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PubMed1998

Extensive genetic diversity among clinical isolates of Streptococcus pyogenes serotype M5.

The genetic diversity of clinical isolates of Streptococcus pyogenes serotype M5 has been characterized. Strain genotypes were defined by macrorestriction profile, 16S ribotype, emm gene subtype, insertion element IS1239 profile, and exotoxin gene determinant. By these criteria, clinical isolates of M5 constituted a multiplicity of strain clusters rather than a homogeneous population as found for certain serotypes. Distance matrices and an unrooted tree were constructed from macrorestriction data with three rarely cutting endonucleases, determined by PFGE. A single IS1239 profile was common to 85% of isolates but there was great diversity of both ribotype and macrorestriction profile, and 18 different emm gene subtypes were detected by PCR-RFLP. DNA sequence analysis of the antigen-coding 5' (hypervariable) region of emm gene amplicons (about 240 bp) showed that 14/18 exhibited up to 6% divergence. Four amplicons had highly divergent sequences--corresponding to those previously determined for emm6, emm11, emm18 and emm77. Further serological and hybridization studies were used to analyse the discrepancy between the Lancefield serotype of these strains (M5) and their emm genotype. Overall, this study shows a high degree of genetic diversity in serotype M5, with implications for the Lancefield scheme itself, for the epidemiology of group A streptococci, and for recombinant DNA strategies for M protein-based vaccine development.

Amino Acid Sequence↗
PubMed2000

Monitoring genome evolution ex vivo: reversible chromosomal integration of a 106 kb plasmid at two tRNA(Lys) gene loci in sequential Pseudomonas aeruginosa airway isolates.

The genome rearrangements in sequential Pseudomonas aeruginosa clone K isolates from the airways of a patient with cystic fibrosis were determined by an integrated approach of mapping, sequencing and bioinformatics. Restriction mapping uncovered an 8.9 kb deletion of PAO sequence between phnAB and oprL in clone K, and two 106 kb insertions either adjacent to this deletion or several hundred kilobases away, close to the pilA locus. These 106 kb blocks of extra DNA also co-existed as the circular plasmid pKLK106 in several clone K isolates and were found to be closely related to plasmid pKLC102 in P. aeruginosa clone C isolates. The breakpoints of the deletion in clone K and the attB-attP sequences for the reversible integration of the plasmid in clones C and K were located within the 3' end of the lysine tRNA structural genes (att site). pKLK106 sequentially recombined with either of the two tRNA(Lys) genes in clone K isolates. The att site of the pilA hypervariable region has been utilized by clone C to target its plasmid pKLC102 into the chromosome; the att site of the phnAB-oprL region has been employed by strain PAO to incorporate a DNA block encoding pyocin, transposases and IS elements. The use of typical phage attachment sites by conjugative genetic elements could be one of the major mechanisms used by P. aeruginosa to generate the mosaic genome structure of blocks of species-, clone- and strain-specific DNA. The example described here demonstrates the potential impact of systematic genome analysis of sequential isolates from the same habitat on our understanding of the evolution of microbial genomes.

Base Sequence↗
PubMed1998

Secondary structure and molecular analysis of interstrain variability in the P5 outer-membrane protein of non-typable Haemophilus influenzae isolated from diverse anatomical sites.

The sequence of the non-typable Haemophilus influenzae (NTHi) P5 outer-membrane protein from a range of clinical isolates is presented and represents the first analysis of the heterogeneity in P5 from NTHi isolates from diverse anatomical sites. The basis of the previously observed inter-strain variation in the electrophoretic mobility is attributed to heterogeneity in three hypervariable regions. Alignment of the P5 sequences identified regions which are highly conserved and align with the transmembrane region predicted for the homologous Escherichia coli protein, OmpA. Variable regions correspond to surface-exposed loops, of which the first loop falls into subclasses. However, these subclasses fail to correlate with anatomical predisposition. Although P5 has been proposed as a fimbrial protein composed of coiled coils, both structural analysis by circular dichroism of purified P5 and computer analysis of the multiply aligned sequences predict a high proportion of beta strand with no evidence of coiled coil structure. A detailed model of P5 is presented.

