peaked during days 3–5 postinoculation in mourning doves and days 2–4 in house finches. Only female Cx. tarsalis with body titers ≥4.6 log10 PFU were capable of transmitting virus. Overall, 38% of females infected by feeding on mourning doves and 22% feeding on house finches were capable of transmission. The quantity of virus expectorated was variable, ranging from 0.8 to 3.4 log10 PFU and was greatest during periods when avian viremias were elevated. Our data indicated that nestling mourning doves and house finches were competent hosts for SLE virus and that the quantity of virus ingested from a viremic avian host varies during the course of the infection and determines transmission rates by the mosquito vector.

A blinded laboratory evaluation compared the accuracy, sensitivity, and specificity of an in situ enzyme immunoassay (EIA), VecTest wicking assay, and reverse transcription-polymerase chain reaction (RT-PCR) to detect and distinguish West Nile (WN) and St. Louis encephalitis (SLE) viruses in pools of 50 mosquitoes. Adult female Culex tarsalis Coquillett were inoculated with either WN or SLE viruses, held for 0-11 d at 28degreesC, killed by freezing, and then were added to 49 or 48 uninfected mosquitoes to make up 14 pools positive for WN virus, 14 positive for SLE virus, 14 positive for both WN and SLE viruses, and 14 negative for virus. Pools were number coded and tested blindly. Virus was not detected in known negative pools. VecTest and RT-PCR assays were comparably sensitive and accurate, detecting virus in pools containing females held for 3 d postinoculation; only RT-PCR detected SLE virus in pools on days 0-1. The VecTest and RT-PCR produced a single false-positive result for WN and SLE, respectively. RT-PCR detected RNA in samples positive by the VecTest, indicating that the detergent in the wicking buffer did not prevent RT-PCR from confirming VecTest results. Detector antibodies used in the in situ EIA cross-reacted between SLE and WN viruses, reducing accuracy. Both the VecTest and RT-PCR provided rapid and specific results, but they detected only those viruses known to be present. Plaque assay on Vero cells was comparably sensitive and had the added benefit of detecting newly emerging viruses, but this method required virus culture followed by identification, thereby delaying reporting.

After-hatching and hatching year, mourning doves were infected by inoculation with either western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses; some birds in each group also were treated with the immunosuppressant cyclophosphamide before and during infection. Cyclophosphamide treatment significantly increased the WEE viremia but did not alter the antibody response. In contrast, cyclophosphamide-treated and -untreated doves did not develop a detectable SLE viremia but became antibody positive. Antibody peaked at 10 wk after inoculation for both viruses and remained detectable in most birds throughout the 26-wk study. When treated with cyclophosphamide the following spring, birds did not relapse and develop a detectable viremia. Previously infected birds were protected when challenged with conspecific virus (i.e., none produced a detectable viremia), but there was no anamnestic antibody response to reinfection. In agreement with our failure to detect relapses, all birds were negative for viral RNA when sera, spleen, lung, and kidney tissues were tested by reverse transcriptase-polymerase chain reaction after necropsy. Our results indicated that adult mourning doves were an incompetent host for SLE virus and probably do not serve as a suitable overwintering or dispersal host for either WEE and SLE viruses.

Abstract Immunosuppression of house finches was attempted by blood feeding Culex tarsalis Coquillett mosquitoes or by injecting birds with the corticosteroid dexamethasone or the immunosuppressant drug cyclophosphamide before and after inoculation with western equine encephalomyelitis or St. Louis encephalitis viruses. Mosquito bites (8–37 females blood feeding on each bird over a 3-d period) did not enhance the viremia response or increase the frequency of chronic infection. In contrast, dexamethasone and cyclophosphamide enhanced the amplitude and duration of the viremia response, but had no consistent effect on the antibody responses as measured by enzyme immunoassay or plaque reduction neutralization assay. Elevated viremias were followed by increases in the frequency of chronic infections with St. Louis encephalitis, but not western equine encephalomyelitis. Immunosuppression may provide a useful tool to study the chronic infection process of flaviviruses in vertebrates.

