Molecular characterization of three enteroviral strains isolated in Kuwait from young children with serious conditions

Introduction: Human enteroviruses are single stranded RNA viruses associated with many serious diseases such as encephalitis and myocarditis. They consist of up to 100 immunologically and genetically distinct types. Three enteroviral isolates, 2104, 3936 and 3988, were previously isolated from patients with neurological disorders or sepsis-like illness. In this study, the molecular characterization of the three isolates was investigated. Methodology: A full genome sequencing was performed by Sanger method, followed by phylogenetic and bootscanning analyses. A detailed analysis of genetic differences between the clinical and prototype isolates were investigated by mapping polymorphisms at nucleotide and amino acid levels, and by comparing RNA secondary structure in the noncoding regions. Results: Based on the phylogenetic analysis of the VP1 gene and complete genome, 2104 was typed as coxsackievirus B1, 3936 as coxsackievirus B5, and 3988 as echovirus 7. Similarity and bootscan plots provided support for intra- and intertypic recombination crossover points occurring mainly along the nonstructural coding regions of the isolates. A sequence divergence of 12 to 14% was detected in the 5’-noncoding region between the clinical isolates and their corresponding prototype strains. Synonymous and nonsynonymous substitutions could be also mapped to different coding regions of the isolates, including those coding for the Puff, Knob and the hydrophobic pocket of the capsid. Examination of relative frequencies of synonymous and nonsynonymous substitutions in different coding regions of enteroviral isolates showed no evidence for selective pressure. Conclusion: The results provided a better understanding of the genetic variations, evolution and adaptation of enteroviruses in Kuwait.

Codon Usage Patterns inCorynebacterium glutamicum: Mutational Bias, Natural Selection and Amino Acid Conservation

The alternative synonymous codons inCorynebacterium glutamicum, a well-known bacterium used in industry for the production of amino acid, have been investigated by multivariate analysis. AsC. glutamicumis a GC-rich organism, G and C are expected to predominate at the third position of codons. Indeed, overall codon usage analyses have indicated that C and/or G ending codons are predominant in this organism. Through multivariate statistical analysis, apart from mutational selection, we identified three other trends of codon usage variation among the genes. Firstly, the majority of highly expressed genes are scattered towards the positive end of the first axis, whereas the majority of lowly expressed genes are clustered towards the other end of the first axis. Furthermore, the distinct difference in the two sets of genes was that the C ending codons are predominate in putatively highly expressed genes, suggesting that the C ending codons are translationally optimal in this organism. Secondly, the majority of the putatively highly expressed genes have a tendency to locate on the leading strand, which indicates that replicational and transciptional selection might be invoked. Thirdly, highly expressed genes are more conserved than lowly expressed genes by synonymous and nonsynonymous substitutions among orthologous genes fromthe genomes ofC. glutamicumandC. diphtheriae. We also analyzed other factors such as the length of genes and hydrophobicity that might influence codon usage and found their contributions to be weak.

Inferring the Evolutionary History of Rice Yellow Mottle Virus from Genomic, Phylogenetic, and Phylogeographic Studies

ABSTRACT Fourteen isolates of Rice yellow mottle virus (RYMV) were selected as representative of the genetic variability of the virus in Africa from a total set of 320 isolates serologically typed or partially sequenced. The 14 isolates were fully sequenced and analyzed together with two previously reported sequences. RYMV had a genomic organization similar to that of Cocksfoot mottle sobemovirus. The average nucleotide diversity among the 16 isolates of RYMV was 7%, and the maximum diversity between any two isolates was 10%. A strong conservative selection was apparent on both synonymous and nonsynonymous substitutions, through the amino acid replacement pattern, on the genome size, and through the limited number of indel events. Furthermore, there was a lack of positive selection on single amino acid sites and no evidence of recombination events. RYMV diversity had a pronounced and characteristic geographic structure. The branching order of the clades correlated with the geographic origin of the isolates along an east-to-west transect across Africa, and there was a marked decrease in nucleotide diversity moving westward across the continent. The insertion-deletion polymorphism was related to virus phylogeny. There was a partial phylogenetic incongruence between the coat protein gene and the rest of the genome. Overall, our results support the hypothesis that RYMV originated in East Africa and then dispersed and differentiated gradually from the east to the west of the continent.

Molecular Evolution of the dotA Gene in Legionella pneumophila

ABSTRACT The molecular evolution of dotA, which is related to the virulence of Legionella pneumophila, was investigated by comparing the sequences of 15 reference strains (serogroups 1 to 15). It was found that dotA has a complex mosaic structure. The whole dotA gene of Legionella pneumophila subsp. pneumophila serogroups 2, 6, and 12 has been transferred from Legionella pneumophila subsp. fraseri. A discrepancy was found between the trees inferred from the nucleotide and deduced amino acid sequences of dotA, which suggests that multiple hits, resulting in synonymous substitutions, have occurred. Gene phylogenies inferred from three different segments (the 5′-end region, the central, large periplasmic domain, and the 3′-end region) showed impressively dissimilar topologies. This was concordant with the sequence polymorphisms, indicating that each region has experienced an independent evolutionary history, and was evident even within the same domain of each strain. For example, the PP2 domain was found to have a heterogeneous structure, which led us hypothesize that the dotA gene of L. pneumophila may have originated from two or more different sources. Comparisons of synonymous and nonsynonymous substitutions demonstrated that the PP2 domain has been under strong selective pressure with respect to amino acid change. Split decomposition analysis also supported the intragenic recombination of dotA. Multiple recombinational exchange within the dotA gene, encoding an integral cytoplasmic membrane protein that is secreted, probably provided increased fitness in certain environmental niches, such as within a particular biofilm community or species of amoebae.

