Search

Background Western Equine Encephalitis (WEE) is a naturally acquired infection and potentially devastating bioweapon, with no specific human countermeasures. An experimental inactivated Western Equine Encephalitis Vaccine (WEEV; WEE TSI-GSD 210) has been used under an IND (investigational New Drug) protocol at the United States Army Medical Research Institute of Infectious Diseases (USAMRIID) since 1976. Methods Over 24 years from 1987 to 2011, 876 subjects received 3 primary vaccine doses under 3 studies with 1,537 booster doses administered (FY87-8, phase 2, laboratory workers, vaccine lots 1-81-1, 1-81-2, and 2-1-91; FY99-12, phase 2 laboratory workers, lot 2-1-91; and FY09-02, phase 1 healthy volunteer, lot 3-1-92). Post-vaccination safety and immunogenicity [plaque reduction neutralization test 80% (PRNT80) > 1:40] were analyzed. Results Overall PRNT80 response to the primary series in FY87-8 was 42% (326/770) but dropped to 16% (14/87) in FY99-12, prompting study FY09-02, which achieved 89% (17/19). The first booster response rate was 68% (814/1194) in FY87-8, 53% (171/324) in FY99-12, and 100% (10/10) in FY09-02. The majority of definitely related adverse reactions (AEs) were mild and local with no definitely related serious AEs. No laboratory acquired WEE infection was documented during this period despite 4 reported exposures in vaccinated subjects. Conclusion The TSI-GSD 210 WEE vaccine was immunogenic, safe and well tolerated. Use of this vaccine could be considered in an emergency setting. Despite decades of safe and effective use under IND, full licensure is not planned due to manufacturing constraints, and a strategic decision to develop alternatives. Clinical trial registration https://clinicaltrials.gov/, identifier NCT01159561.

Following the introduction of West Nile virus into California during the summer of 2003, public health and vector control programs expanded surveillance efforts and were in need of diagnostics capable of rapid, sensitive, and specific detection of arbovirus infections of mosquitoes to inform decision support for intervention. Development of a multiplex TaqMan or real-time semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay in which three virus specific primer-probe sets were used in the same reaction is described herein for the detection of western equine encephalomyelitis, St. Louis encephalitis and West Nile viral RNA. Laboratory validation and field data from 10 transmission seasons are reported. The comparative sensitivity and specificity of this multiplex assay to singleplex RT-PCR as well as an antigen detection (rapid analyte measurement platform) and standard plaque assays indicate this assay to be rapid and useful in providing mosquito infection data to estimate outbreak risk.

The Pantanal hosts diverse wildlife species and therefore is a hotspot for arbovirus studies in South America. A serosurvey for Mayaro virus (MAYV), eastern (EEEV), western (WEEV) and Venezuelan (VEEV) equine encephalitis viruses was conducted with 237 sheep, 87 free-ranging caimans and 748 equids, including 37 collected from a ranch where a neurologic disorder outbreak had been recently reported. Sera were tested for specific viral antibodies using plaque-reduction neutralisation test. From a total of 748 equids, of which 264 were immunised with vaccine composed of EEEV and WEEV and 484 had no history of immunisation, 10 (1.3%) were seropositive for MAYV and two (0.3%) for VEEV using criteria of a ≥ 4-fold antibody titre difference. Among the 484 equids without history of immunisation, 48 (9.9%) were seropositive for EEEV and four (0.8%) for WEEV using the same criteria. Among the sheep, five were sero- positive for equine encephalitis alphaviruses, with one (0.4%) for EEEV, one (0.4%) for WEEV and three (1.3%) for VEEV. Regarding free-ranging caimans, one (1.1%) and three (3.4%), respectively, had low titres for neutralising antibodies to VEEV and undetermined alphaviruses. The neurological disorder outbreak could not be linked to the alphaviruses tested. Our findings represent strong evidence that MAYV and all equine encephalitis alphaviruses circulated in the Pantanal.