A total of 27 bird species from the San Joaquin and Coachella valleys of California were inoculated subcutaneously with sympatric strains of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses. Overall, 133 of 164 birds inoculated with WEE virus developed a viremia detected by plaque assay; significantly greater than 72 of 163 birds inoculated with SLE virus. Host competence was calculated as the average number of days that each avian species had a viremia greater than or equal to 2 log(10) plaque-forming units per 0.1 ml, the threshold for infecting susceptible Culex tarsalis Coquillett, the primary vector of these viruses in California. Eleven of 20 species inoculated with WEE virus had a value greater than or equal to 1 and were considered to be competent hosts, whereas only six of 22 species inoculated with SLE virus had a value greater than or equal to 1. Overall, 133 of 164 birds inoculated with WEE virus and 105 of 163 inoculated with SLE virus produced antibody detectable by enzyme immunoassay and/or plaque reduction neutralization test. Six birds infected with WEE virus (one house finch, three mourning doves, one Brewer's sparrow, and one white-crowned sparrow) and nine birds infected with SLE virus (two house finches, three white-crowned sparrows, one song sparrow, two Western scrub-jays, and one orange crowned warbler) contained viral RNA detected by reverse transcription-polymerase chain reaction at necropsy > 6 wk postinoculation; infectious WEE and SLE viruses were only recovered from three mourning doves and an orange-crowned warbler, respectively, after blind passage in mosquito cells. Our study indicated that birds with elevated field antibody prevalence rates may not be the most competent hosts for encephalitis viruses and that relatively few birds developed chronic infections that could be important in virus persistence and dispersal.

The introduction of a St. Louis encephalitis virus (SLE) genotype new to southeastern California during 2000 was followed by focal enzootic amplification in the Coachella Valley that was detected by seroconversions of 29 sentinel chickens in five of nine flocks of 10 chickens each, isolations of virus from 30 of 538 pools of 50 Culex tarsalis Coquillett females, and collection of 30 positive sera from 2,205 wild birds. This SLE strain over wintered successfully and then amplified during the summer of 2001, with 47 sentinel seroconversions in eight of nine flocks, 70 virus isolations from 719 pools of Cx. tarsalis and Cx. p. quinquefasciatus Say, and 40 positive sera from 847 wild birds. Human illness was not detected by passive case surveillance, despite issuance of a health alert during 2001. Virus amplification during both years was associated with above average temperatures conducive for extrinsic incubation and below average precipitation during spring associated with below average vector abundance. Seroconversions by sentinel chickens provided the timely detection of virus activity, with initial conversions detected before positive mosquito pools or wild bird infections. Vertical infection was not detected among Cx. tarsalis adults reared from immatures collected during the fall-winter of 2000, even though SLE over wintered successfully in this area. Early seroconversions by a sentinel chicken during February 2001 and a recaptured Gambel's quail in April 2001 provided evidence for transmission during winter and spring when ambient temperatures averaged below 17 degrees C, the threshold for SLE replication.

A yearling quarter horse, which was raised in southern California, received routine vaccinations for prevention of infection by Eastern equine encephalomyelitis virus (EEEV). One week later, severe neurologic signs developed, and the horse was humanely destroyed. A vaccine-related encephalomyelitis was later suspected. A final diagnosis of EEEV infection was established on the basis of acute onset of the neurologic signs, histopathologic and serologic testing, and isolation and molecular characterization of EEEV from brain tissue. The vaccine was extensively tested for viral inactivation. Nucleotide sequences from the vaccine and the virus isolated in the affected horse were also compared. In California, arboviral encephalomyelitides are rarely reported, and EEEV infection has not previously been documented. This report describes the occurrence of EEEV infection in the horse and the investigation to determine the source of infection, which was not definitively identified.

