Structural organization of the gene encoding the human iron-sulfur subunit of succinate dehydrogenase.

The iron-sulfur protein (Ip) subunit of succinate dehydrogenase (SDH and complex II) of the respiratory chain is highly conserved in evolution [Gould et al., Proc. Natl. Acad. Sci. USA 86 (1989) 1934-1938]. We have cloned the entire human Ip cDNA, as well as the Ip-encoding gene (SDH-B) from two genomic human libraries. The cDNA contains a coding sequence of 840 nt, flanked by a 5'-UTR of 133 nt and a 3'-UTR of 123 nt. The entire transcript is encoded by eight exons within approx. 40 kb. The seven introns range in size from 0.75 kb to > 11 kb, and they appear to be of the 'late' intron class. Approx. 5 kb of upstream sequence was also cloned, and approx. 2.4 kb of the promoter region were sequenced and analyzed for consensus elements binding potential transcription factors and transcriptional activators.

Evolution of alphaviruses in the eastern equine encephalomyelitis complex.

Evolution of viruses in the eastern equine encephalomyelitis (EEE) complex was studied by analyzing RNA sequences and oligonucleotide fingerprints from isolates representing the North and South American antigenic varieties. By using homologous sequences of Venezuelan equine encephalomyelitis virus as an outgroup, phylogenetic trees revealed three main EEE virus monophyletic groups. A North American variety group included all isolates from North America and the Caribbean. One South American variety group included isolates from the Amazon basin in Brazil and Peru, while the other included strains from Argentina, Guyana, Ecuador, Panama, Trinidad, and Venezuela. No evidence of heterologous recombination was obtained when three separate regions of the EEE virus genome were analyzed independently. Estimates of the overall rate of EEE virus evolution (nucleotide substitution) were 1.6 x 10(-4) substitution per nucleotide per year for the North American group and 4.3 x 10(-4) for the Argentina-Panama South American group. Evolutionary rate estimates for the North American group increased over 10-fold (from about 2 x 10(-5) to 4 x 10(-4)) concurrent with divergence of two monophyletic groups during the early 1970s. The North and South American antigenic varieties diverged roughly 1,000 years ago, while the two main South American groups diverged about 450 years ago. Analysis of multiple strains isolated from an upstate New York transmission focus during the same years suggested that, in certain locations, EEE virus may be relatively isolated for short time periods.

A comparison of the nucleotide sequences of eastern and western equine encephalomyelitis viruses with those of other alphaviruses and related RNA viruses.

The complete nucleotide sequence of a 1982 Florida strain of eastern equine encephalomyelitis (EEE) virus, and partial sequence of the nonstructural protein genes of western equine encephalomyelitis (WEE) virus, were determined. The EEE virus genome was 11,678 nucleotides in length, excluding the cap nucleotide and poly(A) tail, and the nucleotide composition was 28% A, 24% G, 25% C, and 23% U. The organization of both EEE and WEE virus genomes was like that of other alphaviruses and included a termination codon between the nsP3 and nsP4 genes. Codon usage for 10 of 20 amino acids was nonrandom in the EEE genome, and dinucleotide CpG-containing codons were underutilized in both genomes. The slight CpG deficiency was similar to that seen in other alphaviruses and plant viruses in the alphavirus-like group, but less than that of poliovirus and yellow fever virus. This slight deficiency may reflect adaptation for replication in both CpG-deficient vertebrates, as well as insects which do not have CpG-deficient genomes. Phylogenetic analyses using nonstructural protein amino acid sequences indicated that alphaviruses evolved from a common ancestor which existed a few thousand years ago. An intercontinental introduction of an ancestral virus from the Old to New World, or vice versa, probably resulted in two main extant groups: one includes New World (EEE and Venezuelan equine encephalitis) viruses, while the other includes Old World (Sindbis, Middelburg, O'nyong-nyong, Ross River, and Semliki Forest) viruses. The position of WEE virus in the phylogenetic trees indicated that, in addition to its capsid gene (C. S. Hahn et al. (1988) Proc. Natl. Acad. Sci. USA 85, 5997-6001), WEE virus acquired its nonstructural genes from an EEE-like ancestor during recombination.

Diversity within natural populations of eastern equine encephalomyelitis virus.

We evaluated genetic and phenotypic diversity within natural populations of the alphavirus, Eastern equine encephalomyelitis (EEE) virus. RNA fingerprinting revealed that most populations within infected hosts (unpassaged isolates) contained a consensus genotype along with minority genotypes differing in one to three T1-resistant oligonucleotides. Mutation frequencies appeared to be similar to those reported for other RNA viruses, suggesting that the slow rate of EEE virus evolution is not limited by fidelity of genome replication. Within a given year, genetic diversity was generally greater among geographically distant isolates than among those from the same transmission focus, suggesting that dispersal among EEE viruses in North America is not complete annually. Two of three bird isolates from Maryland and New York contained relatively distantly related genotypes, differing in 15-19 oligonucleotides. A 1985 mosquito isolate from Maryland contained stable, small plaque variants which comprised the majority of that population. These small plaque variants differed by up to eight T1-resistant oligonucleotides when compared with their large plaque counterparts. Temperature sensitive virus was not detected in six unpassaged mosquito isolates from Maryland and New York.

Phylogenetic analysis of alphaviruses in the Venezuelan equine encephalitis complex and identification of the source of epizootic viruses.

We studied the evolution of alphaviruses in the Venezuelan equine encephalitis (VEE) complex using phylogenetic analysis of RNA nucleotide sequences from limited portions of the nsP4, E1, and 3' untranslated genome regions of representative strains. The VEE complex constituted a monophyletic group of viruses (descended from a common ancestor); some serologic VEE varieties such as subtype III formed monophyletic groups while subtype I did not. Subtype II Everglades and variety ID enzootic viruses formed a monophyletic group which also included all epizootic variety IAB and IC VEE isolates. Everglades virus diverged from this ID lineage (colonized North America) ca. 100-150 years ago, followed by divergence of variety IAB and IC epizootic viruses. Variety IAB viruses probably emerged from the variety ID lineage once during the early part of this century, while variety IC viruses evolved at least two times. These results identify the source of epizootic VEE viruses as the variety ID enzootic virus lineage which occurs in northern South America and Panama. Even if variety IAB and IC viruses are extinct, recent, multiple emergences of epizootic viruses from an enzootic lineage suggests that other epizootic VEE viruses may evolve again in the future. The close genetic relationship of subtype II Everglades virus to the variety ID lineage also implies the potential for emergence of equine-virulent VEE viruses in Florida.

Genetic characterization of an antigenic subtype of eastern equine encephalomyelitis virus.

A 1983 human Mississippi isolate of eastern equine encephalomyelitis virus (EEEV), recently identified as an antigenic subtype of the North American variety, was genetically characterized using oligonucleotide fingerprinting and sequencing of viral RNA. This strain was found to be very closely related to other North American EEEV isolates from the same time period. Phylogenetic analysis suggested that this subtype belongs to a single EEEV lineage in North America. Two amino acid substitutions in the E2 envelope glycoprotein, not seen in either other isolates sequenced, probably contributed to the antigenic difference with respect to other EEEV strains. These substitutions include threonine for lysine at position 71, resulting in the addition of a potential N-linked glycosylation site, and lysine for glutamic acid at position 147.