Amino Acid Sequence↗
PubMed2004

Reclassification of Brevibacillus brevis strains NCIMB 13288 and DSM 6472 (=NRRL NRS-887) as Aneurinibacillus danicus sp. nov. and Brevibacillus limnophilus sp. nov.

Comparison of the hypervariable region (269-279 bases in length) at the 5' end of the 16S rDNA sequences of 29 bacterial strains that were identified previously as Brevibacillus brevis showed that 13 strains clustered with Aneurinibacillus species, eight strains clustered with Bacillus species and eight strains clustered with Brevibacillus species. Based on DNA-DNA hybridization results, 27 strains, not including [Brevibacillus brevis] NCIMB 13288 and [Brevibacillus brevis] DSM 6472, were reidentified as Aneurinibacillus migulanus, Aneurinibacillus thermoaerophilus, Bacillus methanolicus, Bacillus oleronius, Brevibacillus agri, Brevibacillus brevis and Brevibacillus parabrevis. [Brevibacillus brevis] NCIMB 13288, which was located in the Aneurinibacillus cluster, showed low DNA-DNA relatedness (<14 %) and low 16S rDNA sequence similarity (96.8-97.9 %) to other Aneurinibacillus species. [Brevibacillus brevis] DSM 6472, which was located in the Brevibacillus cluster, also showed low DNA-DNA relatedness (<12 %) and low 16S rDNA sequence similarity (95.4-98.8 %) to other Brevibacillus species. These genotypic and phylogenetic data, plus phenotypic and chemotaxonomic characteristics, suggest that [Brevibacillus brevis] NCIMB 13288 (=IAM 15048) and [Brevibacillus brevis] DSM 6472 (=NRRL NRS-887) represent novel species of the genera Aneurinibacillus and Brevibacillus, respectively, for which the names Aneurinibacillus danicus sp. nov. and Brevibacillus limnophilus sp. nov. are proposed.

Bacillaceae↗
PubMed2003

Intra-chromosomal heterogeneity between the four 16S rRNA gene copies in the genus Veillonella: implications for phylogeny and taxonomy.

Among the seven species characterized within the genus Veillonella, three (Veillonella dispar, Veillonella parvula and Veillonella atypica) have so far been isolated from human flora and during infectious processes. Sequencing and analysis of 16S rDNA (rrs) has been described as the best method for identification of Veillonella strains at the species level since phenotypic characteristics are unable to differentiate between species. rrs sequencing for the three species isolated from humans showed more than 98 % identity between them. Four rrs copies were found in the reference strains and in all the clinical isolates studied. The sequences of each rrs were determined for the clinical strain ADV 360.1, and they showed a relatively high level of heterogeneity (1.43 %). In the majority of cases, polymorphic positions corresponded to nucleotides allowing differentiation between the three species isolated from humans. Moreover, variability observed between rrs copies was higher than that between 16S rDNA sequences of V. parvula and V. dispar. Phylogenetic analysis showed that polymorphism between rrs copies affected the position of strain ADV 360.1 in the tree. Variable positions occurred in stems and loops belonging to variable and hypervariable regions of the 16S rRNA secondary structure but did not change the overall structure of the 16S rRNA. PCR-RFLP experiments performed on 27 clinical isolates of Veillonella sp. suggested that inter-rrs heterogeneity occurs widely among the members of the genus VEILLONELLA: These results, together with the lack of phenotypic criteria for species differentiation, give preliminary arguments for unification of V. dispar and V. parvula.

Base Sequence↗
PubMed2003

Network analysis of human and simian immunodeficiency virus sequence sets reveals massive recombination resulting in shorter pathways.

The intrinsic recombination rate of human immunodeficiency virus (HIV) exceeds the point mutation rate by a factor of 10. As the majority of infected cells in vivo harbour multiple proviruses, the stage is set for rampant recombination. Therefore, it may be presumed that phylogenic relationships and mutation frequencies will probably be affected by recombination. However, the proportion of homoplasies arising from recombination and mutation is not known. By studying the evolution of the hypervariable regions of the simian immunodeficiency virus envelope gene among four macaques, it is shown that homoplasies arise more from recombination than from point mutation. When recombination is accounted for, the minimum number of substitutions in a sequence set may be reduced by as much as 45 %. In fact, the true number of point mutations in a set of HIV sequences tends to the number of discrete substitutions. Hence, lineages are younger than anticipated previously, although not in proportion to the ratio of the intrinsic recombination/point mutation rate. Recombination also inflates codon polymorphisms.