Western equine encephalitis virus (WEEV) naturally cycles between mosquitos and birds or rodents, with a case fatality rate of up to 15% in humans during epizootic outbreaks. There are no medical countermeasures to treat WEEV infection, and accidental aerosol exposure increases the case fatality rate up to 40%. Understanding the pathogenesis of infection is required to develop and assess medical countermeasures. This study describes the clinical and pathological findings of mice infected with WEEV by the aerosol route, and use as a model for WEEV infection in humans. Balb/c mice were infected by the aerosol route with a dose range of high-virulence WEEV strain Fleming to establish the median lethal dose (MLD). The disease course was acute, culminating in severe clinical signs, neuroinvasion, and dose-dependent mortality. Further groups of mice were exposed by the aerosol route, periodically sacrificed, and tissues excised for histopathological examination and virology. Viral titres peaked four days post-challenge in the brain and lungs, corresponding with severe bilateral lesions in rostroventral regions of the encephalon, especially in the olfactory bulb and piriform cortex. Recapitulation of the most serious clinical presentations of human WEEV disease in mice may prove a useful tool in the evaluation of medical countermeasures.

We compared the effect on primary vaccination plaque-reduction neutralization 80% titers (PRNT80) responses of same-day administration (at different injection sites) of two similar investigational inactivated alphavirus vaccines, eastern equine encephalitis (EEE) vaccine (TSI-GSD 104) and western equine encephalitis (WEE) vaccine (TSI-GSD 210) to separate administration. Overall, primary response rate for EEE vaccine was 524/796 (66%) and overall primary response rate for WEE vaccine was 291/695 (42%). EEE vaccine same-day administration yielded a 59% response rate and a responder geometric mean titer (GMT)=89 while separate administration yielded a response rate of 69% and a responder GMT=119. WEE vaccine same-day administration yielded a 30% response rate and a responder GMT=53 while separate administration yielded a response rate of 54% and a responder GMT=79. EEE response rates for same-day administration (group A) vs. non-same-day administration (group B) were significantly affected by gender. A logistic regression model predicting response to EEE comparing group B to group A for females yielded an OR=4.10 (95% CL 1.97-8.55; p=.0002) and for males yielded an OR=1.25 (95% CL 0.76-2.07; p=.3768). WEE response rates for same-day administration vs. non-same-day administration were independent of gender. A logistic regression model predicting response to WEE comparing group B to group A yielded an OR=2.14 (95% CL 1.22-3.73; p=.0077). We report immune interference occurring with same-day administration of two completely separate formalin inactivated viral vaccines in humans. These findings combined with the findings of others regarding immune interference would argue for a renewed emphasis on studying the immunological mechanisms of induction of inactivated viral vaccine protection.

Western equine encephalitis virus (WEEV) is an important pathogen for both humans and equines. The virus is also listed as a bioterrorism agent due to its ability for aerosol transmission with high mortality. No commercial vaccines or antiviral drugs are available for the prevention and treatment of WEEV infection in humans. In this paper, we constructed a recombinant WEEV vaccine, designated as Ad5-WEEV, using a replication defective, human adenovirus serotype 5 (HAd5) as a delivery vector. Ad5-WEEV contains the E3-E2-6K-E1 structural protein gene of the 71V-1658 strain of WEEV and the E1 and E2 proteins were synthesized in cells inoculated with Ad5-WEEV. After intramuscular immunization of mice with two doses of Ad5-WEEV, neutralizing antibodies against WEEV were generated and the mice were completely protected from a lethal dose challenge of 71V-1658. In addition, we showed that passive transfer of serum from the Ad5-WEEV-immunized mice could partially control WEEV infection. These results demonstrate that HAd5 vectors are promising for WEEV vaccine development.

The E1 and E2 glycoproteins of Western Equine Encephalitis (WEE) are candidate antigens for WEE subunit vaccine development. We have cloned the E1 gene of WEE virus and expressed it in Escherichia coli as inclusion bodies. The inclusion bodies were successfully solubilised, refolded and the immunogenicity of this unglycosylated protein was assessed in mice. Immunization of mice with recombinant E1 protein generated both humoral and cell-mediated immune responses, indicating the recombinant E1 protein is immunogenic. Challenge of E1-immunized mice with live WEE virus demonstrated little or no protection from this E. coli-derived non-glycosylated subunit.

House finches, Carpodacus mexicanus, were experimentally infected with high and standard doses of western equine encephalomyelitis virus (WEEV) or St. Louis encephalitis virus (SLEV) to determine whether high doses would produce an elevated viremia response and a high frequency of chronic infections. Finches inoculated with approximately100,000 plaque forming units (PFU) of WEEV or SLEV produced viremia and antibody responses similar to those in finches inoculated with approximately 100 PFU of WEEV or 1000 PFU of SLEV, the approximate quantities of virus expectorated by blood-feeding Culex tarsalis Coquillett. Infected finches were held through winter and then necropsied. Only one finch inoculated with the high dose of SLEV developed a chronic infection. Our data indicated that elevated infectious doses of virus may not increase the viremia level or the frequency of chronic infection in house finches.

