Certain polyomaviruses (PyVs), such as JC and BK, are known to cause severe disease in immunocompromised hosts (Moens et al., 2017Moens U. Krumbholz A. Ehlers B. Zell R. Johne R. Calvignac-Spencer S. et al.Biology, evolution, and medical importance of polyomaviruses: an update.Infect Genet Evol. 2017; 54: 18-38Crossref PubMed Scopus (76) Google Scholar). Human PyV (HPyV)6 and HPyV7 can be shed from skin surfaces without apparent disease (Hashida et al., 2018Hashida Y. Higuchi T. Matsuzaki S. Nakajima K. Sano S. Daibata M. Prevalence and genetic variability of human polyomaviruses 6 and 7 in healthy skin among asymptomatic individuals.J Infect Dis. 2018; 217: 483-493Crossref PubMed Scopus (14) Google Scholar; Schowalter et al., 2010Schowalter R.M. Pastrana D.V. Pumphrey K.A. Moyer A.L. Buck C.B. Merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin.Cell Host Microbe. 2010; 7: 509-515Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar), whereas in immunocompromised patients, the viruses may be associated with a pruritic eruption (Canavan et al., 2018Canavan T.N. Baddley J.W. Pavlidakey P. Tallaj J.A. Elewski B.E. Human polyomavirus-7-associated eruption successfully treated with acitretin.Am J Transplant. 2018; 18: 1278-1284Crossref PubMed Scopus (16) Google Scholar; Ho et al., 2015Ho J. Jedrych J.J. Feng H. Natalie A.A. Grandinetti L. Mirvish E. et al.Human polyomavirus 7-associated pruritic rash and viremia in transplant recipients.J Infect Dis. 2015; 211: 1560-1565Crossref PubMed Scopus (79) Google Scholar; Nguyen et al., 2017Nguyen K.D. Lee E.E. Yue Y. Stork J. Pock L. North J.P. et al.Human polyomavirus 6 and 7 are associated with pruritic and dyskeratotic dermatoses.J Am Acad Dermatol. 2017; 76: 932-940.e3Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar; Smith et al., 2018Smith S.D.B. Erdag G. Cuda J.D. Rangwala S. Girardi N. Bibee K. et al.Treatment of human polyomavirus-7-associated rash and pruritus with topical cidofovir in a lung transplant patient: case report and literature review.Transpl Infect Dis. 2018; 20: e12793Crossref Scopus (11) Google Scholar). We report the case of a 38-year-old woman who developed intractable pruritus and burning associated with a skin eruption 1 year after her second renal transplantation (Supplementary Figure S1a–c). Her immunosuppressive regimen consisted of tacrolimus and mycophenolate mofetil. Histopathology was characteristic of HPyV6- and HPyV7-associated eruptions (Ho et al., 2015Ho J. Jedrych J.J. Feng H. Natalie A.A. Grandinetti L. Mirvish E. et al.Human polyomavirus 7-associated pruritic rash and viremia in transplant recipients.J Infect Dis. 2015; 211: 1560-1565Crossref PubMed Scopus (79) Google Scholar; Nguyen et al., 2017Nguyen K.D. Lee E.E. Yue Y. Stork J. Pock L. North J.P. et al.Human polyomavirus 6 and 7 are associated with pruritic and dyskeratotic dermatoses.J Am Acad Dermatol. 2017; 76: 932-940.e3Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Immunohistochemistry confirmed the expression of PyV large tumor antigen, and immunofluorescence identified HPyV7 tumor antigen and HPyV6 and/or HPyV7 VP1 expression (Supplementary Figure S1d). Shotgun metagenomic sequencing revealed HPyV7 DNA and the absence of HPyV6 (Supplementary Figure S2). The addition of acitretin and reduction of mycophenolate mofetil dose eventually led to the resolution of symptoms (Supplementary Figure S1b and c). Detectable viral protein expression was absent in apparently normal skin after 15 weeks on acitretin and mycophenolate mofetil dose reduction (late-treatment time point) (Supplementary Figure S1d). Shotgun metagenomic sequencing of skin swabs from before treatment and early treatment (3 weeks