Since the arrival of West Nile virus (WNV, Flavivirus, Flaviviridae) into New York City in 1999, the public health community has chronicled the unimpaired spread of this virus across North America from the Atlantic to the Pacific Coasts (1) and from Canada (2) into tropical America (3) and the Caribbean (4,5). Regionally, the epidemic has been characterized by an initial introduction with a few human cases during the first season, followed by explosive amplification and an epidemic during the second season, and then subsidence to maintenance levels. Ongoing or recent transmission of closely related St. Louis encephalitis virus (SLEV) in Florida, Louisiana, and Texas seems to have had little dampening effect on WNV amplification, which contradicts the long-held premise that two closely related flaviviruses cannot co-exist (6). Minimal ecologic resistance or selection pressure has left the strains of WNV intact genetically (7,8), until relatively minor changes may have resulted in attenuation in Mexico (3). In 1999, when WNV was introduced into North America, few encephalitis virus surveillance programs remained intact, and most were structured to protect urban centers (9). Consequently, the initial detection of WNV in most areas occurred after introduction and amplification and frequently was heralded by the discovery of dead crows or horses and humans with neurologic illness. California is somewhat unique in that an extensive arbovirus surveillance program has remained intact statewide. Because of endemic SLEV and western equine encephalomyelitis virus (WEEV, Alphavirus, Togaviridae) transmission and nuisance mosquito problems, California residents have supported special local mosquito and vector control districts that currently protect ca. 33.9 million people (88% of the state's population) over a combined area of ca. 166,107 km2. The associated California Encephalitis Virus Surveillance Program, which has been in place for more than 35 years (10), monitors mosquito abundance and infection rates as well as virus transmission to sentinel chickens. Local surveillance programs are coordinated at the state level by the California Department of Health Services, and supporting diagnostics currently are conducted by that agency and the Center for Vectorborne Diseases at the University of California, Davis. Recent ecologic studies on virus persistence and amplification by that center have been set against this extensive surveillance backdrop and have focused on wetlands along the Salton Sea (11,12). Our current study describes how this surveillance program, extended by associated field research projects, provided an early warning of the arrival of WNV in California and preliminary information on its ecology, surveillance, and dispersal during 2003. Highlighted information includes climatic conditions, the possible route (s) of introduction and subsequent dispersal, abundance of vector populations at the time of invasion, avian populations involved, and the comparative sensitivity of different surveillance indicators in different ecologic settings. Materials and Methods Climate data from Coachella Valley and the Los Angeles basin were downloaded from National Oceanographic and Atmospheric Administration weather stations from the California Integrated Pest Management website (http://www.ipm.ucdavis.edu/). These data were included to describe temperature conditions when virus was active and rainfall events associated with the intrusion of moist monsoon conditions from the Gulf of Mexico. Mosquitoes were collected biweekly at permanent sites by using dry ice-baited CDC-style traps (CO2 traps) operated without light (13) and gravid female traps (14). Sampling effort varied spatially. Six and 42 CO2 traps were operated at wetlands and agricultural habitats in Imperial and Coachella Valleys, respectively, whereas 4–13 CO2 and 6–20 gravid traps were operated per sampling occasion within an 8-km radius of the Whittier Dam area of Los Angeles. Mosquitoes were anesthetized with triethylamine, enumerated by species, grouped into pools of 1 seroconversion, and positive dead birds during each month. Encircled in panel D are the locations of the three foci studied in depth during 2003. Figure 3 Virus temporal dynamics in relation to Culex tarsalis in A) Imperial and B) Coachella Valleys. Shown are female (F) Cx. tarsalis collected per CO2 trap night (TN). West Nile virus minimum infection rates (MIR) per 1,000 tested adjusted for differential sample sizes, and the number of sentinel chicken seroconversions per 2-week period. Table 1 Surveillance data for southern West Nile virus foci and the rest of California, January 1–November 1, 2003a Surveillance data Research areas Imperial Coachella LA Remaining agencies Total Human cases 1 0 0 1 2 Horse cases 0 0 0 1 1 Mosquito pools 238 1,414 1,663 6,416 9,731 Culex tarsalis 150 948 121 3,176 4,395 WNV pos 16 10 0 0 26 SLEV pos 1 3 0 0 4 WEEV pos 0 0 0 1 1 Cx. pipiens complex 0 299 1,036 1,170 2,505 WNV pos 0 0 6 0 6 Othersb 88 167 506 2,070 2,831 Sentinel chickens 6 10 5 191 212 WNV pos 51 18 0 0 69 SLEV pos 3 2 0 8 13 WEEV pos 0 0 0 0 0 Dead birds reported 23 15 1,218 6,294 7,550 Tested 6 5 256 1,118 1,385 WNV pos 0 0 38 21 59 Wild bird sera 0 3,178 1,452 4,502 9,132 WNV pos 51 0 0 51 WEEV pos 2 0 0 2 aLA, Los Angeles; WNV, West Nile virus; SLEV, St. Louis encephalitis virus; WEEV, western equine encephalitis virus; pos, positive.
