Protocolo del Sistema Nacional de Vigilancia Epidemiológica: enfermedades zoonóticas. No. 08 / Protocol of the National Epidemiological Surveillance System: zoonotic diseases. No. 08

Estos protocolos están dirigido a personal médico, paramédico y otros profesionales que realizan acciones gerenciales y operativas de vigilancia epidemiológica en los servicios de salud del país, y están divididos en varios tomos para dar a conocer y actualizar la identificación y medidas de control para diversos padecimientos a fin de continuar con el mejoramiento de las capacidades técnicas de los trabajadores de salud, que permita planificar la prestación de servicios con decisiones partiendo de un enfoque epidemiológico comprobado, para responder a los cambios de tendencias epidemiológicas y con ello contribuir al fortalecimiento de prácticas asertivas de la salud pública de nuestro país.

Susceptibility and Lethality of Western Equine Encephalitis Virus in Balb/c Mice When Infected by the Aerosol Route.

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.

Entry Sites of Venezuelan and Western Equine Encephalitis Viruses in the Mouse Central Nervous System following Peripheral Infection.

UNLABELLED: Venezuelan and western equine encephalitis viruses (VEEV and WEEV; Alphavirus; Togaviridae) are mosquito-borne pathogens causing central nervous system (CNS) disease in humans and equids. Adult CD-1 mice also develop CNS disease after infection with VEEV and WEEV. Adult CD-1 mice infected by the intranasal (i.n.) route, showed that VEEV and WEEV enter the brain through olfactory sensory neurons (OSNs). In this study, we injected the mouse footpad with recombinant WEEV (McMillan) or VEEV (subtype IC strain 3908) expressing firefly luciferase (fLUC) to simulate mosquito infection and examined alphavirus entry in the CNS. Luciferase expression served as a marker of infection detected as bioluminescence (BLM) by in vivo and ex vivo imaging. BLM imaging detected WEEV and VEEV at 12 h postinoculation (hpi) at the injection site (footpad) and as early as 72 hpi in the brain. BLM from WEEV.McM-fLUC and VEEV.3908-fLUC injections was initially detected in the brain's circumventricular organs (CVOs). No BLM activity was detected in the olfactory neuroepithelium or OSNs. Mice were also injected in the footpad with WEEV.McM expressing DsRed (Discosoma sp.) and imaged by confocal fluorescence microscopy. DsRed imaging supported our BLM findings by detecting WEEV in the CVOs prior to spreading along the neuronal axis to other brain regions. Taken together, these findings support our hypothesis that peripherally injected alphaviruses enter the CNS by hematogenous seeding of the CVOs followed by centripetal spread along the neuronal axis. IMPORTANCE: VEEV and WEEV are mosquito-borne viruses causing sporadic epidemics in the Americas. Both viruses are associated with CNS disease in horses, humans, and mouse infection models. In this study, we injected VEEV or WEEV, engineered to express bioluminescent or fluorescent reporters (fLUC and DsRed, respectively), into the footpads of outbred CD-1 mice to simulate transmission by a mosquito. Reporter expression serves as detectable bioluminescent and fluorescent markers of VEEV and WEEV replication and infection. Bioluminescence imaging, histological examination, and confocal fluorescence microscopy were used to identify early entry sites of these alphaviruses in the CNS. We observed that specific areas of the brain (circumventricular organs [CVOs]) consistently showed the earliest signs of infection with VEEV and WEEV. Histological examination supported VEEV and WEEV entering the brain of mice at specific sites where the blood-brain barrier is naturally absent.

Novel indole-2-carboxamide compounds are potent broad-spectrum antivirals active against western equine encephalitis virus in vivo.

