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Many species of Amblyomma ticks are commonly found infesting wild birds in South America, where birds are important hosts for several arboviruses, such as West Nile virus (WNV) and St. Louis encephalitis virus (SLEV). In this study, WNV and SLEV transmission experiments were performed to evaluate the vector competence of three South American tick species: Amblyomma ovale, Amblyomma tigrinum, and Amblyomma tonelliae. Larval and nymphal ticks of each species were allowed to feed on chicks needle inoculated with WNV or SLEV. All three Amblyomma species acquired either WNV or SLEV through larval feeding, with infection rates varying from 3.1% to 100% for WNV and from 0% to 35.7% for SLEV in engorged larvae. Transstadial perpetuation of the viruses was demonstrated in the molted nymphs, with WNV infection rates varying from 0% to 33.7% and SLEV infection rates from 13.6% to 23.8%. Although nymphal ticks also acquired either virus through feeding, transstadial perpetuation to adult ticks was lower, with virus detection in only 3.2% of A. tigrinum and 11.5% of A. tonelliae unfed adult ticks. On the other hand, vector competence for nymphs (exposed to WNV or SLEV through larval feeding) and adult ticks (exposed to WNV or SLEV through larval or nymphal feeding) was null in all cases. Although our results indicate transstadial perpetuation of WNV or SLEV in the three tick species, the ticks were not competent to transmit these agents to susceptible hosts. The role of these ixodid tick species in the epidemiology of WNV and SLEV might be insignificant, even though at least A. ovale and A. tigrinum are frequent bird ticks in Latin America, so the virus could survive winter in the fed larvae. However, future studies are required to determine the implications that this could have, as well as analyze the vector competence of other common bird tick species in South America.

St.Louis encephalitis virus (SLEV) is an emerging human pathogen flavivirus in Argentina. Recently, it has reemerged in the United States. We evaluated the role as amplifying host of six resident bird species and analyzed their capacity as host during the 2005 encephalitis outbreak of SLEV in Córdoba. Eared Dove, Picui Ground Dove, and House Sparrow were the three species with highest host competence index. At a city level, Eared Dove and Picui Ground Dove were themost important amplifying hosts during the 2005 SLEV human outbreak inCórdoba city. This finding highlighted important differences in the SLEV ecology between Argentina and the United States. Characterizing and evaluating the SLEV hosts contribute to our knowledge about its ecology and could help us to understand the causes that promote its emergence as a human pathogen in South America. Fil: Diaz, Luis Adrian. Universidad Nacional de Córdoba. Facultad de Medicina. Laboratorio de Arbovirus y Arenovirus; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina Fil: Flores, Fernando Sebastián. Universidad Nacional de Córdoba. Facultad de Medicina. Laboratorio de Arbovirus y Arenovirus; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Santa Fe. Instituto de Ciencias Veterinarias del Litoral. Universidad Nacional del Litoral. Facultad de Ciencias Veterinarias. Instituto de Ciencias Veterinarias del Litoral; Argentina Fil: Quaglia, Agustín Ignacio Eugenio. Universidad Nacional de Córdoba. Facultad de Medicina. Laboratorio de Arbovirus y Arenovirus; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba; Argentina Fil: Contigiani de Minio, Marta Silvia. Universidad Nacional de Córdoba; Argentina

The California Mosquito-Borne Virus Surveillance and Response Plan recently was developed to provide a semi-quantitative means for assessing risk for western equine encephalomyelitis (WEE) or St. Louis encephalitis (SLE) viruses and to provide intervention guidelines for mosquito control and public health agencies during periods of heightened risk for human infection. West Nile virus recently has arrived in California, and the response plan also will provide a baseline for assessing the risk for human and equine infection with this virus. In the response plan, overall risk is calculated by averaging risk due to 1) environmental conditions, 2) adult mosquito vector abundance, 3) vector infection rates, 4) sentinel chicken seroconversion rates, 5) equine cases (for WEE), 6) human cases, and 7) the proximity of virus activity to populated areas. Overall risk is categorized into three levels: normal season, emergency planning, or epidemic conditions. We evaluated this response plan using historical data from years with no, enzootic, and epidemic activity of WEE and SLE in several areas of California to determine whether calculated risk levels approximated actual conditions. Multiple methods of risk calculation were considered for both viruses. Assessed risk based on cumulative temperature, rainfall, and runoff levels over the entire season provided more or equally accurate assessments than biweekly assessments based solely on the previous half-month. For WEE, during years with enzootic activity or early-season periods of years with WEE epidemic activity, combining horse and human cases as a single risk factor improved the model's ability to forecast pending WEE activity, but separating the two factors allowed a better indication of WEE activity during epidemics and periods with no activity. For SLE, assignment of higher risk to drier conditions as measured by rainfall and runoff yielded the most accurate representation of actual virus activity during all recent study periods.

St. Louis encephalitis (SLE) is endemic in Harris County, Texas. The disease is a public health concern in Houston, the largest city in Harris County, and in the state. Consequently, intensive surveillance for SLE virus in local mosquito populations is carried out by the Harris County Mosquito Control Division each year. In this study, we examined genetic variation among SLE isolates obtained during routine virus surveillance over a 13-year time period (1986-1999). St. Louis encephalitis virus isolates were tested for genetic variation using reverse transcription-polymerase chain reaction followed by single-strand conformation polymorphism (SSCP). The results indicated that multiple genotypes of the virus circulate in Harris County. During several years, the genotypes were restricted in their location, i.e., each general area within the county had a specific genotype of the virus. In other years, the various genotypes were widely distributed throughout the county. The presence of multiple distinct genotypes suggests that viruses with different biological characteristics may be circulating in Harris County, and that discrete foci of SLE virus activity occur simultaneously.

