Whole-Genome Sequences of Four Corynebacterium CDC Group F-1 Strains Isolated from Urine.

Three draft and one complete genome sequence from strains isolated from urine and consistent with Corynebacterium CDC group F-1 were assembled and studied. Genome sizes ranged between 2.3 and 2.44 Mb, with G+C content between 60.4% and 60.7%.

Genome sequence of a chimpanzee herpesvirus and its relation to other primate alphaherpesviruses.

This study reports the complete genome sequence of chimpanzee herpesvirus (ChHV), an alphaherpesvirus isolated from a chimpanzee. Although closely related to human herpes simplex virus type 2 (HSV2), the level of sequence diversity confirms that ChHV is sufficiently distinct to be considered a member of a different virus species rather than a variant strain of HSV2. Phylogenetic comparison with other simplexviruses at several levels supports the hypothesis that HSV2 and ChHV co-evolved with their respective human and chimpanzee hosts and raises questions regarding the evolutionary origins of HSV1.

The attenuated genotype of varicella-zoster virus includes an ORF0 transitional stop codon mutation.

Varicella-zoster virus (VZV) is the first of the human herpesviruses to be attenuated and subsequently approved as a live vaccine to prevent varicella and herpes zoster. Both the attenuated VZV vaccine, called vaccine Oka or vOka, and the parental strain pOka have been completely sequenced. Yet the specific determinants of attenuation are uncertain. The open reading frame (ORF) with the most single nucleotide polymorphisms (SNPs), ORF62, encodes the regulatory protein IE62, but IE62 studies have failed to define a specific SNP associated with attenuation. We have completed next-generation sequencing of the VZV Ellen genome, a strain known to be highly attenuated by its very limited replication in human skin xenografts in the SCID mouse model of VZV pathogenesis. A comparative analysis of the Ellen sequence with all other complete VZV sequences was extremely informative. In particular, an unexpected finding was a stop codon mutation in Ellen ORF0 (herpes simplex virus UL56 homolog) identical to one found in vOka, combined with the absence of polymorphisms in most Ellen ORFs that were known to be mutated in vOka. The mutated ORF0 protein was also imaged in both two dimensions and three dimensions by confocal microscopy. The probability of two VZV strains not connected by a recent common ancestor having an identical ORF0 SNP by chance would be 1 × 10(-8), in other words, extremely unlikely. Taken together, these bioinformatics analyses strongly suggest that the stop codon ORF0 SNP is one of the determinants of the attenuation genotype of live VZV vaccines.

Molecular definition of vaginal microbiota in East African commercial sex workers.

Resistance to HIV infection in a cohort of commercial sex workers living in Nairobi, Kenya, is linked to mucosal and antiinflammatory factors that may be influenced by the vaginal microbiota. Since bacterial vaginosis (BV), a polymicrobial dysbiosis characterized by low levels of protective Lactobacillus organisms, is an established risk factor for HIV infection, we investigated whether vaginal microbiology was associated with HIV-exposed seronegative (HESN) or HIV-seropositive (HIV(+)) status in this cohort. A subset of 44 individuals was selected for deep-sequencing analysis based on the chaperonin 60 (cpn60) universal target (UT), including HESN individuals (n = 16), other HIV-seronegative controls (HIV-N, n = 16), and HIV(+) individuals (n = 12). Our findings indicate exceptionally high phylogenetic resolution of the cpn60 UT using reads as short as 200 bp, with 54 species in 29 genera detected in this group. Contrary to our initial hypothesis, few differences between HESN and HIV-N women were observed. Several HIV(+) women had distinct profiles dominated by Escherichia coli. The deep-sequencing phylogenetic profile of the vaginal microbiota corresponds closely to BV(+) and BV(-) diagnoses by microscopy, elucidating BV at the molecular level. A cluster of samples with intermediate abundance of Lactobacillus and dominant Gardnerella was identified, defining a distinct BV phenotype that may represent a transitional stage between BV(+) and BV(-). Several alpha- and betaproteobacteria, including the recently described species Variovorax paradoxus, were found to correlate positively with increased Lactobacillus levels that define the BV(-) ("normal") phenotype. We conclude that cpn60 UT is ideally suited to next-generation sequencing technologies for further investigation of microbial community dynamics and mucosal immunity underlying HIV resistance in this cohort.

Pyrosequencing of chaperonin-60 (cpn60) amplicons as a means of determining microbial community composition.

