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Adult domestic chickens were infected with West Nile virus (WNV) or St. Louis encephalitis virus (SLEV) and challenged with homologous or heterologous virus at 21 or 56 days postinfection (dpi). Sera were collected at selected time points after infection and assayed by enzyme immunoassay (EIA), plaque reduction neutralization test (PRNT), and a Western blot (WB) alternative to PRNT. EIA results were sensitive and accurate (few false positives) but not specific, requiring a confirmatory test to determine virus infection history. PRNT results generally were specific until challenge, after which test results were frequently equivocal and inadequate to determine first or second infecting virus. WB results confirmed the serologic cross-reactivity between WNV and SLEV envelope protein. Non-structural protein 1 and pre-membrane protein reactivities were highly specific for WNV during SLEV infection, but less specific for SLEV during WNV infection. WB and PRNT specificities were similar for both viruses from 6 to 14 dpi, and sensitivities to WNV were virtually identical.

The spread of West Nile virus (WNV) across the United States into areas with endemic flavivirus activity has complicated serologic surveillance of seasonal virus activity and diagnosis of infected individuals. Here we describe preliminary results from a comparison of serologic assays for flaviviruses: the reference plaque reduction neutralization test (PRNT), enzyme immunoassay (EIA), and a Western blot (WB) in which crude viral lysates were electrophoresed and blotted onto nitrocellulose. Human and chicken sera were tested and compared by each method against WNV and St. Louis encephalitis virus (SLEV). Antibody binding to three viral proteins determined WB interpretation: non-structural protein 1 (NS1), envelope (E), and pre-membrane (prM). WB results for a group of serially collected human plasma samples from WNV seroconverting blood donors were also correlated with transcription mediated amplification (TMA) and polymerase chain reaction (RT-PCR) results. Reactivity with NS1 appeared to be the most useful differentiating marker of WNV and SLEV infection in humans and chickens. Envelope protein was highly cross-reactive and, as indicated by additional results from dengue virus (DENV)-positive human sera, is perhaps useful serologically as a flavivirus group antigen.

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 titersor = 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.

House finches, Carpodacus mexicanus, were experimentally infected with high and standard doses of western equine encephalomyelitis virus (WEEV) or St. Louis encephalitis virus (SLEV) to determine whether high doses would produce an elevated viremia response and a high frequency of chronic infections. Finches inoculated with approximately100,000 plaque forming units (PFU) of WEEV or SLEV produced viremia and antibody responses similar to those in finches inoculated with approximately 100 PFU of WEEV or 1000 PFU of SLEV, the approximate quantities of virus expectorated by blood-feeding Culex tarsalis Coquillett. Infected finches were held through winter and then necropsied. Only one finch inoculated with the high dose of SLEV developed a chronic infection. Our data indicated that elevated infectious doses of virus may not increase the viremia level or the frequency of chronic infection in house finches.

The spread of WNV in California is tracked., West Nile virus (WNV) was first isolated in California during July 2003 from a pool of Culex tarsalis collected near El Centro, Imperial County. WNV transmission then increased and spread in Imperial and Coachella Valleys, where it was tracked by isolation from pools of Cx. tarsalis, seroconversions in sentinel chickens, and seroprevalence in free-ranging birds. WNV then dispersed to the city of Riverside, Riverside County, and to the Whittier Dam area of Los Angeles County, where it was detected in dead birds and pools of Cx. pipiens quinquefasciatus. By October, WNV was detected in dead birds collected from riparian corridors in Los Angeles, west to Long Beach, and through inland valleys south from Riverside to San Diego County. WNV was reported concurrently from Arizona in mid-August and from Baja, Mexico, in mid-November. Possible mechanisms for virus introduction, amplification, and dispersal are discussed.

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 28 degrees C, 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.

