Noninvasive and practical techniques to longitudinally track viral infection are sought after in clinical practice. We report a proof-of-principle study to monitor the viral DNA copy number using a newly established mouse papillomavirus (MmuPV1) mucosal infection model. We hypothesized that viral presence could be identified and quantified by collecting lavage samples from cervicovaginal, anal and oral sites. Nude mice infected at these sites with infectious MmuPV1 were tracked for up to 23 weeks starting at 6 weeks post-infection. Viral DNA copy number was determined by SYBR Green Q-PCR analysis. In addition, we tracked viral DNA load through three complete oestrous cycles to pinpoint whether there was a correlation between the DNA load and the four stages of the oestrous cycle. Our results showed that high viral DNA copy number was reproducibly detected from both anal and cervicovaginal lavage samples. The infection and disease progression were further confirmed by histology, cytology, in situ hybridization, immunohistochemistry and transmission electron microscopy. Interestingly, the viral copy number fluctuated over the oestrous cycle, with the highest level at the oestrus stage, implying that multiple sampling might be necessary to provide a reliable diagnosis. Virus DNA was detected in oral lavage samples at a later time after infection. Lower viral DNA load was found in oral samples when compared with those in anal and vaginal tracts. To our knowledge, our study is the first in vivo study to sequentially monitor papillomavirus infection from mucosal anal, oral and vaginal tracts in a preclinical model.

Papillomavirus disease poses a special challenge to people with compromised immune systems. Appropriate models to study infections in these individuals are lacking. We report here the development of a model that will help to address these deficiencies. The MmuPV1 genome was synthesized and used successfully to produce virus from DNA infections in immunocompromised mice. In these early studies, we have demonstrated both primary and secondary infections, expanded tissue tropism, and extensive dysplasia. Papillomaviruses are double-stranded DNA tumor viruses of about 8 kb. These viruses are ubiquitous in nature, and 241 types have been identified to date in both humans and many other animal species (http://pave.niaid.nih.gov). A subset of human papillomaviruses is linked to cancers, most notably cervical cancer, but also cancers of the head and neck, skin, and other anogenital sites. Papillomaviruses require fully differentiating cells for the completion of the viral life cycle and thus are not easily studied in cell culture systems. For a review of these viruses, see reference 1. The cottontail rabbit papillomavirus (CRPV) in vivo model has proven to be very useful in our hands for the study of cutaneous papillomavirus disease (2-8). An in vivo system to study both cutaneous and mucosal disease, papillomavirus pathology, and