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.     

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.     

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.     

Chromatography of Venezuelan Equine Encephalomyelitis Virus Strains on Calcium Phosphate

Column chromatography of selected Venezuelan equine encephalomyelitis (VEE) viruses on calcium phosphate gel offered a simple and reproducible method for examination of biochemical characteristics and relatedness of strains within the VEE complex. Members of antigenic subgroup I demonstrated a series of elution profiles within a narrow range of 0.22 to 0.25 M phosphate buffer. Members of antigenic subgroups II, III, and IV differed substantially among themselves and viruses of antigenic subgroup I. These differences in elution behavior may contribute to understanding of observed differences in biological behavior and antigenic variation among VEE viruses.     

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.     

Virulence of Venezuelan Equine Encephalomyelitis Virus Subtypes for Various Laboratory Hosts

Mice, guinea pigs, and duck embryo cell cultures were inoculated with known subtypes of Venezuelan equine encephalomyelitis (VEE) virus and the attenuated (TC-83) strain of VEE. With the exception of TC-83, all strains were highly pathogenic for suckling mice by either intracranial or intraperitoneal routes of inoculation used. Virulence for older mice and guinea pigs provided a means to distinguish strains. In addition, virulence or lack of virulence for adult mice or guinea pigs provides a rapid method for separating epizootic subtype IB from TC-83 VEE virus isolates.     

Primary Virus-Cell Interactions in the Immuno-fluorescence Assay of Venezuelan Equine Encephalomyelitis Virus

The conditions under which Venezuelan equine encephalomyelitis (VEE) virus attached to host cells markedly influenced the assay of virus by the fluorescent cell-counting technique. When virus inoculum was centrifuged onto McCoy cell monolayers, approximately 97% of virus was attached to cells within 10 min, in contrast to 34% after stationary incubation at 35 C for 2 hr. Maximal binding of virus occurred only in the presence of 0.1 to 0.15 m NaCl. This salt requirement, added to evidence of (p)H dependence and temperature independence of VEE virus attachment to cells, indicated that the initial union involved electrostatic forces. Virus penetration, measured by the insensitivity of virus-cell complexes to viral antiserum, was complete in 30 min at 35 C. The process was temperature-dependent and un-affected by the ionic content of medium. For assay of VEE virus by the fluorescent cell-counting technique, infected cells may be enumerated as early as 12 hr after infection of cell monolayers. The relationship between virus concentration and cell-infecting units was linear; the distribution of fluorescent cells was random. The virus assay was equivalent in sensitivity but more precise and rapid than that of intracerebral inoculation of mice.     

Acid Phosphatase Activity in Mouse Brain Infected with Venezuelan Equine Encephalomyelitis Virus

The mode of development of Venezuelan equine encephalomyelitis virus and the activity of acid phosphatase in the central nervous system of newborn mice were investigated. Precursor particles appeared to be formed in masses of viroplasm, migrating to the membrane of the Golgi cisterns and vacuoles or to the plasma membrane and being transformed into mature viral particles by budding. Mature viral particles were also found in the lumen of the blood vessels and around the myelin sheath of axons. Increased number of Golgi complexes and depletion of polysomes were the main ultrastructural alterations of the nerve cells. Acid phosphatase activity was found to be increased in the Golgi cisterns, vacuoles, and lysosomes of nerve cells. The presence of acid phosphatase activity in the rough endoplasmic reticulum and perinuclear cisterns suggests increased production of the enzyme in the nerve cells infected with Venezuelan equine encephalomyelitis virus.     

西方马脑炎核酸疫苗表达载体构建及免疫原性研究

为构建西方马脑炎病毒(western equine encephalomyelitis virus,WEEV)结构基因C-E3-E2-6k-E1重组真核表达载体,并研究其作为核酸疫苗的免疫原性.采用PCR方法扩增目的基因,酶切之后连接到pcDNA3.1上构建真核表达载体pcDNA3.1-C-E,用酶切和测序分析方法鉴定正确后,重组质粒被转染到293T细胞,经电镜检测和间接免疫荧光方法证明基因可以表达后,用该重组质粒免疫小鼠,免疫后检测实验组小鼠外周血中CD4+T细胞/CD8+T细胞比例和血清中细胞因子IL-2、IL-4及IFN-γ浓度,以上实验组各项免疫指标与对照组相比差异均显著(P＜ 0.05);ELISA方法检测实验组小鼠血清中WEEV的IgG抗体效价是1∶16.研究结果表明重组质粒pcDNA3.1-C-E可在细胞中获得瞬时表达,并且重组质粒作为核酸疫苗能够刺激小鼠产生免疫反应,具有较强免疫原性,为今后WEEV核酸疫苗研制奠定了良好基础.     

Factors Influencing Virulence and Plaque Properties of Attenuated Venezuelan Equine Encephalomyelitis Virus Populations

A minority of stable large-plaque virus increased proportionally in stored unstable attenuated (9t) Venezuelan equine encephalomyelitis virus populations. L-cell-grown progeny (9t2) of stored 9t showed large amounts of large-plaque virus and increased virulence. Small-plaque virus inhibited large-plaque virus but not the reverse. Serial passage of small-plaque virus from 9t2 yielded a strain (20t) that was more attenuated than 9t.