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.

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

Reptiles can harbor pathogenic microorganisms asymptomatically and serve as potential reservoirs of infection for humans, domestic animals, and other reptiles. Infectious diseases are also problematic for free-ranging reptile populations and are an important consideration in reptile reintroduction and translocation projects. There have been limited serologic studies of free-ranging reptiles for evidence of exposure to potential pathogens. In the present study, serum or plasma samples from five male and five female free-ranging Venezuelan anacondas ( Eunectes murinus) were screened for antibodies to eastern, western, and Venezuelan equine encephalitis viruses, vesicular stomatitis virus, ophidian paramyxovirus, 19 Leptospira interrogansserovars, and Cryptosporidium serpentes . Antibodies to these agents were not detected, or antibody titers were low and possibly nonspecific. These results for the limited number of anacondas surveyed suggest that they do not serve as significant reservoirs for these infectious agents at this location.

Experiments were designed to evaluate the vector competence of Culex tarsalis Coquillet from an area (Sioux City) where Cx. tarsalis is most abundant in Iowa for western equine encephalomyelitis virus (WEE-7738). WEE-7738 was isolated from Aedes trivittatus (Coquillet) collected in Ames, IA, in 1977. Infection rate, dissemination rate, multiplication efficiency, and transmission rate were determined for this virus in the SC strain of Cx. tarsalis. SC strain of Cx. tarsalis was as susceptible to WEE-7738 as Californian strains of Cx. tarsalis were to BFS1703 strain of WEE; OID50 of SC Cx. tarsalis was 2.63 log TCID50 per mosquito and OID50 of Californian strains of Cx. tarsalis were 2.0-4.1 log PFU per mosquito. However, transmission of WEE-7738 (4.2%) by the SC strain of Cx. tarsalis was lower than those (10-60%) reported in other studies.

The incidence of Western Equine Encephalitis (WEE) in humans and equids peaked during the mid-20th century and has declined to fewer than 1–2 human cases annually during the past 20 years. Using the mouse model, changes in WEE virus (WEEV) virulence were investigated as a potential explanation for the decline in the number of cases. Evaluation of 10 WEEV strains representing a variety of isolation locations, hosts, and all decades from the 1940's to the 1990's yielded no evidence of a decline in virulence. These results suggest that ecological factors affecting human and equine exposure should be investigated to explain the decline in WEE.

Western equine encephalitis virus (WEEV) is a naturally occurring recombinant virus derived from ancestral Sindbis and Eastern equine encephalitis viruses. We previously showed that infection by WEEV isolates McMillan (McM) and IMP-181 (IMP) results in high (∼90–100%) and low (0%) mortality, respectively, in outbred CD-1 mice when virus is delivered by either subcutaneous or aerosol routes. However, relatively little is known about specific virulence determinants of WEEV. We previously observed that IMP infected Culex tarsalis mosquitoes at a high rate (app. 80%) following ingestion of an infected bloodmeal but these mosquitoes were infected by McM at a much lower rate (10%). To understand the viral role in these phenotypic differences, we characterized the pathogenic phenotypes of McM/IMP chimeras. Chimeras encoding the E2 of McM on an IMP backbone (or the reciprocal) had the most significant effect on infection phenotypes in mice or mosquitoes. Furthermore, exchanging the arginine, present on IMP E2 glycoprotein at position 214, for the glutamine present at the same position on McM, ablated mouse mortality. Curiously, the reciprocal exchange did not confer mouse virulence to the IMP virus. Mosquito infectivity was also determined and significantly, one of the important loci was the same as the mouse virulence determinant identified above. Replacing either IMP E2 amino acid 181 or 214 with the corresponding McM amino acid lowered mosquito infection rates to McM-like levels. As with the mouse neurovirulence, reciprocal exchange of amino acids did not confer mosquito infectivity. The identification of WEEV E2 amino acid 214 as necessary for both IMP mosquito infectivity and McM mouse virulence indicates that they are mutually exclusive phenotypes and suggests an explanation for the lack of human or equine WEE cases even in the presence of active transmission.