bOther mosquitoes tested: Anopheles franciscanus, An. hermsi, Ae. vexans, Culiseta inornata, Cs. incidens, Cx. erythrothorax, Cx. erraticus, Cx. stigmatosoma, Oc. sierrensis, Oc. dorsalis, Oc. melanimon, Oc. taeniorhynchus, Psorophora columbiae. Serum samples from live free-ranging birds in Coachella Valley showed an increase in Flavivirus prevalence (Figure 4) in resident species (Table 2), with WNV, SLEV, and WEEV detected near sites where these viruses were isolated from mosquitoes or detected by sentinel chicken seroconversions (Figure 2). Confirmatory PRNTs showed that Flavivirus-positive birds were infected with both WNV and SLEV. Of 31 birds with demonstrable PRNT titers, 20 were infected with WNV, 8 were infected with SLEV, and 3 had equivocal titers against both viruses. Live bird sampling programs in Los Angeles, Bakersfield, and Sacramento did not collect antibody-positive birds despite comparable sampling and testing efforts (Table 1). Figure 4 Wild bird Flavivirus seroprevalence rates (Flavi pos %) in Coachella Valley during 2003. Shown are percentages of total serum samples that tested positive each month by enzyme immunoassay. Positives include infections caused by West Nile virus and St. Louis encephalitis. Table 2 Wild birds collected and bled in Coachella Valley, January 1–November 1, 2003 Species Sera % Flavivirus a % WEEVb Abert's Towhee 108 0.9 0.0 House Finch 251 0.4 0.0 Least Bittern 10 10.0 0.0 Gambel's Quail 643 3.3 0.2 Common Ground Dove 95 5.3 0.0 Mourning Dove 729 1.5 0.1 Domestic Pigeon 39 25.6 0.0 White-winged Dove 6 16.7 0.0 58 species 1,297 0.0 0.0 Total 3,178 1.6 0.1 aPositive by enzyme immunoassay (P/N ratio >2). Some EIA-positive sera were negative by plaque reduction neutralization test, whereas some others were postive, but there was 1,200 dead birds by the end of October; 218 of these were tested, and 38 were positive for WNV. As expected because of their susceptibility and large size, most positives were crows, but small-sized passerines also tested positive. In urban Los Angeles, sentinel chickens did not seroconvert to WNV during 2003, despite being situated near recoveries of WNV-positive dead crows and Cx. p. quinquefasciatus pools and being in the vicinity of the large Whittier crow roost. Differences in sentinel chicken sensitivity between rural and urban habitats may relate to vector mosquito dispersal and not to avidity for feeding on chickens. In agreement, of 78 serum specimens taken from backyard chickens of unknown age from this urban area along the Rio Hondo and San Gabriel riparian corridors, 7 had antibody confirmed by PRNT to be WNV. In California, Cx. tarsalis is very dispersive (36,37) and hunts along riparian corridors or vegetative transitions (38,39), whereas Cx. p. quinquefasciatus is less dispersive in urban environments and remains near the point of emergence (40). Therefore, infectious Cx. p. quinquefasciatus may be less likely to disperse in urban environments and encounter confined sentinel flocks than are Cx. tarsalis in rural environments, where farmhouse environs provide widely spaced "islands" of elevated vegetation used by birds for roosting and nesting and by Cx. tarsalis for host-seeking and resting. Southern California environments lack the contiguous canopy found in the eastern deciduous forest, and Culex mosquitoes feed readily at ground level (41,42). Therefore, positioning sentinels at ground level does not appear to have been a critical factor in effectiveness. The number of dead bird reports in Los Angeles increased after WNV was introduced, presumably because of media coverage, public education concerning the dead bird surveillance program, and increased WNV-associated bird deaths. Our laboratory data indicated that approximately 80% of the dead birds tested after the invasion and media publicity were WNV-negative. These data indicated that at low-to-moderate levels of enzootic transmission, dead bird reports alone may not be a true indication of the level and location of WNV transmission. In addition, use of antibody testing of free-ranging birds collected in grain-baited crow traps (mostly House Sparrows and House Finches) did not seem to be a productive surveillance method in Los Angeles, and all birds to date have tested negative, including those trapped at Whittier Narrows. Our data during 2003 clearly showed that WNV introduction, amplification, and widespread dispersal occurred with few human or horse cases, indicating that such cases are insensitive indicators of WNV presence and enzootic activity levels. Most humans in rural southern California reside in homes with some form of air-conditioning and thereby may be protected from mosquito contact during the evening (43). Unknown proportions of horses in California are vaccinated and thereby may be protected from disease. Epidemic transmission of WNV in southern California has been predicted for 2004, and it will be of interest to determine how well enzootic measures of virus activity forecast human infection. Response California health agencies and vector control districts have been preparing for the introduction of WNV since movement into the West seemed eminent, and state guidelines for escalated control responses to surveillance data have been prepared (http://westnile.ca.gov/Publications.htm). Initial responses included enhanced surveillance, expanded larval control operations, and preparation for emergency adult control. Extended surveillance in Imperial County by the Imperial County Health Department, Coachella Valley Mosquito and Vector Control District, and University of California, Davis, and the development of a dead bird surveillance program by the California Department of Health Services during 2002 are examples of new programs that proved useful in tracking WNV during 2003. Detection of WNV in southeastern California during 2003 triggered adult mosquito control operations to interrupt transmission at wetlands and to protect residents of the small towns of Niland in Imperial County and Mecca in Coachella Valley. Dead bird surveillance data in urban Los Angeles were used to direct focal larval control operations and to launch public education programs through various media events. Surveillance activities in southern California continued during the winter of 2003 to 2004 and have included mosquito pool submission, sentinel chicken testing, live bird sampling and testing, and dead bird reporting and testing. All findings have been negative through mid-February 2004, despite surveillance near wetlands along the Salton Sea and at the Whittier Narrows crow roost, perhaps indicating that transmission ceased, despite mild winter conditions. Positive after-hatching-year and second-year resident birds from Coachella Valley have been collected, but these birds presumably were infected during 2003; all winter resident birds, such as White-crowned Sparrows, have remained negative. Planned and ongoing operational responses during spring 2004 have been coordinated at the local, regional, and state levels but necessarily vary among agencies because of local ecology, politics, and funding. The introduction of WNV into California and its anticipated amplification during the next few years will provide a rigorous test of how well a widespread integrated vector management approach to mosquito control can protect the residents of California from mosquito-borne disease.