Animals↗
PubMed2004

Evolutionary study of HVR1 of E2 in chronic hepatitis C virus infection.

Hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) genome was directly sequenced from 12 chronically infected patients who had not responded to interferon (IFN) treatment. Due to the quasispecies nature of HCV circulating genomes, serum samples from four patients showing different evolutionary characteristics were further analysed. Serial samples from each patient were taken before, soon after and 14-23 months after a 6 month IFN treatment. HVR1 from each sample was amplified, cloned and the clones sequenced. For each patient, a phylogenetic analysis of the clones was performed and quasispecies complexity and genetic distances were calculated. The amino acid sequences and predicted antigenic profiles were analysed. The pre-treatment samples of the different patients presented dissimilar genetic quasispecies composition. For three of the patients, we showed that, regardless of the complexity or diversity of the viral populations before treatment, they evolved towards genetic diversification following selective pressure. Once the environment became stable, the entire population tended towards homogeneity. The fourth patient represented a case where different components of the quasispecies coexisted for long periods without replacement. We propose herein that the evolution of HVR1 of E2 is more likely to be directed by selection of clearly different subpopulations (modification of quasispecies equilibrium) than by a continuous mechanism related to the successive accumulation of point mutations. The prevalence of a quasispecies shift mechanism was revealed by the cloning analysis during the follow-up period of the evolutionary process.

Amino Acid Sequence↗
PubMed2004

Evolution of hepatitis C virus variants following blood transfusion from one infected donor to several recipients: a long-term follow-up.

Variants of hepatitis C virus (HCV) from a single infected blood donor and 13 viraemic recipients who were traced were examined by sequencing and cloning to determine the extent of virus diversity in hypervariable region 1. Serum-derived viral isolates were studied from the donor when his HCV infection was discovered in 1993, in his recipients that year (0.3-5 years post-transfusion) and 5 years later in the donor and six viraemic recipients who were still alive. Viral variants of broad diversity were readily demonstrated in the baseline samples of the donor (nucleotide p-distance 0.130), but significantly less (P<0.00003) diversity was observed in the recipients' first samples (p-distances within recipients 0.003-0.062). In the first blood samples of the recipients, many of the viral variants identified were closely related to a strain variant from the donor. In follow-up samples drawn 5 years later from the donor and six recipients, the p-distance among donor clones had increased (0.172, P<0.0005) compared with the recipients, who displayed significantly narrower quasispecies (0.011-0.086). A common finding was that recipients of blood components processed from the same donation differed substantially in persisting HCV infectious sequence. Markedly few changes leading to changes of amino acids had occurred during follow-up in four of six recipients. These results question the significance of the development of viral variants as a necessary phenomenon in the evolution of HCV and pathogenesis of the disease.

Amino Acid Substitution↗
PubMed2004

Hepatitis C virus population analysis of a single-source nosocomial outbreak reveals an inverse correlation between viral load and quasispecies complexity.

The features of Hepatitis C virus (HCV) quasispecies within an envelope segment including the hypervariable region 1 were analysed at an early time point post-infection in seven patients that acquired HCV from a single common donor during a nosocomial outbreak. The grouping of patients according to viral load was reflected in the structure of the quasispecies. A higher viral load correlated with the presence of a predominant HCV genome and a corresponding lower quasispecies complexity. The quasispecies complexity itself was not correlated with HCV clearance or persistence. Thus, the relationship between an intrapatient HCV quasispecies and the clinical outcome of an HCV infection is more complex than previously anticipated.

Amino Acid Sequence↗
PubMed2005

Intra-host evolutionary dynamics of hepatitis C virus E2 in treated patients.

