Sarah S. Wheeler

DIFFERENTIAL IMPACT OF WEST NILE VIRUS ON CALIFORNIA BIRDS

Abstract. The strain of West Nile virus (WNV) currently epidemic in North America contains a genetic mutation elevating its virulence in birds, especially species in the family Corvidae. Although dead American Crows (Corvus brachyrhynchos) have been the hallmark of the epidemic, the overall impact of WNV on North America’s avifauna remains poorly understood and has not been addressed thoroughly in California. Here, we evaluate variation by species in the effect of WNV on California birds from 2004 to 2007 by using (1) seroprevalence in free-ranging birds, (2) percentage of carcasses of each species reported by the public that tested positive for WNV, (3) mortality determined from experimental infections, and (4) population declines detected by trend analysis of Breeding Bird Survey (BBS) data. Using Bayesian linear models, we extrapolate trends in BBS data from 1980–2003 (pre-WNV) to 2004–2007 (post-WNV). We attribute signifcant declines from expected abundance trends in areas supporting epiornitics to WNV transmission. We combine risk assessed from each of the four data sets to generate an overall score describing WNV risk by species. The susceptibility of California avifauna to WNV varies widely, with overall risk scores ranging from low for the refractory Rock Pigeon (Columba livia) through high for the susceptible American Crow. Other species at high risk include, in descending order, the House finch (Carpodacus mexicanus) , Black- crowned N i g h t - Heron ( Nycticorax nycticorax) , Western Scrub - Jay ( Aphelocoma californica), and Yellow-billed magpie (Pica nuttalli). Our analyses emphasize the importance of multiple data sources in assessing the effect of an invading pathogen.

Role of Corvids in Epidemiology of West Nile Virus in Southern California

Abstract The invasion of different southern California landscapes by West Nile virus (WNV) and its subsequent amplification to epidemic levels during 2004 enabled us to study the impact of differing corvid populations in three biomes: the hot Colorado desert with few corvids (Coachella Valley), the southern San Joaquin Valley (Kern County) with large western scrub-jay but small American crow populations, and the cool maritime coast (Los Angeles) with a large clustered American crow population. Similar surveillance programs in all three areas monitored infection rates in mosquitoes, seroconversion rates in sentinel chickens, seroprevalence in wild birds, numbers of dead birds reported by the public, and the occurrence of human cases. Infection rates in Culex tarsalis Coquillett and sentinel chicken seroconversion rates were statistically similar among all three areas, indicating that highly competent mosquito hosts were capable of maintaining enzootic WNV transmission among less competent and widely distributed avian hosts, most likely house sparrows and house finches. In contrast, infection rates in Culex pipiens quinquefasciatus Say were statistically higher in Kern and Los Angeles counties with elevated corvid populations than in Coachella Valley with few corvids. Spatial analyses of dead corvids showed significant clusters near known American crow roosts in Los Angeles that were congruent with clusters of human cases. In this area, the incidence of human and Cx. p. quinquefasciatus infection was significantly greater within corvid clusters than without, indicating their importance in virus amplification and as a risk factor for human infection. In contrast the uniform dispersion by territorial western scrub-jays resulted in a high, but evenly distributed, incidence of human disease in Kern County.

Mosquito Host Selection Varies Seasonally with Host Availability and Mosquito Density

Host selection by vector mosquitoes is a critical component of virus proliferation, particularly for viruses such as West Nile (WNV) that are transmitted enzootically to a variety of avian hosts, and tangentially to dead-end hosts such as humans. Culex tarsalis is a principal vector of WNV in rural areas of western North America. Based on previous work, Cx. tarsalis utilizes a variety of avian and mammalian hosts and tends to feed more frequently on mammals in the late summer than during the rest of the year. To further explore this and other temporal changes in host selection, bloodfed females were collected at a rural farmstead and heron nesting site in Northern California from May 2008 through May 2009, and bloodmeal hosts identified using either a microsphere-based array or by sequencing of the mitochondrial cytochrome c oxidase I (COI) gene. Host composition during summer was dominated by four species of nesting Ardeidae. In addition, the site was populated with various passerine species as well as domestic farm animals and humans. When present, Cx. tarsalis fed predominantly (>80%) upon the ardeids, with Black-crowned Night-Herons, a highly competent WNV host, the most prevalent summer host. As the ardeids fledged and left the area and mosquito abundance increased in late summer, Cx. tarsalis feeding shifted to include more mammals, primarily cattle, and a high diversity of avian species. In the winter, Yellow-billed Magpies and House Sparrows were the predominant hosts, and Yellow-billed Magpies and American Robins were fed upon more frequently than expected given their relative abundance. These data demonstrated that host selection was likely based both on host availability and differences in utilization, that the shift of bloodfeeding to include more mammalian hosts was likely the result of both host availability and increased mosquito abundance, and that WNV-competent hosts were fed upon by Cx. tarsalis throughout the year.