SummaryEither intraperitoneal injection of hyperimmune rabbit serum or deep ether anesthesia administered beginning 18 hours following intracerebral inoculation of virus effects a significant change in survival rate among Swiss mice infected with western equine encephalomyelitis. A combination of the two methods is more effective than either method alone.

Mosquitoes used for virus analysis in Manitoba are obtained by means of hand catches or by the use of light traps, sometimes supplemented with dry ice. A trap is described that has been used successfully for this purpose during the past three years. This trap is simple to operate, the insects are taken in good condition for identification, and newly emerged specimens are killed, thereby eliminating the majority of those that could not possibly be infected with the virus of western equine encephalitis. The methods of shipping, sorting, identifying, and storing specimens are given. These methods are, in large part, standard entomological procedures adapted to present needs.

Anesthesia, by ether, is effective in the treatment of western equine encephalomyelitis in mice. Of mice treated with deep ether anesthesia soon after the intracerebral injection of western equine virus, only 58 per cent developed the disease as compared with 92.4 per cent of control animals. When anesthesia was delayed the approximate length of the incubation period, 60 per cent of the animals developed the disease as compared with 92.4 per cent of the controls. In addition, ether anesthesia delays the development of central nervous system symptoms not only when administered soon after the injection of the virus but also when administered after the disease has progressed far enough to cause objective signs of encephalitis.

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.

In order to establish an accurate, ready-to-use assay for simultaneous detection of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV), we developed one duplex TaqMan real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay, which can be used in human and vector surveillance. First, we selected the primers and FAM-labeled TaqMan-probe specific for WEEV from the consensus sequence of NSP3 and the primers and HEX-labeled TaqMan-probe specific for EEEV from the consensus sequence of E3, respectively. Then we constructed and optimized the duplex real-time RT-PCR assay by adjusting the concentrations of primers and probes. Using a series of dilutions of transcripts containing target genes as template, we showed that the sensitivity of the assay reached 1 copy/reaction for EEEV and WEEV, and the performance was linear within the range of at least 106 transcript copies. Moreover, we evaluated the specificity of the duplex system using other encephalitis virus RNA as template, and found no cross-reactivity. Compared with virus isolation, the gold standard, the duplex real time RT-PCR assay we developed was 10-fold more sensitive for both WEEV and EEEV detection.

Definition of targets for vector control requires an understanding of the relationship between vector abundance and the intensity of arbovirus transmission. Using an extensive surveillance dataset with observations from sentinel chicken flocks and mosquito traps paired in time and space, hierarchical autoregressive logistic regression models were developed to predict the probability of seroconversion in chickens for western equine encephalomyelitis virus (WEEV) based on the relative abundance of the principal vector, Culex tarsalis. After adjustments for confounders, the abundance of Cx. tarsalis 29-42 d before the date of chicken sampling was credibly associated with the risk of WEEV transmission in both the Central and Coachella Valleys, and a doubling of relative Cx. tarsalis abundance was associated with a 58% increase in the odds of seroconversion. The critical time windows identified in our study highlight the need for surveillance of vector populations and forecasting models to guide proactive vector control measures before the detection of transmission to sentinel chickens.

Summary Evidence is presented to show that a heat labile accessory substance which augments the activity of neutralizing antibodies to Western equine encephalomyelitis virus is present in normal serum. This substance loses most of its activity when diluted 1:10. Its possible relationship to complement and its bearing on diagnostic neutralization tests are discussed.

1. Chickens inoculated subcutaneously with 0.2 cc. of a 10(-2) to 10(-7) dilution of Western equine mouse brain virus had the virus in the blood serum between the 12th and the 48th hour in most instances. The fowls showed no signs of illness. 2. Viremia could be induced regularly in chickens by inoculating subcutaneously the least amount of virus which would produce encephalitis in the mouse when inoculated by the intracerebral route. 3. Even the minimal infecting dose for a chicken led to such multiplication of the virus that it was detectable in the serum in a 10(-4) dilution. Moreover, a minimal infecting dose appeared to result in a longer period of viremia than was produced by a larger dose. 4. Virus has not been found to persist for more than 3 days after inoculation in any organ of the chicken tested for it and usually it did not persist over 2 days. Antibodies were present in the blood within at least 15 days after inoculation. 5. It is concluded that chickens may serve as sources of infection for mosquitoes or other blood-sucking ectoparasites for short periods of time after the infecting bite of a similar invertebrate vector. There is no evidence that the chicken serves as a latent carrier of the virus. 6. No virus could be found in the blood of 2 inoculated calves, and virus has not been demonstrated regularly or with the same case in the blood of horses or of men, as it has in that of chickens. It seems unlikely therefore that large mammals serve frequently as sources for mosquito infection. 7. These experimental data on fowls and mammals correlate well with other epidemiological and laboratory findings, in particular with the feeding preference of the mosquitoes found infected in epidemic areas.

