Wounding prior to challenge substantially improves infectivity of cottontail rabbit papillomavirus and allows for standardization of infection

Abstract

The cottontail rabbit papillomavirus (CRPV)/rabbit model has proved useful for the investigation of prophylactic and therapeutic vaccines and for the study of the pathogenesis of papillomavirus infection. It is currently the only animal model in which the entire viral program can be recapitulated, including progression to cancer. CRPV DNA is infectious in domestic rabbits and therefore mutants can be studied without the need to generate corresponding viruses. Although the CRPV animal model is used widely in various laboratories, no optimized or standardized method is used for creating CRPV viral and especially DNA infections. These different methods have made it difficult for investigators to compare results from laboratory to laboratory. A simple and highly efficient method is reported here; it has been refined based on previous methodology for the production of CRPV infections from both virus and plasmid DNA. This method can be adapted easily by other investigators in the field. The resulting standardization will aid in the evaluation of data from different laboratories.

Figure 1

Papilloma outgrowth on sites pre-scarified at day −3 to −10 before DNA challenge. Two sites on each of two rabbits scarified at day −3 to −10 were challenged with 10µg of CRPV DNA. All the pre-scarification time points except day −10 showed consistent papilloma growth as early as one week after DNA challenge. No significant difference in papilloma size was found among these treatments (P>0.05, unpaired student t test)‥

Figure 2

Papilloma outgrowth on sites retaining scab or removing scab. Two sites on each of two rabbits scarified at day −3 were challenged on day 0 with 10µg of CRPV DNA with or without removing the scab. Papillomas on sites from which the scab had been removed were significantly smaller than those on sites on which the scab had been retained (P<0.05, unpaired student t test).

Figure 3

Papilloma outgrowth on sites challenged with different doses of CRPV DNA. Two sites on each of two rabbits were challenged with 10µg and 20µg of CRPV 3 days after scarification. Control sites received 10µg DNA with no pre-scarification. Papillomas on sites challenged with 10µg of CRPV plasmid were comparable to those on sites challenged with 20µg of CRPV plasmid (P>0.05, unpaired student t test).

Figure 4

Papilloma outgrowth on sites challenged with different doses of CRPV DNA with (B) or without (A) pre-scarification. Two sites on each of five rabbits were challenged with from 0.04µg to 10µg of CRPV either 3 days after scarification (B) or without prior scarification (A). Some sites challenged with low amounts of CRPV DNA (0.04µg) grew papillomas following pre-scarification while scattered papillomas were found on sites challenged without pre-scarification (A).

Figure 5

Papilloma outgrowth on sites challenged with 5µg of CRPV was comparable to that on sites challenged with 10µg of CRPV. Two sites on each of four rabbits were challenged with 5µg and 10µg of CRPV three days after scarification. 5µg of CRPV was as efficient as 10 micrograms in inducing papillomas (P>0.05, unpaired student t test).

Figure 6

Papilloma outgrowth on sites challenged with different doses of CRPV infectious virus with (B) or without (A) pre-scarification. Two sites on each of five NZW rabbits were challenged with from 10⁻² to 10⁻⁶ dilutions of H.CRPV virus stock at 3 day after scarification (B) or with no pre-scarification (A). No sites challenged with a low dose (lower than 10⁻³) grew papillomas without pre-scarification treatment (A) while papillomas were found on sites challenged with doses of CRPV as low as 10⁻⁵ with pre-scarification (B).

Figure 7

Papilloma outgrowth on sites challenged with 10µg of CRPV DNA delivered by gene-gun with or without pre-scarification. Two sites on each of five rabbits were challenged with 10µg of CRPV DNA 3 days after scarification or with no pre-scarification. A few sites without pre-scarification treatment grew tiny papillomas whereas most sites with pre-scarification grew significantly larger papillomas (P<0.05, unpaired student t test).