The mechanism for long-term maintenance of St. Louis encephalitis (SLE) virus in California is unknown. Two possibilities are 1) that the virus is maintained locally in discrete enzootic foci by one or more reservoir mechanisms, and/or 2) that the foci are ephemeral in nature and virus is reintroduced periodically from other enzootic areas by migratory birds or movement of vectors. We have investigated these epidemiologic alternatives by studies of genetic variation within a 277 nucleotide portion of the envelope-encoding region among 17 strains of SLE virus isolated since 1952 from different geographic locations in California. Three lineages of virus were detected. One lineage, Group A, consisted of four SLE virus strains isolated in California since 1972 from the Coachella, Sacramento, and San Joaquin Valleys. The group A strains were closely related to strain MSI-7 of SLE virus isolated in Mississippi in 1975. The 13 other strains formed the second and third lineages (Groups B1 and B2) that had geographically overlapping distributions. Group A (BFN 4585) and Group B2 (BFN 4820) appeared to be sympatric in the Sacramento Valley in 1972. Strains from the San Joaquin Valley isolated prior to 1989 (Groups B1 and B2) differed markedly from a 1989 isolate from the same location, Kern 373 (Group A). These results suggest that virus introduction(s) led to changes in genotype, or alternatively that the enzootic virus was subjected to selective pressure leading to rapid emergence of a new genotype. Nucleotide sequences of the envelope and 5′ untranslated region of the viral genome of these virus strains did not correlate with virulence as measured by mortality in weanling mice, nor viremia levels and duration in chickens.

From July 10 through August 4, 1980, five cases of St. Louis encephalitis (SLE) occurred in and near Fort Walton Beach on the Gulf Coast of northwest Florida. These were the first cases of SLE ever reported from the Florida panhandle. To determine the extent of SLE infection in the community, sera (n = 968) were collected from patients at the local hospital and county public health unit and tested for SLE virus antibody. The SLE attack rate was highest in a centrally located impoverished census tract. There was a trend toward decreasing seroprevalence with distance from the central area of the city. Overall, seroprevalence was higher in males (prevalence ratio = 2.7) and in all areas, seroprevalence increased with age. The serosurvey results suggest that SLE has been endemic in the Fort Walton Beach area.

An investigation of the extent of St. Louis encephalitis (SLE) virus activity in the avian population in Pine Bluff, Arkansas was conducted from August 30, 1991 through September 5, 1991, following an SLE epidemic that resulted in 25 human cases. A total of 363 birds of 33 species were captured with ground-level mist nets at four sites along the northern edge of the city. No viruses were isolated from the serum of these birds, but 91 birds (25%) of 11 species had detectable neutralizing antibody against the TBH-28 strain of SLE virus in the constant-virus, serum-dilution, plaque-reduction neutralization test in Vero cell culture. No antibody to eastern equine encephalitis virus was detected. The prevalence of antibody to SLE virus varied among the sites from 11% to 44%, but the prevalence at each site was influenced by the avian species composition. The two most abundant species captured in the city, the American robin (43%) and house sparrow (42%), also had the highest prevalence of antibody. Nine other bird species were serologically positive but at significantly lower rates than for the abundant species. The antibody prevalence was higher in immature birds (27%) than in adult birds (15%), but the antibody titers were higher in adult birds than in immature ones. The overall SLE antibody prevalence and the prevalence for house sparrows were significantly higher than the average prevalence for avian hosts studied during previous SLE epidemics. Therefore, house sparrows would be a good choice for a local sentinel species.

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.

During the monitoring of arbovirus seroprevalence in wild birds collected in California, we inadvertently made two isolates of western equine encephalomyelitis virus (WEEV) from California quail sera being tested by plaque reduction neutralization assay for antibodies against St Louis encephalitis (SLEV) and West Nile (WNV) viruses despite heating the sera at 56 degrees C for 30 minutes. These data prompted us to examine the thermostability of these viruses during heat treatment. The flaviviruses, SLEV and WNV, at titers up to 10(6) plaque-forming units (PFU), were readily inactivated by the standard protocol of heating at 56 degrees C for 30 minutes. In contrast, solutions containing 10(5) and 10(6) PFU of WEEV required 2 hours for complete inactivation. Occasional presence of live virus within sera could lead to false negatives using standard plaque reduction neutralization test protocols.

Adult domestic chickens were infected with West Nile virus (WNV) or St. Louis encephalitis virus (SLEV) and challenged with homologous or heterologous virus at 21 or 56 days postinfection (dpi). Sera were collected at selected time points after infection and assayed by enzyme immunoassay (EIA), plaque reduction neutralization test (PRNT), and a Western blot (WB) alternative to PRNT. EIA results were sensitive and accurate (few false positives) but not specific, requiring a confirmatory test to determine virus infection history. PRNT results generally were specific until challenge, after which test results were frequently equivocal and inadequate to determine first or second infecting virus. WB results confirmed the serologic cross-reactivity between WNV and SLEV envelope protein. Non-structural protein 1 and pre-membrane protein reactivities were highly specific for WNV during SLEV infection, but less specific for SLEV during WNV infection. WB and PRNT specificities were similar for both viruses from 6 to 14 dpi, and sensitivities to WNV were virtually identical.

