Vincent M. Martinez

Effects of Temperature on the Transmission of West Nile Virus by Culex tarsalis (Diptera: Culicidae)

Abstract Culex tarsalis Coquillett females were infected with the NY99 strain of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) and then incubated under constant temperatures of 10–30°C. At selected time intervals, transmission was attempted using an in vitro capillary tube assay. The median time from imbibing an infectious bloodmeal until infected females transmitted WNV (median extrinsic incubation period, EIP50) was estimated by probit analysis. By regressing the EIP rate (inverse of EIP50) as a function of temperature from 14 to 30°C, the EIP was estimated to require 109 degree-days (DD) and the point of zero virus development (x-intercept) was estimated to be 14.3°C. The resulting degree-day model showed that the NY99 WNV strain responded to temperature differently than a lineage II strain of WNV from South Africa and approximated our previous estimates for St. Louis encephalitis virus (family Flaviviridae, genus Flavivirus, SLEV). The invading NY99 WNV strain therefore required warm temperatures for efficient transmission. The time for completion of the EIP was estimated monthly from temperatures recorded at Coachella Valley, Los Angeles, and Kern County, California, during the 2004 epidemic year and related to the duration of the Cx. tarsalis gonotrophic cycle and measures of WNV activity. Enzootic WNV activity commenced after temperatures increased, the duration of the EIP decreased, and virus potentially was transmitted in two or less gonotrophic cycles. Temperatures in the United States during the epidemic summers of 2002–2004 indicated that WNV dispersal and resulting epicenters were linked closely to above-average summer temperatures.

Avian Host and Mosquito (Diptera: Culicidae) Vector Competence Determine the Efficiency of West Nile and St. Louis Encephalitis Virus Transmission

Abstract The ability of the invading NY99 strain of West Nile virus (WNV) to elicit an elevated viremia response in California passerine birds was critical for the effective infection of Culex mosquitoes. Of the bird species tested, Western scrub jays, Aphelocoma coerulescens, produced the highest viremia response, followed by house finches, Carpodacus mexicanus, and house sparrows, Passer domesticus. Most likely, few mourning, Zenaidura macroura, or common ground, Columbina passerine, doves and no California quail, Callipepla californica, or chickens would infect blood-feeding Culex mosquitoes. All Western scrub jays and most house finches succumbed to infection. All avian hosts produced a lower viremia response and survived after infection with an endemic strain of St. Louis encephalitis virus. Culex species varied in their susceptibility to infection with both viruses, with Culex stigmatosoma Dyar generally most susceptible, followed by Culex tarsalis Coquillett, and then Culex p. quinquefasciatus Say. Populations within Culex species varied markedly in their susceptibility, perhaps contributing to the focality of WNV amplification. Transmitting female Cx. tarsalis expectorated from six to 3,777 plaque-forming units (PFU) of WNV during transmission trials, thereby exposing avian hosts to a wide range of infectious doses. Highly susceptible house finches and moderately susceptible mourning doves were infected by subcutaneous inoculation with decreasing concentrations of WNV ranging from 15,800 to <0.3 PFU. All birds became infected and produced comparable peak viremias on days 2–3 postinoculation; however, the rise in viremia titer and onset of the acute phase of infection occurred earliest in birds inoculated with the highest doses. WNV virulence in birds seemed critical in establishing elevated viremias necessary to efficiently infect blood feeding Culex mosquitoes.

Overwintering of West Nile Virus in Southern California

Abstract West Nile virus (family Flaviviridae, genus Flavivirus, WNV) invaded southern California during 2003, successfully overwintered, amplified to epidemic levels, and then dispersed to every county in the state. Although surveillance programs successfully tracked and measured these events, mechanisms that allowed the efficient overwintering and subsequent amplification of WNV have not been elucidated. Our current research provided evidence for three mechanisms whereby WNV may have persisted in southern California during the winters of 2003–2004 and 2004–2005: 1) continued enzootic transmission, 2) vertical transmission by Culex mosquitoes, and 3) chronic infection in birds. WNV was detected in 140 dead birds comprising 32 species, including 60 dead American crows, thereby verifying transmission during the November–March winter period. Dead American crows provide evidence of recent transmission because this species always succumbs rapidly after infection. However, WNV RNA was not detected concurrently in 43,043 reproductively active female mosquitoes comprising 11 species and tested in 1,258 pools or antibody in sera from 190 sentinel chickens maintained in 19 flocks. Although efficient vertical transmission by WNV was demonstrated experimentally for Culex tarsalis Coquillett infected per os, 369 females collected diapausing in Kern County and tested in 32 pools were negative for WNV. Vertical transmission was detected in Culex pipiens quinquefasciatus Say adults reared from field-collected immatures collected from Kern County and Los Angeles during the summer transmission period. Chronic infection was detected by finding WNV RNA in 34 of 82 birds that were inoculated with WNV experimentally, held for >6 wk after infection, and then necropsied. Frequent detection of WNV RNA in kidney tissue in experimentally infected birds >6 wk postinfection may explain, in part, the repeated detection of WNV RNA in dead birds recovered during winter, especially in species such as mourning doves that typically do not die after experimental infection. In summary, our study provides limited evidence to support multiple modes of WNV persistence in southern California. Continued transmission and vertical transmission by Culex p. quinquefasciatus Say seem likely candidates for further study.

