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 IRT-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 titers >1:20, whereas only of 8/13 birds previously infected with SLE were positive by EIA and all had PRNT titers <1:20. 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 horn 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 PRNTT positive before challenge) and was not protective. because 3/6 birds developed an ephemeral viremia on day 1 after infection (mean titer, 10(273) plaque forming units/0.1 ml). When necropsied 7 wk after challenge, 1/10 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 titers >1:10 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 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 BFS1703 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 log(10) plaque-forming units [PFU]/0.1 ml), females of the susceptible high virus producer (HVP) strain rapidly amplified 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 log(10) 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 log(10) 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 Cs. tarsalis vector competence. Females from the WEEV-resistant (WR) strain and two wildtype 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 titers > 4.3 log(10) PFU, and most had titers > 4.8 log(10) 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 log(10) 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 (e(x)), reproductive effort in female eggs per female per generation (R-o), and generation time (T) in days for the infected cohorts were significantly lower than for the uninfected controls, whereas the reproductive rate (r(c)) 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 log(10) 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.

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 titers >1:20, whereas only of 8/13 birds previously infected with SLE were positive by EIA and all had PRNT titers <1:20. 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, 102.73 plaque forming units/0.1 ml). When necropsied 7 wk after challenge, 1/10 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 titers >1:40 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.