Hepatitis C virus (HCV) displays high genetic diversity. Inter-host sequence variability may mainly reflect a neutral drift evolution. In contrast, intra-host evolution may be driven by an adaptive selection to host responses to infection. Here, HCV E2 intra-host evolution in two patients during the course and follow-up of successive treatments with IFN-alpha and IFN-alpha/ribavirin was investigated. Phylogenetic analyses suggested that adaptive pressures prompt a continuous selection of viral variants derived from the previous ones (intra-lineage evolution) and/or a swapping of viral lineages during the course of the infection (inter-lineage evolution). Selection would act not only on the phenotypic features of hypervariable region 1 (HVR1) but also on those of the flanking regions. The pressures operate mainly at the amino acid level, but they also appeared to act on nucleotide sequences. Moreover, HVR1 heterogeneity seemed to be strongly constrained. This work contributes to the knowledge of HCV intra-host evolution during chronicity.

Antiviral Agents↗
PubMed2006

Selective transmission of hepatitis C virus genotypes and quasispecies in humans and experimentally infected chimpanzees.

This study determined whether selective transmission of hepatitis C virus (HCV) species occurred among human and chimpanzee recipients of contaminated blood products or plasma containing multiple genotypes, subgenotypes and quasispecies. Commercially prepared factor VIII concentrate (lot DO56), produced prior to HCV testing and inactivation, was subsequently found by direct cloning to contain the following subgenotypes: 1a and 1b (73 % of clones), 2a (13 % of clones), 2b (11 % of clones) and 3a (4 % of clones). A patient transfused with factor VIII concentrate DO56 was diagnosed with clinical non-A, non-B hepatitis and subsequently found to be infected with HCV subgenotype 1b. Among five chimpanzees inoculated experimentally with the same factor VIII concentrate, two were infected only with HCV subgenotype 1a and three were infected with approximately equivalent clonal proportions of subgenotypes 1a and 1b. HCV hypervariable region 1 (HVR1) quasispecies analysis of the DO56 factor VIII concentrate and a serum specimen from the single chimpanzee that developed a chronic HCV infection following inoculation with DO56 showed 0-56 % nucleotide variation. However, specimens from chimpanzees infected in the second to fourth passages of the DO56 inoculum had 0-8 % HVR1 quasispecies nucleotide variation. The high HVR1 quasispecies variation in the factor VIII concentrate and its first passage in chimpanzees indicates the presence of multiple HCV isolates, whereas the low variation in the second to fourth chimpanzee passages suggests transmission of a single HCV isolate. These findings strongly suggest selective transmission of HCV isolates during experimental chimpanzee infection and among humans exposed to multiple HCV species.

Animals↗
PubMed2006

Broad cellular immunity with robust memory responses to simian immunodeficiency virus following serial vaccination with adenovirus 5- and 35-based vectors.

Adenovirus serotype 35 (Ad35) is a promising vaccine platform for human immunodeficiency virus (HIV) infection and emerging infectious diseases as it is uncommon in humans worldwide and is distinct from Ad5, the major vaccine serotype for which many individuals have pre-existing immunity. The immunogenicity of a first-generation, replication-competent Ad35-based vaccine was tested in the simian immunodeficiency virus (SIV) rhesus macaque model by evaluating its capacity to boost immunity generated by Ad5-based vectors. A series of four immunizations with replication-defective Ad5 vectors expressing SIVmac239 gag induced high-frequency responses mediated by both CD8(+) and CD4(+) T cells directed against several epitopes. Ad5-specific neutralizing antibody responses that did not neutralize Ad35 were rapidly induced but waned over time. Subsequent immunization with Ad5-based vectors was minimally effective, whereas immunization with Ad35-based vectors generated a strong increase in the frequency of Gag-specific T cells with specificities that were unchanged. While this boosting response was relatively transient, challenge with the distinct pathogenic isolate SIV/DeltaB670 generated robust and selective recall responses to Gag with similar specificities as induced by vaccination that were elevated for 25 weeks relative to controls. Vaccination had measurable albeit minor effects on virus load. Unexpectedly, regional hypervariability within the Gag sequence of SIV/DeltaB670 was associated with mutation of the conserved CD8(+) T-cell epitope CM9 without concurrent flanking mutations and in the absence of immune pressure. These findings support the further development of Ad35 as a vaccine vector, and promote vaccine regimens that utilize serial administration of heterologous adenoviruses.