Repeated West Nile Virus Epidemic Transmission in Kern County, California, 2004–2007

ABSTRACT West Nile virus (WNV) has remained epidemic in Kern County, CA, since its introduction in 2004 through 2007 when the human case annual incidence increased from 6–8 to 17 per 100,000, respectively. The 2007 increase in human infection was associated with contradicting surveillance indicators, including severe drought, warm spring but cool summer temperature anomalies, decreased rural and urban mosquito abundance but increased early season infection in urban Culex quinquefasciatus Say, moderate avian “herd immunity,” and declines in the catch of competent (western scrub-jay and house finch) and noncompetent (California quail and mourning dove) avian species. The decline in these noncompetent avian hosts may have increased contact with competent avian hosts and perhaps humans. The marked increase in home foreclosures and associated neglected swimming pools increased urban mosquito production sites, most likely contributing to the urban mosquito population and the WNV outbreak within Bakersfield. Coalescing five surveillance indicators into a risk assessment score measured each half month provided 2- to 6-wk early warning for emergency planning and was followed consistently by the onset of human cases after reaching epidemic conditions. St. Louis encephalitis virus (SLEV) antibody was detected rarely in wild birds but not mosquitoes or sentinel chickens, indicating that previously infected birds were detected in Kern County, but SLEV reintroduction was not successful. In contrast, western equine encephalitis virus (WEEV) was detected during 3 of 5 yr in Culex tarsalis Coquillett, sentinel chickens, and wild birds, but failed to amplify to levels where tangential transmission was detected in Aedes mosquitoes or humans. A comparison of transmission patterns in Kern County to Coachella Valley in the southeastern desert of California showed the importance of mosquito phenology and spatial distribution, corvids, or other avian “super spreaders” and anthropogenic factors in WNV epidemiology.

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.

Does Variation in Culex (Diptera: Culicidae) Vector Competence Enable Outbreaks of West Nile Virus in California

Abstract Since the invasion of California by West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in 2003, we have annually monitored vector competence for the NY99 strain in Culex tarsalis Coquillett, Culex pipiens quinquefasciatus Say, Culex p. pipiens L., and Culex stigmatosoma Dyar populations from four areas: deserts of Coachella Valley, densely urbanized maritime Los Angeles, southern San Joaquin Valley in Kern County, and southern Sacramento Valley near Davis in Sacramento County. Overall, Cx. stigmatosoma was the most competent vector species, followed by Cx. tarsalis and the Cx. pipiens complex. The median infectious dose (ID50) of WNV required to infect 50% of the F1 female progeny reared from wild-caught females, a measure of mesenteronal susceptibility, ranged between 5 and 8 log10 plaque forming units/ml and was not correlated with annual human case incidence or summer maximum likelihood mosquito infection estimates. Odds ratios comparing nonoutbreak years with referent outbreak years were variable and failed to show a distinct pattern for Cx. tarsalis or Cx. pipiens complex females. Apparently factors other than midgut susceptibility within the ranges we measured enabled WNV outbreaks in California. Culex populations remained competent for St. Louis encephalitis virus, indicating that the disappearance of this virus was not related to a loss of vector competence.

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.

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.

Is Nonviremic Transmission of West Nile Virus by Culex Mosquitoes (Diptera: Culicidae) Nonviremic?

Abstract Inter- and intraspecific transfer of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) occurred infrequently when donor Culex tarsalis Coquillett fed concurrently on house finches with recipient Culex quinquefasciatus Say and Cx. tarsalis. Five of six of these house finches had WNV in blood samples collected by jugular venipuncture 30–45 min postfeeding, with titers ranging from 2.3 to 4.2 log10 plaque-forming units (PFU)/ml. After 2-wk incubation at 26°C, three Cx. quinquefasciatus and one Cx. tarsalis of 230 blood-fed recipients were infected, of which one Cx. quinquefasciatus was capable of transmission. Our data indicated that infectious female mosquitoes feeding on small vertebrates create a nonpropagative viremia capable of infecting concurrently cofeeding females. The proportion of recipients infected is likely related to the amount of virus expectorated by donor females, the blood volume of the vertebrate host, and the susceptibility of the cofeeding mosquitoes.