on acitretin) revealed that 99.99% of mappable nonhuman reads were HPyV7, which is a high frequency, even in patients with viral skin disease (Tirosh et al., 2018Tirosh O. Conlan S. Deming C. Lee-Lin S.Q. Huang X. NISC Comparative Sequencing Program, et al.Expanded skin virome in DOCK8-deficient patients.Nat Med. 2018; 24: 1815-1821Crossref PubMed Scopus (42) Google Scholar). Surprisingly, HPyV7 continued to constitute 99.96% of nonhuman reads at the late-treatment time point (Figure 1a). However, the percentage of nonhuman reads among total reads was 59% before treatment compared with 4.5% at late treatment (Supplementary Figure S3), suggesting that the overall HPyV7 DNA burden had decreased. Bulk RNA sequencing revealed a 940-fold decrease in totally normalized HPyV7 transcripts in a late-treatment clinically normal-appearing skin specimen compared with the more affected pretreatment specimen (Figure 1b), demonstrating that clinical resolution was associated with a large reduction in viral transcription. During pathogenic infection with some PyVs, the regulatory region undergoes sequence rearrangements and can accumulate insertion or deletion nucleotide variants (indels) that affect transcription factor binding (Ajuh et al., 2018Ajuh E.T. Wu Z. Kraus E. Weissbach F.H. Bethge T. Gosert R. et al.Novel human polyomavirus noncoding control regions differ in bidirectional gene expression according to host cell, large T-antigen expression, and clinically occurring rearrangements.J Virol. 2018; 92 (e02231-17)Crossref PubMed Scopus (24) Google Scholar). The comparison of HPyV7 DNA and RNA sequences from our patient with a reference viral genome revealed subclonal polymorphisms scattered throughout the viral genome. Of note, a section of the regulatory region contained a high diversity of indels. Interestingly, an 8 bp deletion variant in this region (variant 2) was predominantly expressed in the more affected skin compared with that in the less affected skin, despite representing a minor variant at the DNA level in both samples when compared with the nonindel-containing species (variant 1) (Figure 1c and Supplementary Figure S4a and b). Functional prediction of this variant suggested disruption of a TATA-box, but no other definitive effects on likely transcription factor‒binding sites could be identified. Further investigation revealed that the novel transcript likely encodes a previously unrecognized agnoprotein. In addition to the expression of various agnoprotein sequence variants at this site, there were also multiple splice isoforms (Supplementary Figure S4c). Human BK and JC PyVs express agnoproteins, with little amino acid identity to the proposed HPyV7 agnoprotein, which have been suggested to regulate multiple viral and host functions (De Gascun and Carr, 2013De Gascun C.F. Carr M.J. Human polyomavirus reactivation: disease pathogenesis and treatment approaches.Clin Dev Immunol. 2013; 2013: 373579Crossref PubMed Scopus (53) Google Scholar). We next compared the DNA sequences from this highly variable region with those of the skin of 20 healthy volunteers with asymptomatic HPyV7 colonization (healthy Sequence Read Archive datasets). We identified a small number of common indels in the healthy volunteers, in contrast to the numerous distinct indels in our patient (uncommon indels). Similar distinct regulatory region indels were detected in the skin of a second, previously reported patient with HPyV7-associated eruption (Canavan et al., 2018Canavan T.N. Baddley J.W. Pavlidakey P. Tallaj J.A. Elewski B.E. Human polyomavirus-7-associated eruption successfully treated with acitretin.Am J Transplant. 