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<p>In this section we describe our method of isolation and quantification of MmuPV1 virions from papillomas arising in nude mice that can be serially passaged or used to infect other strains of mice. In our studies we have used both crude virus extracts or purified virus particles. Crude virus extract refers to virus present in supernatants obtained by clarifying papillomatous tissue extracts from nude mice. Our method of generation of crude virus extract has been modified from a previous protocol used to isolate merkel cell polyomavirus from human skin swabs(1). Purified virus particles refer to crude extract clarified over an optiprep gradient. Optiprep gradients have been used to purify papillomavirus generated in <em>vitro</em> (2) and have been described in detail on Dr. Chris Buck’s website: <a href="https://home.ccr.cancer.gov/LCO/pseudovirusproduction.htm#_ENREF_7" target="_blank">https://home.ccr.cancer.gov/LCO/pseudovirusproduction.htm#_ENREF_7</a>. We measure the amount of encapsidated viral genomic DNA to determine the concentration of "viral genome equivalents" (VGE) in any given stock of virus. For this we use a standard agarose gel electrophoresis followed by SYBR-green staining. The same gel can also be used for southern analysis using MmuPV1-specific probes (3) by nick translation as we have described in detail previously(4). Since virus yields from papillomas of nude mice are very high, we have found agarose gel electrophoresis to be sensitive enough to accurately determine virus concentrations. Other methods of virus quantification have also been described in the literature(5, 6).</p>
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<p>References:</p>
<p><strong>        1. Schowalter RM, Pastrana DV, Pumphrey KA, Moyer AL, Buck CB.</strong> 2010. Merkel cell polyomavirus and two                    previously unknown polyomaviruses are chronically shed from human skin. Cell host &amp; microbe <strong>7:</strong>509-515.</p>
<ol start="2">
<li><strong>Buck CB, Thompson CD.</strong> 2007. Production of papillomavirus-based gene transfer vectors. Current protocols in cell biology <strong>Chapter 26:</strong>Unit 26 21.</li>
<li><strong>Uberoi A, Yoshida S, Frazer IH, Pitot HC, Lambert PF.</strong> 2016. Role of Ultraviolet Radiation in Papillomavirus-Induced Disease. PLoS pathogens <strong>12:</strong>e1005664.</li>
<li><strong>Lorenz LD, Rivera Cardona J, Lambert PF.</strong> 2013. Inactivation of p53 rescues the maintenance of high risk HPV DNA genomes deficient in expression of E6. PLoS pathogens <strong>9:</strong>e1003717.</li>
<li><strong>Cladel NM, Budgeon LR, Cooper TK, Balogh KK, Hu J, Christensen ND.</strong> 2013. Secondary infections, expanded tissue tropism, and evidence for malignant potential in immunocompromised mice infected with Mus musculus papillomavirus 1 DNA and virus. Journal of virology <strong>87:</strong>9391-9395.</li>
<li><strong>Handisurya A, Day PM, Thompson CD, Buck CB, Pang YY, Lowy DR, Schiller JT.</strong> 2013. Characterization of Mus musculus papillomavirus 1 infection in situ reveals an unusual pattern of late gene expression and capsid protein localization. Journal of virology <strong>87:</strong>13214-13225.</li>
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