MnPV viral load in skin biopsies.

<p><b>Panel A:</b> The red color shows viral genomes in the suprabasal layers of normal skin visualized by DNA <i>in situ</i> hybridization. The dotted line marks the basal membrane. <b>Panel B:</b> MnPV DNA <i>in situ</i> hybridization of a papilloma. Hyperproliferation of the epidermis can be observed, as well as basal and parabasal cells harboring MnPV DNA. Original magnification panel A: 80×; panel B: 40×. <b>Panel C:</b> qPCR analyses to detect viral DNA were performed by amplifying a fragment of the late L1 ORF. Data were normalized by relating the values to the copy number of β-globin and considering two copies of the gene as a cell equivalent. Tissue samples were taken from normal skin from animals from the naturally infected colony [control (n = 19), vaccinated (n = 19)] and the experimentally infected colony [control (n = 21), vaccinated (n = 22)]. Boxes span the interquartile range and contain the median as a horizontal line. Outliers (•) are depicted outside the 10th and 90th percentile (whiskers). Statistical significance was assessed by the Mann-Whitney test: ***, p<0.001; ****, p<0.0001. Median age for controls: 15.4 months (range: 8.8–20.4 months); vaccinated: 15.1 months (range: 6.7–21.3 months).</p

Humoral immune response to VLP vaccination.

<p><b>Panel A:</b> Antibody titers against the major capsid protein of MnPV were measured by VLP-ELISA two weeks after the third vaccination. The end point titer was determined as the reciprocal of the highest serum dilution with an OD above the blanks. Sera were measured from animals of the naturally and experimentally infected colonies as indicated. Statistical significance was assessed by the Mann-Whitney test: ****, p<0.0001. CTR = unvaccinated controls; VAC = vaccinated animals. <b>Panel B:</b> Correlation between the titer of neutralizing antibodies and anti-L1 antibody titers measured by VLP-ELISA. Sera were obtained from animals of all groups (preimmune sera, n = 20; control animals, n = 32; vaccinated animals, n = 34; immunosuppressed control animals, n = 12; immunosuppressed vaccinated animals, n = 9). The correlation coefficient (r²) was 0.8919 and the slope of the regression line 0.9048, demonstrating a strong correlation between both methods. n: indicates the number of animals.</p

Overview of the vaccination study.

<p><b>Panel A:</b> Vaccination was performed on both naturally infected <i>Mastomys</i> and on virus-free animals, which were subsequently infected. A subgroup of each colony also was kept under immunosuppressive conditions. <b>Panel B:</b> Electron micrograph showing MnPV VLPs with a size of 55 nm. <b>Panel C:</b> Time course of the vaccination study. Numbers indicate the time in months. Animals were vaccinated and bled as depicted. The green asterisk marks when the virus-free animals were experimentally infected. The red line indicates the duration of the treatment with cyclosporine A for the corresponding subgroups (for details, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003924#ppat-1003924-t001" target="_blank">Table 1</a>).</p

Inhibition of virus infection by passive immunization.

<p>To assess the role of neutralizing antibodies, polyclonal serum from vaccinated animals was injected intraperitoneally one day before challenge with MnPV infectious particles. After one week, RNA was extracted from the infected area to detect the MnPV specific E1∧E4 transcript by RT-PCR. GAPDH expression was used as internal control. Three animals were analyzed per group.</p

Tumor histology.

<p>A representative example of a virus-induced keratoacanthoma (A, C, E and G) and an epidermal carcinoma (B, D, F and H) is shown. Epidermal carcinomas appeared in one animal from the immunocompetent naturally infected colony and one animal from the immunocompetent experimentally infected colony. <b>Panel A:</b> Part of keratin filled craters (X) can be seen in the upper left. Irregular epidermal acanthotic proliferations (O) push into the dermis with preservation of a boundary (arrowheads). Apoptotic bodies can be seen (arrow). <b>Panel B:</b> Upper part of an epidermal carcinoma with ulceration, characterized by irregularly shaped strands of atypical keratinocytes invading the inflamed stroma. The inset shows deeper solid parts of tumor with high rates of mitosis and increased nuclear cytoplasmic ratios amongst other cellular atypical features. <b>Panel C:</b> At the lateral margin of a keratoacanthoma, a thickened highly proliferative basal layer can be seen by Ki-67 label. The upper layers also show some Ki-67 positivity. <b>Panel D:</b> In an epidermal carcinoma almost all cells are Ki-67-positive, showing the high proliferative rate of the tumor. Small tumor islets (X) invade the stroma. <b>Panel E:</b> Immunostaining against keratin 14 shows a succinct demarcation of proliferative epidermis from dermal stroma. <b>Panel F:</b> Keratin 14 immunohistology, showing the dropping off of tumor cells into the stroma (arrowheads). <b>Panel G:</b> By laminin immunohistochemistry, a separation of epidermal proliferation from the dermal stroma can be visualized. The inset shows a higher magnification. <b>Panel H:</b> Laminin staining is not continuous, constituting an additional criterion for the invasive nature of the epidermal carcinoma. Original magnification panels A, B and B-inset: 100×; panels C, D, G and H: 200×, panels E, F and G-inset: 400×.</p

Time course of the anti-L1 antibody titer.

