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.     

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.     

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.     

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.     

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).     

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.     

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.     

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.     

Replication of Venezuelan Equine Encephalomyelitis Virus In Vitro: II. Viral Growth Response to Selected Nutritional Additives in Suspension Cultures

An attempt was made to identify nutritional additives that influence the replication of Venezuelan equine encephalomyelitis virus in suspension cultures grown in a defined serum-free medium. Proline, serine, and choline enhanced titers of the virulent PES strain; the progeny population, however, possessed a virulence character that was somewhat different from that of the PES inocula. These nutritional supplements did not appreciably influence the titers of the attenuated 9t and 20t viral strains. When both the PES and 20t strains were employed as a mixed inoculum in culture, the presence of the latter strain appeared to interfere with the growth of the PES strain and reduced its response to the medium supplements.     

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.     

Growth of Venezuelan Equine Encephalomyelitis Virus in Human Diploid Cell Strain WI-38

We demonstrated that Venezuelan equine encephalomyelitis (VEE) virus replicated in and adapted rapidly to human diploid cell strain WI-38. Peak titers of approximately 10(9.8) mouse intracerebral 50% lethal doses were obtained at low passage levels in Eagles basal medium supplemented with calf serum. VEE virus replicated poorly in serum-free medium. Propagation of VEE virus was accompanied by the production of hemagglutinin and cytopathogenic effects.     

Pseudoinfectious Venezuelan Equine Encephalitis Virus: a New Means of Alphavirus Attenuation

Venezuelan equine encephalitis virus (VEEV) is a reemerging virus that causes a severe and often fatal disease in equids and humans. In spite of a continuous public health threat, to date, no vaccines or antiviral drugs have been developed for human use. Experimental vaccines demonstrate either poor efficiency or severe adverse effects. In this study, we developed a new strategy of alphavirus modification aimed at making these viruses capable of replication and efficient induction of the immune response without causing a progressive infection, which might lead to disease development. To achieve this, we developed a pseudoinfectious virus (PIV) version of VEEV. VEE PIV mimics natural viral infection in that it efficiently replicates its genome, expresses all of the viral structural proteins, and releases viral particles at levels similar to those found in wild-type VEEV-infected cells. However, the mutations introduced into the capsid protein make this protein almost incapable of packaging the PIV genome, and most of the released virions lack genetic material and do not produce a spreading infection. Thus, VEE PIV mimics viral infection in terms of antigen production but is safer due to its inability to incorporate the viral genome into released virions. These genome-free virions are referred to as virus-like particles (VLPs). Importantly, the capsid-specific mutations introduced make the PIV a very strong inducer of the innate immune response and add self-adjuvant characteristics to the designed virus. This unique strategy of virus modification can be applied for vaccine development against other alphaviruses.     

Inactivated Venezuelan Equine Encephalomyelitis Vaccine Prepared from Attenuated (TC-83 Strain) Virus

Formalin-inactivated Venezuelan equine encephalomyelitis vaccine was prepared from virus propagated in rolling-bottle cultures of chicken embryo cells. The attenuated, TC-83 strain of virus was propagated in these cultures with a maintenance medium consisting of serum-free medium 199 containing 0.25% human serum albumin and antibiotics. By adjustment of maintenance medium volume (100 to 300 ml) and multiplicity of inoculum (0.04 to 0.00004), high-titered yields of virus were obtained with minimal cell destruction at convenient harvest times, viz, 18 to 24 h postinoculation. Inactivation of virus at 37 C was complete between 8 to 10 h with 0.05% Formalin and within 6 to 8 h with 0.1% Formalin. Antigen extinction potency tests in mice indicated that potent vaccines could be produced at both Formalin concentrations and, furthermore, that potency was not adversely affected by inactivation periods of up to 96 h.