Molecular evidence that epizootic Venezuelan equine encephalitis (VEE) I-AB viruses are not evolutionary derivatives of enzootic VEE subtype I-E or II viruses.

Enzootic strains of Venezuelan equine encephalitis (VEE) virus occur in the United States (Florida), Mexico, Central America and South America. Epizootic VEE first occurred in North and Central America in a widespread outbreak between 1969 and 1972. To investigate the likelihood that this epizootic VEE virus, identified as VEE antigenic subtype I-AB, evolved from enzootic viruses extant in the region, we cloned and sequenced the 26S mRNA region of the genomes of the Florida VEE subtype II virus, strain Everglades Fe3-7c, and the Middle American subtype I-E virus, strain Mena II. This region of the genome encodes the viral structural proteins. The sequences of the 26S mRNA regions of the Everglades and Mena virus genomes differed from that of the reference epizootic VEE subtype I-AB virus, Trinidad donkey strain, by 453 and 887 nucleotides and by 66 and 131 amino acids, respectively. These data confirm previous reports demonstrating significant antigenic and genetic distance between VEE I-AB virus and viruses of subtypes I-E and II. It is unlikely that the epizootic VEE I-AB virus responsible for the 1969 outbreak originated from mutation of enzootic VEE viruses in North or Middle America.

Attenuation of Venezuelan equine encephalitis virus strain TC-83 is encoded by the 5'-noncoding region and the E2 envelope glycoprotein.

The virulent Trinidad donkey (TRD) strain of Venezuelan equine encephalitis (VEE) virus and its live attenuated vaccine derivative, TC-83 virus, have different neurovirulence characteristics. A full-length cDNA clone of the TC-83 virus genome was constructed behind the bacteriophage T7 promoter in the polylinker of plasmid pUC18. To identify the genomic determinants of TC-83 virus attenuation, TRD virus-specific sequences were inserted into the TC-83 virus clone by in vitro mutagenesis or recombination. Antigenic analysis of recombinant viruses with VEE E2- and E1-specific monoclonal antibodies gave predicted antigenic reactivities. Mouse challenge experiments indicated that genetic markers responsible for the attenuated phenotype of TC-83 virus are composed of genome nucleotide position 3 in the 5'-noncoding region and the E2 envelope glycoprotein. TC-83 virus amino acid position E2-120 appeared to be the major structural determinant of attenuation. Insertion of the TRD virus-specific 5'-noncoding region, by itself, into the TC-83 virus full-length clone did not alter the attenuated phenotype of the virus. However, the TRD virus-specific 5'-noncoding region enhanced the virulence potential of downstream TRD virus amino acid sequences.

Genetic evidence that epizootic Venezuelan equine encephalitis (VEE) viruses may have evolved from enzootic VEE subtype I-D virus.

An important question pertaining to the natural history of Venezuelan equine encephalitis (VEE) virus concerns the source of epizootic, equine-virulent strains. An endemic source of epizootic virus has not been identified, despite intensive surveillance. One of the theories of epizootic strain origin is that epizootic VEE viruses evolve from enzootic strains. Likely enzootic sources of VEE virus occur in Colombia and Venezuela where many of the epizootic outbreaks of VEE have occurred. We have determined the nucleotide sequences of the entire genomes of epizootic VEE subtype I-C virus, strain P676, isolated in Venezuela, and of enzootic VEE subtype I-D virus, strain 3880, isolated in Panama. VEE subtype I-D viruses are maintained in enzootic foci in Panama, Colombia, and Venezuela. The genomes of P676 and 3880 viruses differ from that of VEE subtype I-AB virus, strain Trinidad donkey (TRD), by 417 (3.6%) and 619 (5.4%) nucleotides, respectively. The translated regions of P676 and 3880 genomes differ from those of TRD virus by 54 (1.4%) and 66 (1.8%) amino acids, respectively. This study and the oligonucleotide fingerprint analyses of South American I-C and I-D viruses (Rico-Hesse, Roehrig, Trent, and Dickerman, 1988, Am. J. Trop. Med. Hyg. 38, 187-194) provide the most conclusive evidence to date suggesting that equine-virulent strains of VEE virus arise naturally from minor variants present in populations of I-D VEE virus maintained in enzootic foci in northern South America.

Molecular evidence for the origin of the widespread Venezuelan equine encephalitis epizootic of 1969 to 1972.

Venezuelan equine encephalitis (VEE) virus is a mosquito-borne pathogen that has caused encephalitis in equine species and humans during sporadic outbreaks in the western hemisphere. The last, and most widespread, VEE outbreak occurred in South America, Central America, Mexico and the U.S.A. (Texas) during 1969 to 1972. We have cloned and sequenced the genome of a virulent VEE subtype I-AB virus, strain 71-180, isolated in Texas in 1971. Thirty-four nucleotide differences were detected between the genome of 71-180 virus and that of the subtype I-AB Trinidad donkey (TRD) virus isolated during the 1943 VEE epizootic in Trinidad. Fifteen nucleotide changes occurred in the non-structural genes, 16 in the structural genes and three in the 3' non-coding region. Only six of the nucleotide differences resulted in amino acid substitutions: one change in each of non-structural proteins nsP1 and nsP3, two in the E2 envelope glycoprotein, one in the 6K polypeptide and one in the E1 envelope glycoprotein. The close genetic relationship between 71-180 virus and TRD virus, commonly used for production of formalin-inactivated VEE vaccines, suggests that incompletely inactivated virulent vaccine virus may have been the source of this and other VEE outbreaks. Use of formalized virulent virus was discontinued during the 1969 to 1972 panzootic. No VEE epizootics have been reported since the introduction of the live attenuated TC-83 vaccine virus.