2018; 18: 1278-1284Crossref PubMed Scopus (16) Google Scholar), upstream of the agnogene. The variants from this patient were also rare or absent in healthy volunteers, suggesting that these highly mutated viral regions were relatively restricted to diseased skin and may be associated with pathogenicity (Figure 1d and Supplementary Figure S4b). Pathway analysis of host gene expression in the RNA sequencing samples revealed induction of inflammatory genes associated with Type I IFNs, the antiviral response, and NKG2D ligands (Figure 2a). Consistent with this, there was a strong expression of the IFN-induced antiviral protein, MX1, in pretreatment epidermis, which was absent during late treatment (Figure 2b). Two of the most upregulated genes included innate antiviral defense proteins, APOBEC3A and APOBEC3B (Figure 2a). These single-stranded DNA deaminases potentially promote the generation of indels upon DNA repair (Burns et al., 2013Burns M.B. Temiz N.A. Harris R.S. Evidence for APOBEC3B mutagenesis in multiple human cancers.Nat Genet. 2013; 45: 977-983Crossref PubMed Scopus (487) Google Scholar; Green et al., 2016Green A.M. Landry S. Budagyan K. Avgousti D.C. Shalhout S. Bhagwat A.S. et al.APOBEC3A damages the cellular genome during DNA replication.Cell Cycle. 2016; 15: 998-1008Crossref PubMed Scopus (46) Google Scholar). APOBEC3A and/or APOBEC3B exhibited cytoplasmic and nuclear expression, most often within or near virally infected cells (Figure 2c). BK PyV has been shown to upregulate APOBEC3B (Verhalen et al., 2016Verhalen B. Starrett G.J. Harris R.S. Jiang M. Functional upregulation of the DNA cytosine deaminase APOBEC3B by polyomaviruses.J Virol. 2016; 90: 6379-6386Crossref PubMed Scopus (52) Google Scholar), which can produce viral mutations that alter glycan usage for viral entry and conversely facilitate escape from neutralizing antibodies (Peretti et al., 2018Peretti A. Geoghegan E.M. Pastrana D.V. Smola S. Feld P. Sauter M. et al.Characterization of BK polyomaviruses from kidney transplant recipients suggests a role for APOBEC3 in driving in-host virus evolution.Cell Host Microbe. 2018; 23 (628–35.e7)Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Although the RNA sequencing data suggested that affected skin exhibited innate immune activation, the host response was inadequate to achieve viral clearance. Considering the clinical resolution after tapering immunosuppression, we characterized the changes in the host immune response. Immunohistochemistry revealed a moderate CD4+ infiltrate before treatment and during late treatment, but CD8+ T cells infiltrated the epidermis only during late treatment (Figure 2d and Supplementary Figure S5). An HPyV7-like particle ELISA detected a high titer of circulating antiviral antibodies at late treatment but not at earlier time points. In contrast, antibodies to BK virus were present at each time point (Figure 2e). By using multiple orthogonal approaches, these data support a causative role for HPyV7 in this eruption. These results suggest that HPyV7 stimulates an innate immune response, but a protective antiviral response is impaired by treatment with mycophenolate mofetil. We further identified a previously undescribed agnogene within the HPyV7 genome prone to mutation during infection. Many of the observed mutations in the HPyV7 genome are consistent with host activation of APOBEC3 enzymes, as has been seen for other PyVs. Taken together, our findings are consistent with the hypothesis that iatrogenic impairment of immunosurveillance enabled abnormal viral proliferation and innate immune activation that induced APOBEC3A and/or APOBEC3B expression, which in turn led to viral mutagenesis that correlated with increased HPyV7 pathogenicity and