Sequence Imputation of HPV16 Genomes for Genetic Association Studies

Human Papillomavirus type 16 (HPV16) causes over half of all cervical cancer and some HPV16 variants are more oncogenic than others. The genetic basis for the extraordinary oncogenic properties of HPV16 compared to other HPVs is unknown. In addition, we neither know which nucleotides vary across and within HPV types and lineages, nor which of the single nucleotide polymorphisms (SNPs) determine oncogenicity.A reference set of 62 HPV16 complete genome sequences was established and used to examine patterns of evolutionary relatedness amongst variants using a pairwise identity heatmap and HPV16 phylogeny. A BLAST-based algorithm was developed to impute complete genome data from partial sequence information using the reference database. To interrogate the oncogenic risk of determined and imputed HPV16 SNPs, odds-ratios for each SNP were calculated in a case-control viral genome-wide association study (VWAS) using biopsy confirmed high-grade cervix neoplasia and self-limited HPV16 infections from Guanacaste, Costa Rica.HPV16 variants display evolutionarily stable lineages that contain conserved diagnostic SNPs. The imputation algorithm indicated that an average of 97.5±1.03% of SNPs could be accurately imputed. The VWAS revealed specific HPV16 viral SNPs associated with variant lineages and elevated odds ratios; however, individual causal SNPs could not be distinguished with certainty due to the nature of HPV evolution.Conserved and lineage-specific SNPs can be imputed with a high degree of accuracy from limited viral polymorphic data due to the lack of recombination and the stochastic mechanism of variation accumulation in the HPV genome. However, to determine the role of novel variants or non-lineage-specific SNPs by VWAS will require direct sequence analysis. The investigation of patterns of genetic variation and the identification of diagnostic SNPs for lineages of HPV16 variants provides a valuable resource for future studies of HPV16 pathogenicity

Diversity of Skin Infections

The identification of infectious agents in cancer has been one of the most rewarding endeavors in cancer research. Currently about 20% of the global cancer burden is linked to an infection. A common characteristic of virus-induced cancer is an increased incidence in immunosuppressed patients, presumably because of impaired host control of virus. Yet non-melanoma skin cancer (NMSC), the cancer that increases most among the immunosuppressed, does not have an established link to infection. NMSC, including squamous cell carcinoma (SCC) and basal cell carcinoma, is the most common cancer among Caucasians. Ultraviolet radiation is an established risk factor. Human papillomaviruses (HPVs) have been established as the major cause of cervical cancer. Many NMSCs contain one or several cutaneous types of HPV. Exploration of a possible infectious etiology of NMSC requires an unbiased and comprehensive approach for detection of as many infections as possible in the tumor. We examined NMSCs and other presumably HPV-associated lesions for the presence of unidentified HPV types or other microorganisms, using a combination of multiple displacement amplification (MDA), which amplifies all DNA in a sample without any requirement of prior knowledge of the nucleotide sequence, degenerate “general HPV primers” PCR and high-throughput sequencing. The most common microbial DNA in NMSC was Staphylococcus aureus (S. aureus). We also identified sequences from at least 40 previously not described putative HPV types, of which three novel types (HPV 109, 112 and 114) and an HPV 88 isolate were cloned and completely sequenced. Prevalences and viral loads were investigated in skin and genital samples from different patient groups. S. aureus DNA was more commonly detected in SCC compared to healthy skin (odds ratio, 6.23; 95% confidence interval, 3.10 – 12.53). However, the study design could not determine the causality of the association. HPV 88, 109 and 112 were almost only found in their index patients, whereas HPV114 was found in 1.7% of the female genital samples. In summary, we find that there is a wide diversity of HPV types in the skin. The association of S. aureus with SCC raises the possibility of general susceptibility to infection in SCC. An association of NMSC with a specific infection remains to be found

Ancient papillomavirus-host co-speciation in Felidae

The evolutionary rate of feline papillomaviruses is inferred from the phylogenetic analysis of their hosts, providing evidence for long-term virus-host co-speciatio

