Laila Sara Arroyo Mühr

Deep sequencing extends the diversity of human papillomaviruses in human skin

Most viruses in human skin are known to be human papillomaviruses (HPVs). Previous sequencing of skin samples has identified 273 different cutaneous HPV types, including 47 previously unknown types. In the present study, we wished to extend prior studies using deeper sequencing. This deeper sequencing without prior PCR of a pool of 142 whole genome amplified skin lesions identified 23 known HPV types, 3 novel putative HPV types and 4 non-HPV viruses. The complete sequence was obtained for one of the known putative types and almost the complete sequence was obtained for one of the novel putative types. In addition, sequencing of amplimers from HPV consensus PCR of 326 skin lesions detected 385 different HPV types, including 226 previously unknown putative types. In conclusion, metagenomic deep sequencing of human skin samples identified no less than 396 different HPV types in human skin, out of which 229 putative HPV types were previously unknown.

Diversity of human papillomaviruses in skin lesions

Pools of frozen biopsies from patients with squamous cell carcinoma (SCC) (n=29) actinic keratosis (AK) (n=31), keratoacanthoma (n=91) and swab samples from 84 SCCs and 91 AKs were analysed with an extended HPV general primer PCR and high-throughput sequencing of amplimers. We found 273 different HPV isolates (87 known HPV types, 139 previously known HPV sequences (putative types) and 47 sequences from novel putative HPV types). Among the new sequences, five clustered in genus Betapapillomavirus and 42 in genus Gammapapillomavirus. Resequencing of the three pools between 21 to 70 times resulted in the detection of 283 different known or putative HPV types, with 156 different sequences found in only one of the pools. Type-specific PCRs for 37 putative types from an additional 296 patients found only two of these putative types. In conclusion, skin lesions contain a large diversity of HPV types, but most appeared to be rare infections.

Detection of DNA viruses in prostate cancer

We tested prostatic secretions from men with and without prostate cancer (13 cases and 13 matched controls) or prostatitis (18 cases and 18 matched controls) with metagenomic sequencing. A large number (>200) of viral reads was only detected among four prostate cancer cases (1 patient each positive for Merkel cell polyomavirus, JC polyomavirus and Human Papillomavirus types 89 or 40, respectively). Lower numbers of reads from a large variety of viruses were detected in all patient groups. Our knowledge of the biology of the prostate may be furthered by the fact that DNA viruses are commonly shed from the prostate and can be readily detected by metagenomic sequencing of expressed prostate secretions.

Viremia during pregnancy and risk of childhood leukemia and lymphomas in the offspring: Nested case-control study

A possible role for infections of the pregnant mother in the development of childhood acute leukemias and lymphomas has been suggested. However, no specific infectious agent has been identified. Offspring of 74,000 mothers who had serum samples taken during pregnancy and stored in a large‐scale biobank were followed up to the age of 15 years (750,000 person years) through over‐generation linkages between the biobank files, the Swedish national population and cancer registers to identify incident leukemia/lymphoma cases in the offspring. First‐trimester sera from mothers of 47 cases and 47 matched controls were retrieved and analyzed using next generation sequencing. Anelloviruses were the most common viruses detected, found in 37/47 cases and in 40/47 controls, respectively (OR: 0.6, 95% CI: 0.2–1.9). None of the detected viruses was associated with leukemia/lymphoma in the offspring. Viremia during pregnancy was common, but no association with leukemia/lymphoma risk in the offspring was found.

Are human papillomavirus DNA prevalences providing high-flying estimates of infection? An international survey of HPV detection on environmental surfaces

Most epidemiological studies of human papillomavirus (HPV) infections rely on HPV DNA detection. Recent studies have reported very high prevalence and acquisition rates in men.1 However, presence of HPV DNA is not proof of infection, as it might represent environmental contamination. Studies of HPV DNA on environmental surfaces could provide insights of the possible magnitude of this problem. We studied surfaces that frequently contact anogenital skin: toilet seats in airport restrooms. Apparently clean seats in 23 airports located in 13 countries (Belgium, Denmark, Finland, France, Germany, Italy, Jordan, the Netherlands, Russia, …