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.

The Western equine encephalitis virus (WEEV) is a potential Biological Warfare (BW) agent. The WEEV is endemic in western Canada and has caused epidemics of "sleeping sickness" with a mortality rate of 7-9%. The E2 glycoprotein is a structural component of the WEEV and elicits production of neutralizing antibodies against the virus following an infection event. The envelope glycoprotein E2 is considered as the major target protein for the development of vaccines because it includes epitopes that elicit neutralizing antibodies. This report describes the successful cloning of the E2 gene of WEEV and expression in Escherichia coli as inclusion bodies. The inclusion bodies were successfully solubilized, refolded and the immunogenicity of this non-glycosylated protein was assessed in BALB/c mice. Recombinant E2 (rE2) protein was specifically and strongly recognized by inactivated WEEV-immunized mice serum sample on ELISA, suggesting that E. coli derived rE2 protein retained at least some functional characteristics of its native conformation. The immunogenicity of the refolded rE2 protein was demonstrated by strong humoral and cell mediated immune (CMI) responses in rE2-immunized BALB/c mice. The current study also demonstrated that rE2-immunized mice could be partially protected from lethal challenge of WEEV.

Context Dengue, Japanese encephalitis, tick-borne encephalitis, yellow fever, and West Nile viruses cause substantial morbidity and mortality each year. Modern transportation and the relaxation of mosquito-control measures are largely responsible for the increase of disease caused by flaviviruses. Without effective antiviral drugs, vaccination offers the best chance of decreasing the incidence of these diseases, and live virus vaccines are the most promising and cost effective. However, flaviviruses can recombine, which raises the possibility of recombination between a vaccine strain and wild-type virus resulting in a new virus with potentially undesirable properties. Starting point Recently, Arunee Sabchareon and colleagues reported up to 90% seroconversion rates in a phase I trial of live-attenuated dengue-virus vaccines in children (Pediatr Infect Dis J 2004; 23: 99-109). Other live flavivirus vaccines have also been tested against dengue, Japanese encephalitis, and West Nile viruses. Thus far, efficacy seems promising. Where next Safety issues with the live flavivirus vaccines need to be recognised and addressed. The theoretical possibility of untoward recombination events can never be entirely dismissed, but steps can be taken to minimise risk. The development of non-live flavivirus vaccines should be encouraged.

A prospective study was performed to determine the serologic response of previously vaccinated horses to revaccination against eastern and western equine encephalomyelitis (EEE and WEE). Horses responded variably to each antigen, and some horses had low or undetectable antibodies 6 months after vaccination. Some horses did not develop increasing titers to EEE or WEE despite recent vaccination. Geometric mean titers peaked 2 weeks after revaccination and were significantly increased from before revaccination. Except for one horse, EEE:WEE titer ratios ranged from 0.25 to 2.0. Regular vaccination against EEE and WEE did not interfere with testing for Saint Louis encephalitis.

To determine the classification, phylogenetic and genetic type of XJ-90260 and XJ-91006 viruses isolated in China.Two pairs of specific primers were designed to amplify the NSP4 and E1-3' UTR genes of XJ-90260 and XJ-91006 viruses and the nucleotide sequences between them and with that of other Alphavirus were compared.The nucleotide sequences identity between XJ-90260 and XJ-91006 viruses was 100% and has showed the highest with WEE, comparing with the other 26 Alphaviruses in 3'-UTR. NSP4 gene of XJ-90260 and XJ-91006 viruses high homologous with EEE virus and E-1 gene high homologous wih SIN virus. XJ-90260 and XJ-91006 viruses belong to the B group of WEE and are closest to the Russian strain (Y62-33) by phylogenetic analysis.XJ-90260 and XJ-91006 viruses isolated in China are recombinant Alphaviruses, they belong to the same genetic type as WEE does.

