Diagnostic applications of molecular and serological assays for bluetongue and African horse sickness.

The availability of rapid, highly sensitive and specific molecular and serologic diagnostic assays, such as competitive enzyme-linked immunosorbent assay (cELISA), has expedited the diagnosis of emerging transboundary animal diseases, including bluetongue (BT) and African horse sickness (AHS), and facilitated more thorough characterisation of their epidemiology. The development of assays based on real-time, reverse-transcription polymerase chain reaction (RT-PCR) to detect and identify the numerous serotypes of BT virus (BTV) and AHS virus (AHSV) has aided in-depth studies of the epidemiology of BTV infection in California and AHSV infection in South Africa. The subsequent evaluation of pan-serotype, real-time, RT-PCR-positive samples through the use of serotype-specific RT-PCR assays allows the rapid identification of virus serotypes, reducing the need for expensive and time-consuming conventional methods, such as virus isolation and serotype-specific virus neutralisation assays. These molecular assays and cELISA platforms provide tools that have enhanced epidemiologic surveillance strategies and improved our understanding of potentially altered Culicoides midge behaviour when infected with BTV. They have also supported the detection of subclinical AHSV infection of vaccinated horses in South Africa. Moreover, in conjunction with whole genome sequence analysis, these tests have clarified that the mechanism behind recent outbreaks of AHS in the AHS-controlled area of South Africa was the result of the reversion to virulence and/or genome reassortment of live attenuated vaccine viruses. This review focuses on the use of contemporary molecular diagnostic assays in the context of recent epidemiologic studies and explores their advantages over historic virus isolation and serologic techniques.

La disponibilité d'essais diagnostiques moléculaires et sérologiques rapides, hautement sensibles et spécifiques tels que l'épreuve immuno-enzymatique de compétition (ELISAc), a accéléré le diagnostic des maladies animales transfrontalières émergentes, dont la fièvre catarrhale ovine (FCO) et la peste équine, et contribué à dresser un tableau épidémiologique plus complet de ces maladies. Grâce à la mise au point d'essais basés sur l'amplification en chaîne par polymérase en temps réel couplée à une transcription inverse (RT­PCR) qui permettent de détecter et d'identifier les nombreux sérotypes du virus de la fièvre catarrhale du mouton et du virus de la peste équine, des études approfondies ont pu être conduites sur l'épidémiologie de l'infection par le virus de la fièvre catarrhale du mouton en Californie et de l'infection par le virus de la peste équine en Afrique du Sud. L'évaluation postérieure des échantillons positifs à une RT­PCR en temps réel de groupe (détectant le virus quel que soit le sérotype) au moyen de RT­PCR spécifiques de chaque sérotype permet d'identifier rapidement le sérotype causal et de limiter le recours à des méthodes classiques onéreuses et chronophages comme l'isolement viral ou les essais de neutralisation virale spécifiques de chaque sérotype. Les outils fournis par ces essais moléculaires et par les plateformes ELISAc ont renforcé les stratégies de surveillance épidémiologique et permis de mieux connaître les altérations potentielles de comportement chez les tiques Culicoides infectées par le virus de la fièvre catarrhale du mouton. Ils ont également contribué à détecter les cas d'infection asymptomatique par le virus de la peste équine chez des chevaux vaccinés en Afrique du Sud. En outre, associés avec l'analyse de séquences du génome entier, ces tests ont révélé que le mécanisme sous-jacent aux récents foyers de peste équine dans la zone de contrôle en Afrique du Sud correspondait à une réversion vers la virulence et/ou à un réassortiment du génome des souches de vaccin à virus vivant atténué. Les auteurs passent en revue l'utilisation des essais de diagnostic moléculaire de nouvelle génération dans le contexte de récentes études épidémiologiques et cherchent à établir leurs avantages par rapport aux techniques classiques d'isolement viral et de recherche sérologique.