ABSTRACT Diagnostic assays for the detection of St. Louis encephalitis (SLE) and western equine encephalomyelitis (WEE) viruses in mosquito pools and avian tissues were compared for sensitivity, accuracy and specificity. The in situ enzyme immunoassay (EIA), plaque assay on Vero cells, passage in Aedes albopictus Skuse C6/36 and C7/10 cells, antigen capture enzyme immunoassay (AC-EIA), and single and multiplex reverse transcription-polymerase chain reactions (RT-PCR) were evaluated using pools of 50 mosquitoes containing 1-2 experimentally infected individuals. RT-PCR was the most sensitive assay, with a detection limit of <0.1 plaque forming unit. AC-EIA was the fastest and most economical procedure, but was the least sensitive, detecting only 38% of positive pools. The in situ EIA included initial virus amplification on Vero cells, thereby improving assay sensitivity to detect 68% of positive pools. Passage in C6/36 and/or C7/10 cell culture revealed the presence of infectious virus in samples positive by RT-PCR, but initially negative by plaque assay on Vero cell culture, indicating that detection was related to assay sensitivity and not to the absence of intact infectious virus. Combining WEE and SLE RT-PCR assays into a multiplex assay reduced sensitivity, but stilldetected viral RNA at titers below plaque assay sensitivity. Plaque assay on Vero cells, mosquito cell passage, and several RT-PCR procedures were evaluated for their ability to detect WEE and SLE in white-crowned sparrow tissues during acute and chronic stages of infection. All assays detected virus during acute infection at times of high viremia; however, only RT-PCR assays were positive by day 7 when virus was not detected in sera. RT-PCR detected SLE RNA in spleen tissue from one bird 51 d after infection. Assay sensitivity also was compared using extracts of homogenized bird organs spiked with known titers of WEE and SLE. Trizol RNA extraction followed by Qiagen one-step RT-PCR was the most sensitive method, but occasionally resulted in the presence of secondary bands confounding interpretation and requiring confirmatory assays. A balanced surveillance program should combine systems that allow the detection of new agents and the sensitive monitoring of endemic agents to provide an early warning of pending health risks.

Western equine encephalomyelitis and St. Louis encephalitis viral RNA can be detected 20 days after death of infected Culex tarsalis in the absence of a cold chain. Viral RNA was detected with the reverse transcription-polymerase chain reaction in mosquitoes infected either parenterally or perorally in the laboratory and then killed and held for up to 20 days at 27 degrees C. Cell culture assay and in situ enzyme immunoassay did not detect infectious virus in the same mosquitoes.

A recent case of California encephalitis, a rare mosquito-borne viral disease, represents only the fourth ever reported and the first since the initial three cases in 1945. This case was diagnosed retrospectively on the basis of a rise in antibody titer between acute- and convalescent-phase serum samples.

Adult house finches from Kern County were inoculated subcutaneously with recent sympatric and allopatric isolates of western equine encephalomyelitis and St. Louis encephalitis (SLE) viruses made from Culex tarsalis Coquillett collected in Kern County and Coachella Valley, CA, respectively. Virulence, as measured by the amplitude of the viremia response during days 1 and 2 postinfection, varied significantly among strains, but independently of geographic origin. The intensity of the immune response, as measured by an enzyme immunoassay and a plaque reduction neutralization test, seemed to be independent of virulence, especially for SLE where some strains failed to produce a detectable viremia but elicited a strong antibody response. Our preliminary data indicated that strain Virulence may be associated with the level of enzootic activity during the year of isolation.

