Genetic Conservation of Highlands J Viruses

Abstract

We studied molecular evolution of the mosquito-borne alphavirus Highlands J (HJ) virus by sequencing PCR products generated from 19 strains isolated between 1952 and 1994. Sequences of 1200 nucleotides including portions of the E1 gene and the 3' untranslated region revealed a relatively slow evolutionary rate estimated at 0.9-1.6 x 10(-4) substitutions per nucleotide per year. Phylogenetic trees indicated that all HJ viruses descended from a common ancestor and suggested the presence of one dominant lineage in North America. However, two or more minor lineages probably circulated simultaneously for periods of years to a few decades. Strains isolated from a horse suffering encephalitis, and implicated in a recent turkey outbreak, were not phylogenetically distinct from strains isolated in other locations during the same time periods. Our findings are remarkably similar to those we obtained previously for another North American alphavirus, eastern equine encephalomyelitis virus, with which Highlands J shares primary mosquito and avian hosts, geographical distribution, and ecology. These results support the hypotheses that the duration of the transmission season affects arboviral evolutionary rates and vertebrate host mobility influences genetic diversity.

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

“…All estimates of evolutionary rates for alphaviruses circulating continuously in enzootic transmission foci are at least four times greater, and rates in the tropics are believed to be at least 10 times greater. 14,18,35,37 These estimates argue against a continuous, enzootic-like cycle for IAB viruses. However, the recent finding of a 1983 mosquito isolate of a subtype IC VEE virus in Venezuela during an interepidemic period, which is genetically indistinguishable from epidemic isolates made in 1962 and 1995, 7 has raised the possibility of maintenance of epizootic viruses in a genetically static manner between outbreaks.…”

“…6,000 ϫ g for 30 min, RNA was extracted using Trizol (BRL Laboratories, Bethesda, MD), as described previously. 18 Reverse transcription-polymerase chain reactions (PCRs) were used to amplify overlapping segments of the 3Ј-terminal 525 nucleotides of the nonstructural protein 4 (nsP4) gene and the 26S structural genome region. Amplicons were generated from viral RNA between genome nucleotide position 6963 and the 3Ј terminus of the genome using forward/reverse primers NS4A.…”

“…The PCRs were done following a previously described procedure 18 with 30 amplification cycles as follows: heat-denaturation at 95ЊC for 30 sec, primer annealing at 49ЊC for 30 sec, and extension at 72ЊC for 1 min.…”

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

“…19 The maximum likelihood model included a transition:transversion ratio of 4:1-5:1 and a gamma distribution of 0.25, based on empirical alphavirus estimates. [20][21][22] Bootstrapping 23 was performed to place confidence estimates on selected clades within trees. The outgroup consisted of representative homologous sequences of four major lineages of EEE virus.…”

Trocara virus: a newly recognized Alphavirus (Togaviridae) isolated from mosquitoes in the Amazon Basin.

“…RNA was extracted from one half of each virus/Trizol suspension according to manufacturer's protocols, as described previously. 10 cDNAs were synthesized from the RNA using a poly(T) oligonucleotide primer (T 25 V-Mlu; 5Ј-TTAC-GAATTCACGCGT 25 V-3Ј). The polymerase chain reaction (PCR) amplification was performed on the cDNA using the poly(T) primer and a forward primer designated ␣10247 (5Ј-TACCCNTTYATGTGGGG-3Ј) that corresponds to a conserved alphavirus sequence near the N-terminus of the E1 glycoprotein gene.…”