La existencia de ensayos moleculares y serológicos de diagnóstico rápidos y de gran sensibilidad y especificidad, como el ensayo inmunoenzimático de competición (ELISAc), ha acelerado el diagnóstico de enfermedades animales transfronterizas emergentes, como la lengua azul o la peste equina, y facilitado una caracterización más exhaustiva de su epidemiología. La creación de ensayos basados en la reacción en cadena de la polimerasa acoplada a transcripción inversa (RT?PCR) en tiempo real para detectar y caracterizar los numerosos serotipos de los virus de la lengua azul y la peste equina ha ayudado a estudiar a fondo la epidemiología de sendos episodios infecciosos causados por el virus de la lengua azul en California y por el virus de la peste equina en Sudáfrica. El subsiguiente análisis de las muestras positivas a la prueba de RT?PC en tiempo real de cualquier serotipo con empleo de ensayos RT?PCR dirigidos específicamente contra uno u otro serotipo permite identificar rápidamente los serotipos víricos, lo que hace menos necesario el uso de métodos convencionales más caros y largos, como el aislamiento del virus o técnicas de neutralización vírica adaptadas específicamente a un serotipo. Estos dispositivos de ensayo molecular o de ELISAc ponen a nuestra disposición herramientas que potencian las estrategias de vigilancia epidemiológica y ayudan a conocer mejor las eventuales alteraciones del comportamiento de los jejenes Culicoides al ser infectados por el virus de la lengua azul. Estas técnicas han ayudado también a detectar en Sudáfrica casos de infección asintomática por el virus de la peste equina en caballos vacunados. Estas pruebas, además, empleadas en combinación con el análisis de secuencias genómicas completas, han servido para aclarar que el mecanismo subyacente a los recientes brotes de peste equina surgidos en la zona de Sudáfrica donde la enfermedad estaba bajo control fue fruto de la reversión a la virulencia y/o el reordenamiento genómico de virus vacunales atenuados. Los autores, centrándose en el uso de modernos ensayos moleculares de diagnóstico como parte de recientes estudios epidemiológicos, examinan las ventajas que ofrecen en comparación con las tradicionales técnicas serológicas y de aislamiento vírico.

West Nile Virus Activity in a Winter Roost of American Crows (Corvus brachyrhynchos): Is Bird-To-Bird Transmission Important in Persistence and Amplification?

Since its emergence in North America, West Nile virus (WNV) has had a large impact on equines, humans, and wild bird communities, yet gaps remain in our understanding of how the virus persists at temperate latitudes when winter temperatures preclude virus replication and host-seeking activity by mosquito vectors. Bird-to-bird transmission at large communal American Crow roosts could provide one mechanism for WNV persistence. Herein, we describe seasonal patterns of crow and Culex mosquito abundance, WNV infection rates, and the prevalence of WNV-positive fecal samples at a winter crow roost to test the hypothesis that bird-to-bird transmission allows WNV to persist at winter crow roosts. Samples were collected from large winter crow roosts in the Sacramento Valley of California from January 2013 until August 2014, encompassing two overwintering roost periods. West Nile virus RNA was detected in local crow carcasses in both summer [13/18 (72% WNV positive)] and winter [18/44 (41% WNV positive)] 2013-2014. Winter infections were unlikely to have arisen by recent bites from infected mosquitoes because Culex host-seeking activity was very low in winter and all Culex mosquitoes collected during winter months tested negative for WNV. Opportunities existed for fecal-oral transfer at the overwintering roost: most carcasses that tested positive for WNV had detectable viral RNA in both kidney and cloacal swabs, suggesting that infected crows were shedding virus in their feces, and >50% of crows at the roost were stained with feces by mid-winter. Moreover, 2.3% of fecal samples collected in late summer, when mosquitoes were active, tested positive for WNV RNA. Nevertheless, none of the 1,119 feces collected from three roosts over two winters contained detectable WNV RNA. This study provided evidence of WNV infection in overwintering American crows without mosquito vector activity, but did not elucidate a mechanism of WNV transmission during winter.

Lack of Evidence for Laboratory and Natural Vertical Transmission of Bluetongue Virus in Culicoides sonorensis (Diptera: Ceratopogonidae).

Culicoides sonorensis (Wirth & Jones) is the principal North American vector of bluetongue virus (BTV). BTV infection of livestock is distinctly seasonal (late summer and fall) in temperate regions of the world such as California, which has led to speculation regarding vertical transmission of the virus within the midge vector as a potential mechanism for interseasonal maintenance ("overwintering") of the virus. To evaluate potential vertical transmission of BTV in its midge vector, we fed adult midges BTV-spiked blood and used a BTV-specific quantitative reverse transcriptase polymerase chain reaction assay to evaluate parent, egg, and progeny stages of laboratory-reared C. sonorensis for the presence of viral nucleic acid. Whereas BTV nucleic acid was weakly detected in egg batches of virus-fed female midges, virus was never detected in subsequent progeny stages (larvae, pupae, and F1 generation adults). Similarly, BTV was not detected in pools of larvae collected from the waste-water lagoon of a BTV-endemic dairy farm in northern California during the seasonal period of virus transmission. Collectively, these results indicate that BTV is not readily transmitted vertically in C. sonorensis, and that persistence of the virus in long-lived parous female midges is a more likely mechanism for overwintering of BTV in temperate regions.