Identification of a novel bat papillomavirus by metagenomics

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Infections in skin cancer

The increasing prevalence of skin cancer results in that it will soon equal that of all other cancers combined. Sun exposure is a well-known risk factor for its development, but despite the growing public awareness of the harmful consequences of ultraviolet radiation, the cancer incidence continues to increase, implying that other factors might also have a role in promoting this disease. Data from immunosuppressed patients reveals a 100-fold increased incidence of nonmelanoma skin carcinoma (NMSC), but an infectious etiology has not been established. However, certain human papillomaviruses (HPVs) have previously been detected in this type of cancer. We applied high throughput sequencing to different skin lesions in order to assess which organisms were present. Most viral reads (>95%) belonged to human papillomavirus. Traditionally, viral detection was performed using PCR methods. We used degenerate “general” HPV primers and multiplexed novel “specific” HPV primers in order to amplify a broad number of HPVs by PCR. This method showed a very high sensitivity, but the HPV types with low similarity to the primer sequences might have escaped amplification. Therefore, we performed an unbiased approach based on non-PCR whole genome amplification, independent of sequence information, in order to detect those “escaping” HPV types, as well as to determine if other viruses were present in the samples. Overall, we identified almost 100 putative novel HPV types in total, and characterized 4 novel HPV types (HPV 197, 200, 201 and 202). Most of the HPV types were detected in very few patients each, and at a very low viral load (below 0.5 copies/cell), except for HPV 197, which was the most commonly found virus in skin tumors (37.4% of skin lesions). Despite the higher sensitivity of PCR methods, the unbiased approach detected HPV in 37/40 condyloma acuminata that had been reported as “HPV-negative” with specific PCR techniques. Certain HPV types, including HPV 197, were not detected by PCR and only by non-PCR based methods. Therefore, more unbiased PCR-independent methods are needed to describe which organisms are most commonly present in skin lesions. The work in this thesis has expanded our knowledge of the wide genomic diversity of HPV on the skin, and finds that PCR-independent methods are needed to describe which organisms are most commonly present in skin lesions. Further studies are needed to assess any possible role of viral infections in skin cancer, elucidation of mechanistic effects and determine the direction of causality of any associations

Metagenomic profiling of placental tissue suggests DNA virus infection of the placenta is rare.

It is widely recognized that pathogens can be transmitted across the placenta from mother to foetus. Recent re-evaluation of metagenomic studies indicates that the placenta has no unique microbiome of commensal bacteria. However, viral transmission across the placenta, including transmission of DNA viruses such as the human herpesviruses, is possible. A fuller understanding of which DNA virus sequence can be found in the placenta is required. We employed a metagenomic analysis to identify viral DNA sequences in placental metagenomes from full-term births (20 births), pre-term births (13 births), births from pregnancies associated with antenatal infections (12 births) or pre-term births with antenatal infections (three births). Our analysis found only a small number of DNA sequences corresponding to the genomes of human herpesviruses in four of the 48 metagenomes analysed. Therefore, our data suggest that DNA virus infection of the placenta is rare and support the concept that the placenta is largely free of pathogen infection

Review on Viral Metagenomics and Its Future Perspective in Zoonotic and Arboviral Disease Surveillance

Viral metagenomics or full-length genome sequencing of enriched viral particle preparations has been frequently applied for viral discovery resulting in the genetic characterization of numerous human and animal viruses. The  non- specific nature of viral metagenomics endows it with great potential as a unversal virus detection assay. Historical methods includes, electron microscopy, cell culture, inoculation in suckling mice and serology. Many viruses cannot be cultivated, excluding the use of cell line isolation and serologic techniques, and can only be characterized by molecular methods. The molecular-based techniques provide sensitive and rapid means of virus detection and identification. Most of these tests are designed to be virus-specific that makes them unsuitable for detection of unexpected and/or completely new viruses, as well as novel viral variants that poses great challenge due their re-emrgince property. The recently developed approaches of viral metagenomics provide an effective novel way to screen samples and detect viruses without previous knowlege of the infectious agent, thereby enabling a better diagnosis and disease control. The basic steps involved in viral metagenomics include preparation of viral nucleic acid that is free from host and contaminating nuceic acids, sequence-independent amplification of viral nucleic acid,sequencing and use of bioinformatics tools for analysis of sequnce generated. Viral metagenomics aims to provide the genetic composition of the complete viral population of a sample in an unbiased and culture-independent manner. Viral metagenomics has been successfully used to investigate viral populations in different environments such as seawater, gastreointestinal tracts, and respiratory samples and have demonstrated that there is a high diversity among viruses. Many potential emerging viruses of concern might already be infecting humans, domestic animals or wlidlife but awailt their detection by disease surveilla-nce which can be possible through application of viral metagenomics. This review aims to de-scribes the different possible steps of a viral metagenomics and its future application in viral zonootic and arboviral disease surveillance. Keywords: Arboviral disease, Bioinformatics,  Emerging infectious disease, Sequencing, Surveillance, Viral metagenomics.Begna Bulch

Characterisation and evolutionary dynamics of ten novel Gammapapillomavirus types from South African penile swabs

