Alberto Peretti

The ancient evolutionary history of polyomaviruses

Polyomaviruses are a family of DNA tumor viruses that are known to infect mammals and birds. To investigate the deeper evolutionary history of the family, we used a combination of viral metagenomics, bioinformatics, and structural modeling approaches to identify and characterize polyomavirus sequences associated with fish and arthropods. Analyses drawing upon the divergent new sequences indicate that polyomaviruses have been gradually co-evolving with their animal hosts for at least half a billion years. Phylogenetic analyses of individual polyomavirus genes suggest that some modern polyomavirus species arose after ancient recombination events involving distantly related polyomavirus lineages. The improved evolutionary model provides a useful platform for developing a more accurate taxonomic classification system for the viral family Polyomaviridae.

Improved detection reveals active β -papillomavirus infection in skin lesions from kidney transplant recipients

The aim of this study was to determine whether detection of β-HPV gene products, as defined in epidermodysplasia verruciformis skin cancer, could also be observed in lesions from kidney transplant recipients alongside the viral DNA. A total of 111 samples, corresponding to 79 skin lesions abscised from 17 kidney transplant recipients, have been analyzed. The initial PCR analysis demonstrated that β-HPV-DNA was highly present in our tumor series (85%). Using a combination of antibodies raised against the E4 and L1 proteins of the β-genotypes, we were able to visualize productive infection in 4 out of 19 actinic keratoses, and in the pathological borders of 1 out of 14 squamous cell carcinomas and 1 out of 31 basal cell carcinomas. Increased expression of the cellular proliferation marker minichromosome maintenance protein 7 (MCM7), that extended into the upper epithelial layers, was a common feature of all the E4-positive areas, indicating that cells were driven into the cell cycle in areas of productive viral infections. Although the present study does not directly demonstrate a causal role of these viruses, the detection of E4 and L1 positivity in actinic keratosis and the adjacent pathological epithelium of skin cancer, clearly shows that β-HPV are actively replicating in the intraepidermal precursor lesions of kidney transplant recipients and can therefore cooperate with other carcinogenic agents, such as UVB, favoring skin cancer promotion.

The Nuclear DNA Sensor IFI16 Acts as a Restriction Factor for Human Papillomavirus Replication through Epigenetic Modifications of the Viral Promoters

ABSTRACT The human interferon-inducible IFI16 protein, an innate immune sensor of intracellular DNA, was recently demonstrated to act as a restriction factor for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1) infection by inhibiting both viral-DNA replication and transcription. Through the use of two distinct cellular models, this study provides strong evidence in support of the notion that IFI16 can also restrict human papillomavirus 18 (HPV18) replication. In the first model, an immortalized keratinocyte cell line (NIKS) was used, in which the IFI16 protein was knocked down through the use of small interfering RNA (siRNA) technology and overexpressed following transduction with the adenovirus IFI16 (AdVIFI16) vector. The second model consisted of U2OS cells transfected by electroporation with HPV18 minicircles. In differentiated IFI16-silenced NIKS-HPV18 cells, viral-load values were significantly increased compared with differentiated control cells. Consistent with this, IFI16 overexpression severely impaired HPV18 replication in both NIKS and U2OS cells, thus confirming its antiviral restriction activity. In addition to the inhibition of viral replication, IFI16 was also able to reduce viral transcription, as demonstrated by viral-gene expression analysis in U2OS cells carrying episomal HPV18 minicircles and HeLa cells. We also provide evidence that IFI16 promotes the addition of heterochromatin marks and the reduction of euchromatin marks on viral chromatin at both early and late promoters, thus reducing both viral replication and transcription. Altogether, these results argue that IFI16 restricts chromatinized HPV DNA through epigenetic modifications and plays a broad surveillance role against viral DNA in the nucleus that is not restricted to herpesviruses. IMPORTANCE Intrinsic immunity is mediated by cellular restriction factors that are constitutively expressed and active even before a pathogen enters the cell. The host nuclear factor IFI16 acts as a sensor of foreign DNA and an antiviral restriction factor, as recently demonstrated by our group for human cytomegalovirus (HCMV) and herpes simplex virus 1 (HSV-1). Here, we provide the first evidence that IFI16 inhibits HPV18 replication by repressing viral-gene expression and replication. This antiviral restriction activity was observed in immortalized keratinocytes transfected with the religated genomes and in U2OS cells transfected with HPV18 minicircles, suggesting that it is not cell type specific. We also show that IFI16 promotes the assembly of heterochromatin on HPV DNA. These changes in viral chromatin structure lead to the generation of a repressive state at both early and late HPV18 promoters, thus implicating the protein in the epigenetic regulation of HPV gene expression and replication.