clinical disease. Datasets related to this article can be found at https://www.ncbi.nlm.nih.gov/nuccore/MG674199.1 (human polyomavirus 7 complete genome), hosted at Genbank; https://submit.ncbi.nlm.nih.gov/subs/bioproject/SUB6080707/overview (metagenomic data), hosted at National Center for Biotechnology Information BioProject; https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE148169 (RNA sequencing data), hosted at Gene Expression Omnibus. There will be no restrictions on data availability. Rachel K. Rosenstein: http://orcid.org/0000-0001-6375-6129 Diana V. Pastrana: http://orcid.org/0000-0002-8084-5665 Gabriel J. Starrett: http://orcid.org/0000-0001-5871-5306 Matthew R. Sapio: http://orcid.org/0000-0002-8855-5419 Natasha T. Hill: http://orcid.org/0000-0001-9043-4643 Jay-Hyun Jo: http://orcid.org/0000-0002-2039-3425 Chyi-Chia R. Lee: http://orcid.org/0000-0002-5306-7781 Michael J. Iadarola: http://orcid.org/0000-0001-7188-9810 Christopher B. Buck: http://orcid.org/0000-0003-3165-8094 Heidi H. Kong: http://orcid.org/0000-0003-4424-064X Isaac Brownell: http://orcid.org/0000-0002-0090-9914 Edward W. Cowen: http://orcid.org/0000-0003-1918-4324 The authors state no conflict of interest. We sincerely thank the patient for participating in these investigations. We appreciate the technical support of the National Cancer Institute Center for Cancer Research genomics core facility and the National Institutes of Health Intramural Sequencing Center. We thank R. Wang and B. Elewski for their assistance with the sequencing of human polyomavirus 7 from the patient reported previously (Canavan et al., 2018Canavan T.N. Baddley J.W. Pavlidakey P. Tallaj J.A. Elewski B.E. Human polyomavirus-7-associated eruption successfully treated with acitretin.Am J Transplant. 2018; 18: 1278-1284Crossref PubMed Scopus (16) Google Scholar). We thank M. Taylor, T. Pillai, and the Segre lab for their underlying contributions. This work utilized the computational resources of the National Institutes of Health High Performing Computation Biowulf cluster (http://hpc.nih.gov). Conceptualization: RKR, CBB, HHK, IB, EWC; Data Curation: RKR, CCRL, HHK, EWC; Formal Analysis: RKR, DVP, GJS, MRS, NTH, JHJ; Funding Acquisition: MJI, CBB, HHK, IB, EWC; Investigation: RKR, DVP, GJS, MRS, NTH, JHJ, CCRL; Methodology: DVP, MRS, MJI, HHK, CBB, IB; Project Administration: RKR; Resources: MJI, CBB, HHK, IB, EWC; Software: GJS, MRS, JHJ; Supervision: MJI, HHK, CBB, IB, EWC; Validation: RKR, MRS; Visualization: RKR, DVP, GJS, MRS, NTH, JHJ, CCRL, IB; Writing - Original Draft Preparation: RKR, IB, EWC; Writing - Review and Editing: RKR, DVP, GJS, MRS, NTH, JHJ, CCRL, MJI, CBB, HHK, IB, EWC Experiments were performed in accordance with approved protocols by the institutional review board at the National Cancer Institute (NCT02471352 and NCT00001506). The patient provided written informed consent for clinical photography, sample acquisition, and subsequent analyses including sequencing studies. Download .xlsx (1.3 MB) Help with xlsx files Supplementary Table S1Host RNA-seq expression values as sFPKM and raw counts. RNA-seq, RNA sequencing; sFPKM, significant fragments per kilobase per million aligned reads.Supplementary Table S2. SRA datasets containing HPyV7 reads. HPyV, human polyomavirus; SRA, Sequence Read Archive. Patient care and sample collection were performed in accordance with approved protocols by the institutional review board at the National Cancer Institute (NCT02471352 and NCT00001506). The individual provided written informed consent for clinical photography, sample acquisition, and subsequent analyses including sequencing studies. QOL measurements such as the Dermatology Life Quality