Antibody titers against St. Louis encephalitis virus (SLE) measured by a plaque reduction neutralization test (PRNT) decreased rapidly in house finches (Capodacus mexicanus) after initial infection, whereas antibodies measured by enzyme immunoassay (EIA) remained detectable in all birds for the length of the experiment, indicating long-term persistence and greater assay sensitivity of the EIA. After 52 wk, birds were challenged by subcutaneous inoculation with the same strain of SLE virus. Virus was not detected for 1-4 d postchallenge in blood samples tested by plaque assay and RT-PCR or by xenodiagnosis in Culex tarsalis fed concurrently and then held for 11 d at 26 degrees C. Virus was detected by all three methods in control birds infected concurrently for the first time. Challenge with SLE produced a rapid and marked ananmestic rise in both neutralizing and EIA antibody titers that exceeded the primary response in the same birds or in concurrently inoculated control birds. At necropsy 4 wk postchallenge, 3 of 7 challenged and 1 of 2 positive control birds were chronically infected, with viral RNA detected by RT-PCR in brain, spleen, lung, and/or kidney tissues. Our results indicated that persistence of protective antibody prevents reinfection during the following season and may prevent the recrudescence of infectious virus in chronically infected birds.

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.

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

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.

Field-collected house finches of mixed sex and age were infected experimentally with either western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses during the summer or fall of 1998 and maintained over the winter under ambient conditions. To detect natural relapse during the spring, 32 birds were bled weekly from February through June 1999, and then necropsied 1 yr after infection to detect chronic infections using a reverse transcription polymerase chain reaction (RT-PCR). After 10 mo, 13/14 surviving birds previously infected with WEE were antibody positive by enzyme immunoassay (EIA), and 11/14 had plaque reduction neutralization test (PRNT) antibody titers1:20, whereas only of 8/13 birds previously infected with SLE were positive by EIA and all had PRNT titers1:20. When necropsied, 1/14 and 1/13 birds had WEE and SLE RT-PCR positive lung or spleen tissue, respectively; blood, brain, and liver tissues were negative as were all previous blood samples. All tissues from these birds including weekly blood samples tested negative for infectious virus by plaque assay on Vero cell culture. To determine if persistent antibody was protective, birds infected initially with WEE or SLE in November 1998 were challenged 6 mo later with homologous virus. WEE antibody persisted well (5/6 birds remained PRNT positive before challenge) and remained protective, because 0/6 birds were viremic after challenge. In contrast, SLE antibody decayed rapidly (0/6 birds remained PRNT positive before challenge) and was not protective, because 3/6 birds developed an ephemeral viremia on day 1 after infection (mean titer, 10(2.73) plaque forming units/0.1 ml). When necropsied 7 wk after challenge, 1/110 birds infected with WEE and 1/10 birds infected with SLE exhibited an RT-PCR positive spleen, despite the fact that both birds had PRNT antibody titers1:40 at this time. To determine if immunosuppression would cause a chronic infection to relapse, eight birds initially infected with either WEE or SLE were treated with cyclophosphamide and then tested repeatedly for viremia; all samples were negative for virus by plaque assay. Collectively, our results indicated that a low percentage of birds experimentally infected with WEE or SLE developed chronic infections in the spleen or lung that could be detected by RT-PCR, but not by plaque assay. Birds did not appear to relapse naturally or after immunosuppression. The rapid decay of SLE, but not WEE, antibody may allow the relapse of chronic infections of SLE, but not WEE, to produce viremias sufficiently elevated to infect mosquitoes.

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.