Detection of Persistent West Nile Virus RNA in Experimentally and Naturally Infected Avian Hosts

To determine whether West Nile virus (WNV) persistent infection in avian hosts may potentially serve as an overwintering mechanism, House Sparrows and House Finches, experimentally and naturally infected with several strains of WNV, and two naturally infected Western Scrub-Jays were held in mosquito-proof outdoor aviaries from 2007-March 2008. Overall, 94% (n = 36) of House Sparrows, 100% (n = 14) of House Finches and 2 Western Scrub-Jays remained WNV antibody positive. When combined by species, 37% of the House Sparrows, 50% of the House Finches, and 2 Western Scrub-Jays were WNV RNA positive at necropsy, up to 36 weeks post-infection. Infectious WNV was not detected. Our study supports the hypothesis that some avian hosts support the long-term persistence of WNV RNA, but it remains unresolved whether these infections relapse to restart an avian-arthropod transmission cycle and thereby serve as an overwintering mechanism for WNV.

Dynamics of West Nile Virus Persistence in House Sparrows (Passer domesticus)

West Nile Virus (WNV) is now endemic throughout North America, with annual recurrence dependent upon successful overwintering when cold temperatures drive mosquito vectors into inactivity and halt transmission. To investigate whether avian hosts may serve as an overwintering mechanism, groups of eight to ten House Sparrows were experimentally infected with a WN02 genotype of WNV and then held until necropsy at 3, 5, 7, 9, 12, 15, or 18 weeks post-infection (pi) when they were assessed for the presence of persistent infection. Blood was collected from all remaining birds every two weeks pi, and sera tested for WNV RNA and WNV neutralizing antibodies. West Nile virus RNA was present in the sera of some birds up to 7 weeks pi and all birds retained neutralizing antibodies throughout the experiment. The detection of persistently infected birds decreased with time, from 100% (n = 13) positive at 3 weeks post-infection (pi) to 12.5% (n = 8) at 18 weeks pi. Infectious virus was isolated from the spleens of birds necropsied at 3, 5, 7 and 12 weeks pi. The current study confirmed previous reports of infectious WNV persistence in avian hosts, and further characterized the temporal nature of these infections. Although these persistent infections supported the hypothesis that infected birds may serve as an overwintering mechanism, mosquito-infectious recrudescent viremias have yet to be demonstrated thereby providing proof of principle.

Evidence for Co-evolution of West Nile Virus and House Sparrows in North America

West Nile virus (WNV) has been maintained in North America in enzootic cycles between mosquitoes and birds since it was first described in North America in 1999. House sparrows (HOSPs; Passer domesticus) are a highly competent host for WNV that have contributed to the rapid spread of WNV across the U.S.; however, their competence has been evaluated primarily using an early WNV strain (NY99) that is no longer circulating. Herein, we report that the competence of wild HOSPs for the NY99 strain has decreased significantly over time, suggesting that HOSPs may have developed resistance to this early WNV strain. Moreover, recently isolated WNV strains generate higher peak viremias and mortality in contemporary HOSPs compared to NY99. These data indicate that opposing selective pressures in both the virus and avian host have resulted in a net increase in the level of host competence of North American HOSPs for currently circulating WNV strains.

Migratory Birds and the Dispersal of Arboviruses in California

Each spring large numbers of neotropical migrants traversing the Pacific flyway pass through the Coachella Valley enroute to northern destinations, providing an opportunity to test the hypothesis that mosquito-borne encephalitis viruses are introduced annually into California by migratory birds. A total of 5,632 sera were collected from 43 species of migrants during spring (April-June), of which 34 (0.61%) comprised of 14 species tested positive by enzyme immunoassay; only 10 were confirmed by plaque reduction neutralization tests (PRNT). In addition, of 1,109 migrants comprised of 76 species that were reported dead by the public and necropsied, 126 (11%) were positive for West Nile virus (WNV) RNA; however, only three (0.7%) of 428 birds tested during the spring were positive. Limited experimental infection studies with WNV showed that Orange-crowned Warblers were highly susceptible and frequently died, whereas most Yellow Warblers survived. Our results indicated that birds entering California rarely exhibited a history of infection and that most birds probably became infected after entering California.

