Eight serotypes of human astroviruses (HAstV-1 to HAstV-8) have been described. To date, the entire genomes of HAstV-1 and HAstV-2 as well as the ORF2 sequences of HAstV-1-6 and 8 have been reported. In this study, the ORF2 sequences of seventeen strains of HAstVs originating from different countries were determined, as well as the sequence ORF2 of one porcine astrovirus (PAstV) strain. Afterwards, comparison of the capsid protein precursors encoded by ORF2 of 46 strains of HAstVs, PAstV, and feline astrovirus (FAstV) was carried out. A phylogenetic tree showed eight genogroups of HAstVs that corresponded exactly to the serotypes. HAstV-3 and 7 were the most closely related, whereas HAstVs, FAstV, and PAstV segregated from each other. Compared to a PAstV, a FAstV is closer to HAstVs. Furthermore, the capsid protein precursors were divided into four regions (after amino acid residues 424, 688, and 776, respectively) based on sequence identity. Region I was the most conserved, and FAstV was very close in identity to HAstVs. Two amino acid motifs in region I were predicted to contain the common antigenic epitopes. Region II was relatively variable. Deletions and insertions were characteristic of region III, and region IV was relatively conserved. To our knowledge, this is the first comparative sequence analysis of the capsid protein precursors of eight serotypes of HAstVs as well as two animal astroviruses (FAstV and PAstV).

In recent years, various polymorphic loci and multicopy insertion elements have been discovered in the Mycobacterium tuberculosis genome, such as the direct repeat (DR) locus, the major polymorphic tandem repeats, the polymorphic GC-rich repetitive sequence, IS6110, and IS1081. These, especially IS6110 and the DR locus, have been widely used as genetic markers to differentiate M. tuberculosis isolates and will continue to be so used, due to the conserved nature of the genome ofM. tuberculosis. However, little is known about the processes involved in generating these or of their relative rates of change. Without an understanding of the biological characteristics of these genetic markers, it is difficult to use them to their full extent for understanding the population genetics and epidemiology of M. tuberculosis. To address these points, we identified a cluster of 7 isolates in a collection of 101 clinical isolates and investigated them with various polymorphic genetic markers, which indicated that they were highly related to each other. This cluster provided a model system for the study of IS6110 transposition, evolution at the DR locus, and the effects of these on the determination of evolutionary relationships among M. tuberculosis strains. Our results suggest that IS6110 restriction fragment length polymorphism patterns are useful in grouping closely related isolates together; however, they can be misleading if used for making inferences about the evolutionary relationships between closely related isolates. DNA sequence analysis of the DR loci of these isolates revealed an evolutionary scenario, which, complemented with the information from IS6110, allowed a reconstruction of the evolutionary steps and relationships among these closely related isolates. Loss of the IS6110 copy in the DR locus was noted, and the mechanisms of this loss are discussed.

The present study investigated the prevalence and diagnostic potential of the most commonly reported mutations associated with isoniazid resistance, katG 315Thr, katG 315Asn, inhA ؊15T, inhA ؊8A, and the oxyR-ahpC intergenic region, in a population sample of 202 isoniazid-resistant Mycobacterium tuberculosis isolates and 176 randomly selected fully sensitive isolates from England and Wales identified by using a directed oligonucleotide array and limited DNA sequencing. The strains were recovered from patients originating from 29 countries; 41 isolates were multidrug resistant. Mutations affecting katG 315, the inhA promoter, and the oxyR-ahpC intergenic region were found in 62.7, 21.9, and 30% of 169 genotypically distinct isoniazid-resistant isolates, respectively, whereas they were found in 0, 0, and 8% of susceptible strains, respectively. The frequency of mutation at each locus was unrelated to the resistance profile or previous antituberculous drug therapy. The commonest mutation in the oxyR-ahpC intergenic region, ahpC ؊46A, was present in 23.7% of isoniazid-resistant isolates and 7.5% of susceptible isolates. This proved to be a phylogenetic marker for a subgroup of M. tuberculosis strains originating on the Indian subcontinent, which shared IS6110-based restriction fragment length polymorphism and spoligotype features with the Delhi strain and Central Asian strain CAS1; and this marker is strongly associated with isoniazid resistance and the katG 315Thr mutation. In total, 82.8% of unrelated isoniazid-resistant isolates could be identified by analysis of just two loci: katG 315 and the inhA promoter. Analysis of the oxyR-ahpC intergenic region, although phylogenetically interesting, does not contribute significantly to further identification of isoniazid-resistant isolates.Tuberculosis (TB) is one of the leading causes of death due to an infectious agent throughout the world, and the increasing rates of drug-resistant TB are of global concern. In England and Wales the incidence of TB has risen from 11.3/100,000 population to 12.7/100,000 population over the last decade (36), although the incidence of drug resistance has remained stable, with isoniazid and rifampin resistance occurring in 6 to 7% and 1% of isolates from new patients, respectively. International and regional studies report an incidence of drug resistance in new cases of TB to any first-line antituberculous drug ranging from 2 to 37% (median, 10.7%) (13), with resistance to isoniazid being the commonest form. Resistance is considerably higher among patients previously treated for TB (median, 23.3%). The present quadruple anti-TB therapy regimens are designed to treat isoniazid-resistant isolates while clinicians await the results of drug susceptibility testing; however, administration of isoniazid is not without risk of side effects. Phenotypic drug susceptibility testing takes between 4 and 6 weeks from the time of receipt of clinical samples. Genotypic analysis, in contrast, can be performed in a matter of hours. Commercial and in-house s...

SUMMARYExtensive DNA sequence diversity was noted in Helicobacter pylori flagellin genes flaA and flaB. PCR amplified sequences from 49 isolates were digested with Alul, Hindlll, Mbol or MspI, the resultant patterns were compared between the different isolates and these used to differentiate the isolates from each other. Evidence that the extensive diversity that was found in these genes is the result of reassortment of sequences between strains in the bacterial population is presented, such that a comparatively small number of individual sequence mutations can recombine together in random combinations to form a greater number of distinct alleles. Geographical differences in the predominant patterns in theftaA alleles were also observed and could reflect regional differences either in the human host population or in the bacterial population. In view of the genetic complexity of this species, molecular typing schemes designed to identify related strains may falsely associate strains if the methods do not characterize sufficient genetic sites to exclude chance associations of genetic markers in strains which are actually not closely related to each other.

A 267-nucleotide Mycobacterium tuberculosis genomic sequence (ipl, the IS6110 preferential locus) which can harbor the insertion sequence IS6110 at six alternative locations has been identified in some three-quarters of the isolates tested. Only one IS6110 copy was observed at this locus in the ipl::IS6110 ؉-containing isolates tested, and all insertions had the same orientation. The implications of this finding for IS6110 fingerprint typing methods is discussed in this work.