Maximum Likelihood (ML) tree of papillomaviruses (PV).

<p>ML tree comprising a representative set of 79 types including our two novel types (AsPV1, RnPV2) and one variant (MmuPV1 variant), as inferred from predicted E6, E7, E1, E2, L2, and L1 aa sequence analysis (3,720 aa positions, of which 69% were parsimony-informative). Generic PV clades <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047164#pone.0047164-Bernard1" target="_blank">[11]</a> are indicated by Greek lettering. Supertaxa are colored red (Alpha+Omikron-PV), green (Beta+Xi-PV), blue (Delta+Zeta-PV), and ocher (Lamda+Mu-PV), respectively. Branch lengths are drawn to scale, with the scale bar indicating the number of nt substitutions per site. Numbers on branches are bootstrap support values under the ML criterion; values under 50 are not shown. The novel PV types and variant of our present study are highlighted by arrows. According to the common PV-nomenclature our novel variant of <i>Mus musculus</i> is referred to MmuPV1 variant.</p

Genome organization of RnPV2, MmuPV1 variant, and AsPV1.

<p>Boxes indicating PV genes are drawn to scale and genes are drawn in three lines representing three putative open reading frames relative to nt position zero at the beginning of the upstream regulatory region. The polyadenylation sites are indicated with triangles.</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

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

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

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

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

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

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