L'A., professeur de philosophie, decrit une experience pedagogique effectuee avec une de ses classes a l'universite. Le but du jeu etait que chaque etudiant ecrive un papier dans lequel il exprime ses pensees philosophiques personnelles avant d'avoir eu de cours sur le sujet. Un second papier, ecrit en cours de semestre, complete le premier et permet de comparer les changements advenus apres que les etudiants aient suivis des cours

Adult domestic chickens were infected with West Nile virus (WNV) or St. Louis encephalitis virus (SLEV) and challenged with homologous or heterologous virus at 21 or 56 days postinfection (dpi). Sera were collected at selected time points after infection and assayed by enzyme immunoassay (EIA), plaque reduction neutralization test (PRNT), and a Western blot (WB) alternative to PRNT. EIA results were sensitive and accurate (few false positives) but not specific, requiring a confirmatory test to determine virus infection history. PRNT results generally were specific until challenge, after which test results were frequently equivocal and inadequate to determine first or second infecting virus. WB results confirmed the serologic cross-reactivity between WNV and SLEV envelope protein. Non-structural protein 1 and pre-membrane protein reactivities were highly specific for WNV during SLEV infection, but less specific for SLEV during WNV infection. WB and PRNT specificities were similar for both viruses from 6 to 14 dpi, and sensitivities to WNV were virtually identical.

The spread of West Nile virus (WNV) across the United States into areas with endemic flavivirus activity has complicated serologic surveillance of seasonal virus activity and diagnosis of infected individuals. Here we describe preliminary results from a comparison of serologic assays for flaviviruses: the reference plaque reduction neutralization test (PRNT), enzyme immunoassay (EIA), and a Western blot (WB) in which crude viral lysates were electrophoresed and blotted onto nitrocellulose. Human and chicken sera were tested and compared by each method against WNV and St. Louis encephalitis virus (SLEV). Antibody binding to three viral proteins determined WB interpretation: non-structural protein 1 (NS1), envelope (E), and pre-membrane (prM). WB results for a group of serially collected human plasma samples from WNV seroconverting blood donors were also correlated with transcription mediated amplification (TMA) and polymerase chain reaction (RT-PCR) results. Reactivity with NS1 appeared to be the most useful differentiating marker of WNV and SLEV infection in humans and chickens. Envelope protein was highly cross-reactive and, as indicated by additional results from dengue virus (DENV)-positive human sera, is perhaps useful serologically as a flavivirus group antigen.

The vector competence of Culex tarsalis Coquillett for the BFS 1703 strain of western equine encephalomyelitis virus (WEEV) changed significantly as a function of time after infection, mosquito genotype, and infectious virus dose. After ingesting a high virus dose (5 log10 plaque-forming units [PFU]/0.1 ml), female of the susceptible high virus producer (HVP) strain rapidly amplified the virus, developed a disseminated infection, and efficiently transmitted WEEV by 4 days postinfection (dpi). The quantity of virus expectorated peaked at 4 dpi (mean 3.4 log10 PFU), and the percentage of females transmitting per os peaked at 7 dpi (80%); both measures of transmission subsequently decreased to low levels throughout the remainder of infected life. HVP females imbibing a low virus dose (3 log10 PFU/0.1 ml) were infected less frequently and took longer to amplify virus to levels recorded for the high virus dose group and did not transmit virus efficiently, thereby indicating midgut infection and escape barriers were dose and time dependent. These data emphasized the importance of elevated avian viremias in Cx. tarsalis vector competence. Females from the WEEV-resistant (WR) strain and two wild-type strains from Kern and Riverside counties were significantly less susceptible to infection at both high and low doses than was the HVP strain. Overall, females with a high virus titer more frequently had a disseminated infection, but there did not seem to be a distinct threshold demarcating this relationship. In marked contrast, all infected females transmitting virus had body titers4.3 log10 PFU, and most had titers4.8 log10 PFU. These data indicated that not all females with a disseminated infection transmitted virus because of the presence of one or more salivary gland barriers.