In North America, West Nile and St. Louis encephalitis viruses have been detected in a wide range of vector species, but the majority of isolations continue to be from pools of mixed mosquitoes in the Culex subgenus Culex. Unfortunately, the morphologic identification of these important disease vectors is often difficult, particularly in regions of sympatry. We developed a sensitive real-time TaqMan polymerase chain reaction assay that allows reliable identification of Culex mosquitoes including Culex pipiens pipiens, Cx. p. quinquefasciatus, Cx. restuans, Cx. salinarius, Cx. nigripalpus, and Cx. tarsalis. Primers and fluorogenic probes specific to each species were designed based on sequences of the acetylcholinesterase gene (Ace2). Both immature and adult mosquitoes were successfully identified as individuals and as mixed species pools. This identification technique provides the basis for a rapid, sensitive, and high-throughput method for expounding the species-specific contribution of vectors to various phases of arbovirus transmission.

To further study the phenomenon of flavivirus persistent infection, golden hamsters (Mesocricetus auratus) were inoculated intraperitoneally with a low pathogenicity strain of St. Louis encephalitis virus (SLEV). After inoculation, the animals remained asymptomatic and developed high levels of specific neutralizing antibodies in their sera. However, about one half of the hamsters continued to shed infectious SLEV in their urine for prolonged periods of time. By co-cultivation, SLEV was recovered from selected tissues (kidney, lung, and brain) of some of the animals for up to 185 days after initial infection. Although no specific histopathologic changes were observed in these tissues, SLEV antigen was shown by immunohistochemistry in the interstitium and tubular epithelium of the renal cortex and in a few large neurons of the cerebral cortex. Seventeen SLEV isolates from urine and tissues of the chronically infected hamsters were sequenced. In comparison with the infecting parent SLEV strain, two common mutations and amino acid substitutions were observed in all of the hamster isolates. The findings of this study were very similar to previous descriptions of chronic West Nile, Modoc, and tick-borne encephalitis virus infections in mammals, and they re-emphasize the potential importance of persistent flavivirus infection in vertebrates.

St. Louis encephalitis (SLE) and West Nile (WN) flaviviruses are genetically closely related and cocirculate in the United States. Virus neutralization tests provide the most specific means for serodiagnosis of infections with these viruses. However, use of wild-type SLE and WN viral strains for laboratory testing is constrained by the biocontainment requirements. We constructed two highly attenuated yellow fever (YF) virus chimeras that contain the premembrane-envelope (prM-E) protein genes from the virulent MSI-7 (isolated in the United States) or the naturally attenuated CorAn9124 (Argentina) SLE strains. The YF/SLE (CorAn version) virus and the previously constructed YF/WN chimera were shown to specifically distinguish between confirmed human SLE and WN cases in a virus neutralization test using patient sera. These chimeras have the potential for use as diagnostic reagents and vaccines against SLE and WN.

Using a dynamic hydrology model, we simulated land surface wetness conditions at 42 sites in 28 counties in southcentral Florida from 1990 to 1998 and compared these simulations with the incidence of human cases of St. Louis encephalitis (SLE) within these counties. Within counties, drought four months prior and wetting one-half month prior were significantly associated with human cases of SLE. Simulated land surface wetness conditions resolved transmission loci in both space and time, and May drought was significantly associated with the subsequent occurrence of human SLE cases. These findings are consistent with previous results associating simulated land surface wetness conditions with the transmission of SLE virus as measured in sentinel chickens, and support our working hypothesis that springtime drought facilitates SLE virus amplification in mosquito and wild bird populations.

Harris County, Texas, is an endemic area of St. Louis encephalitis (SLE); and an active surveillance program that monitors SLE virus activity in mosquitoes, birds, and humans has been in place there for the past 28 years. In June of 2002, West Nile (WN) virus appeared in Houston and quickly spread throughout the region. This report describes the results of 12 years of SLE surveillance in Harris County and the contrasting pattern of WN virus activity, when it arrived in 2002. Our data indicate that both SLE and WN viruses can coexist, despite their ecologic, antigenic, and genetic similarities, and that both viruses will probably persist in this geographic region.

Sera from outpatients attending county health department clinics in areas of California with consistent (Imperial Valley) and sporadic (Sacramento Valley) enzootic transmission of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses exhibited neutralizing antibody prevalence rates of 1.3% (n = 690) and 0.5% (n = 1,066) for WEE and 11.0% and 0.8% for SLE, respectively. Seroprevalence for SLE virus in Imperial County increased as a function of both age and years of residence, indicating that this virus was endemic with a low rate of annual infection. Of 26 sera that tested positive for SLE virus antibody by an enzyme immunoassay, but were negative by plaque reduction neutralization test, 14 (53%) had neutralizing antibody that reacted with > or = one type of dengue (DEN) virus. The DEN virus infections presumably were acquired elsewhere because neither the vectors nor DEN virus transmission occurs in California. The low prevalence of neutralizing antibody for WEE and SLE in the California human population indicated that despite recent increases in enzootic transmission, contact between humans and infectious mosquitoes have remained low.

Sera from 19 (2.6%) and 118 (16.4%) of 719 outpatients attending clinics in the southeastern Coachella Valley, California during 1993 and 1994 exhibited IgG antibodies to western equine encephalomyelitis and St. Louis encephalitis (SLE) viruses, respectively, using enzyme immunoassays. However, only seven (1.0%) and 36 (5.0%) outpatients were positive by plaque-reduction neutralization tests (PRNTs), and seven (1.0%) and 84 (11.7%) outpatients were positive by sera hemagglutination inhibition assays, respectively. None were positive for IgM antibodies indicative of recent infection or were diagnosed clinically with central nervous system disease. Prevalence of PRNT antibody to SLE increased as a function of patient age, but did not vary significantly in relation to years of residence, sex, race, postal zip code, occupation, or month of collection.

