Morphogenesis of Venezuelan Equine Encephalomyelitis Virus

Morphogenesis of Venezuelan equine encephalomyelitis virus was studied by means of electron microscopy. Virus-specific structures (factories, viroplasts) were found at early stages of infection; these structures were composed of fibrillar and cylindrical formations, aggregates of ribosomes, and viral nucleoids. The latter emerged from fibrillar and cylindrical structures. Aggregates of viral nucleoids were found in the cytoplasm and occasionally in the nuclei of virus-infected cells. Viral envelopes and mature virions were formed on the cell membranes and on the membranes of intracellular vacuoles. Anomalous forms of virions (both polygenomic and oligogenomic) were observed.     

Exposure to 2500 Lux Increases Serum Melatonin in Venezuelan Equine Encephalomyelitis

When mice infected with the Venezuelan equine encephalomyelitis (VEE) virus were exposed to 2500 lux with a 12 h light: 12 h dark photoperiod, the serum levels of melatonin (MLT) remained constantly elevated. In mice exposed to 400 lux low levels of serum MLT were detected during the day and high levels during the night. An increase in the survival rate of the infected mice from 6 to 13 days after virus inoculation was also observed. The significant increment in the concentration of serum MLT produced by the high intensity light could be responsible for the longer survival rate of mice infected with the VEE virus.     

Mouse Potency Assay for Western Equine Encephalomyelitis Vaccines

A potency assay for Western equine encephalomyelitis vaccine was developed which utilized mice as the test animal instead of guinea pigs or hamsters. By immunizing several groups of mice with dilutions of the vaccine and challenging them intracerebrally with virulent virus, it was possible to determine mathematically a dose of vaccine capable of protecting 50% of the animals (ED(50)). When log dilutions of virulent virus were used to challenge mice which were immunized with dilutions of the vaccine, there was no difference among the ED(50) values for the dilutions of challenge virus. In a direct comparison of ED(50) values determined from the immunization of mice and those determined from the immunization of guinea pigs, there were no differences in the rankings of the vaccines.     

Inactivation of Venezuelan Equine Encephalomyelitis Virus by gamma-Radiation

Exposure of Venezuelan equine encephalomyelitis (VEE) virus (at -70 C) to 6 × 10⁶ r γ-radiation (⁶⁰Co) resulted in loss of lethality for young adult mice and guinea pigs, and loss of capacity to produce plaques or cytopathic effects in tissue culture. The suckling mouse was more sensitive for detecting live virus in radiated suspensions than was the adult mouse or guinea pig. Live virus was demonstrable in preparations exposed to 6 × 10⁶ r but not in suspensions exposed to 8 × 10⁶ r and more. The rate of inactivation of VEE virus by γ-radiation was an exponential function of the dosage.     

Properties of Venezuelan Equine Encephalomyelitis Virus Grown In Vivo

One intracerebral passage of either the parent egg seed (PES) or an attenuated variant (10t) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus in young adult mice produced progeny that were no longer differentiated unequivocally on the basis of plaque size. Plaques averaging about 2 mm in diameter, which was somewhat smaller than those formed by the PES virus and larger than those of the 10t strain, were formed by both strains. Seven serial passages of the PES virus in mouse brain failed to alter its virulence appreciably. In contrast, passage in mouse brain progressively changed the properties of the attenuated 10t strain. A substrain was isolated that possessed virulence similar to that of the PES virus and formed small plaques similar to those of the 10t strain. These findings showed a unique dissociation between the plaque size and virulence of the 10t strain. The new substrain differed from the PES virus and the 10t strain in its capacity for growth in mouse tissues after intraperitoneal inoculation. The substrain multiplied poorly in splenic tissue, which supports growth of the PES and 10t strains, but grew to high titers in the brain, which does not support appreciable growth of the 10t strain.     

Inactivation of Venezuelan Equine Encephalomyelitis Virus by γ-Radiation

Exposure of Venezuelan equine encephalomyelitis (VEE) virus (at -70 C) to 6 × 10⁶ r γ-radiation (⁶⁰Co) resulted in loss of lethality for young adult mice and guinea pigs, and loss of capacity to produce plaques or cytopathic effects in tissue culture. The suckling mouse was more sensitive for detecting live virus in radiated suspensions than was the adult mouse or guinea pig. Live virus was demonstrable in preparations exposed to 6 × 10⁶ r but not in suspensions exposed to 8 × 10⁶ r and more. The rate of inactivation of VEE virus by γ-radiation was an exponential function of the dosage.     

