Robert E. Chiles

West Nile Virus in California

The spread of WNV in California is tracked.

Experimental Infection of California Birds with Western Equine Encephalomyelitis and St. Louis Encephalitis Viruses

Abstract 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 ≥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 value ≥1 and were considered to be competent hosts, whereas only six of 22 species inoculated with SLE virus had a value ≥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.

Patterns of Avian Seroprevalence to Western Equine Encephalomyelitis and Saint Louis Encephalitis Viruses in California, USA

Abstract 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-seekingCulex tarsalisCoquillett 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 222,455Cx. tarsalisfemales 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 whereCx. tarsalishost-seeking females hunt most frequently. These serosurveys provided important background information for planned host competence and chronic infection studies.

Detection of Encephalitis Viruses in Mosquitoes (Diptera: Culicidae) and Avian Tissues

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 still detected 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 Virus Infection Affects the Life Table Characteristics of Culex tarsalis (Diptera: Culicidae)

Abstract 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.

Method of Infection Does Not Alter Response of Chicks and House Finches to Western Equine Encephalomyelitis and St. Louis Encephalitis Viruses

Abstract 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.

Encephalitis Virus Persistence in California Birds: Preliminary Studies with House Finches

Abstract 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.

Role of Nestling Mourning Doves and House Finches as Amplifying Hosts of St. Louis Encephalitis Virus

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.

Persistence and Amplification of St. Louis Encephalitis Virus in the Coachella Valley of California, 2000-2001

Abstract 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 2001provided evidence for transmission during winter and spring when ambient temperatures averaged below 17°C, the threshold for SLE replication.

Effects of time after infection, mosquito genotype, and infectious viral dose on the dynamics of Culex tarsalis vector competence for western equine encephalomyelitis virus.

ABSTRACT 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 log10 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 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 titers >4.3 log10 PFU, and most had titers >4.8 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.