An epidemic of St. Louis encephalitis (SLE) occurred in Jefferson County, Arkansas during July–August 1991. At least 26 human cases were involved, with 25 cases in the town of Pine Bluff. Twelve isolates of SLE virus were obtained from mosquitoes collected in Pine Bluff between August 13 and 24: 11 from pools of Culex pipiens quinquefasciatus, resulting in a minimum infection rate of 1.6 per 1,000 (n = 6,768) for this subspecies, and one isolate from a pool of 22 mosquitoes identified as Cx. (Culex) spp. Three of the SLE-positive pools, two from Cx. p. quinquefasciatus and one from Cx. (Cux.) spp., also yielded isolates of Flanders virus. Larval surveys resulted in the collection of seven species in four genera from 28 larva-positive habitats and the identification of one significant site of Cx. p. quinquefasciatus production. Ecologic assessments conducted at 12 randomly selected residences resulted in the identification of 17 larva-positive habitats, for an average mosquito-positive habitat rate of 1.4 per residence, and a Cx. p. quinquefasciatus larva-positive habitat rate of 0.6 per residence. Aedes albopictus and Cx. p. quinquefasciatus were the species most frequently encountered in larval surveys in residential neighborhoods.

Flocks of sentinel domestic pigeons (Columbia livia) detected increases in St. Louis encephalitis (SLE) and western equine encephalomyelitis (WEE) virus activity in southern California concurrently with flocks of sentinel chickens. However, occasional low-titered, transient seroconversions to both WEE and SLE viruses also occurred in pigeons during periods when virus activity was not detected by seroconversions in sentinel chickens, by virus isolation from Culex mosquitoes, or by human disease. Moreover, SLE virus seroconversions detected in pigeons by a hemagglutination inhibition (HI) test frequently could not be confirmed either by a plaque-reduction neutralization test (PRNT) on the same sera, or by an HI test on the next monthly serum sample from the same bird. Experimental infection studies, in which pigeons were inoculated subcutaneously with SLE (SOUE 16-84) virus, confirmed that pigeons developed low-titered and transient HI antibodies that were detectable infrequently by PRNT. In contrast, experimental infection with WEE (BFS 1703) virus produced elevated antibody responses that were detectable by HI for 8-12 weeks and by PRNT for at least 25 weeks. Pigeons infected with SLE virus rarely developed detectable viremias, whereas most birds infected with WEE virus developed viremias on postinfection day 1 that persisted for two or three days. Host-preference studies indicated that pigeons were less attractive as bait in lard can traps to host-seeking Culex mosquitoes than were chickens, and that blood-engorged Culex females collected near sentinel locations fed more frequently upon galliform than columbiform birds. Collectively, these results indicated that sentinel pigeons would not provide an adequate replacement for sentinel chickens to monitor WEE or SLE viruses, and would be a dead-end host for SLE virus.

A semi-isolated population of Culex tarsalis in Kern County, CA was found to vary significantly in its seasonal and yearly susceptibility to peroral infection with western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses during the breeding seasons of 1975-1981. Female cohorts of the population were significantly more resistant to WEE virus from 1975 through 1977 than from 1978 through 1981. On the average, females were 40 times more susceptible to WEE virus during May than during August. Increases in resistance correlated significantly with the number of days accrued from April through June, when daily ambient air temperatures equaled or exceeded 26.7 degrees C or 32.2 degrees C, respectively. Analyses of data on WEE viral susceptibility, temperature, and rainfall for the 7 year period suggested that Cx. tarsalis should be most susceptible to infection in years with wet winter-early spring periods followed by cool springs. However, inconsistencies in the expression of WEE viral susceptibility during mid- to late summer of some years indicated that other unidentified extrinsic factors, in addition to temperature and rainfall, may induce changes during preimaginal development that affect the peroral susceptibility of adult females. Seasonal and yearly changes in the susceptibility of the Cx. tarsalis population to per os infection with SLE virus did not necessarily occur at the same time as those observed with WEE virus and did not appear to correlate with changes in ambient air temperature. Thus, extrinsic factors that affect the expression of viral susceptibility of Cx. tarsalis, and perhaps the genetic basis of viral susceptibility, apparently differ for WEE and SLE viruses.

In 1980 Reeves wrote that epidemics of St. Louis encephalitis (SLE) are preventable by means of surveillance and vector abatement. This view is examined in the light of epidemic activity during the last decade (1977–1986), in which 9 discrete outbreaks occurred. In addition, 5 interactive factors (virus, vector, viremic host, human immunity, environmental temperature) described by Reeves as essential to the development of an SLE epidemic are reviewed in the light of recent research. Although much progress has been achieved, many questions remain about SLE epidemiology and ecology. Among the most important are: Do Culex pipiens complex mosquitoes play a significant role in SLE virus transmission in the western United States? Is there a sylvatic cycle of SLE virus transmission in the east-central United States? What are the most sensitive and specific predictors of SLE virus activity in the east-central United States? What are the overwintering mechanisms which assure SLE virus recrudescence, and what role does transovarial transmission of virus play?

St. Louis encephalitis (SLE) virus was isolated from a 21-year-old female hospitalized on 4 October 1978 in Belém, Brazil. Symptomatology on admission included fever, chills, severe headache, abdominal pain, myalgia, arthralgia and jaundice. SLE virus was isolated from her blood drawn on the 8th day of illness and subsequent seroconversion was documented. Serological tests showed the isolate to be closely related to the Belém prototype of SLE virus but distinct from other flaviviruses tested. The patient was discharged without sequelae after 16 days of hospitalization. Epidemiological investigations where the patient worked and lived revealed no evidence of extensive transmission of SLE virus.

