A novel interpretation of structural dot plots of genomes derived from the analysis of two strains of Neisseria meningitidis.

Neisseria meningitidis is the agent of invasive meningococcal disease, including cerebral meningitis and septicemia. Because the diseases caused by different clonal groups (sequence types) have their own epidemiological characteristics, it is important to understand the differences among the genomes of the N. meningitidis clonal groups. To this end, a novel interpretation of a structural dot plot of genomes was devised and applied; exact nucleotide matches between the genomes of N. meningitidis serogroup A strain Z2491 and serogroup B strain MC58 were identified, leading to the specification of various structural regions. Known and putative virulence genes for each N. meningitidis strain were then classified into these regions. We found that virulence genes of MC58 tend more to the translocated regions (chromosomal segments in new sequence contexts) than do those of Z2491, notably tending towards the interface between one of the translocated regions and the collinear region. Within the col-linear region, virulence genes tend to occur within 16 kb of gaps in the exact matches. Verification of these tendencies using genes clustered in the cps locus was sufficiently supportive to suggest that these tendencies can be used to focus the search for and understanding of virulence genes and mechanisms of pathogenicity in these two organisms.

Role of positive selection pressure on the evolution of H5N1 hemagglutinin.

The surface glycoprotein hemagglutinin (HA) helps the influenza A virus to evade the host immune system by antigenic variation and is a major driving force for viral evolution. In this study, the selection pressure on HA of H5N1 influenza A virus was analyzed using bioinformatics algorithms. Most of the identified positive selection (PS) sites were found to be within or adjacent to epitope sites. Some of the identified PS sites are consistent with previous experimental studies, providing further support to the biological significance of our findings. The highest frequency of PS sites was observed in recent strains isolated during 2005-2007. Phylogenetic analysis was also conducted on HA sequences from various hosts. Viral drift is almost similar in both avian and human species with a progressive trend over the years. Our study reports new mutations in functional regions of HA that might provide markers for vaccine design or can be used to predict isolates of pandemic potential.

Systematic analysis of host immunological pressure on the envelope gene of human immunodeficiency virus type 1 by an immunobioinformatics approach.

As the number of HIV-1 sequences has increased in the public database and new tools of immunological bioinformatics have become available, making it possible to better understand at a population level how host immune response drives the evolution of HIV-1 envelope (Env). We analyzed 1100 unique full-length envelope sequences and systematically determined positive selection (PS) sites by QUASI analysis and found that PS sites were widely dispersed across Env. The frequency of Env PS sites appears to be relatively stable over time. Moreover, between 25% and 61% of PS sites are shared between subtypes A, B, C, and D, suggesting that host immune responses target the same regions of Env gene across different clades at the population level. Significant correlations were observed between PS sites and Neutralizing antibody (NAb) response, as well as PS sites and Th epitopes. Furthermore, NAb sites in combination with cytotoxic-T lymphocyte (CTL) epitopes and proteasome cleavage sites were also significantly associated with PS sites, suggesting NAb may be the major force driving the evolution of HIV-1 Env. We also identified regions that are free from PS, but heavily targeted by CTL or NAb, implying that functional constraints may be responsible for the lack of positive selection in these regions. These findings should help researchers to identify epitopes or regions of HIV-1 that may aid in designing vaccines.

Serological diagnosis of syphilis: enzyme-linked immunosorbent assay to measure antibodies to individual recombinant Treponema pallidum antigens.

We standardized an indirect ELISA for measurement of serum antibody levels to four individual treponemal recombinant proteins that have been commonly used in a number of commercial EIAs, mostly as a mixture of antigens. When tested with 127 syphilis-negative and 37 secondary syphilis sera, ELISA O.D.s obtained for each of the four antigens clearly distinguished between these two groups of samples. Sensitivity and specificity of 100% was obtained with the current set of samples. Further evaluations with sera from different stages of syphilis can help to define the applications of this ELISA test for each of the four antigens studied.

Phylogenetic relationships of Campylobacter jejuni based on porA sequences.

Campylobacter porins are the dominant major outer membrane protein (MOMP) of these bacteria. They are composed of hypervariable, surface-exposed, peptide loops and membrane-embedded, conserved peptide regions. Porins are functionally important and may also be useful for molecular subtyping methods but have not yet been well characterized. We therefore sequenced the porA gene from 39 Campylobacter isolates, including multilocus sequence type (MLST) reference strains, isolates from patients with the Guillain-Barré syndrome, other clinical isolates, and serotyping reference strains. These were compared with additional sequences available from GenBank. Three distinct porA lineages were observed after phylogenetic analysis. Both Campylobacter coli and Campylobacter jejuni were found with group 3 porA sequences, and this was the only group showing any evidence of recombination among porA genes. There was no recombination between porA genes from C. jejuni groups 1 and 2, suggesting there may be functional constraints on changes at this locus. Most of the amino acid differences among the three groups were present in surface-exposed loops, and dissimilar substitutions were found when groups 1 and 2 MOMP were compared. Different MOMP sequence groups may have different biological or antigenic properties, which in turn may be associated with survival in different environments, host adaptation, or virulence.

Use of the oxford multilocus sequence typing protocol and sequencing of the flagellin short variable region to characterize isolates from a large outbreak of waterborne Campylobacter sp. strains in Walkerton, Ontario, Canada.

The Walkerton (Ontario, Canada) outbreak of waterborne Escherichia coli O157:H7 and Campylobacter jejuni was quite limited in both space and time, making it a good model for exploring the utility of different typing and subtyping methods for the characterization of relationships among isolates of these organisms. We have extended previous work with these organisms through analysis by the Oxford multilocus sequence typing (MLST) and the flagellin short variable region (fla-SVR) sequencing methods. Additional isolates not epidemiologically related to the Walkerton outbreak have also been included. Both sequencing methods identified and differentiated between Walkerton outbreak strains 1 and 2. When these strains were compared with isolates that were not part of the outbreak, the information produced by the fla-SVR method more often correlated with epidemiological findings than that produced by MLST, though both methods were required for optimal discrimination. The MLST data were more relevant in terms of the overall population structure of the organisms. Both mutation and recombination appeared to be responsible for generating diversity among the isolates tested.