Markers of Venezuelan encephalitis virus which distinguish enzootic strains of subtype I-D from those of I-E.

Strains of Venezuelan encephalitis virus isolated from enzootic habitats during interepizootic periods in Middle America and northern South America can be distinguished from each other antigenically by hemagglutination inhibition. This test has provided the basis for the classification of these virus strains into subtypes I-E and I-D, respectively. Virus strains of these two subtypes have been found to differ profoundly with respect to virulence for English short hair guinea pigs. Studies are described which confirm that virus strains of the I-D subtype are guinea pig virulent, and that virulence is not the result of cocycling subpopulations of epizootic subtype I-AB or I-C virions. Two additional markers were found which distinguish subtype I-D and I-E Venezuelan encephalitis virus strains. Firstly, hydroxylapatite chromatography of intact virions at pH 6.5 showed differential elution of I-D and I-E prototype strains. Virions of subtype I-D strains eluted at 0.08 to 0.11 M phosphate, while those of subtype I-E strains eluted at 0.15 to 0.20 M phosphate. Secondly, the isoelectric points of the E1 envelope glycoproteins of the I-D and I-E prototype strains were significantly different; pH 6.85 to 7.00 and pH 7.25 to 7.30, respectively. There was no significant difference in the isoelectric points of the E2 envelope glycoproteins. These distinguishing characteristics most likely reflect a fundamental difference in virion surface structure.

Marker characteristics of Venezuelan encephalitis virus strains isolated before and after epidemics and equine epizootics in Middle America.

Ninety-four strains of Venezuelan encephalitis (VE) virus isolated from sentinel hamsters exposed in the Middle American countries of Mexico, Guatemala, Belize, and Honduras were examined for the presence of virions with marker characteristics of strains that cause large epidemics and equine epizootics. Thirty-four strains came from before and 60 strains came from after the Middle American epidemics and equine epizootics of 1966 and 1969-1972. Twenty-three virion clones that resembled epizootic strains by hydroxylapatite chromatography and Vero monkey kidney cell plaque size determinations were characterized further. However, the predominant virions in these clones were like enzootic strains from Middle America north of the Panama Canal region, and not like Middle American epizootic VE strains, since they were in hemagglutination-inhibition antigenic subtype IE, usually had optimal pH of hemagglutination at 6.2, and were avirulent for English shorthair guinea pigs inoculated subcutaneously. These results provide evidence against the theory of origin of epidemic-equine epizootic VE virus strains that posits that epizootic virions emerge in Middle America from strains containing mixtures of enzootic and epizootic virions in enzootic habitats.

Virion envelope glycoproteins as epidermiological markers of Venezuelan encephalitis virus isolates.

Virion polypeptide compositions of 26 isolates of Venezuelan encephalitis virus were analyzed by a reproducible and comparative technique of discontinuous sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. Although the molecular weight of the core polypeptide for each isolate was 36,000, numbers and molecular weights of envelope glycoproteins were heterogeneous. Isolates associated with human, but not equine, disease usually had two glycoproteins of 50,000 to 51,000 and 51,000 to 55,000 molecular weight, whereas isolates associated with both human and equine disease usually had an additional, third polypeptide band of either 45,000 to 46,000 or 56,000 to 58,000 molecular weight. The former isolates were in hemagglutination inhibition subtypes I-D, I-E, III, or IV, and the latter were in subtypes I-A, I-B, I-C, or II. Thus virion envelope glycoproteins should be useful markers of Venezuelan encephalitis virus isolates in epidemiological investigations.

A hamster-attenuated, temperature-sensitive mutant of Venezuelan encephalitis virus.

Pathogenicities of 10 temperature-sensitive mutants of Venezuelan encephalitis virus were studied using the hamster model of human virulence. The parental strain and nine of the temperature-sensitive mutants produced lethal infections in hamsters. Strain ts 126 showed reduced hamster virulence. Deaths with the lethal mutants usually occurred 1 to 3 days later than with parental virus. Nine mutants produced lower levels of viremia than parental virus. Attenuation of ts 126 was related to restriction of viral growth in spleen and probably bone marrow and to absence of the usual pathological lesions in hemopoietic tissues and brain, but was functionally unrelated to temperature sensitivity since temperatures of both normal and infected hamsters remained within the permissive range of the mutant. Deaths did not correlate with titers of the 10 mutants in blood at permissive temperatures or with reversions of four temperature-sensitive mutants to non-temperature-sensitive virus in hamsters.