Longitudinal analysis of genotype distribution of influenza A virus from 2003 to 2005.

Influenza A viruses cause yearly epidemics, in part, due to their ability to overcome immunity from previous infections through acquisition of mutations. Amino acid sequences encoded by genes 4 (HA), 6 (NA), 7 (M), and 8 (NS) from 77 H3N2 influenza A isolates, collected between November 2003 and March 2005, were analyzed to determine the extent to which the viruses mutated within epidemic periods and between the epidemics. Nucleotide and amino acid sequences were stable throughout the epidemics but experienced substantial changes between epidemics. Major changes occurred in the HA gene in 5 to 7 amino acids and the NA gene in 11 to 13 amino acids and changes of 5 amino acids occurred in the M and NS genes. In the HA gene, changes occurred in sites known to be epitopes that determine the hemagglutination inhibition reactivity, and these were shown to be associated with a change of strain from A/Fujian/411/2002-like to A/California/7/2004-like viruses. Our findings indicate that genotype determination promises to be a rapid approach for detecting new strains of influenza A viruses in a population.

Identification of Campylobacter spp. and discrimination from Helicobacter and Arcobacter spp. by direct sequencing of PCR-amplified cpn60 sequences and comparison to cpnDB, a chaperonin reference sequence database.

A robust method for the identification of Campylobacter spp. based on direct sequencing of PCR-amplified partial cpn60 sequences and comparison of these to a reference database of cpn60 sequences is reported. A total of 53 Campylobacter isolates, representing 15 species, were identified and distinguished from phenotypically similar Helicobacter and Arcobacter strains. Pairwise cpn60 sequence identities between Campylobacter spp. ranged from 71 to 92 %, with most between 71 and 79 %, making discrimination of these species obvious. The method described overcomes limitations of existing PCR-based methods, which require time-consuming and complex post-amplification steps such as the cloning of amplification products. The results of this study demonstrate the potential for use of the reference chaperonin sequence database, cpnDB, as a tool for identification of bacterial isolates based on cpn60 sequences amplified with universal primers.

Coordinated response to SARS, Vancouver, Canada.

Two Canadian urban areas received travelers with severe acute respiratory syndrome (SARS) before the World Health Organization issued its alert. By July 2003, Vancouver had identified 5 cases (4 imported); Toronto reported 247 cases (3 imported) and 43 deaths. Baseline preparedness for pandemic threats may account for the absence of sustained transmission and fewer cases of SARS in Vancouver.

Identification of pathogenic Helicobacter species by chaperonin-60 differentiation on plastic DNA arrays.

A microarray method for bacterial species identification based on cpn60 and 16S rDNA hybridization was developed. Specific cpn60 or 16S rDNA oligonucleotides from various Helicobacter or Campylobacter species were printed and immobilized onto a proprietary plastic solid support. Using universal primers, fragments derived from either cpn60 or 16S rDNA genes from single isolates or from a complex human waste sludge DNA sample spiked with Helicobacter pylori were biotinylated and hybridized to the plastic slide. Subsequent querying with a streptavidin-horseradish peroxidase conjugate followed by color development using tetramethylbenzidine resulted in accurate Helicobacter species identification with no cross-hybridization to either the 16S rDNA or the cpn60 sequence of a closely related strain of Campylobacter jejuni. The combination of a nonfluorescence visual detection system with a polymer-based DNA microarray slide has resulted in a molecular tool that should prove useful in numerous applications requiring rapid, low-cost bacterial species identification.

An Outbreak of Human Coronavirus OC43 Infection and Serological Cross-reactivity with SARS Coronavirus.

BACKGROUND: In summer 2003, a respiratory outbreak was investigated in British Columbia, during which nucleic acid tests and serology unexpectedly indicated reactivity for severe acute respiratory syndrome coronavirus (SARS-CoV). METHODS: Cases at a care facility were epidemiologically characterized and sequentially investigated for conventional agents of respiratory infection, SARS-CoV and other human CoVs. Serological cross-reactivity between SARS-CoV and human CoV-OC43 (HCoV-OC43) was investigated by peptide spot assay. RESULTS: Ninety-five of 142 residents (67%) and 53 of 160 staff members (33%) experienced symptoms of respiratory infection. Symptomatic residents experienced cough (66%), fever (21%) and pneumonia (12%). Eight residents died, six with pneumonia. No staff members developed pneumonia. Findings on reverse transcriptase-polymerase chain reaction assays for SARS-CoV at a national reference laboratory were suspected to represent false positives, but this was confounded by concurrent identification of antibody to N protein on serology. Subsequent testing by reverse transcriptase-polymerase chain reaction confirmed HCoV-OC43 infection. Convalescent serology ruled out SARS. Notably, sera demonstrated cross-reactivity against nucleocapsid peptide sequences common to HCoV-OC43 and SARS-CoV. CONCLUSIONS: These findings underscore the virulence of human CoV-OC43 in elderly populations and confirm that cross-reactivity to antibody against nucleocapsid proteins from these viruses must be considered when interpreting serological tests for SARS-CoV.

Characterization of vaginal microflora of healthy, nonpregnant women by chaperonin-60 sequence-based methods.