Index and Visual Analog Scales were administered. We have complied with all relevant ethical regulations. Relevant pretreatment medications: 1,000 mg mycophenolate mofetil (MMF) twice daily, 5 mg tacrolimus twice daily. Early-treatment medications included 500 mg MMF twice daily, 5 mg tacrolimus twice daily, and 25 mg acitretin twice daily. Late-treatment medications included 500 mg MMF daily, 5 mg tacrolimus twice daily, and 25 mg acitretin twice daily. After skin swabs were collected as described in each section below, 4–6 mm skin punch biopsy specimens were obtained from the patient and were frozen for RNA sequencing or placed in formalin for H&E and immunohistochemical analyses. The more affected specimen was a site of severe itch with hyperpigmentation and scale over the lower back. The less affected specimen was a site of mild itch, with normal pigmentation and scale over the flank. The late-treatment specimen was from a clinically normal-appearing site over the lower back that was previously affected. Skin swabs (from the abdomen, lower back, posterior thigh) were collected and frozen at pretreatment, early treatment (3 weeks on acitretin and 1 week on a tapered dose of 500 mg MMF twice daily), and late-treatment (15 weeks on acitretin and 9 weeks on a tapered dose of 500 mg MMF daily) time points. Peripheral blood was processed by the clinical laboratory for research purposes. After antigen retrieval (CC1 buffer, Ventana Medical Systems), tissue sections were incubated with the following primary antibodies: anti-CD4 antibody (Clone SP35, Prediluted, Catalog #790-4423, Roche, Basel Switzerland, RRID:AB_2335982), anti-CD8 antibody (Clone SP57, Prediluted, Catalog #790-4460, Roche, RRID:AB_2335985), and anti-simian virus 40 tumor antigen antibody (Clone PAb416, 1:300 dilution, Catalog #DP02, Calbiochem, San Diego, CA, RRID: AB_212848). PAb416 cross reacts with several human polyomavirus (HPyV) types. For the CD4 and/or CD8 double stain, unstained tissue sections were incubated with anti-CD4 antibody and labeled with a brown chromogen; the sections were subsequently incubated with anti-CD8 antibody and labeled with a red chromogen. The staining was performed on the Roche Ventana Medical Systems BenchMark ULTRA automated immunohistochemistry platform using the ultraview Universal DAB Detection Kit (Ventana Medical Systems, Oro Valley, AZ), following standard laboratory protocols established by the histology section of the Laboratory of Pathology at the National Institutes of Health. Microscopy and image acquisition were performed with Olympus BX41 microscope, Olympus UPlanSApo lenses, and Nikon NIS Elements Imaging software (Nikon 5.10.01 [Build, 1307] 64 bit) with Nikon 6 MP camera (Nikon, Tokyo, Japan) (calibrated per manufacturer’s specifications). Saline premoistened swabs were collected and stored at −80 oC; swabs were processed as described (Pastrana et al., 2018Pastrana D.V. Peretti A. Welch N.L. Borgogna C. Olivero C. Badolato R. et al.Metagenomic discovery of 83 new human papillomavirus types in patients with immunodeficiency.mSphere. 2018; 3 (e00645-18)Crossref PubMed Scopus (39) Google Scholar). Briefly, the swabs were soaked in 150 μl of nuclease buffer (Dulbecco’s PBS, 10 mM magnesium chloride, 0.5% Brij-58 [#P5884, Sigma-Aldrich, St. Louis, MO], and 0.1% Benzonase [#E1014, Sigma-Aldrich]) and incubated at room temperature for 15 minutes inside 2 ml of low protein-binding polypropylene centrifuge columns (#89896, Pierce Manufacturing, Appleton, WI). Columns were spun at 1,000g for 5 minutes and the flow through transferred to 2-ml siliconized microcentrifuge tubes. The columns were washed three times with wash buffer (Dulbecco’s