Present strategies for surveillance, prevention, and control of arbovirus dis eases in western North America have been developed from more than 4 decades of epi demiological research and development of mosquito control technology. Methods of pre diction of outbreaks remain imprecise, although our understanding of sources of variation associated with indicators used for prediction is improving. Well organized and funded systematic mosquito abatement remains the most effective method of prevention of human cases of mosquito-borne virus disease, although emergency methods must be employed when outbreaks are imminent. The development of information management systems technology, use of recent developments of sampling theory, and research on vector com petency and related areas should permit much better precision in estimates of impending outbreaks. The development of strategies for the surveil lance, prevention, and control of arthropod-borne virus diseases in North America grew out of stud ies of the epidemiology of these diseases con ducted during the period preceding World War II. Approaches have been refined over the years, and materials and equipment have been im proved, but the basic concepts remain relatively unchanged since those times. I will examine these concepts and discuss their validity in light of the present state of knowledge of arbovirus disease ecology. If the name “Reeves” appears frequent ly in this discussion it is because his contribu tions to this area have been so extensive.

A dot enzyme-linked immunosorbent assay utilizing a novel membrane, polyvinylidene difluoride, is described. This assay was developed for the rapid detection of serum antibodies to eastern equine encephalomyelitis virus and St. Louis encephalitis virus in sentinel chickens. Antigens were spot-filtered through the membrane. Membranes were dipped into small vials of sera. Antigen-antibody complexes were detected with enzyme-conjugated antiglobulin which, when exposed to substrate, produced a colored insoluble product. The antibody detection protocol was completed within 50 min and was compared with a standard plate enzyme immunoassay. Chickens were experimentally infected with eastern equine encephalomyelitis and St. Louis encephalitis and bled on a daily basis. The dot immunoassay correctly identified 99% (123/124) of the eastern equine encephalomyelitis virus and 100% (67/67) of the St. Louis encephalitis virus antisera. Sera from sentinel chicken flocks in Maryland were also assayed. These data indicate that the dot immunoassay should be considered as an alternative to current assays for the screening of sera for antibodies to virus antigens. This assay could easily be performed in the field and allows for the screening of antibodies to several different viruses in one test.

Female Aedes albopictus and Aedes epactius infected by inoculation with St. Louis encephalitis (SLE) virus transmitted virus transovarially to their F1 progeny. With Ae. epactius, minimal infection rates (MIR) in F1 larvae were similar for progeny reared at 18 (+/- 1) degree C or 27 (+/- 1) degree C, whereas, MIR's were significantly higher in F1 adults reared at 18 degrees C, than they were in F1 adults reared at 27 degrees C. Use of different strains of SLE virus appeared to influence transovarial transmission of virus by Ae. albopictus, but not by Ae. epacticus. Transovarial transmission of SLE virus was not demonstrated with colonized strains of Aedes dorsalis, Ades sierrensis, Ades triseriatus, Ades vexans, Culex pipiens, Culex quinquefasciatus and Culex tarsalis, and field strains of Ae. vexans.

Birds are the primary hosts for St. Louis encephalitis (SLE) virus in most of North America. Because the increased prevalence of antibody in House Sparrows (Passer domesticus) has been related to human cases, this species has been frequently used as a sentinel of SLE virus activity in urban areas. This study investigated the susceptibility of House Sparrows to two strains of SLE virus, measured antibody profiles, and evaluated the use of House Sparrows in an urban surveillance system. House Sparrows were susceptible to both strains of SLE virus inoculated, although not equally, and produced viremias sufficient to infect vector mosquitoes. Both hemagglutination-inhibiting (HI) and neutralizing (N) antibody developed rapidly and to high titers within 2 weeks after inoculation. Detectable humoral antibody began to disappear by 3 months, but persisted for 2 years in 27% for HI and 36% for N antibody of the surviving birds. However, all of the surviving birds were resistant to reinfection with SLE virus at 2 years after inoculation. The titer of HI antibody appeared to be useful in determining recent exposure to SLE virus. The experimental data on HI antibody development and persistence was related to field serologic data from House Sparrows. The monthly prevalences of SLE antibody for independent samples of sera from House Sparrows collected in Memphis, Tennessee, in 1980 were similar. SLE amplification in the House Sparrow population was delayed until September. The Memphis arbovirus surveillance system detected the amplification quickly, and responded with increased adult mosquito control in the focal areas. Urban surveillance of SLE utilizing House Sparrows as sentinels is discussed.

Experiments were conducted to determine whether transovarial transmission (TOT) of St. Louis encephalitis (SLE) virus occurs in Culex pipiens complex mosquitoes, the principal vectors of SLE virus in the central-eastern United States. In 1978, field-collected mosquitoes from Memphis, Tennessee, and McLeansboro, Illinois, were used; during 1979, colonized mosquitoes from Chicago, Illinois, and Memphis, Tennessee, were used. Mosquitoes were infected by feeding on viremic chicks inoculated with an SLE virus strain isolated from Cx. pipiens complex mosquitoes collected from Memphis, Tennessee, in 1976. During the 1979 experiments, progeny larval and adult mosquitoes were held at two temperatures, 18 and 25 degrees C. Progeny were tested for virus by plaque assay in duck embryo cell cultures and by inoculation of Aedes albopictus C6/36 cells and examination by immunofluorescence. In 1978, most of the progeny tested were from the first ovarian cycle, and a single occurrence of TOT was documented. In 1979, a single TOT occurred from 46,856 first ovarian cycle progeny, whereas 7 of 9,234 progeny of the second ovarian cycle were infected. The rate of TOT was higher for progeny of Memphis than Chicago mosquitoes, and for mosquitoes held at 18 degrees C than at 25 degrees C; however, these differences were not statistically significant. Four positive pools were females, and three were fed on chicks for transmission attempts. The positive Chicago mosquito pool failed to transmit, but both Memphis pools successfully transmitted virus. The overall rates of TOT of SLE virus in progeny of the first and second ovarian cycle were, respectively, 1/45, 151 and 1/1,460. The significance of these results as they relate to the natural history of SLE virus is discussed.