Neurotropic alphaviruses, including western, eastern, and Venezuelan equine encephalitis viruses, cause serious and potentially fatal central nervous system infections in humans for which no currently approved therapies exist. We previously identified a series of thieno[3,2-b]pyrrole derivatives as novel inhibitors of neurotropic alphavirus replication, using a cell-based phenotypic assay (W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275), and subsequently developed second- and third-generation indole-2-carboxamide derivatives with improved potency, solubility, and metabolic stability (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). In this report, we describe the antiviral activity of the most promising third-generation lead compound, CCG205432, and closely related analogs CCG206381 and CCG209023. These compounds have half-maximal inhibitory concentrations of ∼1 µM and selectivity indices of >100 in cell-based assays using western equine encephalitis virus replicons. Furthermore, CCG205432 retains similar potency against fully infectious virus in cultured human neuronal cells. These compounds show broad inhibitory activity against a range of RNA viruses in culture, including members of the Togaviridae, Bunyaviridae, Picornaviridae, and Paramyxoviridae families. Although their exact molecular target remains unknown, mechanism-of-action studies reveal that these novel indole-based compounds target a host factor that modulates cap-dependent translation. Finally, we demonstrate that both CCG205432 and CCG209023 dampen clinical disease severity and enhance survival of mice given a lethal western equine encephalitis virus challenge. These studies demonstrate that indole-2-carboxamide compounds are viable candidates for continued preclinical development as inhibitors of neurotropic alphaviruses and, potentially, of other RNA viruses. IMPORTANCE There are currently no approved drugs to treat infections with alphaviruses. We previously identified a novel series of compounds with activity against these potentially devastating pathogens (J. A. Sindac et al., J. Med. Chem. 55:3535-3545, 2012, doi:http://dx.doi.org/10.1021/jm300214e; W. Peng et al., J. Infect. Dis. 199:950-957, 2009, doi:http://dx.doi.org/10.1086/597275; J. A. Sindac et al., J. Med. Chem. 56:9222-9241, 2013, http://dx.doi.org/10.1021/jm401330r). We have now produced third-generation compounds with enhanced potency, and this manuscript provides detailed information on the antiviral activity of these advanced-generation compounds, including activity in an animal model. The results of this study represent a notable achievement in the continued development of this novel class of antiviral inhibitors.

Molecular determinants of mouse neurovirulence and mosquito infection for Western equine encephalitis virus.

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.

Neurotropic arboviruses induce interferon regulatory factor 3-mediated neuronal responses that are cytoprotective, interferon independent, and inhibited by Western equine encephalitis virus capsid.

Cell-intrinsic innate immune responses mediated by the transcription factor interferon regulatory factor 3 (IRF-3) are often vital for early pathogen control, and effective responses in neurons may be crucial to prevent the irreversible loss of these critical central nervous system cells after infection with neurotropic pathogens. To investigate this hypothesis, we used targeted molecular and genetic approaches with cultured neurons to study cell-intrinsic host defense pathways primarily using the neurotropic alphavirus western equine encephalitis virus (WEEV). We found that WEEV activated IRF-3-mediated neuronal innate immune pathways in a replication-dependent manner, and abrogation of IRF-3 function enhanced virus-mediated injury by WEEV and the unrelated flavivirus St. Louis encephalitis virus. Furthermore, IRF-3-dependent neuronal protection from virus-mediated cytopathology occurred independently of autocrine or paracrine type I interferon activity. Despite being partially controlled by IRF-3-dependent signals, WEEV also disrupted antiviral responses by inhibiting pattern recognition receptor pathways. This antagonist activity was mapped to the WEEV capsid gene, which disrupted signal transduction downstream of IRF-3 activation and was independent of capsid-mediated inhibition of host macromolecular synthesis. Overall, these results indicate that innate immune pathways have important cytoprotective activity in neurons and contribute to limiting injury associated with infection by neurotropic arboviruses.

Variation in western equine encephalomyelitis viral strain growth in mammalian, avian, and mosquito cells fails to explain temporal changes in enzootic and epidemic activity in California.

The decrease in western equine encephalomyelitis virus (WEEV; Togaviridae, Alphavirus) activity in North America over the past 20-30 years has prompted research to determine if there have been concurrent declines in virulence. Six (WEEV) strains isolated from Culex tarsalis mosquitoes from California during each of the six preceding decades failed to show a consistent declining temporal trend in virus titer using mosquito (C6/36), avian (duck embryo fibroblast), or mammalian (Vero) cells, results similar to our recent in vivo studies using birds and mosquitoes. Titers measured by Vero cell plaque assay were consistently highest on mosquito cell culture, followed by avian and mammalian cell cultures. Similar to previous in vivo results in house sparrows and mice, titers for the IMP181 strain isolated in 2005 were significantly lower in both avian and mammalian cells. Real-time monitoring of changes in cell growth measured by electrical impedance showed consistent differences among cell types, but not WEEV strains. Collectively, these in vitro results failed to explain the decrease in WEEV enzootic and epidemic activity. Results with the IMP181 strain should be verified by additional sequencing, cell growth, and pathogenesis studies using concurrent or 2006 isolates of WEEV from California.

Effect of exogenous interferon and an interferon inducer on western equine encephalitis virus disease in a hamster model.