The effects of method of infection and virus dose on the viremia and antibody responses of 1-wk-old chicks and after-hatching-year house finches to infection with western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses were studied under laboratory conditions. Using a capillary tube technique, females from 2 strains of Culex tarsalis Coquillett mosquitoes were estimated to expectorate from 1.0 to 1.7 log(10) plaque forming units (PFU) of WEE and from 1.9 to 2.2 log(10) PFU of SLE. Based on the proportion of parenterally infected females that transmitted and the number that blood fed during each experiment, virus doses per bird were estimated to be 1.0-1.9 log(10) PFU for WEE and 1.4-2.3 log(10) PFU for SLE. When infected with comparable doses of WEE by subcutaneous inoculation, there was no significant difference in the duration or magnitude of the viremia response between birds infected by mosquito bite or syringe; few birds developed a viremia response after infection with SLE, precluding analysis. In chickens, increasing the syringe dose of WEE from 0.3 to 1.7 log(10) PFU/0.1 ml shortened the time when viremia first appeared from 3 to 1 d postinfection and increased the duration of the viremia period from 1 to 3 d, but did not alter the maximum viremia titer. In house finches, increasing the syringe dose of WEE from 2.6 to 3.3 log(10) PFU/0.1 ml did not alter markedly the viremia response. Most birds developed antibody detected by enzyme immunoassay (EIA) or plaque reduction neutralization test (PRNT). In chickens, WEE EIA levels and PRNT titers were higher for birds infected by syringe than by mosquito bite, whereas in house finches the pattern was reversed. For birds infected with SLE, there was overlap among groups infected by mosquito bite or syringe. These results indicate that subcutaneous syringe inoculation provides a biologically sound mode of infection that did not alter viremia and antibody responses when compared with infection by mosquito bite.

Temporal and spatial changes in the enzootic activity of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses were monitored at representative wetland study sites in the Coachella, San Joaquin, and Sacramento valleys of California from 1996 to 1998 using three methods: (1) virus isolation from pools of 50 host-seeking Culex tarsalis Coquillett females, (2) seroconversions in flocks of 10 sentinel chickens, and (3) seroprevalence in wild birds collected by mist nets and, grain baited traps. Overall, 74 WEE and one SLE isolates were obtained from 222,455 Cx. tarsalis females tested in 4,988 pools. In addition, 133 and 40 seroconversions were detected in 28 chicken flocks, and 143 and 27 of 20,192 sera tested from 149 species of wild birds were positive for antibodies to WEE and SLE, respectively. WEE was active in all three valleys, whereas SLE only was detected in Coachella Valley. Seroconversions in sentinel chickens provided the most sensitive indication of enzootic activity and were correlated with seroprevalence rates in wild birds. Avian seroprevalence rates did not provide an early warning of pending enzootic activity. in chickens, because positive sera from after hatching year birds collected during spring most probably were the result of infections acquired during the previous season. Few seroconversions were detected among banded recaptured birds collected during spring and early summer. Age and resident status, but not sex, were significant risk factors for wild bird infection, with the highest seroprevalence rates among after hatching year individuals of permanent resident species. Migrants (with the exception of mourning doves) and winter resident species rarely were positive. House finches, house sparrows, Gambel's quail, California quail, common ground doves, and mourning doves were most frequently positive for antibodies. The initial detection of enzootic activity each summer coincided closely with the appearance of hatching year birds of these species in our study areas, perhaps indicating their role in virus amplification. Bird species most frequently positive roosted or nested in elevated upland vegetation, sites where Cx. tarsalis host-seeking females hunt most frequently. These serosurveys provided important background information for planned host competence and chronic infection studies.

In laboratory vector competence studies, Aedes dorsalis (Meigen) collected from Morro Bay, CA, did not vertically transmit sympatric strains of western equine encephalomyelitis virus (WEE). This population of Ae. dorsalis was highly susceptible to oral infection and was a competent horizontal vector of WEE. The E2 region of the viral genome of the 3 virus strains isolated from Ae. dorsalis in Morro Bay were closely related genetically to a strain of WEE isolated in 1953 from a geographically separate location that is used regularly in the laboratory. These laboratory findings support recent field research and indicate that Ae. dorsalis probably does not play a significant role in WEE persistence in coastal California.

The ecology of western equine encephalomyelitis virus (WEE) was studies during 1994-1996 along a portion of the north shore of the Salton Sea in Coachella Valley, California, known to support a focal Aedes dorsalis (Meigen) population. WEE was detected during 1995 by the seroconversion of sentinel chickens concurrently at sites within and outside of the area supporting Ae. dorsalis. WEE was not detected during 1994 or 1996; neither was WEE detected by seroconversion of sentinel rabbits nor by isolation from host-seeking females of either the primary vector, Culex tarsalis Coquillett (42,083 females tested in 913 pools), or Ae. dorsalis (10,804 females tested in 245 pools and 1,940 adults reared from field-collected immatures tested in 72 pools). Collectively, the results of this and previous investigations indicate that Ae. dorsalis may not be essential for the maintenance or amplification of WEE virus in southeastern California.