The virulence characteristics of 67 strains of St. Louis encephalitis (SLE) virus isolated from various sources in North, Middle, and South America were compared in mice and rhesus monkeys. Each virus strain was titrated in mice exactly 21 days old and virulence was expressed as the ratio of intracerebral (ic)/intraperitoneal (ip) LD50. Virus strains fell into three groups: 1) high virulence (ic/ip LD50 ratio approximately 1.0); 2) intermediate virulence (variable mortality over a wide dose range); and 3) low virulence (ic/ip LD50 less than or equal to 0.00002). Virus strains isolated during Culex pipiens and Cx. nigripalpus--borne epidemics in the eastern United States were highly virulent for mice, whereas a high proportion of the endemic virus strains isolated from Cx. tarsalis in the western United States were attenuated. Virus strains isolated from birds (the usual host for SLE virus) were highly virulent, in contrast to strains from rodents and carnivores, which were attenuated. Isolates from humans exhibited variable virulence characteristics. In experimentally-infected mice, virulence correlated with high viremia, replication in extraneural tissues, and earlier neuroinvasion. Mouse virulence correlated with clinical and histopathologic markers of pathogenicity for ic inoculated rhesus monkeys. Monkeys immunized with nonpathogenic strains by subcutaneous inoculation were partially protected against ic challenge with a virulent virus strain. The virulence classification of SLE virus strains is discussed in terms of epidemiologic correlations. This classification provides a framework for future studies on the antigenic, genetic, and biochemical bases for SLE virus strain variation.

Mosquito population indices from California for the period 1953-1973 were analyzed to determine their association with activity of St. Louis encephalitis (SLE) and western equine encephalomyelitis (WEE) viruses. Culex tarsalis female populations, as measured by New Jersey light trap indices (LTI), correlated positively with the incidence rates of encephalitis in humans, and were a reliable means of forecasting the years of highest incidence. The critical level of C. tarsalis in urban areas below which no human cases of SLE and WEE were detected was an LTI of 0.1. Critical urban levels of C. tarsalis associated with significant human SLE or WEE incidence ranged between LTIs of 6.4 (for rural mosquito abatement districts [MADs] with large resident human populations). Peaks in annual incidence of SLE and WEE in humans occurred during years when seasonal average C. tarsalis female populations in urban areas reached a LTI of 21. Peaks in weekly incidence of SLE and WEE were associated, respectively, with weekly LTIs of 21 and 81 in urban traps. Isolation rates of SLE virus from mosquito pools and transmission of the virus to enzootic hosts were highest when urban LTIs were between 10 and 19.9 and between 5 and 9.9, respectively. The WEE viral isolations and enzootic transmission rates were highest when LTIs in urban areas were 1-4.9.

Summary St. Louis encephalitis virus was isolated on 23 occasions from mosquitoes collected between September 7 and October 25 at widely separated localities of the four-county Tampa Bay area during the St. Louis encephalitis epidemic of 1962. Twenty-two of these isolations were obtained from 485 pools of Culex nigripalpus; the 23rd was from 91 pools of Melanoconion, a subgenus of Culex. The mosquitoes were collected alive in battery-powered light traps and traps baited with ducklings or chicks. These isolations indicate that infected vectors were prevalent over a vast area exposing the human population to infection throughout the region.

Summary Experience with twenty-five cases of acute encephalitis believed to be of viral origin has been analyzed. There is serological evidence that three of these cases were due to St. Louis virus. An unknown virus was isolated from the serum of one patient. In most of the cases, onset of illness was sudden. Fever, headache and mental change were the main symptoms. There was a mortality rate of 16 percent. It is significant that in about 35 percent of the cases, there was some residual defect after illness.

Summary The transovarian passage of hemagglutination-inhibition (HI) antibodies to St. Louis encephalitis (SLE) virus was studied in 18 naturally infected hens and 51 of their chicks. HI antibody was readily detected in the 24-hour-old chick in quantitative relationship to maternal antibody titers. The loss of this inhibitor was an exponential function of time, and was below detectable levels at the 4th week. Serum-neutralizing antibody studies on the hens indicated the SLE-HI inhibitor was specific antibody. In contrast, the Eastern encephalitis-HI inhibitor appeared to be a non-specific substance related to the ovulatory activity of the bird. The interpretation of SLE-HI antibodies in chicks under 4 weeks of age requires consideration of the presence of maternal antibody as a contributing cause.