Adenoviruses, Human↗
PubMed2026

The genetic control of rapid genome content divergence in Arabidopsis thaliana.

Genome evolution in eukaryotes is predominantly driven by the dynamics of repetitive sequences, which vary widely in both copy number and sequence composition. Rates of repeat evolution differ between and within species and are likely modulated by both genetics and environment. To uncover factors shaping the rate of genome content evolution, we analyzed 1,142 resequenced Arabidopsis thaliana genomes using a novel K-mer based approach to characterize genome content variation and identify hypervariable regions underlying differences in repeat abundance. We next treated repeat abundance as a quantitative trait and performed genome-wide association analyses across more than 400 repeat families to identify the genetic basis of copy number variation. Integrating these results through a meta-GWAS approach revealed both cis-acting variants and more than 50 trans-acting loci that regulate repeat abundance genome-wide. Cis-acting variation was predominantly localized to pericentromeric and centromeric regions, whereas trans-acting loci were enriched for candidate genes involved in DNA replication, DNA repair, DNA methylation regulation. Finally, we found evidence that purifying selection acts against mutations that accelerate genome content divergence, favoring alleles that constrain repeat expansion. Together, these findings provide new insights into the genetic architecture and evolutionary forces shaping genome evolution in A. thaliana and establish a framework for investigating these processes in other plant species.

Journal Article↗
PubMed2025

Transition of Staphylococcus aureus tetracycline resistance plasmid pT181 from independent multicopy replicon to predominantly integrated chromosomal element over 65 years.

Mobile genetic elements (MGEs), including plasmids, phages and genome islands, are major sources of bacterial genetic diversity. The small plasmid pT181 confers tetracycline resistance in bacterial pathogen Staphylococcus aureus via an efflux pump, TetK. pT181 was one of the earliest sequenced S. aureus plasmids, and has been isolated in both clinical and livestock-associated strains for decades, both as an independent replicon and integrated in the chromosome as part of staphylococcal cassette chromosome mec (SCCmec). Bacterial genome analysis tools and high-quality sequences with metadata are publicly available, but these resources remain underleveraged for examining historical data, especially when studying the spread of MGEs across a species and over time. Using publicly available reads and metadata, we explored the evolution of pT181 over almost seven decades of samples to identify temporal trends in sequence evolution, copy number changes, and spread across S. aureus and beyond. pT181 was prevalent across S. aureus (found in 9.5% of 83,366 genomes tested), with a conserved sequence outside of three hypervariable regions. The history of pT181 since 1954 is characterized by spread across strains, significant variation in plasmid copy number of the independent replicon, and increasing frequency of integration of the plasmid into the S. aureus chromosome. We have identified multiple chromosomal integration locations of the plasmid, including outside of the previously characterized SCCmec. We find that pT181 has been transferred across staphylococcaceae and into a Gram-negative species. The repeated integration of pT181 into the chromosome may indicate co-evolution of the plasmid and the host, potentially to facilitate increased antibiotic resistance.

Journal Article↗
PubMed1994

Characterization of human AFLP systems apolipoprotein B, phenylalanine hydroxylase, and D1S80.

Methodology is presented for amplified fragment length polymorphism (AFLP) typing using a nonisotopic, PCR protocol. Human variable number tandem repeat (VNTR) loci used for identification in forensic and paternity testing were optimized for reaction and thermal-cycling parameters. Loci analyzed were the apolipoprotein B (APOB) 3' hypervariable region (HVR), phenylalanine hydroxylase 3' HVR (PAH), and D1S80. Coamplification of a monomorphic beta-globin fragment serves as an amplification control. Biotin is integrated into PCR amplicon through primer incorporation. AFLP products undergo agarose gel electrophoresis and Southern transfer to a nylon membrane. Amplicons were detected using a streptavidin-enzyme conjugate. Either colorimetric- or chemiluminescent-developed bands are genotyped using locus-specific allele ladders with known VNTR repeat numbers. Using this methodology, we have successfully typed > 500 individuals from three population groups for each locus during data basing and casework.