PBS, 0.8 M of sodium chloride, and 0.5% Brij-58) by centrifugation as mentioned earlier. Swab extracts were loaded on top of iodixanol (#D1556, Optiprep, Sigma-Aldrich) step gradients consisting of 1.3 ml of each 27%, 33%, and 39% solutions of iodixanol mixed with Dulbecco’s PBS/0.8 M sodium chloride. The gradients were formed on Beckman ½ × 2 inch ml polyallomer centrifuge tubes (#326819) and spun at 234,000g for 3.5 hours at 16 oC. Six fractions of 730 μl were collected, and DNA was extracted from the individual fractions. For the extraction, 200 μl of each fraction was mixed with 50 μl of 5× digest buffer (250 mM Tris pH 8.0, 125 mM EDTA, 2.5% SDS, 2.5% Proteinase K (#191331, Qiagen, Hilden, Germany) and 50 mM dithiothreitol (#20291, Pierce Manufacturing). The samples were allowed to digest at 50 oC for 15 minutes and then heat inactivated at 72 oC for 10 minutes. DNA was precipitated from each sample with 125 μl of 7.5 M ammonium acetate and 2.6 volumes of 95% ethanol for 1 hour at room temperature and then overnight at 4 oC. The next day, samples were spun at 16,000g for 1 hour at room temperature and washed once with 70% ethanol. After air drying, samples were prepared for rolling circle amplification by resuspending with 10 μl of sample buffer from a TempliPhi kit (#GE25-6400-10, Sigma-Aldrich), placed at 95 oC for 3 minutes, allowed to cool down, and mixed with 10 μl of reaction buffer and 0.4 μl of enzyme mix from the TempliPhi kit. Rolling circle amplification reactions were carried out at 30 oC for 16–24 hours and then heat inactivated at 65 oC for 10 minutes. Reactions were ethanol precipitated as before and resuspended in 75 μl of diluted 2 mM Tris-hydrochloric acid pH and 0.2 mM EDTA solution. Samples were processed with the Nextera XT DNA sample kit (Illumina, San Diego, CA) and sent to the National Cancer Institute Center for Cancer Research genomics core facility for deep sequencing with the Illumina MiSeq platform. Reads were assembled into contigs using CLC Genomics Workbench 10.1.1 (RRID: SCR_011853, Qiagen). The contigs were compared with the National Center for Biotechnology Information nr database using BLASTX, and a contig with 99% identity to HPyV7 (GenBank NC_014407) was identified. Of a total of 1,567,812 sample reads, 17% (271,381) successfully mapped to the HPyV7 reference genome (Schowalter et al., 2010Schowalter R.M. Pastrana D.V. Pumphrey K.A. Moyer A.L. Buck C.B. Merkel cell polyomavirus and two previously unknown polyomaviruses are chronically shed from human skin.Cell Host Microbe. 2010; 7: 509-515Abstract Full Text Full Text PDF PubMed Scopus (429) Google Scholar). The complete consensus sequence of the HPyV7 of this patient was generated from this alignment and deposited to GenBank, receiving the accession number MG674199. Formalin-fixed, paraffin-embedded tissue sections were stained for tumor antigen, VP1, APOBEC3A and/or APOBEC3B, and MX1 as previously described (Kommagani et al., 2009Kommagani R. Leonard M.K. Lewis S. Romano R.A. Sinha S. Kadakia M.P. Regulation of VDR by deltaNp63alpha is associated with inhibition of cell invasion.J Cell Sci. 2009; 122: 2828-2835Crossref PubMed Scopus (31) Google Scholar; Leonard et al., 2011Leonard M.K. Kommagani R. Payal V. Mayo L.D. Shamma H.N. Kadakia M.P. ΔNp63α regulates keratinocyte proliferation by controlling PTEN expression and localization.Cell Death Differ. 2011; 18: 1924-1933Crossref PubMed Scopus (46) Google Scholar). mAbs 2t10, 6V32, and 10-87-13 were produced as reported previously (Pastrana et al., 2010Pastrana D.V. Pumphrey K.A. Cuburu N. Schowalter R.M. Buck C.B. Characterization of monoclonal antibodies specific for the Merkel cell polyomavirus capsid.Virology. 