Summary The present report concerns the susceptibility of three species of insectivorous bats (Tadarida brasiliensis mexicana, Myotis lucifugus lucifugus, Eptesicus fuscus fuscus) to experimental infection with several strains of Japanese B encephalitis (JBE) and St. Louis encephalitis viruses. All three bat species proved to be susceptible to subcutaneous inoculation with one or more virus strains, the intensity of the infection depending on the mouse-passage level and origin of the strain. In general, high mouse-passage strains were less infective for bats than recent isolates. The characteristics of experimental arbovirus infection in bats include a persistent viremia (15–30 days) and viral invasion and multiplication in interscapular brown adipose tissue and, to a lesser degree, in brain and kidney tissue. Intracerebral inoculation of JBE virus produced a more intense and widespread infection in the big brown bat (Eptesicus f. fuscus) than subcutaneous inoculation, yet evidence of viral pathology was not observed in sections of infected brain or brown fat. None of the infected bats showed evidence of disease, indicating that these viruses are capable of replicating in various tissues of the bat without noticeably damaging cells or producing overt signs of encephalitis. Reasons why bats might be effective reservoirs of arboviruses in various phases of the year-round transmission cycles are discussed and the application of these experimental studies in the planning of field work is indicated.

Summary Laboratory studies were conducted in attempts to infect three common species of bird mites, Dermanyssus gallinae, Ornithonyssus bursa and O. sylviarum, with the virus of St. Louis encephalitis and to transmit infection by the bite of these mites to newly hatched chickens. The virus tests were performed in three-week-old white Swiss mice, inoculated intracerebrally. Detectable virus persisted less than 2 days in the mites, and no transmissions by bite were shown. These negative results support those previously reported by Reeves et al. (1955) and indicate a lack of importance of bird mites in transmission of active St. Louis encephalitis infection.

Summary A method is described for collecting mosquito saliva in oil and measuring its volume and virus content. Preliminary results indicate that Culex pipiens may discharge 104.5 cubic microns of saliva per minute and that St. Louis encephalitis virus reaches a concentration of 109.9 suckling mouse LD50 per ml in the saliva. The concentration of virus was observed to change significantly in periods of time as short as one hour.

Summary A total of 25,240 mosquitoes was captured in the Tampa Bay area during September and October 1962, using miniature, battery-operated light traps. These were tested in suckling mice in 1,114 pools. A total of 19 isolations of St. Louis encephalitis virus was made, 18 from Culex nigripalpus and a single one from Anopheles crucians. Twenty-nine other virus isolations were made. Twenty-eight were from An. crucians and have been identified as a new virus related to Cache Valley virus of the Bunyamwera arbovirus group; the other was an unidentified virus of the California arbovirus group from Aedes atlanticustormentor.

Summary Seventeen percent of 238 wild birds and 36 percent of 162 domestic birds at Caymanas, Jamaica, were found to be positive for St. Louis encephalitis virus infection by hemagglutination-inhibition and neutralization tests in 1962. Conversions detected in local and sentinel chickens suggest that between March and August there probably was an increase in the transmission rate of SLE virus. This increased rate coincided with the breeding period of birds in the area. An isolation of St. Louis encephalitis virus, confirmed by the Trinidad Regional Virus Laboratories, was made from a mockingbird nestling in August. The isolate seems to be closely related to Tr 9464 and Ja 7532 viruses, which are both closely related to the Parton strain of SLE virus.

Summary A total of 287 sera collected from children under 15 years (104) and adults residing in eight communities in Monagas State, Venezuela, was examined for presence of antibodies to arthropodborne viruses by hemagglutination-inhibition and neutralization tests. No immunity to eastern and western equine encephalitis viruses was encountered, but, with the exception of Maturin, each community had some adults who were immune to Venezuelan equine encephalitis virus. Only three sera from adults were found to neutralize Mayaro. No evidence of recent activity of yellow fever and dengue type 2 viruses was discovered. Eight sera neutralized Ilheus but no other group B agent used, and two sera neutralized St. Louis encephalitis virus only. Neutralization test results with a Caraparu-like agent, TRVL 34053-1, indicate that infections with this or a closely related agent have occurred occasionally. In each community there were some sera that neutralized Cache Valley virus. Limited tests with a Manzanilla antigen gave negative results.

Eight strains of a flavivirus identified as St. Louis encephalitis (SLE) virus were isolated from wild rodents, birds, and sentinel mice in three locations in the State of São Paulo, Brazil from 1967--1969. No illness attributable to SLE virus infection was detected among the local inhabitants, although about 5% of the local population had neutralizing antibodies to this virus.

The potential of triatomines to maintain arboviruses was demonstrated by the ability of Rhodinius prolixus with experimentally punctured abdomen to harbor Venezuelan equine encephalitis (VEE) virus for at least 4 months and St. Louis encephalitis virus for 1 month. At 30 days after infection VEE virus was found at low titers in Trypanosoma cruzi-infected R. prolixus and at moderate titers in T. rangeli-infected R. neglectus. Transmission of VEE virus by bite of punctured bugs was successful 2 weeks after virus ingestion; attempts at 30 days failed.

Experimental infection of 11 Bradypus variegatus and Choloepus hoffmanni sloths with St. Louis encephalitis (SLE) virus produced detectable viremias of seven to 27 (median 13) days duration and maximum titers of 2.7 to 6.5 (median 5.1) log10 median suckling mouse intracranial lethal doses (SMicLD50) per ml. Experimental SLE viremia onset was delayed and maximum titer depressed in two sloths concurrently infected with naturally acquired viruses. SLE viremias in four experimentally inoculated cormorants Phalacrocorax olivaceus were shoter, and of equal or lower titer, than in sloths. Colonized Culex pipiens quinquefasciatus mosquitoes were infected by feeding on sloths circulating at least 4.8 log10 SMicLD50 of SLE virus per ml, and subsequently transmitted the infection to mice and chicks. An uninoculated baby Bradypus became infected by contact transmission from its mother. The antibody response of sloths to SLE virus was slow, being undetectable until several weeks post-inoculation. However, both sloth species developed high and long-lasting neutralizing and hemagglutination-inhibition antibody titers. The complement-fixation antibody response in Bradypus was lower and slower to develop than in Choloepus. Sloths with naturally acquired SLE virus antibody did not become detectably viremic after experimental inoculation. Neither sloths nor cormorants become overtly ill from SLE virus infection.