Spatial variation in host feeding patterns of Culex tarsalis and the Culex pipiens complex (Diptera: Culicidae) in California.

West Nile virus (family Flaviviridae, genus Flavivirus, WNV) is now endemic in California across a variety of ecological regions that support a wide diversity of potential avian and mammalian host species. Because different avian hosts have varying competence for WNV, determining the blood-feeding patterns of Culex (Diptera: Culicidae) vectors is a key component in understanding the maintenance and amplification of the virus as well as tangential transmission to humans and horses. We investigated the blood-feeding patterns of Culex tarsalis Coquillett and members of the Culex pipiens L. complex from southern to northern California. Nearly 100 different host species were identified from 1,487 bloodmeals, by using the mitochondrial gene cytochrome c oxidase I (COI). Cx. tarsalis fed on a higher diversity of hosts and more frequently on nonhuman mammals than did the Cx. pipiens complex. Several WNV-competent host species, including house finch and house sparrow, were common bloodmeal sources for both vector species across several biomes and could account for WNV maintenance and amplification in these areas. Highly competent American crow, western scrub-jay and yellow-billed magpie also were fed upon often when available and are likely important as amplifying hosts for WNV in some areas. Neither species fed frequently on humans (Cx. pipiens complex [0.4%], Cx. tarsalis [0.2%]), but with high abundance, both species could serve as both enzootic and bridge vectors for WNV.

Development of a high-throughput microsphere-based molecular assay to identify 15 common bloodmeal hosts of Culex mosquitoes.

For vectorborne infections, host selection by bloodfeeding arthropods dictates the interaction between host and pathogen. Because Culex mosquitoes that transmit West Nile virus (WNV) feed both on mammalian and avian hosts with varying competence, understanding the bloodfeeding patterns of these mosquitoes is important for understanding the transmission dynamics of WNV. Herein, we describe a new microsphere-based assay using Luminex xMAP® technology to rapidly identify 15 common hosts of Culex mosquitoes at our California study sites. The assay was verified with over 100 known vertebrate species samples and was used in conjunction with DNA sequencing to identify over 125 avian and mammalian host species from unknown Culex bloodmeals, more quickly and with less expense than sequencing alone. In addition, with multiplexed labelled probes, this microsphere array identified mixed bloodmeals that were difficult to discern with traditional sequencing. The microsphere set was easily expanded or reduced according to host range in a specific area, and this assay has made it possible to rapidly screen thousands of Culex spp. bloodmeals to extend our understanding of WNV transmission patterns.

Complexity of the Culex pipiens complex in California.

In California, the Culex pipiens complex consists of Culex pipiens, Cx. quinquefasciatus, their hybrids and Culex pipiens form molestus. Using 15 microsatellite markers and a variety of statistical analyses of within- and among-population variation, there is widespread introgression throughout the Central Valley with mostly quinquefasciatus genotypes in the south and pipiens in the north. Those specimens in the Sacramento County area consisted primarily of pipiens-quinquefasciatus and pipiens-molestus hybrids. Populations in Coachella Valley and Los Angeles, CA and Benton, WA were Cx. quinquefasciatus and Cx. pipiens, respectively. Studies are underway to relate these genotypes to phenotypes of autogeny, diapause and vector competence for West Nile Virus.

Potential for the emergence of Japanese encephalitis virus in California.

The potential risk for the introduction and establishment of Japanese encephalitis virus (JEV) within California is described based on the literature. JEV is a mosquito-borne arbovirus endemic to Asia that when transmitted to humans can lead to Japanese encephalitis (JE), a disease affecting mostly children with a fatality rate up to 30%. The geographical expansion of JEV in Asia along with the recent introduction and rapid spread of West Nile virus (WNV) across the United States, demonstrates the ability of arboviruses to rapidly extend their distributions. California is at particular risk for the introduction of JEV because it is a large state functioning as a hub for international travel and commerce with Asia, potentially allowing the introduction of mosquitoes infected with JEV. If JEV is introduced into California, the virus might become established due to the significant number of susceptible mosquito vectors and vertebrate hosts. Once introduced, the lack of active surveillance for JEV, the ambiguous clinical presentation of JE, the cross reactivity of serological testing between JEV and other flaviviruses, and the probability that clinicians and laboratories would not consider JE as a possible diagnosis would likely delay recognition. A significant delay in detection of JEV in California would make control and eradication of the virus very difficult and costly. Public health authorities should consider the need for future control efforts if JEV emerges in the United States.

Deposition of pyrethrins and piperonyl butoxide following aerial ultra-low volume applications in the Coachella Valley, California.