Evaluation of Accumulation of Hepatitis C Virus Mutations in a Chronically Infected Chimpanzee: Comparison of the Core, E1, HVR1, and NS5b Regions

ABSTRACT Four hepatitis C virus genome regions (the core, E1, HVR1, and NS5b) were amplified and sequenced from yearly samples obtained from a chronically infected chimpanzee over a 12-year span. Nucleotide substitutions were found to accumulate in the core, E1, and HVR1 regions during the course of chronic infection; substitutions within the NS5b region were not detected for the first 8 years and were found to be minimal during the last 4 years. The rate of accumulation of mutations in the core and E1 regions, based on a direct comparison between the first 1979 sequence and the last 1990 sequence, was 1.120 × 10−3, while phylogenetic ancestral comparison using the 12 yearly sequences showed a rate of 0.816 × 10−3bases per site per year. Temporal evaluation of the sequences revealed that there appeared to be periods in which substitutions accumulated and became fixed, followed by periods with relative stasis or random substitutions that did not persist. Synonymous and nonsynonymous substitutions within the core, E1, and HVR1 regions were also analyzed. In the core and E1 regions, synonymous substitutions predominated and gradually increased over time. However, within the HVR1 region, nonsynonymous substitutions predominated but gradually decreased over time.

Allelic Variation within Helicobacter pylori babA and babB

ABSTRACT Helicobacter pylori strains show both geographic and disease-associated allelic variation. We investigated the diversity present in two genes, babA and babB, which are members of a paralogous family of outer membrane proteins. Eleven family members within a single H. pylori strain, predicted to encode proteins with substantial N- and C-terminal similarity to each other, were classified as babA paralogues. In their central regions, most are less than 54% related to one another. Examining the babA and babB central regions in 42 H. pylori strains from different geographic locales, we identified five different allele groups of babA (AD1 to AD5) and three different allele groups of babB (BD1 to BD3). Phylogenetic analysis revealed that the allelic groupings ofbabA and babB are independent of one another and that, for both, geographic variation is present. Analysis of synonymous and nonsynonymous substitutions in these regions showed thatbabA is more diverse, implying an earlier origin than that of the same region of babB, but that the babAdiversity region may have more functional constraints. Although recombination has been central to the evolution of both genes, withbabA and babB showing low mean compatibility scores and homoplasy ratios of 0.71 and 0.67, respectively, recombination is not sufficient to obscure evidence of clonal descent. Despite the involvement of babA in binding to the host blood group antigen Lewis B, neither the presence of differentbabA allele groups nor that of different babBallele groups is a determining factor in Lewis B binding of H. pylori strains.

Reevaluation of Amino Acid Variability of the Human Immunodeficiency Virus Type 1 gp120 Envelope Glycoprotein and Prediction of New Discontinuous Epitopes

ABSTRACT To elucidate the evolutionary mechanisms of the human immunodeficiency virus type 1 gp120 envelope glycoprotein at the single-site level, the degree of amino acid variation and the numbers of synonymous and nonsynonymous substitutions were examined in 186 nucleotide sequences for gp120 (subtype B). Analyses of amino acid variabilities showed that the level of variability was very different from site to site in both conserved (C1 to C5) and variable (V1 to V5) regions previously assigned. To examine the relative importance of positive and negative selection for each amino acid position, the numbers of synonymous and nonsynonymous substitutions that occurred at each codon position were estimated by taking phylogenetic relationships into account. Among the 414 codon positions examined, we identified 33 positions where nonsynonymous substitutions were significantly predominant. These positions where positive selection may be operating, which we call putative positive selection (PS) sites, were found not only in the variable loops but also in the conserved regions (C1 to C4). In particular, we found seven PS sites at the surface positions of the α-helix (positions 335 to 347 in the C3 region) in the opposite face for CD4 binding. Furthermore, two PS sites in the C2 region and four PS sites in the C4 region were detected in the same face of the protein. The PS sites found in the C2, C3, and C4 regions were separated in the amino acid sequence but close together in the three-dimensional structure. This observation suggests the existence of discontinuous epitopes in the protein's surface including this α-helix, although the antigenicity of this area has not been reported yet.

The Correlation Between Synonymous and Nonsynonymous Substitutions in Drosophila: Mutation, Selection or Relaxed Constraints?

Codon usage bias, the preferential use of particular codons within each codon family, is characteristic of synonymous base composition in many species, including Drosophila, yeast, and many bacteria. Preferential usage of particular codons in these species is maintained by natural selection acting largely at the level of translation. In Drosophila, as in bacteria, the rate of synonymous substitution per site is negatively correlated with the degree of codon usage bias, indicating stronger selection on codon usage in genes with high codon bias than in genes with low codon bias. Surprisingly, in these organisms, as well as in mammals, the rate of synonymous substitution is also positively correlated with the rate of nonsynonymous substitution. To investigate this correlation, we carried out a phylogenetic analysis of substitutions in 22 genes between two species of Drosophila, Drosophila pseudoobscura and D. subobscura, in codons that differ by one replacement and one synonymous change. We provide evidence for a relative excess of double substitutions in the same species lineage that cannot be explained by the simultaneous mutation of two adjacent bases. The synonymous changes in these codons also cannot be explained by a shift to a more preferred codon following a replacement substitution. We, therefore, interpret the excess of double codon substitutions within a lineage as being the result of relaxed constraints on both kinds of substitutions in particular codons.