Varicella-zoster virus (VZV) is a remarkably stable virus that until recently was thought to exhibit nearuniversal 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 fullgenome 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. Varicella-zoster virus (VZV) is a member of the genusVaricellovirus in the Alphaherpesvirinae subfamily of the Herpesviridae. It is the causative agent of chicken pox (varicella) in children, after which it establishes latency in the sensory ganglia with the potential to reactivate at a later time to cause shingles (zoster). The sequencing of the first complete VZV genome, VZV-Dumas, by Davison and Scott (10) opened the door for genetic analysis of this extremely stable virus. The genome is comprised of ϳ125 kb of linear double-stranded DNA containing approximately 71 open reading frames (ORFs). The viral structure is similar to that of other alphaherpesviruses, consisting of two unique regions, unique long and unique short, each flanked by inverted repeats; short repeats termed terminal repeat long and internal repeat long border the unique long region, while larger repeats termed terminal repeat short (TR S ) and internal repeat short (IR S ) border the unique short region.A number of factors likely contribute to the overall stability of the VZV genome, one of which is the efficient proofreading activity of the DNA polymerase, which exhibits 3Ј to 5Ј exonuclease activity as documented in herpes simplex virus type 1 (HSV-1) (2). The synonymous and nonsynonymous mutation rates among the herpesviruses have been es...

Objective-In Canada, opportunistic screening programs have successfully reduced mortality from cervical cancer; however, minority or disadvantaged groups, as well as women in northern and rural areas, are inadequately recruited by this approach. Hence, we set out to examine the structural barriers that prevent First Nations women's participation in cervical cancer screening.Methods-Using a participatory action research approach and semistructured interview guides, we conducted in-depth interviews with 18 experienced health care professionals, 12 of whom were also community members. These individuals included nurses, nurse practitioners, community health representatives, social workers and physicians who provide care to women in our First CIHR Author Manuscript CIHR Author Manuscript CIHR Author ManuscriptNations partner communities. In the current report, we explored perceived barriers to cervical cancer screening through the lens of service providers.Results-Structural barriers to cervical cancer screening for First Nations women included shortage of appropriate health care providers, lack of a recall-based screening system, geographic and transportation barriers; health literacy and socioeconomic inequalities, generational effects, and the colonial legacy.Conclusion-Existing, opportunistic cervical cancer screening programs do not perform well for First Nations women who experience significant screening-related health inequalities that are largely influenced by structural barriers. Sustainable screening interventions in First Nations communities require approaches that resolve these structural barriers, explore new ways of screening, and provide education for both women and health care providers. Many of the structural barriers are rooted in colonial history. Given the negative impact of the consequences of colonization on indigenous women worldwide, many of our findings strongly resonate with marginalized populations in other countries.

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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.

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 ( . 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.

Herpes simplex virus type 1 (HSV-1) and varicella zoster virus (VZV) are closely related viruses causing lifelong infections. They are typically associated with mucocutaneous or skin lesions, but may also cause severe neurological or ophthalmic diseases, possibly due to viral- and/or host-genetic factors. Although these viruses are well characterized, genome-wide evolutionary studies have hitherto only been presented for VZV. Here, we present a genome-wide study on HSV-1. We also compared the evolutionary characteristics of HSV-1 with those for VZV. We demonstrate that, in contrast to VZV for which only a few ancient recombination events have been suggested, all HSV-1 genomes contain mosaic patterns of segments with different evolutionary origins. Thus, recombination seems to occur extremely frequent for HSV-1. We conclude by proposing a timescale for HSV-1 evolution, and by discussing putative underlying mechanisms for why these otherwise biologically similar viruses have such striking evolutionary differences.