The life table attributes of Culex tarsalis Coquillett females infected experimentally by feeding on 4 and 6 log10 plaque-forming units (PFU) of western equine encephalomyelitis virus (WEEV) per milliliter of heparinized chicken blood were compared with an uninfected control group. Females continually were offered 10% sucrose and an oviposition substrate and daily a blood meal through a biomembrane feeder. Mortality (dead females) and fecundity (female eggs per female) were monitored daily until all females died. Overall, 94% of 198 females in the two virus-infected groups were positive for WEEV at death when tested by plaque assay; the average body virus titer at death did not differ between groups. WEEV infection significantly altered the life table characteristics of Cx. tarsalis. Life expectancy at infection in days (ex), reproductive effort in female eggs per female per generation (Ro), and generation time (T) in days for the infected cohorts were significantly lower than for the uninfected controls, whereas the reproductive rate (rc) in female eggs per female per day was higher for infected than uninfected cohorts. In agreement with the WEEV infection data that showed similar body titers, there were few differences between the life table parameters for the 4 and 6 log10 PFU treatment groups. Greatest differences were observed for survivorship between days 17-40 when virus titers in infected dying females were greatest. Our data extend recent studies that indicate mosquito infection with encephalitis viruses has a cost of reduced life expectancy and fitness.

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.

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 viremiaor = 2 log10 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 valueor = 1 and were considered to be competent hosts, whereas only six of 22 species inoculated with SLE virus had a valueor = 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 necropsy6 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.

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.

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.

Female Culex tarsalis Coquillett in reproductive diapause were infected per os or by intrathoracic inoculation with western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses during "fall," maintained over a simulated "winter," and then tested for virus infection and transmission in vitro and in vivo after "vernal" termination. Exposure of F1 progeny of field-collected females to cool temperatures and short daylength produced females in reproductive diapause that were reluctant to imbibe infectious virus from pledgets soaked with suspensions of virus, blood and sucrose (2.5% by volume). Those infected per os maintained virus at very low or undetectable titers. Some females that originally tested negative for WEE by plaque assay on Vero cell culture tested positive by reverse transcriptase-polymerase chain reaction (RT-PCR) and by Vero cell culture after passage in mosquito cells. Few females became infected orally with SLE, but these infected females developed elevated titers. Females inoculated with SLE retained their infection through winter and then transmitted readily in vitro and in vivo. Feeding on a vertebrate host after diapause termination significantly increased the titer of SLE in previously infected females. These experiments simulated how infections acquired either horizontally or vertically may provide mechanisms for WEE and SLE overwintering. Attempts to detect infected females during winter following a summer with enzootic WEE activity were negative by both RT-PCR and plaque assay.

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.

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 log10 plaque forming units (PFU) of WEE and from 1.9 to 2.2 log10 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 log10 PFU for WEE and 1.4–2.3 log10 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 log10 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 log10 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.

The ecology of western equine encephalomyelitis virus (WEE) was studied 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.

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.

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.

This paper reports the first isolation of Jamestown Canyon (JC) virus from coastal California and the results of tests for antibody to JC virus in mammals living in coastal California. The virus isolation was made from a pool of 50 Aedes dorsalis females collected as adults from Morro Bay, San Luis Obispo County, California. The virus isolate was identified by two-way plaque reduction-serum dilution neutralization tests done in Vero cell cultures. Sera from the mammals were tested for antibody to JC virus by a plaque-reduction serum dilution neutralization method. A high prevalence of JC virus-specific antibody was found in horses and cattle sampled from Morro Bay. This finding is additional evidence for the presence of a virus antigenically identical or closely related to JC virus in Morro Bay and indicates that the vectors of the virus in Morro Bay feed on large mammals. A high prevalence of virus-specific antibody was also found in horses sampled from Marin and San Diego counties. This finding suggests that viruses antigenically identical or closely related to JC virus are geographically widespread in coastal California.