OBJECTIVE: The purpose of this study was to use a novel method that was based on the application of chaperonin-60 sequencing to describe the vaginal microflora of 16 healthy women. STUDY DESIGN: Asymptomatic women consented for vaginal swabs to be collected at the time of a clinical pelvic examination. Total genomic DNA was isolated from the vaginal swabs. Degenerate, universal polymerase chain reaction primers were used to amplify an approximately 555 base pair region of the universal chaperonin-60 gene, which is found in all eubacteria and eukaryotes, from the total genomic DNA and libraries of cloned polymerase chain reaction products were constructed. Library clones were sequenced, and the resulting sequences were assigned to taxonomic groups on the basis of similarity to reference sequence data. Presence of Chlamydophila psittaci sequences in the samples was confirmed by species-specific polymerase chain reaction. RESULTS: Sixteen of the 23 women who were enrolled had normal flora by Nugent's score of <4 and had adequate polymerase chain reaction product for assessment. Vaginal flora libraries were dominated by a variety of sequences with similarity to Lactobacillus spp L. crispatus, L. iners, L. gasseri, L. jensenii, and L. buchneri. Other sequences that were identified included representatives of Gardnerella spp, sequences with similarity to Porphyromonas spp and Megasphaera spp and sequences identical to C psittaci. CONCLUSION: Culture-independent, chaperonin-60 sequence-based molecular methods can lead to the identification of greater diversity within defined taxa compared with those that are identified by standard culture-based methods and to the identification of novel organisms that were not previously associated with vaginal flora.

Biochemical analysis, cpn60 and 16S rDNA sequence data indicate that Streptococcus suis serotypes 32 and 34, isolated from pigs, are Streptococcus orisratti.

Streptococcus suis serotypes have traditionally been identified by morphology, biochemical profiling and serotyping. Analysis of the sequences of 16S rRNA and cpn60 genes of the 35 characterized serotypes of S. suis led to the observation that two serotypes 32 and 34, are significantly distinct from other S. suis serotypes and may represent a distinct species. Here we present DNA sequence data and biochemical profiles which indicate that S. suis serotypes 32 and 34, isolated from pigs, are clustered with Streptococcus orisratti, a Voges-Proskauer negative, alpha-haemolytic, aesculin-hydrolytic, Lancefield group A streptococcus isolated from the teeth of rats.

Comparison of ileum microflora of pigs fed corn-, wheat-, or barley-based diets by chaperonin-60 sequencing and quantitative PCR.

We have combined the culture-independent methods of high-throughput sequencing of chaperonin-60 PCR product libraries and quantitative PCR to profile and quantify the small-intestinal microflora of pigs fed diets based on corn, wheat, or barley. A total of 2,751 chaperonin-60 PCR product clones produced from samples of ileum digesta were examined. The majority (81%) of these clones contained sequences independently recovered from all three libraries; 372 different nucleotide sequences were identified, but only 14% of the 372 different sequences were recovered from all three libraries. Taxonomic assignments of the library sequences were made by comparison to a reference database of chaperonin-60 sequences combined with phylogenetic analysis. The taxa identified are consistent with previous reports of pig ileum microflora. Frequencies of each sequence in each library were calculated to identify taxa that varied in frequency between the corn, barley, and wheat libraries. The chaperonin-60 sequence inventory was used as a basis for designing PCR primer sets for taxon-specific quantitative PCR. Results of quantitative PCR analysis of ileum digesta confirmed the relative abundances of targeted taxa identified with the library sequencing approach. The results of this study indicate that chaperonin-60 clone libraries can be valid profiles of complex microbial communities and can be used as the basis for producing quantitative PCR assays to measure the abundance of taxa of interest during experimentally induced or natural changes in a community.

cpnDB: a chaperonin sequence database.

Type I chaperonins are molecular chaperones present in virtually all bacteria, some archaea and the plastids and mitochondria of eukaryotes. Sequences of cpn60 genes, encoding 60-kDa chaperonin protein subunits (CPN60, also known as GroEL or HSP60), are useful for phylogenetic studies and as targets for detection and identification of organisms. Conveniently, a 549-567-bp segment of the cpn60 coding region can be amplified with universal PCR primers. Here, we introduce cpnDB, a curated collection of cpn60 sequence data collected from public databases or generated by a network of collaborators exploiting the cpn60 target in clinical, phylogenetic, and microbial ecology studies. The growing database currently contains approximately 2000 records covering over 240 genera of bacteria, eukaryotes, and archaea. The database also contains over 60 sequences for the archaeal Type II chaperonin (thermosome, a homolog of eukaryotic cytoplasmic chaperonin) from 19 archaeal genera. As the largest curated collection of sequences available for a protein-encoding gene, cpnDB provides a resource for researchers interested in exploiting the power of cpn60 as a diagnostic or as a target for phylogenetic or microbial ecology studies, as well as those interested in broader subjects such as lateral gene transfer and codon usage. We built cpnDB from open source tools and it is available at http://cpndb.cbr.nrc.ca.

Neisseria meningitidis carriage during an outbreak of serogroup C disease.

During 2001, an outbreak of serogroup C meningococcal disease led to immunization of individuals aged 13-29 years in Abbotsford, British Columbia, Canada. This study addresses the distribution of Neisseria meningitidis carriage in this population and the implications of that distribution for the targeting of the immunization campaign. Pharyngeal swabs were obtained at immunization from 2004 people. Colonies were identified and serogrouped using standard agglutination methods and by PCR. Isolates were characterized using pulsed-field gel electrophoresis (PFGE). The prevalence of N. meningitidis carriage was 153 carriers per 2004 subjects (7.6%; 95% confidence interval, 6.5%-8.9%). Only 6 (4%) of the isolates from these carriers were found to be serogroup C by agglutination or PCR testing, and all of these were from individuals within the age group targeted for immunization. Only 1 of these 6 isolates was found to be identical to the outbreak strain by PFGE. The observation that a virulent strain is not circulating widely suggests the possibility of low background immunity in the population at risk and emphasizes the importance of vaccination in controlling epidemic group C meningococcal disease.