Background : Cervical dysplasia, also referred to as squamous intraepithelial lesion (SIL) in cytology or cervical intraepithelial neoplasia in histopathology, is thought to have the potential to advance in progressive stages to cervical cancer. However, not all cases of SIL progress, and most of the mild lesions spontaneously regress. Factors that govern regression, persistence, and progression of SIL are poorly understood. Purpose: Our analysis sought to identify factors that determined persistence or regression of SIL. Methods: Seventy subjects with histopathologically confirmed cervical dysplasia were followed at 3-month intervals for 15 months. At each visit, the cervix was evaluated by Pap smear and colposcopy, and exfoliated cervicovaginal cells were analyzed for human papillomavirus (HPV) DNA. For each subject, data from every two consecutive visits were grouped as a pair. Persistent SIL was considered present if a lesion was detected at a visit (t) as well as at the next visit (t + 1) and absent if a lesion was detected at visit t but not at visit t + 1. A statistical model for time-dependent data correlated persistent SIL with various risk factors. Results: Age, ethnicity, education, sexual behavior, smoking, and the use of oral contraceptives did not correlate with persistent SIL. The risk of persistent SIL was associated with continual HPV infection in visits / and t + 1 (HPV positive by Southern blot analysis: odds ratio [OR] = 3.91, and 95% confidence interval [CI] = 1.58–9.65; HPV positive by polymerase chain reaction [PCR]: OR = 2.42, and 95% CI = 1.03–5.67) and a persistent high viral load (OR = 4.07, and 95% CI = 1.35–12.30). When typed by PCR, individuals with type-specific persistent infection in visits t and t + 1, and particularly those with a continual high viral load (OR = 4.97; 95% CI = 1.45–17.02), had the highest risk for persistent SIL compared with those with a low level of type-specific persistent infection or non-type-specific persistent infection. The presence of persistent HPV infection in visits t − 1 and t (the preceding time interval) was also predictive of persistent SIL in visits t and t + 1, although the strength of association was weaker, suggesting that persistent HPV and SIL occur synchronously. Conclusion: HPV infection and its associated cervical lesions tend to occur concurrently, and type-specific persistent HPV infection, particularly with a high viral load, produces chronic cervical dysplasia. Implications: The natural history of genital HPV infection directly influences the prognosis of cervical dysplasia as measured by persistence of the lesion. Testing for HPV infection may be valuable in the clinical management of women with cervical dysplasia. [J Natl Cancer Inst 1995;87: 1365–71]

Human papillomavirus (HPV) infection is one of the most important etiologic causes of oropharyngeal head and neck squamous cell carcinoma (HNSCC). Patients with HPV-positive HNSCC were reported to have a better clinical outcome than patients with HPV-negative cancers. However, little is known about the possible causes of different clinical outcomes. In this study, we analyzed a detailed immune profile of tumor samples from HNSCC patients with respect to their HPV status. We analyzed the characteristics of immune cell infiltrates, including the frequency and distribution of antigen-presenting cells and naïve, regulatory and effector T cells and the cytokine and chemokine levels in tumor tissue. There was a profound difference in the extent and characteristics of intratumoral immune cell infiltrates in HNSCC patients based on their HPV status. In contrast to HPV-negative tumor tissues, HPV-positive tumor samples showed significantly higher numbers of infiltrating IFNγ+ CD8+ T lymphocytes, IL-17+ CD8...

Human cytomegalovirus is a widespread pathogen of major medical importance. It causes significant morbidity and mortality in immunocompromised individuals, and congenital infections can result in severe disabilities or stillbirth. Development of a vaccine is prioritized, but no candidate is close to release. Although correlations of viral genetic variability with pathogenicity are suspected, knowledge about the strain diversity of the 235-kb genome is still limited. In this study, 96 full-length human cytomegalovirus genomes from clinical isolates were characterized, quadrupling the amount of information available for full-genome analysis. These data provide the first high-resolution map of human cytomegalovirus interhost diversity and evolution. We show that cytomegalovirus is significantly more divergent than all other human herpesviruses and highlight hot spots of diversity in the genome. Importantly, 75% of strains are not genetically intact but contain disruptive mutations in a diverse set of 26 genes, including the immunomodulatory genes UL40 and UL111A. These mutants are independent of culture passage artifacts and circulate in natural populations. Pervasive recombination, which is linked to the widespread occurrence of multiple infections, was found throughout the genome. The recombination density was significantly higher than those of other human herpesviruses and correlated with strain diversity. While the overall effects of strong purifying selection on virus evolution are apparent, evidence of diversifying selection was found in several genes encoding proteins that interact with the host immune system, including UL18, UL40, UL142, and UL147. These residues may present phylogenetic signatures of past and ongoing virus-host interactions. IMPORTANCE Human cytomegalovirus has the largest genome of all viruses that infect humans. Currently, there is a great interest in establishing associations between genetic variants and strain pathogenicity of this herpesvirus. Since the number of publicly available full-genome sequences is limited, knowledge about strain diversity is highly fragmented and biased toward a small set of loci. Combined with our previous work, we have now contributed 101 complete genome sequences. We have used these data to conduct the first high-resolution analysis of interhost genome diversity, providing an unbiased and comprehensive overview of cytomegalovirus variability. These data are of major value to the development of novel antivirals and a vaccine and to identify potential targets for genotype-phenotype experiments. Furthermore, these data have enabled a thorough study of the evolutionary processes that have shaped cytomegalovirus diversity.

Background: Estimating evolutionary rates for slowly evolving viruses such as papillomaviruses (PVs) is not possible using fossil calibrations directly or sequences sampled over a time-scale of decades. An ability to correlate their divergence with a host species, however, can provide a means to estimate evolutionary rates for these viruses accurately. To determine whether such an approach is feasible, we sequenced complete feline PV genomes, previously available only for the domestic cat (Felis domesticus, FdPV1), from four additional, globally distributed feline species: Lynx rufus PV type 1, Puma concolor PV type 1, Panthera leo persica PV type 1, and Uncia uncia PV type 1. Results: The feline PVs all belong to the Lambdapapillomavirus genus, and contain an unusual second noncoding region between the early and late protein region, which is only present in members of this genus. Our maximum likelihood and Bayesian phylogenetic analyses demonstrate that the evolutionary relationships between feline PVs perfectly mirror those of their feline hosts, despite a complex and dynamic phylogeographic history. By applying host species divergence times, we provide the first precise estimates for the rate of evolution for each PV gene, with an overall evolutionary rate of 1.95 × 10^-8 (95% confidence interval 1.32 × 10^-8 to 2.47 × 10^-8) nucleotide substitutions per site per year for the viral coding genome. Conclusion: Our work provides evidence for long-term virus-host co-speciation of feline PVs, indicating that viral diversity in slowly evolving viruses can be used to investigate host species evolution. These findings, however, should not be extrapolated to other viral lineages without prior confirmation of virus-host co-divergence.

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 10 ⁴ -fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.