<p>The time course of antibody production was studied in the naturally infected colony, both in control (A) and vaccinated animals (B). Additionally, the same was measured in control (C) and vaccinated animals (D) from the former virus-free colony. Antibody titers were measured by VLP-ELISA at different time-points and are displayed as box plots. Boxes span the interquartile range and contain the median as a horizontal line. Outliers (•) are depicted outside the 10th and 90th percentile (whiskers). NAT = naturally infected colony; EXP = originally virus-free colony after experimental infection; IS = immunosuppressed animals; CTRL = vaccinated virus-free animals (for number of animals, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003924#ppat-1003924-t001" target="_blank">Table 1</a>).</p

Incidence of virus-induced tumors.

<p>Immunocompetent and immunosuppressed animals were monitored over a period of 20 months for the occurrence of skin tumors (for number of animals, see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003924#ppat-1003924-t001" target="_blank">Table 1</a>). The Kaplan-Meier plots (<b>Panels A–D</b>) show the tumor incidence for each group in vaccinated (dashed line) and control animals (full line). Tumor-bearing animals displayed 1–4 skin tumors each (mean = 1.4). Green arrows indicate the time points of vaccination. The red arrow indicates the time of experimental infection, the blue arrow the start of the CsA feed, the purple arrow of both. The small vertical bars indicate censored animals which died for unknown reasons before tumor development. Differences between vaccinated and control animals were evaluated using the log rank test. The number of animals is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003924#ppat-1003924-t001" target="_blank">Table 1</a>. <b>Panels E/F:</b> Papillomas and keratoacanthomas in the lower back (infection site) of control animals from the experimentally infected colony. <b>Panels G/H:</b> Papillomas and keratoacanthomas arising in control animals from the naturally infected colony. Bars: 1 cm.</p

Molecular analyses of tumor-bearing animals.

<p><b>A)</b> Viral load in tissue samples from UV-irradiated and control animals from the MnPV-infected colony analyzed by qPCR and normalized to a plasmid standard. Samples were grouped according to their origin as indicated (ctrl skin: skin from unirradiated animals; ui skin/UV skin: unirradiated or UV-irradiated skin from irradiated animals; KSCC/nKSCC: UV-induced SCCs; non-UV tumor: tumors from non-UV sites of irradiated animals and spontaneous tumors from unirradiated animals). UV^+/- indicates whether the animal was UV-exposed or not (Kruskal-Wallis test, *p<0.05, ***p<0.001, ^nsp>0.05). <b>B)</b> Southern blot analysis of unirradiated and UV-irradiated skins, a KSCC and a non-UV tumor. DNA was digested with ApaI (no cleavage site in MnPV), XbaI (one site) or XhoI (two sites) as indicated (Form I: supercoiled; Form II: relaxed circular; Form III: linear form of MnPV). <b>C)</b> Semi-quantitative RT-PCR for the most abundant MnPV <i>E1^E4</i> transcript in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>. <b>D)</b> Semi-quantitative RT-PCR for MnPV <i>E6</i>, <i>E7</i> and <i>L1</i> transcripts in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>.</p

Schematic overview of the mechanism suggested for UV-induced NMSC development in <i>Mastomys coucha</i>.

<p><b>A)</b> MnPV infects basal epithelial cells of the skin of young animals via small injuries. <b>B)</b> MnPV genome is amplified in stratified skin layers (pink and red nuclei) and new virions are released. <b>C)</b> UVB irradiation of the skin. <b>D)</b> UVB-irradiated skin is hyperproliferative, favoring viral replication and virion formation. UVB-induced photoproducts, e.g. in <i>Trp53</i>, occur in keratinocytes (altered nuclei). In uninfected cells, damages are repaired. In infected cells, MnPV-E6/E7 reduce chromosomal stability and inhibit DNA repair. Mutations can accumulate and altered cells become neoplastic. <b>E)</b> Neoplastic squamous cells (light blue) start forming a well-differentiated keratinizing SCC, still representing a permissive system that allows viral replication and formation of virions. <b>F)</b> When neoplastic squamous cells accumulate further mutations (dark blue), a spindle cell phenotype is acquired, forming a poorly differentiated SCC that may become ulcerated. MnPV cannot replicate in dedifferentiated cells and the viral DNA is subsequently lost.</p

Summary of the absolute numbers of tumors in the different groups, percentage of tumor-bearing animals and median time of tumor development.

<p>Summary of the absolute numbers of tumors in the different groups, percentage of tumor-bearing animals and median time of tumor development.</p