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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.

Monitoring serum antibodies against natural infections or after immunizations has been a standard clinical diagnostic procedure. However, collecting blood samples requires trained personnel, and may cause discomfort and increase the risk of complications. In this study, we investigated whether tear samples could serve as a surrogate for serum samples to measure specific antibodies. A widely used preclinical cottontail rabbit papillomavirus (CRPV)/rabbit model has been a surrogate model for high-risk human papillomavirus (HPV) infections. New Zealand white rabbits, either naturally infected with CRPV or immunized with two clinically available HPV vaccines (Gardasil and Cervarix), were examined for antibody generation in both tear and serum samples. We demonstrated that antibodies were detectable in tears from both naturally infected as well as vaccinated animals. Overall, the antibody levels in tears were ~10-fold lower than those from the corresponding serum samples, but background noise was lower in tear samples. The isotypes of antibodies in tears were predominantly IgA and IgG. These findings showed clearly that tears could be a surrogate for serum samples for monitoring antibody responses. As collecting tears causes no discomfort and poses no risk to patients, it represents a novel and promising method for monitoring future HPV epidemiological studies as well as for use in clinical practice.