The summer abundance of Culex tarsalis in Kern County, California, during 1990-98 was related quantitatively to rainfall, snow depth and water content, and runoff of the Kern River. Total monthly rain that fell during winter, lagged by 4-6 months, explained only 13% of the variability in the number of host-seeking females collected per trap night per month during summer. In contrast, regression analysis showed that river runoff 1 month earlier explained 67% of the variability in mosquito abundance. The water content of snowpack measured within the Kern River watershed during winter explained 70% of the variation in average mosquito abundance during the following summer. After being absent from Kern County since 1983, western equine encephalomyelitis virus (WEE) returned during the wet years of 1996-98 after the flow of the Kern River exceeded 150,000 acre-ft (450 hectare-meters) per month. Water content of snow in the Sierra Nevada during winter provided an excellent early warning of vernal river runoff, mosquito abundance, and enzootic WEE activity levels on the floor of the San Joaquin Valley.

Western equine encephalitis virus (WEEV; Togaviridae , Alphavirus ) is an enveloped RNA virus that is typically transmitted to vertebrate hosts by infected mosquitoes. WEEV is an important cause of viral encephalitis in humans and horses in the Americas, and infection results in a range of disease, from mild flu-like illnesses to encephalitis, coma, and death. In addition to spreading via mosquito vectors, human WEEV infections can potentially occur directly via aerosol transmission. Due to its aerosol infectivity and virulence, WEEV is thus classified as a biological safety level 3 (BSL-3) agent. Because of its highly infectious nature and containment requirements, it has not been possible to investigate WEEV's structure or assembly mechanism using standard structural biology techniques. Thus, to image WEEV and other BSL-3 agents, we have constructed a first-of-its-kind BSL-3 cryoelectron microscopy (cryoEM) containment facility. cryoEM images of WEEV were used to determine the first three-dimensional structure of this important human pathogen. The overall organization of WEEV is similar to those of other alphaviruses, consistent with the high sequence similarity among alphavirus structural proteins. Surprisingly, the nucleocapsid of WEEV, a New World virus, is more similar to the Old World alphavirus Sindbis virus than to other New World alphaviruses.

Summary 1. Evidence for transplacental transmissionof antibodies and viruses is reviewed. 2. Two clinical and serologicallyproved cases of Western equine encephalomyelitis in twin infants 5 days old are presented. Evidence strongly suggests transplacental transmission of the virus. 3. Maternal infection with Western equine encephalomyelitis in the late stages of pregnancy does not invariably infect the infant. In utero infection may be dependent upon local placental defects.

Lockart, Royce Z., Jr. (The University of Texas, Austin). Production of an interferon by L cells infected with Western equine encephalomyelitis virus. J. Bacteriol.85:556–566. 1963.—Two strains of Western equine encephalomyelitis virus (WEE), WEE (L+) and WEE (L−), which differed with respect to their cytopathogenicity for L cells were isolated. Both strains reproduced in L cells, and both induced the production of an interferon distinct from virus particles. L-cell monolayers were protected from degeneration by prior addition of interferon. By use of the absence of cytopathic effects (CPE) as an end point, interferon content was assayed. Monolayers failing to show CPE consistently produced less than 2% as much virus as control monolayers, indicating that virus synthesis was also inhibited. The use of this assay method was facilitated by the use of horse serum that appeared to contain antibodies against WEE and that permitted interferon to act selectively in the presence of active virus. It was found that interferon was produced during the time in which active virus was produced, and not significantly later. No interferon could be found in fluids from cells treated with inactive virus, although these are known to act as interfering agents. Interferon production was inhibited by pretreatment of L cells with sufficient amounts of interferon. It is concluded that interferon production is closely connected with WEE virus synthesis in L cells. The question is raised as to whether interferon need be a necessary intermediate for interference in L cells.

