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. 2010 Apr;165(1):36-41.
doi: 10.1016/j.jviromet.2009.12.014. Epub 2009 Dec 29.

Differences in methodology, but not differences in viral strain, account for variable experimental outcomes in laboratories utilizing the cottontail rabbit papillomavirus model

Nancy M Cladel  1 , Jiafen Hu, Karla K Balogh, Neil D Christensen
Affiliations

Differences in methodology, but not differences in viral strain, account for variable experimental outcomes in laboratories utilizing the cottontail rabbit papillomavirus model

Nancy M Cladel et al. J Virol Methods. 2010 Apr.

Abstract

The cottontail rabbit papillomavirus (CRPV) animal model is used in several laboratories worldwide to investigate immunogenicity, carcinogenicity and life cycle aspects of papillomaviruses. It is the only animal model in which the full life cycle of the virus from initiation of infection to malignant progression can be studied. A major strength of the model is that the viral DNA is infectious. This feature allows for the study of mutant genomes without the need to create infectious mutant virus. Results from laboratory to laboratory have not always been consistent. Different laboratories use different methods for creating infections from DNA and it was postulated that the different challenge methods could play a role in the differential outcomes. Because different laboratories use different strains of CRPV, it was also desirable to test if the difference in CRPV genomes contributed to the differential outcomes. In this study, three of the CRPV strains used most widely (Washington B, Orth CRPV and Hershey CRPV) were cloned into PUC19; the E8 ATG ko mutants for each strain were also generated. We employed the infection technique reported previously in which scarification is done first and is followed with delivery of DNA by pipette 3 days later. The papilloma outgrowth generated by these three wild type constructs and their E8 ATG ko mutants was compared. No significant difference was found among the three strains or their E8 ATG ko mutants. E8 ATG ko mutants induced significantly smaller but persistent papillomas when compared to their respective wild type CRPVs. The gene gun was also used to create infections with both Hershey CRPV DNA and the corresponding E8 ATG ko and was found to lead to less vigorous growth as well as some regressions. Further studies suggested that gene gun delivery might have induced an immune response which then resulted in compromised growth of papillomas. It was concluded that the E8 gene is not required for infection. We suggest that standardized infection methods should be used in laboratories so that inconsistencies in conclusions will be minimized.

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Figures

Figure 1
Figure 1
Comparison of the three vial strains and their E8 ATG ko mutants. Growth was comparable for the three viral strains. The respective E8 mutants grew at similar rates but slower than wild type virus.
Figure 2
Figure 2
Figure 2A Influence of the gene gun on the growth of both wild type H. CRPV and E8ko papillomas initiated by gene gun. Sites were either scarified at day -3 or were not scarified prior to infection. The scale is the same as that used in Figure 1 in order to facilitate comparison. Figure 2B Influence of the gene gun on the growth of both wild type CRPV and E8ko papillomas initiated by pipette. Sites were either scarified at day -3 or were not scarified prior to infection. The scale is the same as that used in Figure 1 in order to facilitate comparison. Note that all animals also had gene gun infections (figure 2A) and thus were influenced by the gene gun even though the infections represented in this graph were initiated by pipette.
Figure 2
Figure 2
Figure 2A Influence of the gene gun on the growth of both wild type H. CRPV and E8ko papillomas initiated by gene gun. Sites were either scarified at day -3 or were not scarified prior to infection. The scale is the same as that used in Figure 1 in order to facilitate comparison. Figure 2B Influence of the gene gun on the growth of both wild type CRPV and E8ko papillomas initiated by pipette. Sites were either scarified at day -3 or were not scarified prior to infection. The scale is the same as that used in Figure 1 in order to facilitate comparison. Note that all animals also had gene gun infections (figure 2A) and thus were influenced by the gene gun even though the infections represented in this graph were initiated by pipette.
Figure 3
Figure 3
Papilloma volumes at week 8 of papilloma growth. A. Wild type H. CRPV, pipette delivery, no gene gun influence. B. E8ko H. CRPV, pipette delivery, no gene gun influence. C. Wild type, gene gun infection. D. E8ko gene gun infection. E. Wild type, pipette delivery in animals also experiencing the gene gun. F. E8ko, pipette delivery in animals also experiencing the gene gun.
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