EVALUATION OF PYRETHRIN AND PERMETHRIN GROUND ULTRA-LOW VOLUME APPLICATIONS FOR ADULT CULEX CONTROL IN RURAL AND URBAN ENVIRONMENTS OF THE COACHELLA VALLEY OF CALIFORNIA

ABSTRACT Six experimental ground ultra-low volume (ULV) applications of Pyrenone® 25-5 (0.0025 lb/acre) and Aqua-Reslin® (0.007 lb/acre) were made by truck-mounted Pro-Mist® or London Fog® equipment over 1-mi2 study areas in rural and residential environments of the Coachella Valley, Riverside County, California. Efficacy of replicate applications was evaluated by measuring mortality among caged sentinel mosquitoes, by evaluating changes in host-seeking abundance at replicated dry ice–baited traps positioned along intersecting east–west and north–south transects, and by differential recapture patterns of marked females released near traps in the sprayed central core and unsprayed control areas. Sentinel mortality agreed well with estimates of droplet density measured by “slide spinners” and was affected by 1) distance of cages from the truck route; 2) landscape features, such as tree lines that created wind shadows; 3) irregular landscape that disrupted the particle cloud; 4) low wind speed that failed to carry the droplet cloud through the environment; and 5) failure of the droplets to penetrate dead airspace within stands of vegetation. Despite variable sentinel mortality, Culex tarsalis relative abundance in rural landscapes within and around our study areas always declined after ULV applications. Concurrent decreases in abundance at traps within sprayed and adjoining unsprayed areas confounded our estimates of percentage of control using Mullas formula, which compares abundance in sprayed and unsprayed areas pre- and postspray. ULV applications significantly affected recapture patterns, in that recapture rates within the spray zone usually were significantly less than in the unsprayed zone. Collectively, our data indicated that ground ULV applications measurably reduced Cx. tarsalis abundance in rural areas of the Coachella Valley but that further evaluations may be necessary to validate efficacy on Culex quinquefasciatus populations in residential communities.

Efficacy of Three Vaccines in Protecting Western Scrub-Jays (Aphelocoma californica) from Experimental Infection with West Nile Virus: Implications for Vaccination of Island Scrub-Jays (Aphelocoma insularis)

The devastating effect of West Nile virus (WNV) on the avifauna of North America has led zoo managers and conservationists to attempt to protect vulnerable species through vaccination. The Island Scrub-Jay (Aphelocoma insularis) is one such species, being a corvid with a highly restricted insular range. Herein, we used congeneric Western Scrub-Jays (Aphelocoma californica) to test the efficacy of three WNV vaccines in protecting jays from an experimental challenge with WNV: (1) the Fort Dodge West Nile-Innovator(®) DNA equine vaccine, (2) an experimental DNA plasmid vaccine, pCBWN, and (3) the Merial Recombitek(®) equine vaccine. Vaccine efficacy after challenge was compared with naïve and nonvaccinated positive controls and a group of naturally immune jays. Overall, vaccination lowered peak viremia compared with nonvaccinated positive controls, but some WNV-related pathology persisted and the viremia was sufficient to possibly infect susceptible vector mosquitoes. The Fort Dodge West Nile-Innovator DNA equine vaccine and the pCBWN vaccine provided humoral immune priming and limited side effects. Five of the six birds vaccinated with the Merial Recombitek vaccine, including a vaccinated, non-WNV challenged control, developed extensive necrotic lesions in the pectoral muscle at the vaccine inoculation sites, which were attributed to the Merial vaccine. In light of the well-documented devastating effects of high morbidity and mortality associated with WNV infection in corvids, vaccination of Island Scrub-Jays with either the Fort Dodge West Nile-Innovator DNA vaccine or the pCBWN vaccine may increase the numbers of birds that would survive an epizootic should WNV become established on Santa Cruz Island.

Use of Scented Sugar Bait Stations to Track Mosquito-Borne Arbovirus Transmission in California

ABSTRACT Laboratory and field research was conducted to determine if Culex tarsalis Coquillett expectorated West Nile virus (WNV) during sugar feeding and if a lure or bait station could be developed to exploit this behavior for WNV surveillance. Experimentally infected Cx. tarsalis repeatedly expectorated WNV onto filter paper strips and into vials with wicks containing sucrose that was readily detectable by a quantitative reverse transcriptase-polymerase chain reaction assay. Few females (33%, n = 27) became infected by imbibing sugar solutions spiked with high concentrations (107 plaque forming units/ml) of WNV, indicating sugar feeding stations probably would not be a source of WNV infection. In nature, sugar bait stations scented with the floral attractant phenyl acetaldehyde tracked WNV transmission activity in desert but not urban or agricultural landscapes in California. When deployed in areas of the Coachella Valley with WNV activity during the summer of 2011, 27 of 400 weekly sugar samples (6.8%) tested positive for WNV RN A by reverse transcriptase-polymerase chain reaction. Prevalence of positives varied spatially, but positive sugar stations were detected before concurrent surveillance measures of infection (mosquito pools) or transmission (sentinel chicken seroconversions). In contrast, sugar bait stations deployed in urban settings in Los Angeles or agricultural habits near Bakersfield in Kern County supporting WNV activity produced 1 of 90 and 0 of 60 positive weekly sugar samples, respectively. These results with sugar bait stations will require additional research to enhance bait attractancy and to understand the relationship between positive sugar stations and standard metrics of arbovirus surveillance.