Mice are used as models for western equine encephalitis virus (WEEV) infection, but high mortality is generally only seen with intracranial or intranasal challenge, while peripheral inoculation results in approximately 50% mortality and is not dose-dependent. Hamsters were therefore studied as a model for WEEV infection. Hamsters were highly sensitive to intraperitoneal (i.p.) infection with WEEV. Disease progression was rapid, and virus titers in serum, brain, liver, and kidney of infected hamsters peaked between 2 and 4 days post-virus inoculation (dpi). Foci of virus infection were detected in neurons of the cerebral cortex and midbrain. Pre-treatment i.p. with either interferon alfacon-1 (5 microg/kg/day) or with Ampligen (3.2 mg/kg/day) resulted in complete survival, reduced brain titers, and improved weight gain. This model of WEEV infection in hamsters appears to serve as a suitable model for the evaluation of potential therapeutic agents for the treatment of WEE disease.

Complete genomic RNA sequence of western equine encephalitis virus and expression of the structural genes.

The complete nucleotide sequence of the 71V-1658 strain of western equine encephalitis virus (WEE) was determined (minus 25 nucleotides from the 5' end). A 5' RACE reaction was used to sequence the 5' terminus from WEE strain CBA87. The deduced WEE genome was 11508 nucleotides in length, excluding the 5' cap nucleotide and 3' poly(A) tail. The nucleotide composition was 28% A, 25% C, 25% G and 22% U. Comparison with partial WEE sequences of strain 5614 (nsP2-nsP3 of the nonstructural region) and strain BFS1703 (26S structural region) revealed comparatively little variation; a total of 149 nucleotide differences in 8624 bases (1.7% divergence), of which only 28% (42 nucleotides) altered the encoded amino acids. Comparison of deduced nsP1 and nsP4 amino acid sequences from WEE with the corresponding proteins from eastern equine encephalitis virus (EEE) yielded identities of 84.9 and 83.8%, respectively. Previously uncharacterized stem-loop structures were identified in the nontranslated terminal regions. A cDNA clone of the 26S region encoding the structural polyprotein of WEE strain 71V-1658 was placed under the control of a cytomegalovirus promoter and transfected into tissue culture cells. The viral envelope proteins were functionally expressed in tissue culture, as determined by histochemical staining with monoclonal antibodies that recognize WEE antigens, thus, forming the initial step in the investigation of subunit vaccines to WEE.

Vector competence of Culex tarsalis (Diptera: Culicidae) from Iowa for a sympatric strain of western equine encephalomyelitis (WEE-7738) virus.

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.

Vector competence of Aedes dorsalis (Diptera: Culicidae) from Morro Bay, California, for western equine encephalomyelitis virus.

In laboratory vector competence studies, Aedes dorsalis (Meigen) collected from Morro Bay, CA, did not vertically transmit sympatric strains of western equine encephalomyelitis virus (WEE). This population of Ae. dorsalis was highly susceptible to oral infection and was a competent horizontal vector of WEE. The E2 region of the viral genome of the 3 virus strains isolated from Ae. dorsalis in Morro Bay were closely related genetically to a strain of WEE isolated in 1953 from a geographically separate location that is used regularly in the laboratory. These laboratory findings support recent field research and indicate that Ae. dorsalis probably does not play a significant role in WEE persistence in coastal California.

Characterization of modulation of western equine encephalomyelitis virus by Culex tarsalis (Diptera: Culicidae) maintained at 32 degrees C following parenteral infection.

Two lines of Culex tarsalis Coquillett genetically selected for low or high western equine encephalomyelitis (WEE) virus production (low viral producer [LVP] or high viral producer [HVP], respectively) modulated WEE (i.e., decreased the concentration of virus to < 10(4) plaque-forming units after intrathoracic inoculation). The LVP line modulated WEE more than HVP, and modulation was most pronounced at 32 degrees C. At 15 degrees C, viral replication to high titers occurred in both lines. When infected LVP were transferred to 15 degrees C after 4 d extrinsic incubation at 32 degrees C, replication of WEE to high titers did not occur. Mosquitoes transferred from 15 degrees C after replication to high titers occurred; to 32 degrees C did significantly modulate WEE titer. Incubation at 32 degrees C prior to infection had no effect on the degree or timing of WEE modulation in both LVP and HVP lines. Most LVP infected following feeding on a high dose of WEE had salivary gland infection barriers. Viral modulation by Cx. tarsalis was an alphavirus phenomenon, and was not restricted to WEE.

Effect of temperature on the transmission of western equine encephalomyelitis and St. Louis encephalitis viruses by Culex tarsalis (Diptera: Culicidae).