The purpose of this paper is to record an interesting phenomenon observed in a number of experiments which were begun early in 1945. These experiments were prompted primarily by the writer's curiosity and desire to answer for himself the following question: Since so many of the antibiotic agents derived from bacteria have failed to show antiviral activity, would bacteria behave differently if they were allowed to grow in a medium containing a virus? In satisfying this curiosity a few preliminary experiments were carried out in which bacteria were inoculated into a virus suspension of known potency. After 48 hours of incubation most of the bacteria that had grown out were removed from the mixture of centrifugation, and the quantity of virus remaining in the suspension was determined by titration in susceptible animals. For comparison a portion of the same virus suspension without bacterial inoculation was similarly incubated and titrated at the same time. The findings obtained from these early experiments were entirely unexpected. It was found that while the virus when present alone was almost inactive after 48 hours of incubation, the virus in the presence of bacterial growth still retained most of its initial potency after the same period of incubation. Further experiments were carried out in which

SummaryGarter snakes were inoculated in September and November with Western equine encephalomyelitis (WEE) virus and were caused to hibernate under simulated natural conditions. They emerged during March, April, May and June. After varying periods when no virus was detectable in their blood, virus was detected in concentrations as high as 106, and for a period up to 70 days following emergence of snakes. Normal mosquitoes became infected by feeding on these snakes and after an extrinsic incubation period of approximately 3 weeks transmitted WEE virus to 1-day-old chicks. These data demonstrate that snakes may serve as a natural overwintering mechanism for WEE virus.The technical assistance of Jack Cory and Edward Patzer is gratefully acknowledged.

SummaryIf 2 cc of rabbit hyperimmune antiserum of high titer were given intraperi-toneally to mice of about 17 g weight— prodigious amount since it represents twice the total blood volume of the host—encephalitis was prevented from developing in animals receiving intracerebrally ordinarily lethal doses of the virus of Western equine encephalitis. The preventive effect was noted when the virus was multiplying but had not as yet reached its maximal titer. Thus, if animals were treated with antiserum 2 hours before, at the same time, or from 4-16 hours after the virus was inoculated, it was effective in preventing an attack of encephalitis. Even if serum was administered 24, 36 or 48 hours after the virus certain but not all mice survived without recognizable signs of the disease. At 72 hours, however, only an exceptional mouse survived after serum treatment. Finally, at 73 or more hours after virus inoculation, when definite manifestations of experimental Western equine encephalitis could be observed, injec...

Summary The influence of underfeeding, deficiency of the whole “B” complex, thiamin-deficiency, riboflavin-deficiency, carbohydrate-deficiency and protein-deficiency was studied with respect to its effect on the development of immunity in mice vaccinated with formalinized Western equine encephalomyelitis mouse brain virus. Immunity was determined by the development of cerebral resistance and of neutralizing antibodies. The latter were quantitatively determined by the method of serum dilution as well as the usual method employing undiluted serum. Mice were given three doses of vaccine of 0.3 ml each intraperitoneally on three alternate days and were tested two weeks after the first dose. Underfed mice developed considerable immunity but not to the same extent as did those on an adequate diet. The well-nourished vaccinated mice resisted between 100 and 200 times more virus intracerebrally than did the underfed animals. The test for neutralizing antibodies likewise showed a significant difference between undernourished and well-nourished mice, with the well-nourished animals neutralizing between 10 and 100 times as much virus as did the starved mice. The serum of the adequately nourished animals could be diluted between 3 and 8 times more than that of the serum obtained from the undernourished mice and still neutralize the effects of a constant amount of virus (50 LD50 doses). Prolonging the period of starvation for an additional two weeks did not accentuate the depressed immunity response. There was no difference in the immune response of animals on a synthetic diet and those on a stock diet. Removal of the whole “B” complex from the synthetic diet yielded equivocal results regarding the immune response in mice, but a decreased ability on the part of “B”-deficient animals to produce antibodies appeared possible. In one case the “B”-deficient animals developed a neutralization index which was 15 fold less than that of the control animals and no difference when the diluted serum was tested against a constant amount of virus and in the other case no difference between neutralization indexes occurred but this time the serum from adequately fed animals could be diluted six times more than that of the “B”-deficient animals and still neutralize the effects of a constant amount of virus. Thiamin- or riboflavin-deficiency had no effect either on the production of neutralizing antibodies or on the development of cerebral resistance. Protein- or carbohydrate-deficiency did not cause a marked failure on the part of deficient animals to develop immunity but there was a quantitative difference. The cerebral immunity was about ten fold greater in the well-nourished than in the deficient mice. The neutralization index of the serum from the well-nourished animals was twenty fold greater and the serum could be diluted three to four times more and still neutralize the effect of 50 LD50 doses of virus in mice.