Genome Sequence of a Fish-Associated Polyomavirus, Black Sea Bass (Centropristis striata) Polyomavirus 1

ABSTRACT All known polyomaviruses are associated with mammals or birds. Using virion enrichment, random-primed rolling circle amplification, and deep sequencing, we identified a polyomavirus associated with black sea bass (Centropristis striata). The virus has a variety of novel genetic features, suggesting a long evolutionary separation from polyomaviruses of terrestrial animals.

α- and β-Papillomavirus infection in a young patient with an unclassified primary T-cell immunodeficiency and multiple mucosal and cutaneous lesions

BACKGROUND Correlating human papillomavirus (HPV) type with the clinical and histopathological features of skin lesions (from genital and nongenital sites) can present a diagnostic challenge. OBJECTIVE In this study, HPV infection patterns were correlated with pathology and clinical presentation in lesional and nonlesional body sites from a young patient with a primary T-cell immunodeficiency. METHODS HPV infection was evaluated at both DNA and protein levels by polymerase chain reaction and immunohistochemistry. RESULTS The patients genital lesions were caused exclusively by α-genotypes (high-risk type HPV-51 in the anal and low-risk type HPV-72 in the penile condylomas). The opposite was true for the skin lesions, which were infected by β-genotypes alone (HPV-8 and HPV-24). HPV-24 was the predominant type in terms of viral load, and the only one found in productive areas of infection. The patient had already developed high-grade dysplasia in the anal condyloma-like lesions, and showed areas of early-stage dysplasia in the lesions caused by the β-genotype HPV-24. LIMITATIONS The basic origin of the immunodeficiency is not yet defined. CONCLUSION These findings provide proof of principle that both α- and β-genotypes can cause overt dysplastic lesions when immunosurveillance is lost, which is not restricted to epidermodysplasia verruciformis.

Analysis of human β-papillomavirus and Merkel cell polyomavirus infection in skin lesions and eyebrow hair bulbs from a cohort of patients with chronic lymphocytic leukaemia.

Research demonstrates an increased incidence of skin cancer in immunocompromised hosts, including patients with chronic lymphocytic leukaemia (CLL) and organ transplant recipients (OTRs). Active human β‐papillomavirus (β‐HPV) infection has been found in OTR skin lesions, suggesting its possible involvement in skin carcinogenesis. Merkel cell polyomavirus (MCPyV) has also been reported in cases of skin cancer.

Identification of the skin virome in a boy with widespread human papillomavirus-2-positive warts that completely regressed after administration of tetravalent human papillomavirus vaccine