More than 75,000 immature mosquitoes in three genera were collected from coastal California, reared to the adult stage, and tested for virus by plaque assay in Vero cell cultures. Twenty-six strains of Morro Bay (MB) virus, a newly recognized member of the California (CAL) serogroup, were isolated from Aedes squamiger, a pestiferous salt marsh mosquito species restricted to intertidal salt marshes in coastal California and Baja California. The geographic distribution of the isolates was 10 from San Luis Obispo County, one each from Santa Barbara and Orange Counties, and 14 from San Diego County. No virus isolations were made from 23,157 Ae. squamiger collected north of San Luis Obispo County (midpoint in the geographic range of this species in California). Thus, MB virus infection in Ae. squamiger appears to be restricted to the southern range of this species in California. Serum dilution neutralization tests indicated that MB virus represents a novel subtype of the California encephalitis (CE) serotype within the CAL serogroup. Comparative analyses of genomic sequence data from four geographically distinct MB virus isolates indicated that the isolates are genetically similar to each other and distinct from other CE serotype bunyaviruses. Phylogenetic analysis of nucleocapsid protein gene sequence data indicated that MB virus represents a distinct lineage within the CE serotype and thus supports the serologic classification of MB virus as a distinct CAL serogroup virus.

Histochemical staining of the midgut epithelial cell surface complex carbohydrates of the mosquito Culex tarsalis was examined electron microscopically. The microvillar surface is composed primarily of neutral vic -glycoconjugates; positively stained by silver methenamine and silver protein. Lanthanum and alcian blue staining indicate that the microvilli contain a minimal anionic component; possibly phosphoglycoconjugates. Similarly, the intercellular junctions contain a predominance of neutral vic -glycoconjugates. In addition, the intercellular junctions contain fixed positive charges, based on en bloc phosphotungstic acid staining. The midgut basolateral membrane system and the basal lamina are both highly anionic; stained by ruthenium red, tannic acid, alcian blue and periodic acid-chromic acid-phosphotungstic acid. The basolateral plasma membrane also contains some vic -glycoconjugates. Selective staining indicates that the anionic component of the basolateral plasma membrane and the basal lamina is predominantly carboxyl groups; no specific staining for sulfo- or phosphoglycoconjugates was observed.

The infection and morphogenetic events associated with the replication of Western equine encephalomyelitis (WEE) virus within the mesenterons of Aedes dorsalis and three strains of Culex tarsalis are compared and contrasted. WEE virus apparently penetrates mesenteronal epithelial cells in vivo through membrane fusion. Profiles of apparent membrane fusion events were observed between virus particles and the microvillar surface of the mesenteron and naked nucleocapsids are observed intracellularly along the apical margin of the mesenteronal epithelial cell within 3 h of ingestion of the bloodmeal. Further, no viral particles were found in association with endocytotic nor lysosomal vacuoles during the initial phases of infection. In those strains of Cx. tarsalis that supported viral replication and in Ae. dorsalis , accumulations of nucleocapsids and maturation of WEE virus were evident along basolateral membranes of the mesenteron by 22–24 h after ingestion of the bloodmeal. Maximal extracellular nascent virus occurred between 30–36 hrs. The Knights Landing strain of Cx. tarsalis revealed no subcellular morphological alteration in response to infection throughout the period of study. However, distinct morphological structures associated with the infection were observed in strains or species with enhanced susceptibility compared to Knights Landing (i.e., Cx. tarsalis WS-3 and Ae. dorsalis ). In both, apical accumulations of nucleocapsids were apparent by 29 h post infection. These nucleocapsids were most often embedded in a rather amorphous matrix and occasionally in association with membrane profiles; presumably endoplasmic reticulum. Ae. dorsalis also demonstrated some alterations in response to WEE viral infection that were unique relative to Cx. tarsalis and some of these may be considered cytopathological. First, progeny virions were observed repeatedly within lysosomal figures. Second, extensive cytoplasmic vacuolization was noted and occasionally it appeared that these vacuolated cells were being sloughed off into the lumen of the mesenteron.

BASIC medical services are the right of every citizen. The recently enacted Medicare and Medicaid programs now guarantee competent care and adequate facilities to previously neglected segments of American society. These programs are understandably silent, however, about some essential human rights that are infrequently recognized and respected. Indeed, these important rights are often violated, not only by family and friends but also by many in the medical profession. Four such rights that are essential to the human spirit need some defense: the rights to privacy, pain and truth, and the right to die. The legitimacy of these rights may be . . .