From 8 August through 5 December 1977, 110 laboratory-documented cases (78 confirmed, 32 presumptive) of St. Louis encephalitis (SLE) occurred in 20 counties throughout central-south Florida. Most cases were initially identified through an active surveillance system involving repeated contact of hospital infection control officers. This outbreak had three characteristics that are unusual for SLE epidemics in the eastern United States: it lacked an urban focus, a disproportionate number of cases affected young males, and the epidemic period extended into December. There was no significant difference between attack rates for blacks and whites. Males were significantly more often affected than females. Because of the time required for laboratory confirmation of cases, most outbreak control measures were initiated after the outbreak was declining. Reporting of clinically suspected SLE cases to initiate epidemic mosquito control should improve the timeliness of control measures in future outbreaks.

This is the introductory paper to a series on the ecology of arboviruses in Argentina. Epizootics of equine encephalitis have occurred since at least 1908, principally in the Pampa and Espinal biogeographic zones, with significant economic losses; human cases of encephalitis have been rare or absent. Both western equine and eastern equine encephalitis viruses have been isolated from horses during these epizootics, but the mosquitoes responsible for transmission have not been identified. A number of isolations of Venezuelan equine encephalitis (VEE) virus were reported between 1936 and 1958 in Argentina, but the validity of these findings has been seriously questioned. Nevertheless, serological evidence exists for human infections with a member of the VEE virus complex. Serological surveys conducted in the 1960s indicate a high prevalence of infection of humans and domestic animals with St. Louis encephalitis (SLE), and 2 SLE virus strains have been isolated from rodents. Human disease, however, has rarely been associated with SLE infection. Only 7 isolations of other arboviruses have been described (3 of Maguari, 1 of Aura, 2 of Una, and 1 of an untyped Bunyamwera group virus). In 1977, we began longitudinal field studies in Santa Fe Province, the epicenter of previous equine epizootics, and in 1980 we extended these studies to Chaco and Corrientes provinces. The study sites are described in this paper.

We studied the capacity of 44 strains of St. Louis encephalitis (SLE) virus to induce viremia in an epidemiologically important wild avian host, the House Sparrow (Passer domesticus). Selected virus strains were also inoculated at varying doses into 3-week-old chicks. Viremic responses were analyzed in terms of the proportion of inoculated nestling and adult birds which became viremic, the mean duration and the mean peak titer of viremia. Infectivity of avian sera was determined by plaque assay in primary duck embryo cell cultures. The susceptibility of the House Sparrow to viremic infection with different SLE virus strains varied markedly. Nestling sparrows 6-10 days of age were generally more susceptible than adult birds. All virus strains isolated during Culex pipiens-borne epidemics in the eastern United States were highly viremogenic [viremia in 80% of birds with a mean duration of greater than or equal to 1.6 days in adults or greater than or equal to 2.7 days in nestlings and a mean peak titer of greater than or equal to 10(3.0) plaque-forming units (PFU)/ml in adults and 10(4.0) PFU/ml in nestlings]. All virus strains isolated from Culex tarsalis in the western United States, strains isolated from rodents in South America. and six of 16 strains isolated from various sources elsewhere in tropical America were partially or fully attenuated. A high degree of concordance was demonstrated between experimental viremia in sparrows, viremia in 3-week-old chicks, and neurovirulence for weanling mice. The epidemilogic significance of these findings is discussed.

Both sexes of Toxorhynchites amboinensis, an unusually large, non-blood sucking mosquito, were found to be as susceptible to infection with each of the four types of dengue virus by intrathoracic inoculation as Aedes albopictus. Tx. amboinensis infected with dengue virus could be identified easily by fluorescent antibody staining of head squashes and had advantages of size, hardiness, and safety (for females) as compared with Ae. albopictus. Tx. amboinensis also were more susceptible to infection with Japanese and St. Louis encephalitis viruses than were Vero cell cultures and appeared useful for detection and propagation of other flaviviruses and arthropod-borne viruses of other taxonomic groups.

The largest laboratory-documented outbreak of St. Louis encephalitis (SLE) in the United States occurred in the Chicago metropolitan area in the summer and early fall of 1975. Of 1,456 illnesses investigated, 326 cases of confirmed or probable SLE and 420 cases of suspected SLE were found in the six-county area. The outbreak peaked in early September, but cases continued to occur through October. Cases clustered geographically in the south-western suburbs. There was a direct correlation between age and attack rate, severity of illness, and mortality rate—all of which increased with successive age groups. Thirty-six persons died. Males and females were equally affected. This epidemic marked the first time that St. Louis encephalitis has been documented in the Chicago area.

In a reveiw of 2,963 patients with signs of infections of the central nervous system in New York State in 1966--1977, arboviruses were found to be the confirmed or presumptive etiologic agents in 60 patients. California encephalitis (CE) virus was the most common (44 patients), followed by Powassan (POW) virus (8), St. Louis encephalitis virus (7), and eastern equine encephalitis (EEE) virus (1). Most patients (47) were children. The incidence of encephalitis was higher in patients with arbovirus findings than in infections with any other of the common neurotropic viruses. The disease was fatal in two patients, one infected with POW virus, the other with EEE virus. Most patients with CE virus infections resided in suburban areas. All POW infections were contracted in six rural counties known for their recreational facilities.