The extrinsic incubation rate (inverse of the time in days from infection to median transmission) of western equine encephalomyelitis (WEE) and St. Louis encephalitis (SLE) viruses by laboratory strains of Culex tarsalis Coquillett increased as a linear function of incubation temperatures from 10 to 30 degrees C. The estimated temperatures for zero transmission thresholds (intercept of the X axis) were 10.9 and 14.9 degrees C, and the number of degree days above these thresholds required for median transmission (inverse of the slope) was 67.6 and 115.2, respectively. Although the bodies of most Cx. tarsalis females remained infected and the WEE viral titer did not decrease significantly throughout the incubation periods at all temperatures, transmission rates by females incubated at 20 to 30 degrees C decreased markedly after peaking at 7-10 d after infection. In contrast, midgut escape and salivary gland infection barriers limited the transmission rates of SLE virus at all temperatures, but these rates did not decrease markedly as a function of incubation time, indicating that virus modulation did not occur. Degree-day models were used to calculate monthly changes in the duration of the extrinsic incubation period for WEE and SLE viruses in the San Joaquin and Coachella valleys based on mosquito temperatures estimated by combining nocturnal air and diurnal resting site temperatures. Temperatures in the San Joaquin Valley averaged 5 degrees C cooler than in the Coachella Valley, proportionately shortening the duration of the potential transmission season for WEE virus from 10 to 8 mo and for SLE virus from 8 to 5 mo, respectively.

Effect of the anesthetizing agent triethylamine on western equine encephalomyelitis and St. Louis encephalitis viral titers in mosquitoes (Diptera: Culicidae).

Triethylamine (TEA) is a chemical compound that provides an effective means to anesthetize mosquitoes. The mosquitoes remain alive but are incapacitated for several hours; they do not recover following treatment. There was no effect on the titers of infectious virus recovered from western equine encephalomyelitis or St. Louis encephalitis virus-infected Culex tarsalis Coquillett and Aedes dorsalis (Meigen) that were anesthetized with TEA prior to storage at -70 degrees C. Furthermore, TEA had no effect on isoenzyme profiles of uninfected Cx. tarsalis.

Antiviral activities of pyrimidine nucleoside analogues: some structure--activity relationships.

Seventeen nucleoside derivatives (derived from arabinosylcytosine, resp. cytidine, 5-fluorouracil and uracil) were tested by agar-diffusion plaque-inhibition test for their antiviral activity with herpes simplex, vaccinia, fowl plague, Newcastle disease and western equine encephalomyelitis viruses. The highest antiviral activity against DNA viruses exhibited arabinosylcytosine, N4-acylarabinosylcytosines, arabinosylthiouracil, cyclocytidine and its 5'-chloroderivative. RNA viruses were inhibited by 5-fluorouridine only, whereas other tested compounds were ineffective or showing marginal activity only. By search for relationship between chemical structure and antiviral activity a tendency was found of higher antiviral activity at lower lipophilicity. This is probably due to better transport of the studied compounds into cell. The chemical structure, however, is the main reason of antiviral activity.

Diel changes in adult mosquito microhabitat temperatures and their relationship to the extrinsic incubation of arboviruses in mosquitoes in Kern County, California.

Microhabitat temperatures experienced by nocturnally active Culex tarsalis Coquillett mosquitoes were measured in the southern San Joaquin Valley of California. Temperatures of the diurnal resting shelter and nocturnal activity air space were measured by digital recorder at three Cx. tarsalis habitats in Kern County. Temperatures measured by digital recorders agreed well with temperatures recorded concurrently at nearby California Irrigation Management and Inspection System weather stations. Monthly temperatures among habitats were combined to depict the diel temperature regimen experienced by female Cx. tarsalis as a consequence of daily movement between the diurnal resting shelter and nocturnal air space. Transitions between the two microhabitats corresponded to the times of mosquito ingress and egress (i.e., sunrise and sunset). The composite daily temperature mean ranged from 7.4 degrees C in December to 21.5 degrees C in July, and temperature extremes seldom exceeded a daily maximum of 25.0 degrees C during the summer or dropped below a daily minimum of 5.0 degrees C during the winter. Overall, mosquitoes occupied a composite thermal environment where the extrinsic incubation of an arbovirus would have occurred at ambient temperatures that were cooler than either the diurnal resting shelter or activity air space.

Temporal variations in the susceptibility of a semi-isolated population of Culex tarsalis to peroral infection with western equine encephalomyelitis and St. Louis encephalitis viruses.

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.

Characterization of the mesenteronal infection with Western equine encephalomyelitis virus in an incompetent strain of Culex tarsalis.