Human papillomaviruses (HPVs) are genetically diverse, belonging to five distinct genera: Alpha, Beta, Gamma, Mu and Nu. We discovered ten novel Gammapapillomaviruses (Gamma-HPVs). Genomic characterisation and phylogenetic evaluation of the ten novel Gamma-HPV types were done: HPV211, HPV212, HPV213, HPV214, HPV215, HPV216, HPV219, HPV220, HPV221 and HPV222. These HPVs were previously identified in a study that was done on 218 penile samples (104 HIV negative and 114 HIV positive) using high throughput sequencing (Roche 454) of amplimers obtained using FAP59/64 primers which were designed to detect “cutaneous” or Beta- and Gamma-HPVs. Fifteen putative novel HPV types were identified from the short HPV L1 FAP fragments HPV211 (CT02, KY063000), HPV212 (CT03, KY063001), HPV213 (CT04, KY063002), HPV214 (CT06, KY063004), HPV215 (CT07, KY063005), HPV216 (CT12, KY063010), HPV219 (CT01, KY062999), HPV220 (CT08, KY063006), HPV221 (CT09, KY063007) and HPV222 (CT155, AY009886) with prevalences varying from 0.5% to 4.1% of men sampled. Multiple full genome clones for each novel type were generated through whole genome amplification, cloning and next generation sequencing. Complete genome sizes were: HPV211 (7253 bp), HPV212 (7208 bp), HPV213 (7096 bp), HPV214 (7357 bp), HPV215 (7186 bp), HPV216 (7233 bp), HPV219 (7108 bp), HPV220 (7381 bp), HPV221 (7326 bp) and HPV222 (7275 bp). Phylogenetically the novel Papillomaviruses (PVs) all clustered with Gamma-HPVs: HPV211 is most closely related to HPV168 (72% identity in the L1 nucleotide sequence) of the Gamma-8 species, HPV212 is most closely related to HPV144 (82.9%) of the Gamma-17 species, HPV213 is most closely related to HPV153 (71.8%) of the Gamma-13 species, HPV214 is most closely related to HPV103 (75.3%) of the Gamma-6 species, HPV215 and HPV216 are most closely related to HPV129 (76.8% and 79.2% respectively) of the Gamma-9 species. HPV219 is phylogenetically most closely related to HPV213 (87% identity in L1 gene) of the Gamma-13 species, HPV220 to HPV212 (72%) of Gamma-17, HPV221 to HPV142 (80%) of Gamma-10, HPV222 to HPV162 (73%) of Gamma-19. The novel HPV types demonstrated the classical genomic organisation of Gamma-HPVs, with seven open reading frames (ORFs) encoding five early (E1, E2, E4, E6 and E7) and two late (L1 and L2) proteins. Typical of Gamma-HPVs, the novel types all lacked the E5 ORF and HPV214 also lacked the E6 ORF. We further examined variation of the novel types in clinical specimens from which they were identified. All the clones of HPV211, HPV214, HPV216, HPV219 and HPV221 were identical and showed 100% pairwise identity. The clones of HPV213, HPV215, HPV212, HPV220 and HPV222 had several differences. Analysis of mismatches between the nine genomic clones of HPV212 showed a total of 67 mismatch positions that varied along the 7208 bp genome and all the clones were unique. Analysis of mismatches between the 10 genomic clones of HPV213 showed a total of 51 mismatch positions that varied along the 7096 bp genome and it had 5 unique clones. The 6 genomic clones of HPV215 showed a total of 50 mismatch positions along a 7186 bp genome and it had 3 identical and 3 different clones. HPV220 had 4 different genomic clones that showed 17 mismatch positions along a 7381 bp genome. The 5 different clones of HPV222 showed a total of 24 mismatch positions along the 7275 bp genome. Conserved domains observed among the novel types were the Zinc finger binding Domain and PDZ domains. A retinoblastoma binding protein (pRB) binding domain in the E7 protein was additionally identified in HPV214 and HPV222. PVs are thought to evolve slowly because they co-opt high-fidelity host cellular DNA polymerases for their replication. Despite extensive efforts to catalogue all the HPV species that infect humans, it is likely that many still remain undiscovered. We used the genome sequences of the ten novel viruses and related HPVs to analyse the evolutionary dynamics of these viruses at the whole genome and individual gene scales. We found statistically significant incongruences between the phylogenetic trees of different genes which imply gene-to-gene variation in the evolutionary processes underlying the diversification of Gamma-PVs. We were, however, only able to detect convincing evidence of a single recombination event which, on its own, cannot explain the observed incongruences between gene phylogenies. The divergence times of the last common ancestor (LCA) of the Alpha, Beta, Mu, Nu and Gamma genera was predicted to have existed between 49.7-58.5 million years ago before splitting into the five main lineages. The LCA of the presently sampled Gamma-PVs was predicted to have existed between 45.3 and 67.5 million years ago: approximately at the time when the simian and tarsier lineages of the primates diverged. The discovery, characterisation and classification of HPV211, HPV212, HPV213, HPV214, HPV215 HPV216, HPV219, HPV220, HPV221 and HPV222 add these novel types to the repertoire of the ever expanding Gamma-HPVs genus hence expanding our knowledge of these viruses