2010; 405: 20-25Crossref PubMed Scopus (16) Google Scholar; Leonard et al., 2015Leonard B. McCann J.L. Starrett G.J. Kosyakovsky L. Luengas E.M. Molan A.M. et al.The PKC/NF-κB signaling pathway induces APOBEC3B expression in multiple human cancers.Cancer Res. 2015; 75: 4538-4547Crossref PubMed Scopus (79) Google Scholar). The 2t10 antibody recognizes an epitope present in HPyV7 large tumor and small tumor antigens. The 6V32 antibody recognizes both HPyV6 and HPyV7 VP1. The 10-87-13 antibody was generated against APOBEC3B and cross reacts with APOBEC3A and APOBEC3G and was a generous gift from Dr Reuben Harris (also available from the National Institutes of Health acquired immunodeficiency syndrome reagent program [https://antibodyregistry.org/search.php?q=AB_2721202]). Anti-MX1 (ab95926) was purchased from Abcam (Catalog #, RRID:AB_10677452, Abcam Biotechnology company, Cambridge, United Kingdom). Antibodies were used at a 1:100 dilution (2t10 and 6V32), 1:200 dilution (ab95926), or 1:1,000 dilution (10-87-13) and incubated overnight at 4 oC. The secondary antibodies were anti-mouse Alexa Fluor-488 (Catalog #1858182, RRID: AB_2536161) and anti-rabbit Alexa Fluor-568 (Catalog #A11011, RRID: AB_143157) (ThermoFisher Scientific, Carlsbad, CA). Slides were imaged using a Zeiss A1 Fluorescent Microscope at ×20 magnification (Carl Zeiss AG, Oberkochem, Germany) and an AxioCam MRm with AxioVision Rel 4.8 software. The acquisition time for Supplementary Figure S1d tumor antigen images was FITC 300 ms. The acquisition time for Supplementary Figure S1d VP1 images was FITC 300 ms. The acquisition time for Figure 2b was Texas Red (TR) 150 ms. Acquisition times for Figure 2c VP1 images were DAPI 100 ms for before treatment and 75 ms for late treatment, FITC 750 ms, TR 150 ms. Acquisition times for Figure 2c tumor antigen images were DAPI 40 ms, FITC 300 ms, TR 600 ms. Skin swabs were collected after premoistening with yeast cell lysis buffer (MasterPure Yeast DNA Purification Kit, Epicentre, Madison, WI) and stored at −80 oC. Procedures for DNA extraction, library generation, and sequencing were performed as previously described (Oh et al., 2014Oh J. Byrd A.L. Deming C. Conlan S. NISC Comparative Sequencing Program Kong H.H. et al.Biogeography and individuality shape function in the human skin metagenome.Nature. 2014; 514: 59-64Crossref PubMed Scopus (538) Google Scholar). Samples were incubated in yeast cell lysis buffer and Ready lyse (Epicentre) for 30 minutes at 37 oC and then mechanically disrupted using 5 mm stainless steel beads (Qiagen) in a Tissuelyser (Qiagen) for 2 minutes, 30 Hz. Samples were incubated for 30 minutes at 65 oC and placed on ice for 5 minutes, and debris was spun down after treatment with MPC protein precipitation reagent. Samples were combined with 350 μl of 100% ethanol and column purified using the Invitrogen PureLink Genomic DNA. Finally, samples were eluted in 30 μl of water (Qiagen). Control swabs were collected at the time of sample collection after premoistening with yeast cell lysis buffer and were waved in the ambient environment without contacting the patient’s skin for a similar duration of time as experimental swabs. Control swabs also underwent the same DNA extraction processes and sequencing along with experimental samples, and no apparent contamination from either reagents or experimental procedures were observed. Nextera XT library kits were used to generate Illumina libraries per the manufacturer’s instructions. Libraries were sequenced on an Illumina HiSeq at the National Institutes of Health Intramural Sequencing Center to a target of 30–100 million clusters of 2 × 125 bp reads. In total, for three time points, we obtained 554 million total reads and 172 million of nonhuman, quality-filtered paired-end total reads (median 26 million reads per sample) compared with those of an effective negative control. To assess the abundance of each taxon within the metagenome, MetaPhlAn (RRID: SCR_004915), version 2.0, was used (Truong et al., 2015Truong D.T. Franzosa E.A. Tickle T.L. Scholz M. Weingart G. Pasolli E. et al.MetaPhlAn2 for enhanced metagenomic taxonomic profiling.Nat Methods. 