Cancer risks after solid organ transplantation and after long‐term dialysis

Immunosuppression involves an inability to control virus infections and increased incidence of virus‐associated cancers. Some cancers without known viral etiology are also increased, but data on exactly which cancer forms are increased has been inconsistent. To provide a reliable and generalizable estimate, with high statistical power and long follow‐up time, we assessed cancer risks using comprehensive, population‐based registries in two different countries and from two different immunosuppressed patient groups (solid organ transplant recipients (OTRs) and long‐term dialysis patients (LDPs)). National registries in Denmark and Sweden identified 20,804 OTRs and 31,140 LDPs that were followed up using national cancer registries. Standardized incidence ratios (SIR) compared to the general population were estimated. We found highly similar results, both for the two different countries and for the two different immunosuppressed cohorts, namely an increased incidence for the following specific cancer forms: Non‐melanoma skin cancer (NMSC), non‐Hodgkins lymphoma and cancers of the lip, kidney, larynx and thyroid. The SIR for overall cancer among OTRs was 3.5 [n = 2,142, 95% CI, 3.4–3.7] in Sweden, 2.9 [n = 1,110, 95% CI, 2.8–3.1] in Denmark and 1.6 [n = 1,713, 95% CI, 1.5–1.6] among LDP. The SIR for NMSC among OTRs was 44.7 [n = 994, 95% CI, 42–47.5] in Sweden and 41.5 [n = 445, 95% CI, 37.8–45.5] in Denmark. The increased SIR for NMSC among LDPs was 5.3 [n = 304, 95% CI, 4.7–5.9]). In summary, an increased SIR for a specific, similar set of cancer forms is consistently found among the immunosuppressed. Conceivable explanations include surveillance bias and immunosuppression‐related susceptibility to viral infections.

Towards quality and order in human papillomavirus research

The International Human Papillomavirus (HPV) Reference Center supports quality and order in HPV research and diagnostics. Notably, the center assigns HPV type numbers to novel HPV types, maintains a reference clone repository, and issues international proficiency panels for HPV genotyping. The established HPV types, currently up to HPV225, belong to 5 different genera: alpha (65 types), beta (54 types), gamma (98 types), mu (3 types) and nu (1 type). Since 2014, 23 novel types have been established, 82.6% of which belong to the gamma genus. Reference clones have been provided to 44 different research laboratories and the global proficiency program for HPV genotyping has seen an increasing participation (currently 146 laboratories) and complete proficiency has increased over time (from 26% to 59% of datasets). In summary, an increasing complexity of the HPVs requires international efforts to support a recognized quality and order among HPV types.

Viruses in cancers among the immunosuppressed

Most cancer forms known to be caused by viruses are increased among the immunosuppressed, but several cancer forms without established viral etiology are also increased, notably nonmelanoma skin carcinoma (NMSC). We followed all 13,429 solid organ transplantation patients in Sweden for cancer occurrence after transplantation. We requested these tumor specimens and sequenced the first 89 specimens received (62 NMSCs, 27 other cancers). The sequences were analyzed for viruses based on two bioinformatics algorithms (paracel‐blast (sensitive for detection of known viruses) and hidden Markov model (HMM; sensitive for distantly related viruses)). Among the 62 NMSCs, the virus family detected in the largest proportion of specimens was Mimiviridae (9/62 NMSCs). The majority of the virus‐related reads belonged to Papillomaviridae. The HMM analysis identified 86 additional previously not described viral contigs related to 11 virus families, with reads related to Mimiviridae being the most common (detected in 28/62 NMSCs) with the most prevalent contig (Mimivirus SE906, 1937 bp) detected in 17/62 NMSCs. Among the 27 other cancers, viral sequences were detected in only 5 specimens by blast analysis, compared to in all 27 specimens by HMM (Mimiviridae, Poxviridae, Phycodnaviridae and virus‐related sequences yet unclassified to any family). 99% of the virus reads belonged to a single previously not described sequence (Mimivirus SE996, 911 bp). A multitude of viruses is readily detectable in specimens with cancers occurring among the immunosuppressed, with sequences related to Mimiviridae being the most prevalent. Further research would be needed to elucidate the biological significance of the viruses.

Viruses in case series of tumors: Consistent presence in different cancers in the same subject.

Studies investigating presence of viruses in cancer often analyze case series of cancers, resulting in detection of many viruses that are not etiologically linked to the tumors where they are found. The incidence of virus-associated cancers is greatly increased in immunocompromised individuals. Non-melanoma skin cancer (NMSC) is also greatly increased and a variety of viruses have been detected in NMSC. As immunosuppressed patients often develop multiple independent NMSCs, we reasoned that viruses consistently present in independent tumors might be more likely to be involved in tumorigenesis. We sequenced 8 different NMSCs from 1 patient in comparison to 8 different NMSCs from 8 different patients. Among the latter, 12 different virus sequences were detected, but none in more than 1 tumor each. In contrast, the patient with multiple NMSCs had human papillomavirus type 15 and type 38 present in 6 out of 8 NMSCs.

Viremia preceding multiple sclerosis: Two nested case-control studies

Infections have been suggested to be involved in Multiple Sclerosis (MS). We used metagenomic sequencing to detect both known and yet unknown microorganisms in 2 nested case control studies of MS. Two different cohorts were followed for MS using registry linkages. Serum samples taken before diagnosis as well as samples from matched control subjects were selected. In cohort1 with 75 cases and 75 controls, most viral reads were Anelloviridae-related and >95% detected among the cases. Among samples taken up to 2 years before MS diagnosis, Anellovirus species TTMV1, TTMV6 and TTV27 were significantly more common among cases. In cohort2, 93 cases and 93 controls were tested under the pre-specified hypothesis that the same association would be found. Although most viral reads were again related to Anelloviridae, no significant case-control differences were seen. We conclude that the Anelloviridae-MS association may be due to multiple hypothesis testing, but other explanations are possible.