We have used the endogenous reverse transcriptase reaction of viral core particles from duck liver to elucidate the mechanism of inhibition of duck hepatitis B virus (DHBV) replication by the nucleoside analog (؊)-␤-L-2,3-dideoxy-3-thiacytidine (3TC). As is the case in human immunodeficiency virus replication, 3TC-5-triphosphate (3TC-TP) acts as a chain terminator for the DNA polymerase activities. The results of several different experiments support this conclusion, which explains the potent activity of 3TC against the hepadnaviruses. In isolated DHBV core particles, 3TC-TP inhibited the reverse transcriptase in a manner that resembled competitive inhibition with respect to dCTP. However, the kinetics of inhibition was not linear on a double-reciprocal plot for the highest concentrations of 3TC-TP and the lowest concentration of dCTP. This anomaly would be expected if binding to the nucleotide site was followed by DNA chain termination. Calculations that used only the linear part of the curve yielded a K i of 0.78 ؎ 0.10 M 3TC-TP. The inhibition of core particles incubated in vitro with 3TC-TP was not reversed by removal of the free inhibitor. 3TC-TP inactivated the reverse transcriptase activity in a concentration-dependent manner. The K m of the chain termination reaction was calculated at 0.71 ؎ 0.05 M. Similar competitive kinetics and irreversible inhibition were also obtained on the endogenous DNA polymerase from viral particles from serum, suggesting that 3TC-TP also acts as a chain terminator of the DNA-directed DNA polymerase of DHBV replication. Core particles purified from DHBV-infected hepatocytes treated with 3TC also showed irreversible inhibition of the endogenous reverse transcriptase activity, indicating that chain termination is the mechanism of inhibition in the presence of the normal deoxynucleotide pools within cells. Endogenous sequencing of duck liver viral core particles with 3TC-TP as a chain terminator shows a pattern of bands similar to that obtained with ddCTP, and [ 32 P]3TC-TP labeled the growing DNA chain at the C position.) is a deoxycitidine analog in which the 3Ј carbon is replaced by a sulfur atom with loss of the 3Ј OH group necessary for the elongation of the DNA chain. 3TC was shown to be an effective inhibitor of human immunodeficiency virus (HIV) replication (7,13,28,33), and it was recently approved for compassionate use as an anti-HIV agent.Previous work showed that 3TC is also a very effective agent against human hepatitis B virus (5, 10, 32) and duck hepatitis B virus (DHBV [32]). In vivo and in infected hepatocytes, 3TC rapidly reduces the amount of viral DNA within days of the start of treatment, and it does not show significant toxicity (reference 5 and our unpublished results). 3TC is currently undergoing phase II clinical trials as an antiviral agent for human hepatitis B virus.For both HIV and hepadnaviruses, the mechanism of action of 3TC requires phosphorylation to 3TC-5Ј-triphosphate (3TC-TP), which in turn specifically inhibits the viral polymerases (3, 6, 15...

In order to gain a better perspective on the true variability of varicella-zoster virus (VZV) and to catalogue the location and number of differences, 11 new complete genome sequences were compared with those previously in the public domain (18 complete genomes in total). Three of the newly sequenced genomes were derived from a single strain in order to assess variations that can occur during serial passage in cell culture. The analysis revealed that while VZV is relatively stable genetically it does posses a certain degree of variability. The reiteration regions, origins of replication and intergenic homopolymer regions were all found to be variable between strains as well as within a given strain. In addition, the terminal viral sequences were found to vary within and between strains specifically at the 3' end of the genome. Analysis of single nucleotide polymorphisms (SNPs) identified a total of 557 variable sites, 451 of which were found in coding regions and resulted in 187 different in amino acid substitutions. A comparison of the SNPs present in the two gE mutant strains, VZV-MSP and VZV-BC, suggested that the missense mutation in gE was primarily responsible for the accelerated cell spread phenotype. Some of the variations noted with high passage in cell culture are consistent with variations seen in the IE62 gene of the vaccine strains (S628G, R958G and I1260V) that may help in pinpointing variations essential for attenuation. Although VZV has been considered to be one of the most genetically stable human herpesviruses, this initial assessment of genomic VZV cartography provides insight into ORFs with previously unreported variations.