2015; 12: 902-903Crossref PubMed Scopus (931) Google Scholar). The sequence data from this study have been submitted to the National Center for Biotechnology Information BioProject under accession number PRJNA556827. Skin biopsies were flash frozen on dry ice and stored at −80 °C. Qiazol (Qiagen N.V., Venlo, Netherlands) solution was added to frozen samples, and RNA extraction was performed using a Fastprep 24 bead-beating homogenizer (MPBio, Santa Ana, CA; Lysing Matrix D) and the RNeasy Minikit (Qiagen). Stranded paired-end libraries were generated from 1 μg total RNA using the TruSeq Stranded Total RNA Library Prep Gold kit (Illumina) and sequenced on an Illumina HiSeq4000 (2 × 76 bp read length). Adapters were Integrated DNA Technologies (IDT, Coralville, IA) for Illumina - TruSeq RNA UD Indexes to eliminate index misassignment. Amplification was performed using 10 cycles, which was optimized for the input amount and to minimize overamplification. Libraries were pooled in equimolar amounts for sequencing. Sequencing was performed in two batches according to when the skin biopsy specimens were collected. In the first batch, more affected and less affected sites were biopsied, and two technical replicates of each were sequenced at an average of 45 million reads per technical replicate. For the second batch, a late-treatment biopsy specimen was collected and sequenced together with a third replicate of the original two biopsy specimens at an average read depth of 235 million reads. Alignment and quantification of count data were performed using MAGIC, version 2017.05.10, as previously described (LaPaglia et al., 2018LaPaglia D.M. Sapio M.R. Burbelo P.D. Thierry-Mieg J. Thierry-Mieg D. Raithel S.J. et al.RNA-seq investigations of human post-mortem trigeminal ganglia.Cephalalgia. 2018; 38: 912-932Crossref PubMed Scopus (44) Google Scholar). Expression levels were estimated by calculating significant fragments per kilobase per million aligned reads (Supplementary Table S1), a metric of expression calculated to correct for library and/or protocol biases (Zhang et al., 2015Zhang W. Yu Y. Hertwig F. Thierry-Mieg J. Zhang W. Thierry-Mieg D. et al.Comparison of RNA-seq and microarray-based models for clinical endpoint prediction.Genome Biol. 2015; 16: 133Crossref PubMed Scopus (198) Google Scholar). Host RNA sequencing was displayed with heatmaps. The flame scale illustrates the range of expression ratios from 1.0 (yellow) to 0.0 (blue). To study viral gene expression, all RNA sequencing reads were aligned against a fusion reference containing the hg38 and HPyV7 (GenBank Accession #: NC_014407) reference genomes using STAR 2.5.3ab (RRID: SCR_015899) with the --chimSegmentMin 36 argument (Dobin et al., 2013Dobin A. Davis C.A. Schlesinger F. Drenkow J. Zaleski C. Jha S. et al.STAR: ultrafast universal RNA-seq aligner.Bioinformatics. 2013; 29: 15-21Crossref PubMed Scopus (15203) Google Scholar). Alignments and junctions were visualized using IGV (RRID: SCR_011793) (Robinson et al., 2011Robinson J.T. Thorvaldsdóttir H. Winckler W. Guttman M. Lander E.S. Getz G. et al.Integrative genomics viewer.Nat Biotechnol. 2011; 29: 24-26Crossre
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