Genetic evidence for the origins of Venezuelan equine encephalitis virus subtype IAB outbreaks.

Abstract

Abstract. Epizootics of Venezuelan equine encephalitis (VEE) involving subtype IAB viruses occurred sporadically in South, Central and North America from 1938 to 1973. Incompletely inactivated vaccines have long been suspected as a source of the later epizootics. We tested this hypothesis by sequencing the PE2 glycoprotein precursor (1,677 nucleotides) or 26S/nonstructural protein 4 (nsP4) genome regions (4,490 nucleotides) for isolates representing most major outbreaks. Two distinct IAB genotypes were identified: 1) 1940s Peruvian strains and 2) 1938-1973 isolates from South, Central, and North America. Nucleotide sequences of these two genotypes differed by 1.1%, while the latter group showed only 0.6% sequence diversity. Early VEE virus IAB strains that were used for inactivated vaccine preparation had sequences identical to those predicted by phylogenetic analyses to be ancestors of the 1960s-1970s outbreaks. These data support the hypothesis of a vaccine origin for many VEE outbreaks. However, continuous, cryptic circulation of IAB viruses cannot be ruled out as a source of epizootic emergence.

Ecological studies of enzootic Venezuelan equine encephalitis in north-central Venezuela, 1997-1998.

“…Nucleotide sequences of reverse transcription-polymerase chain reaction (RT-PCR) products totaling 1,677 base pairs, excluding the terminal primers, were generated for the complete P62 gene as described previously. 17 Viral RNA was extracted from the supernatant of first passage Vero cell stocks using Trizol LS (Bethesda Research Laboratories, Bethesda, MD) according to the manufacturer's protocol. The RT-PCR protocol and primers were described previously.…”

“…The RT-PCR protocol and primers were described previously. 17 Nucleotide sequences generated directly from amplicons using the Applied Biosystems (Foster City, CA) Prism sequencing kit and 377 sequencer, according to the manufacturer's protocol, were aligned using the PILEUP program 18 in the Wisconsin Package (Genetics Computer Group, Madison, WI) with default settings. Phylogenetic analyses were performed using the complete 1,677 nucleotide amplicon sequences with maximum likelihood, maximum parsimony and neighbor joining programs implemented in PAUP 4.0 (Sinauer Associates, Sunderland, MA).…”

“…The species captured and the results of the virology studies are shown in Table 3. Proechimys guairae (17) and Didelphis marsupiales (14) were abundant throughout the study period. There was no virus isolation from wild mammal specimens, but HI antibodies to VEE virus were detected in 5 of 17 (29%) of the Proechimys guairae captured.…”

“…Complete sequences of 1,677 nucleotides covering the P62 gene were determined directly from RT-PCR amplicons and compared to homologous VEE complex sequences found in the GenBank library. 12,17,[25][26][27][28] The Miranda isolates exhibited a minimum nucleotide sequence divergence of 8% (3% divergence of deduced amino acid sequences) versus all other VEE complex viruses sequenced previously. Phylogenetic analyses using all methods generated trees with nearly identical topologies, with the exception of a minor difference in groupings within the subtype IAB clade in two equally parsimonious trees.…”

Genetic diversity and relationships among Venezuelan equine encephalitis virus field isolates from Colombia and Venezuela.

“…We extracted RNA from cell culture medium as described previously. 25 A 250-L volume of cell culture medium was mixed with 0.75 mL of Trizol LS reagent (Gibco BRL, Gaithersburg, MD), and RNA was extracted according to the manufacturer's protocol; transfer RNA was added as a carrier to enhance RNA precipitation.…”