DEAR EDITOR, Human papillomavirus (HPV) infections are normally controlled by an intact cell-mediated and humoral immune system. Therefore patients with cell-mediated immunodeficiencies – whether primary, secondary or iatrogenic – are all at increased risk of developing extensive, persistent and recurrent warts. However, the sequestration of HPV in epithelial cells can provide protection for the virus, resulting in inefficient activation of innate immunity, poor priming of the adaptive response and persistence of infections, even in immunocompetent individuals. In addition, genome variations, especially those enhancing viral promoter activities, have been associated with enhanced viral replication and unusual clinical manifestations. A 16-year-old boy presented in October 2012 with a 4-year history of recalcitrant multiple large, hyperkeratotic, fissured, cauliflower-like plantar warts on the right foot. Some small warts were also developing on his hands (Fig. 1, upper row). He had no relevant personal or family history of other illnesses or dermatological conditions, and was HIV negative. He had previously been treated with numerous therapies, including keratolytics, diathermics, cryotherapy, photodynamic therapy and topical imiquimod without any improvement. As the warts kept enlarging and spreading, affecting the patient’s quality of life, we decided to treat the patient with intravenous cidofovir (5 mg kg 1 for five cycles). The treatment was well tolerated, with no sign of wart regression 3 months posttreatment. Immunophenotype analysis of the patient’s peripheral blood mononuclear cells failed to reveal any abnormalities, and his immunoglobulin levels were also shown to lie within the normal range. The DNA extracted from swabs obtained from the skin overlying the plantar warts, from unaffected sites (pooled from different sites and from the forehead separately) and from plucked eyebrows was analysed by polymerase chain reaction (PCR) for the aand b-HPV genotypes. Positivity for a-types was found for the pooled unaffected skin swabs and for the plantar warts. Biopsies from the plantar warts were available as formalinfixed paraffin-embedded blocks. As detection of the anti-E4 protein provides a well-established biomarker of productive HPV infection, tissue sections from these blocks were stained by immunofluorescence using a panel of in-house anti-E4 antibodies against the cutaneous genotypes a2, l1 and c4. As shown in the lower panels of Figure 1, only anti-E4 HPV2 showed cytoplasmic staining in the more superficial layers. HPV2 infection, with a reported prevalence in multiple common warts of 28–34% in the European population, was confirmed by fluorescence in situ hybridization using the HPV2 genome as a probe, with many cells staining positive for both nuclear HPV2 and cytoplasmic E4 (Fig. 1). Consistent with these findings, HPV2-specific real-time quantitative PCR (Data S1; see Supporting Information) revealed very high viral loads (up to 417 copies per cell) in skin swabs from the plantar warts, as well as in the unaffected skin sites (180 copies per cell). According to the clinical presentation, the histology of the warts showed prominent papillomatosis, acanthosis, hypergranulosis and hyperkeratosis of the horny layer. A marked clearing of the cytoplasm in granular cells (‘koilocytosis’) and numerous keratohyalin granules were also present (Fig. 1, bottom left). The decision was taken to vaccinate the patient, based on the following: (i) sporadic literature reports on the cross-protective potential of the quadrivalent HPV vaccine (types 6, 11, 16 and 18); (ii) 65% identity between the L1 protein from HPV2 and the vaccine types, which supports a possible low-level cross-protection; and (iii) the failure of conventional therapies. The vaccine was administered in the patient’s arm in three shots (August and October 2013 and February 2014). Improvement was noticed as early as 6 weeks after receiving the first injection. When the patient returned for a follow-up assessment in November 2013, the warts had significantly decreased in size and thickness, and by March 2014 they had completely regressed. Accordingly, the viral load values of skin swabs from the plantar region had also dropped to very low levels (2 copies per cell). To gain more insight into the patient’s skin viral microbiota, DNA extracted from skin swabs from the affected foot was rolling-circle amplified and deep sequenced. Sequencing reads were cleaned to remove any human sequences and compared with genomes available in the Papillomavirus Episteme (PaVE) database (http://pave.niaid.nih.gov/). Those that could not be assigned to HPV genomes were further compared with whole viral and bacterial genome datasets available in the National Center for Biotechnology Information databases. Thirty percent of all reads (45 343) could be assigned to HPV genomes. A very small fraction (156 reads) displayed matches

Erratum: Improved detection reveals active β-papillomavirus infection in skin lesions from kidney transplant recipients (Modern Pathology (2014) 27 (917) DOI:10.1038/modpathol.2014.2)

We gratefully acknowledge ‘Fondazione Banca Popolare di Novara per il territorio’ for their contribution in purchasing the digital scanner Pannoramic MIDI, and ‘Stichting Pathologie Onderzoek en Ontwikkeling’ for supporting the HPV genotyping. This work was supported by grants from Compagnia di San Paolo (CSP2012 to MG) and Associazione Italiana per la Ricerca sul Cancro—AIRC (IG 2012 n. 12850 to MG), ESCMID Research Grant 2013 (ESCMID 2013 to CB), and from the Ministry of Education, University and Research—MIUR (FIRB 2008 to MDA). The PhD Fellowships of Simone Lanfredini and Alberto Peretti are funded by Compagnia San Paolo and by ‘Fondazione Franca Capurro per Novara Onlus’, respectively. Modern Pathology (2014) 27, 917