The chaperonin-60 universal target (cpn60 UT) is generated from a set of PCR primers and provides a universally conserved, phylogenetically informative sequence signature for determining the composition of microbial communities by DNA sequencing. Pyrosequencing of cpn60 UT amplicons is emerging as a next-generation tool for providing unprecedented sequencing depth and resolution of microbial communities in individual samples. Owing to the increase in sequencing depth, the dynamic range across which the presence and abundance of individual species can be sampled experimentally also increases, significantly improving our ability to investigate microbial community richness and diversity. The flexible format of the pyrosequencing reaction setup combined with the ability to pool samples through the use of multiplexing IDs makes the generation of microbial profiles based on the cpn60 UT both feasible and cost-effective. We describe here the methods we have developed for determining microbial community profiles by pyrosequencing of cpn60 UT amplicons, from generating amplicons to sequencing and data analysis.

Pyrosequencing of the chaperonin-60 universal target as a tool for determining microbial community composition.

We compared dideoxy sequencing of cloned chaperonin-60 universal target (cpn60 UT) amplicons to pyrosequencing of amplicons derived from vaginal microbial communities. In samples pooled from a number of individuals, the pyrosequencing method produced a data set that included virtually all of the sequences that were found within the clone library and revealed an additional level of taxonomic richness. However, the relative abundances of the sequences were different in the two datasets. These observations were expanded and confirmed by the analysis of paired clone library and pyrosequencing datasets from vaginal swabs taken from four individuals. Both for individuals with a normal vaginal microbiota and for those with bacterial vaginosis, the pyrosequencing method revealed a large number of low-abundance taxa that were missed by the clone library approach. In addition, we showed that the pyrosequencing method generates a reproducible profile of microbial community structure in replicate amplifications from the same community. We also compared the taxonomic composition of a vaginal microbial community determined by pyrosequencing of 16S rRNA amplicons to that obtained using cpn60 universal primers. We found that the profiles generated by the two molecular targets were highly similar, with slight differences in the proportional representation of the taxa detected. However, the number of operational taxonomic units was significantly higher in the cpn60 data set, suggesting that the protein-encoding gene provides improved species resolution over the 16S rRNA target. These observations demonstrate that pyrosequencing of cpn60 UT amplicons provides a robust, reliable method for deep sequencing of microbial communities.

A full-genome phylogenetic analysis of varicella-zoster virus reveals a novel origin of replication-based genotyping scheme and evidence of recombination between major circulating clades.

Varicella-zoster virus (VZV) is a remarkably stable virus that until recently was thought to exhibit near-universal genetic homogeneity among circulating wild-type strains. In recent years, the expanding knowledge of VZV genetics has led to a number of groups proposing sequence-based typing schemes, but no study has yet examined the relationships between VZV genotypes at a full-genome level. A central hypothesis of this study is that VZV has coevolved with humankind. In this study, 11 additional full VZV genomic sequences are presented, bringing the current number of complete genomic sequences publicly available to 18. The full-genome alignment contained strains representing four distinct clades, but the possibility exists that a fifth clade comprised of African and Asian-like isolates was not represented. A consolidated VZV genotyping scheme employing the origin-associated region between reiteration region R4 and open reading frames (ORFs) 63 and 70 is described, one which accurately categorizes strains into one of four clades related to the geographic origin of the isolates. The full-genome alignment also provided evidence for recombination having occurred between the major circulating VZV clades. One Canadian clinical isolate was primarily Asian-like in origin, with most of the genome showing strong sequence identity to the Japanese-like clade B, with the exceptions being two putative recombination regions, located in ORFs 14 to 17 and ORFs 22 to 26, which showed clear similarity to the European/North American clade A. The very low rate of single-nucleotide polymorphisms scattered across the genome made full-genome sequencing the only definitive method for identifying specific VZV recombination events.

Complete genome sequence of cercopithecine herpesvirus 2 (SA8) and comparison with other simplexviruses.

We have obtained the complete sequence of the herpesvirus simian agent 8 (SA8; cercopithecine herpesvirus 2) a baboon simplexvirus closely related to the monkey B virus and herpes simplex virus types 1 and 2. The genome of SA8 is 150,715 bp long, with an overall G/C content of 76%, the highest among the simplexviruses sequenced so far. The sequencing has confirmed that the genomic arrangement of SA8 is similar to that of other simplexviruses: unique long and unique short regions bordered by two sets of inverted repeats. All genes identified in SA8 are homologous and collinear with those of the monkey B virus, including the lack of the RL1 open reading frame, a gene responsible for neurovirulence in human herpes simplex viruses. This latter finding supports the hypothesis that a different pathogenetic mechanism may have developed in human simplexviruses, after their divergence from monkey simplexviruses.