Multiple HPV infection: microanatomy by in situ hybridization and immunohistochemistry

Specific human papillomavirus (HPV) types have been shown to be associated with proliferative epithelial lesions with variable biological consequences in infected patients. Simultaneous infection by more than one HPV type has been infrequently reported, and its clinical significance is unknown. We have examined four biopsies of cervical and vulvar tissue, each with evidence of infection by two different HPVs. Using both in situ hybridization and immunohistochemical techniques, we determined the cellular distribution of the viral infections. Using biotinylated type-specific probes and stringent conditions we were able to demonstrate that in each case the two HPVs occupied distinct, non-overlapping foci within the lesions. The condylomatous tissues contained DNA from HPV types that are associated with high-grade neoplasia and invasive cancer (16 and 18), as well as types commonly associated with benign proliferative lesions. Immunohistochemical analysis of the lesions with antibody to bovine papillomavirus capsid antigen failed to detect HPV in regions shown by in situ hybridization to contain HPV 16 and 18 DNA, whereas type 6 and 11 infected areas were readily identified. These results provide indirect evidence of viral interference between HPV types and indicate that interference may limit the number of HPV types that produce active infections within a single cell.