Early Protection in Hamsters Immunized with Attenuated Venezuelean Equine Encephalomyelitis Vaccine

The rapid onset and persistence of homologous and heterologous protection induced by attenuated Venezuelan equine encephalomyelitis (VEE) vaccine (TC-83) were studied in the hamster, by using challenge response as the index of protection. At 8 hr postvaccination with 10³ median immunizing doses of TC-83 vaccine, 15 to 20% of animals were protected against challenge with VEE virus as well as Eastern and Western equine encephalomyelitis viruses. The percentage of protection increased with time postvaccination until 80 to 90% homologous and heterologous protection was achieved by 18 hr postvaccination. Temporal studies indicated that early protection (days 1 to 6) correlated with vaccine viremia, and that the percentage of protection against heterologous challenge decreased with the cessation of viremia. Data are presented to indicate that the early protection phenomenon is one of interference, since little or no replication of a challenge virus occurred when it was administered during the vaccine viremia stage.     

Properties of Venezuelan Equine Encephalomyelitis Virus Accompanying Attenuation In Vitro

Virus obtained during serial plaque passage of the virulent parent egg seed (PES) of the Trinidad strain of Venezuelan equine encephalomyelitis (VEE) virus produced only large plaques during either 3 serial plaque passages in chick fibroblasts or 10 plaque passages in L cells, and was lethal for mice by the intraperitoneal route. Virus showing these characteristics was designated the stable large-plaque (Ls) type. In contrast, virus obtained during serial plaque passage of the attenuated 9t strain in chick fibroblasts formed only very small plaques and was not lethal for mice by the intraperitoneal route. Virus showing these properties was designated the stable small-plaque (Ss) type. Under other passage conditions, however, large-plaque virus that yielded about 90% large and 10% small plaques was obtained; this virus was designated the unstable large or Lu type because it differed from the Ls type, which yielded only large plaques. The Lu type continued to yield the same ratio of large to small plaques for several plaque-to-plaque passages. In addition, small-plaque virus that yielded both large and small plaques and that showed a reduced capability to infect mice was also recovered. This virus was designated the unstable small or Su type because it differed from the Ss type in its higher level of virulence and in its plaque-forming properties. Thus, based upon the properties of virulence for mice and plaque size, four viral types could be discerned. The evidence suggests that serial passage in cell culture imposed environmental pressures that sequentially selected the following viral types: Ls, Lu, Su, and Ss.     

Effect of NO2 on Airborne Venezuelan Equine Encephalomyelitis Virus

Studies were conducted to determine the effect of nitrogen dioxide (NO(2)) on aerosol survival and biological decay rate of Venezulean equine encephalomyelitis (VEE) virus and spores of Bacillus subtilis var. niger. The NO(2) concentrations used in the experiments were 0.5, 5, and 10 ppm at 24 C and 85% RH. The survival of airborne VEE virus disseminated as particles 1 to 5 mum in diameter was significantly influenced by the presence of 5 ppm of NO(2). At this concentration, the biological decay rate increased threefold and the aerosol recovery and aerosol survival of the VEE virus were significantly lower than at 0.5 ppm or in the absence of NO(2). Airborne spores of B. subtilis were not significantly affected by as much as 10 ppm of NO(2).     

Hemagglutination-Inhibition Method and Immunofluorescence Staining with Venezuelan Equine Encephalomyelitis Virus

Hemagglutination and fluorescent antibody (FA) are compared for the direct detection of virus devoid of host cells. A determination was made of the minimal number of tissue plaque-forming units of Venezuelan equine encephalomyelitis virus that could be detected by the hemagglutination technique. Similar concentrations of the virus in bovine albumin borate saline, Brain Heart Infusion broth (Difco), and demineralized water were tested by the FA technique. Somewhat higher concentrations of the virus in bovine albumin borate saline were used in the hemagglutination-inhibition test. The quantitative hemagglutination procedure employed for these studies was carried out at 37 C for 75 min with variations in concentration of goose red cells. As a result of lowering the red cell concentration, smaller concentrations of virus were detected. The direct FA staining procedure applied to slide preparations containing known numbers of tissue culture plaque-forming units of virus was negative. Adsorbed viral antigen on agglutinated goose erythrocytes was visualized by direct and indirect FA techniques.