A new indirect enzyme immunoassay (EIA) was developed to screen wild bird sera for antibodies against western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses. The detector antibody was made by immunizing rabbits with serum proteins pooled from single species representatives of four bird orders and was conjugated with horseradish peroxidase to allow visualization with the ABTS substrate in an EIA plate reader set at 405 nm. The detector antibody recognized a wide range of bird species and was more accurate, sensitive, and specific than a hemaglutination inhibition test when compared to a plaque reduction neutralization test (PRNT). EIA positive sera frequently could not be confirmed by PRNT; however, practically all sera positive by PRNT also were positive by EIA. The new EIA has been incorporated into our field research program and has been used to economically screen over 10,000 wild bird sera from 124 species for antibodies against WEE and SLE.

Eight enzootic strains of western equine encephalomyelitis (WEE) virus isolated from Culex tarsalis or Aedes melanimon collected in several geographic areas of California were evaluated for their virulence in suckling mice, adult mice, and one-day-old baby chickens. The epidemic Fleming strain and the cloned B628(Cl 15) variant were used as virulent and avirulent control viruses, respectively, in adult mice. Enzootic strains of WEE virus were grouped into three phenotypes on the basis of their neurovirulent and neuroinvasive properties in adult mice. Three strains possessed high neurovirulence and were neuroinvasive; three strains had intermediate neurovirulence and lacked neuroinvasiveness; and two strains had low to nil neurovirulence and were non-neuroinvasive. In fact, five of the eight enzootic strains lacked neuroinvasiveness. Interestingly, highly virulent enzootic strains of WEE virus were isolated from Cx. tarsalis collected in the Sacramento Valley during 1994 and 1995 in the absence of identified human disease. The Fleming strain, the B628(Cl 15) variant, and four enzootic strains from the Sacramento Valley were virulent for baby chickens following subcutaneous inoculation. Thus, inoculation into baby chicks cannot discriminate between WEE viruses that are virulent and avirulent for adult mice.

Sera from outpatients attending county health department clinics in areas of California with consistent (Imperial Valley) and sporadic (Sacramento Valley) enzootic transmission of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses exhibited neutralizing antibody prevalence rates of 1.3% (n = 690) and 0.5% (n = 1,066) for WEE and 11.0% and 0.8% for SLE, respectively. Seroprevalence for SLE virus in Imperial County increased as a function of both age and years of residence, indicating that this virus was endemic with a low rate of annual infection. Of 26 sera that tested positive for SLE virus antibody by an enzyme immunoassay, but were negative by plaque reduction neutralization test, 14 (53%) had neutralizing antibody that reacted with > or = one type of dengue (DEN) virus. The DEN virus infections presumably were acquired elsewhere because neither the vectors nor DEN virus transmission occurs in California. The low prevalence of neutralizing antibody for WEE and SLE in the California human population indicated that despite recent increases in enzootic transmission, contact between humans and infectious mosquitoes have remained low.

Sera from 19 (2.6%) and 118 (16.4%) of 719 outpatients attending clinics in the southeastern Coachella Valley, California during 1993 and 1994 exhibited IgG antibodies to western equine encephalomyelitis and St. Louis encephalitis (SLE) viruses, respectively, using enzyme immunoassays. However, only seven (1.0%) and 36 (5.0%) outpatients were positive by plaque-reduction neutralization tests (PRNTs), and seven (1.0%) and 84 (11.7%) outpatients were positive by sera hemagglutination inhibition assays, respectively. None were positive for IgM antibodies indicative of recent infection or were diagnosed clinically with central nervous system disease. Prevalence of PRNT antibody to SLE increased as a function of patient age, but did not vary significantly in relation to years of residence, sex, race, postal zip code, occupation, or month of collection.