Interactions of western equine encephalomyelitis (WEE) virus are compared with mesenterons of 2 genetically selected susceptible (WS) and refractory (WR) lines of Culex tarsalis. Both WS and WR females had similar susceptibility when parenterally inoculated with virus, thus it was initially thought that resistance in WR Cx. tarsalis was associated with a mesenteronal infection barrier. Present data on viral growth in mesenterons dissected from females fed on virus-soaked pledgets suggest that virus infected and multiplied in some WR mesenterons, but to significantly lower titers than in WS mesenterons. The proportion of WR females with infected mesenterons varied depending on the time after feeding, incubation temperature, and whether mesenterons were incubated with WEE viral antibody before viral assay. The percentage of WR mesenterons infected did not increase significantly when diethylaminoethyl dextran was added to the infectious bloodmeal, the pH of the infectious bloodmeal was altered, or virus was introduced by intrathoracic inoculation into the hemocoel. It was concluded that the low titers of virus detected in both WR mesenterons and whole mosquitoes were influenced by the genetic ability of WR Cx. tarsalis to modulate WEE viral titers to low or undetectable levels after peroral or parenteral infection. These findings make it difficult to determine what proportion of the WR mesenterons are resistant to infection with WEE virus. WS and WR Cx. tarsalis were equally susceptible to peroral infection with the flavivirus St. Louis encephalitis and the bunyavirus Turlock.

Infection of a poikilothermic cell line (XL-2) with eastern equine encephalitis and western equine encephalitis viruses.

Eastern Equine Encephalitis (EEE) was in Cuba before the 1940s; the virus has been isolated from horses, birds, and rodents during epizootic as well as interepizootic periods. The only isolation of Western Equine Encephalitis (WEE) virus was from a sick pigeon found in the vicinity of Havana University. Both viruses can cause human disease; the isolation of WEE virus from the centre of an urban area emphasises the need for the prompt isolation and rapid identification of these agents. The object of this work was to compare the sensitivity of a continuous cell line (XL-2) from the toad, Xenopus laevis, with primary chick embryo cell cultures (CEC) routinely used for isolation as well as assay of these viruses. Both cell systems were infected with EEE virus isolated from horse brain and WEE virus isolated from a sick pigeon. A clear cytopathic effect (CPE) consisting of rounding and detachment of cells was observed in both cell cultures infected with the two viruses. By 18 hours post-infection, there was partial destruction of the cell monolayer and by 24 hours the CPE was total. The infectious titre of EEE and WEE viruses in XL-2 and CEC were similar. Both viruses produced small plaques (0.5-1.0 mm diameter) in XL-2 cells. Studies on the sensitivity of the XL-2 cells for direct isolation of the two viruses from field samples and for the detection of Cuban flaviviruses by the immunofluorescence test are in progress.

Western equine encephalomyelitis virus: in vivo infection and morphogenesis in mosquito mesenteronal epithelial cells.

The infection and morphogenetic events associated with the replication of Western equine encephalomyelitis (WEE) virus within the mesenterons of Aedes dorsalis and three strains of Culex tarsalis are compared and contrasted. WEE virus apparently penetrates mesenteronal epithelial cells in vivo through membrane fusion. Profiles of apparent membrane fusion events were observed between virus particles and the microvillar surface of the mesenteron and naked nucleocapsids are observed intracellularly along the apical margin of the mesenteronal epithelial cell within 3 h of ingestion of the bloodmeal. Further, no viral particles were found in association with endocytotic nor lysosomal vacuoles during the initial phases of infection. In those strains of Cx. tarsalis that supported viral replication and in Ae. dorsalis, accumulations of nucleocapsids and maturation of WEE virus were evident along basolateral membranes of the mesenteron by 22-24 h after ingestion of the blood-meal. Maximal extracellular nascent virus occurred between 30-36 hrs. The Knights Landing strain of Cx. tarsalis revealed no subcellular morphological alteration in response to infection throughout the period of study. However, distinct morphological structures associated with the infection were observed in strains or species with enhanced susceptibility compared to Knights Landing (i.e., Cx. tarsalis WS-3 and Ae. dorsalis). In both, apical accumulations of nucleocapsids were apparent by 29 h post infection. These nucleocapsids were most often embedded in a rather amorphous matrix and occasionally in association with membrane profiles; presumably endoplasmic reticulum. Ae. dorsalis also demonstrated some alterations in response to WEE viral infection that were unique relative to Cx. tarsalis and some of these may be considered cytopathological. First, progeny virions were observed repeatedly within lysosomal figures. Second, extensive cytoplasmic vacuolization was noted and occasionally it appeared that these vacuolated cells were being sloughed off into the lumen of the mesenteron.