Data on adulticide deposition were collected during studies optimizing aerial ultra-low volume applications and droplet size in the desert environment of the Coachella Valley, Riverside County, California. Pyrenone 25-5 and BVA Spray 13 oil were applied by a single-engine, fixed wing aircraft equipped with 2 Micronair AU5000 atomizers. Data recorded by a portable weather station documented that weather conditions were suitable for application. Adulticide residue was collected using 24-cm-diameter filter papers positioned along 2-3 transects, with 3 positive controls held outside of the treated zone. The trace amounts of 2 major insecticidal components (pyrethrin I and II) and the synergist piperonyl butoxide (PBO) were detected from samples near the center of the spray zone by high-performance liquid chromatography (HPLC); pyrethrin deposition was highest at the center, 156 microg/m2, and it was not detectable 60 m beyond the center of the transect, whereas PBO deposition was 5,000 microg/m2 at the center but was not detectable beyond 150 m. Droplet diameters on spinning Teflon slides were larger than expected for the rated output of the atomizers. For these single swath trials, the lack of swath overlap due to drift resulted in low mortality in sentinel mosquitoes. Detection of residues was limited to the centroid of droplet densities on spinning glass slides and with mortality among sentinel mosquitoes, indicating HPLC may be useful in detecting postspray residues.

Ability of transstadially infected Ixodes pacificus (Acari: Ixodidae) to transmit West Nile virus to song sparrows or western fence lizards.

The hypothesis that Ixodes pacificus Cooley & Kohls (Acari: Ixodidae) may serve as a reservoir and vector of West Nile virus (family Flaviviridae, genus Flavivirus, WNV) in California was tested by determining the ability of this tick species to become infected with the NY99 strain of WNV while feeding on viremic song sparrows, to maintain the infection transstadially, and then to transmit WNV to recipient naive song sparrows and western fence lizards during the nymphal stage. The percentage of ticks testing positive by reverse transcription-polymerase chain reaction (RT-PCR) decreased from 77% of 35 larvae at day 6 after ticks were transferred to donor song sparrows (day of detachment) to 23% of 35 nymphs at 59 d postinfestation (approximately 19 d after molting to the nymphal stage). However, the percentage of ticks positive by RT-PCR from which infectious virus was recovered by Vero cell assay decreased from 59% on day 6 to 12% on day 59, even though there was no statistically significant decrease in the quantity of RNA within positive ticks. Attempts to improve the sensitivity of plaque assays by blind passage through C6/36 cell cultures were unsuccessful. These data indicated that ticks maintained viral RNA but not necessarily infectious virus over time. Nymphs from larvae that fed on song sparrows with peak viremias ranging from 7.2 to 8.5 log10 plaque-forming units (PFU) per ml were used in transmission attempts. From one to seven RNA-positive nymphal ticks engorged and detached from each of four recipient song sparrows or western fence lizards. Blood samples from sparrows and lizards remained negative, indicating that transmission did not occur. An additional four lizards inoculated with 1,500 PFU of WNV developed moderate viremias, ranging from 4.2 to 5.6 log10 PFU/ml. Our data and data from previous studies collectively indicated that ixodid ticks were not able to experimentally transmit WNV and therefore most likely would not be important vectors in WNV transmission cycles.

Nesting Ardeid colonies are not a focus of elevated West Nile virus activity in southern California.

A large nesting colony of Ardeid birds at the Finney-Ramer Wildlife Refuge in Imperial County, California, did not appear to be a focus of West Nile virus (WNV) amplification during the summer of 2004. Blood samples taken during June and July from 155 nestlings of four species of Ardeid birds (cattle egrets, black-crowned night herons, great egrets, and snowy egrets) and five nestling double-crested cormorants yielded a single WNV isolation from a 3-week-old cattle egret. Antibody was detected by enzyme immunoassay from 20 nestlings (13%), 14 (70%) of which were confirmed as positive by plaque reduction neutralization test (PRNT). However, titration end points against WNV and St. Louis encephalitis virus (SLEV) were similar precluding viral identification. The grouping of positives within few nests, highest PRNT titers in youngest birds (<1 weeks of age), the decline of titer with nestling age, and the lack of antibody specificity indicated that antibody may have been acquired maternally and did not represent new infections. Infection rates in Culex tarsalis mosquitoes collected near the Ardeid colony at Ramer Lake (3.1 per 1,000) were statistically similar to rates estimated at the nearby Wister Unit wetlands (5.3 per 1,000) that lacked an Ardeid nesting colony. Black-crowned night heron nestlings experimentally infected with the NY99 strain of WNV produced viremias >5 log10 plaque forming units (PFU)/mL and were considered moderately competent hosts, whereas cattle egret nestlings had viremias that remained <5 log10 PFU/mL and were incompetent hosts.