The rate of adsorption of WEE virus onto chicken embryo cells in vitro was determined both on a cell layer and on a cell suspension. One-step growth curves were determined in cell suspensions and on cell layers. The latent period varied between 2 and 3½ hours; it was shorter on cell layers and decreased with higher multiplicity of infection. The shortest period is probably the real latent period. The growth curves of the virus showed an initial exponential rise and reached a maximal constant value after 6 to 8 hours. The maximum virus yield per cell varied between 200 and 1000 on the cell layer, and between 100 and 200 in suspended cells. The yield of single infected cells was determined. An analysis of the distributions of the individual yields obtained after various periods of virus growth led to two main conclusions: (1) that virus is released from the same cell over a long period of time; (2) that one phase of the intracellular virus growth is exponential.

Summary The North American distribution of the mosquito Culex tarsalis Coq. is presented in detail and it compares well with the range of the severe 1941 epizootic of the Western equine encephalomyelitis virus in horses. This mosquito has been found to be naturally infected with this virus in six widely distributed areas over its range which extends throughout the great plains, prairies, and western irrigated areas. During the war period 1942–1946 the known distribution of C. tarsalis was extended 500 miles eastward to South Carolina. The species may have been discovered in this area due to more intensive collecting during that time, or the species may have been introduced due to increased airplane and vehicular traffic in that area. The mosquito is most abundant in the western states and becomes less common eastward. The peak of adult abundance in the western United States is in July and August, and eastward the population maximum occurs progressively later until in the southeast, adults are found only in late fall and winter. The main hosts are wild and domestic animals which are attacked at dusk and night. Human beings are not important hosts. The larvae breed in a large variety of habitats and under a wide range of conditions

SummaryIf Japanese B encephalitis virus is instilled into the noses of 3- to 4-week-old rats 72 hours prior to the inoculation of Western equine encephalomyelitis virus by the same route a high percentage of the animals resist infection with the latter virus. If one inoculation of Japanese B virus preceded the Western equine virus 50% of the rats survived whereas 67% of the animals survived when 3 inoculations of Japanese B virus preceded the equine encephalomyelitis virus. Rats which survived when equine encephalomyelitis virus was instilled into their noses 72 hours following inoculation of Japanese B encephalitis virus by the same route were found to be susceptible to a second intranasal inoculation of equine encephalomyelitis virus.

It has been shown by many workers in the United States that wild birds are associated with the natural history of Western equine encephalitis (WEE). They have also demonstrated that birds can be infected with WEE virus through the bite of an infected mosquito. Wild ducks of many species make up a large part of the bird population to be found in Saskatchewan from April to November each year. By using the neutralization technique, we have found that WEE antibodies are present in the blood of many wild ducks, indicating previous infection with the virus. Further studies carried out at this laboratory showed that wild ducks can be infected with the virus by the oral route, suggesting another possible means by which birds could be naturally infected.

SummaryGarter snakes were infected with western equine encephalomyelitis (WEE) virus by the bites of experimentally infected Culex tarsalis. The snakes hibernated overwinter under simulated natural conditions and viremia was detected up to 69 days after emergence the following spring. Snakes which were maintained at room temperature after emergence developed viremia within a few days, while those maintained under simulated natural conditions required up to approximately 5 weeks. As shown in previous studies, circulating virus was detected in concentrations sufficient to infect C. tarsalis. The use of fish tags proved satisfactory in assuring positive identification of individual snakes.

Single-step growth curves of Western equine encephalomyelitis (WEE) virus in cultures of chick-embryo cells showed that infectious virus titers began to increase by 2.5 hours post-inoculation (PI). The amounts of infectious ribonucleic acid (IRNA) isolated by the use of cold phenol began to increase about 30 minutes earlier. The effect of inhibiting protein synthesis on the time at which IRNA and virus began to increase was determined. Puromycin was used at a concentration of 49 μg/ml. This concentration of puromycin inhibited by 98–99% the incorporation of H 3 -histidine into chick-embryo cell proteins. When chick-embryo cells infected with WEE virus were incubated with puromycin from 1 to 3 hours PI, a subsequent delay of 1.5 and 2 hours occurred before IRNA and virus titers, respectively, began to increase. However, when infected cultures were incubated with puromycin from 3 to 5 hours PI, a time when virus synthesis was already underway, there was only a 30-minute delay before IRNA and virus titers began to increase again. It is concluded that puromycin-sensitive events must occur prior to the synthesis of viral IRNA and virus.