Alleles↗
PubMed2001

Genetic evidence on the origins of Indian caste populations.

The origins and affinities of the approximately 1 billion people living on the subcontinent of India have long been contested. This is owing, in part, to the many different waves of immigrants that have influenced the genetic structure of India. In the most recent of these waves, Indo-European-speaking people from West Eurasia entered India from the Northwest and diffused throughout the subcontinent. They purportedly admixed with or displaced indigenous Dravidic-speaking populations. Subsequently they may have established the Hindu caste system and placed themselves primarily in castes of higher rank. To explore the impact of West Eurasians on contemporary Indian caste populations, we compared mtDNA (400 bp of hypervariable region 1 and 14 restriction site polymorphisms) and Y-chromosome (20 biallelic polymorphisms and 5 short tandem repeats) variation in approximately 265 males from eight castes of different rank to approximately 750 Africans, Asians, Europeans, and other Indians. For maternally inherited mtDNA, each caste is most similar to Asians. However, 20%-30% of Indian mtDNA haplotypes belong to West Eurasian haplogroups, and the frequency of these haplotypes is proportional to caste rank, the highest frequency of West Eurasian haplotypes being found in the upper castes. In contrast, for paternally inherited Y-chromosome variation each caste is more similar to Europeans than to Asians. Moreover, the affinity to Europeans is proportionate to caste rank, the upper castes being most similar to Europeans, particularly East Europeans. These findings are consistent with greater West Eurasian male admixture with castes of higher rank. Nevertheless, the mitochondrial genome and the Y chromosome each represents only a single haploid locus and is more susceptible to large stochastic variation, bottlenecks, and selective sweeps. Thus, to increase the power of our analysis, we assayed 40 independent, biparentally inherited autosomal loci (1 LINE-1 and 39 Alu elements) in all of the caste and continental populations (approximately 600 individuals). Analysis of these data demonstrated that the upper castes have a higher affinity to Europeans than to Asians, and the upper castes are significantly more similar to Europeans than are the lower castes. Collectively, all five datasets show a trend toward upper castes being more similar to Europeans, whereas lower castes are more similar to Asians. We conclude that Indian castes are most likely to be of proto-Asian origin with West Eurasian admixture resulting in rank-related and sex-specific differences in the genetic affinities of castes to Asians and Europeans.

Adult↗
PubMed2003

Structure of a single-chain Fv fragment of an antibody that inhibits the HIV-1 and HIV-2 proteases.

The monoclonal antibody 1696, which was raised against the HIV-1 protease, inhibits the catalytic activity of the enzyme from both the HIV-1 and HIV-2 strains. The antibody cross-reacts with peptides containing the N-terminus of the enzyme, which is highly conserved between these strains. The crystal structure of a single-chain Fv fragment of 1696 (scFv-1696) in the non-complexed form, solved at 1.7 A resolution, is compared with the previously reported non-complexed Fab-1696 and antigen-bound scFv-1696 structures. Large conformational changes in the third hypervariable region of the heavy chain and differences in relative orientation of the variable domains are observed between the different structures.

Antibodies, Monoclonal↗
PubMed2005

Solution structure of the N-terminal A domain of the human voltage-gated Ca2+channel beta4a subunit.

Ca2+ channel beta subunits regulate trafficking and gating (opening and closing) of voltage-dependent Ca2+ channel alpha1 subunits. Based on primary sequence comparisons, they are thought to be modular structures composed of five domains (A-E) that are related to the large family of membrane associated guanylate-kinase (MAGUK) proteins. The crystal structures of the beta subunit core, B-D, domains have recently been reported; however, very little is known about the structures of the A and E domains. The N-terminal A domain is a hypervariable region that differs among the four subtypes of Ca2+ channel beta subunits (beta1-beta4). Furthermore, this domain undergoes alternative splicing to create multiple N-terminal structures within a given gene class that have distinct effects on gating. We have solved the solution structure of the A domain of the human beta4a subunit, a splice variant that we have shown previously to have alpha1 subunit subtype-specific effects on Ca2+ channel trafficking and gating.

Amino Acid Sequence↗