Avian host and mosquito (Diptera: Culicidae) vector competence determine the efficiency of West Nile and St. Louis encephalitis virus transmission.

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.

Previous infection protects house finches from re-infection with St. Louis encephalitis virus.

Antibody titers against St. Louis encephalitis virus (SLE) measured by a plaque reduction neutralization test (PRNT) decreased rapidly in house finches (Capodacus mexicanus) after initial infection, whereas antibodies measured by enzyme immunoassay (EIA) remained detectable in all birds for the length of the experiment, indicating long-term persistence and greater assay sensitivity of the EIA. After 52 wk, birds were challenged by subcutaneous inoculation with the same strain of SLE virus. Virus was not detected for 1-4 d postchallenge in blood samples tested by plaque assay and RT-PCR or by xenodiagnosis in Culex tarsalis fed concurrently and then held for 11 d at 26 degrees C. Virus was detected by all three methods in control birds infected concurrently for the first time. Challenge with SLE produced a rapid and marked ananmestic rise in both neutralizing and EIA antibody titers that exceeded the primary response in the same birds or in concurrently inoculated control birds. At necropsy 4 wk postchallenge, 3 of 7 challenged and 1 of 2 positive control birds were chronically infected, with viral RNA detected by RT-PCR in brain, spleen, lung, and/or kidney tissues. Our results indicated that persistence of protective antibody prevents reinfection during the following season and may prevent the recrudescence of infectious virus in chronically infected birds.

Experimental infection of California birds with western equine encephalomyelitis and St. Louis encephalitis viruses.

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

Comparison of dry ice-baited centers for disease control and New Jersey light traps for measuring mosquito abundance in California.

Mosquito catch in New Jersy light traps (NJLTs) has been declining in recent years, compromising the sensitivity of the California mosquito monitoring program. Centers for Disease Control traps (CDCTs) operated without light and augmented with dry ice have been considered for replacement or augmentation. To provide information on comparative sensitivity and ability to measure abundance over time and space, catch of mosquitoes in NJLTs was compared to catch in CDCTs operated concurrently at 8-10 sites within the Coachella Valley, Kern, San Joaquin County, and Sacramento-Yolo Mosquito and Vector Control Districts. The CDCTs always collected more female mosquitoes than did NJLTs; however, differences in sensitivity varied markedly over time and space precluding the calculation of a universal conversion factor. Regressions of the catch of female Culex tarsalis in CDCTs as a function of catch in NJLTs within districts indicated that the slopes varied markedly, again precluding the derivation of a universal function. Therefore, we recommend that mosquito surveillance programs replace or supplement NJLTs with systematically operated CDCTs to enhance sampling sensitivity for females of most mosquito species. However, both trap types should be operated concurrently at several sites within each district to derive regression functions to convert historical relative abundance data from NJLTs to equivalent counts in CDCTs for retrospective analyses.

Persistence and amplification of St. Louis encephalitis virus in the Coachella Valley of California, 2000-2001.

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

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 stilldetected 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.

Encephalitis virus persistence in California birds: preliminary studies with house finches.

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, 10(2.73) plaque forming units/0.1 ml). When necropsied 7 wk after challenge, 1/110 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.

Landscape affects the host-seeking patterns of Culex tarsalis (Diptera: Culicidae) in the Coachella Valley of California.

Effective arbovirus transmission requires that the principal vertebrate hosts and vectors have frequent contact. Vegetation and other landscape features used by roosting or nesting birds at night dictate their exposure to nocturnally active host-seeking Culex tarsalis Coquillett and therefore to western equine encephalomyelitis and St. Louis encephalitis viruses. Precipitin tests on 645 Cx. tarsalis that were collected resting and host-seeking near the Salton Sea in Coachella Valley, CA, indicated that passeriform birds (64%) and rabbits (25%) were the most frequent bloodmeal hosts and that the percentage of females feeding on birds varied temporally as an inverse fuction of mosquito abundance. Blood meals were not taken from communally roosting water birds. The spatial distribution of host-seeking females then was investigated by deploying dry ice baited traps within seven sites representative of habitats found along the Salton Sea. Mosquito catch was greatest at traps within elevated vegetation such as Tamarisk, mesquite, cattails, and orchards and lowest at traps positioned at snags over water, sand bars, open fields, and within housing in a small rural community. These data indicate that host-seeking Cx. tarsalis females congregated at specific landscape features that were not necessarily associated with large concentrations of potential bloodmeal hosts.