Production of infectious bovine papillomavirus from cloned viral DNA by using an organotypic raft/xenograft technique

Abstract

Bovine papillomavirus type 1 (BPV-1) induces fibropapillomas in its natural host and can transform fibroblasts in culture. The viral genome is maintained as an episome within fibroblasts, which has allowed extensive genetic analyses of the viral functions required for DNA replication, gene expression, and transformation. Much less is known about BPV-1 gene expression and replication in bovine epithelial cells because the study of the complete viral life cycle requires an experimental system capable of generating a fully differentiated stratified bovine epithelium. Using a combination of organotypic raft cultures and xenografts on nude mice, we have developed a system in which BPV-1 can replicate and produce infectious viral particles. Organotypic cultures were established with bovine keratinocytes plated on a collagen raft containing BPV-1-transformed fibroblasts. These keratinocytes were infected with virus particles isolated from a bovine wart or were transfected with cloned BPV-1 DNA. Several days after the rafts were lifted to the air interface, they were grafted on nude mice. After 6-8 weeks, large xenografts were produced that exhibited a hyperplastic and hyperkeratotic epithelium overlying a large dermal fibroma. These lesions were strikingly similar to a fibropapilloma caused by BPV-1 in the natural host. Amplified viral DNA and capsid antigens were detected in the suprabasal cells of the epithelium. Moreover, infectious virus particles could be isolated from these lesions and quantitated by a focus formation assay on mouse cells in culture. Interestingly, analysis of grafts produced with infected and uninfected fibroblasts indicated that the fibroma component was not required for productive infection or morphological changes characteristic of papillomavirus-infected epithelium. This system will be a powerful tool for the genetic analysis of the roles of the viral gene products in the complete viral life cycle.

Figure 1

Time course of bovine organotypic raft development. Organotypic rafts were cultured by using mouse 3T3 fibroblasts and bovine keratinocytes. Samples were taken at the times shown after lifting the raft to the air–liquid interface. Sections of the rafts were stained with H&E. The stratum corneum (sc), stratum granulosum (sg), stratum spinosum (ss), and stratum basale (sb) are indicated by arrows.

Figure 2

BPV-1 replicates in cutaneous bovine xenografts on nude mice. Fetal bovine skin was grafted onto nude mice and infected with BPV-1 wart extract. a shows an uninfected graft, and b shows a BPV-infected graft.

Figure 3

Infectious BPV-1 can be produced in organotypic rafts grafted on nude mice. Examples of an uninfected (a and b) and a BPV-1 infected (d and e) xenograft are shown. The dermal equivalent contained BALB/BPV fibroblasts that resulted in a fibroma in both cases. a and d show the gross pathology of the graft, and b and e show an H&E-stained section. The flanking mouse skin can be observed at the margins. The plates shown in c and f are 100-mm dishes of C127 cells stained with methylene blue. Foci derived from infectious BPV-1 obtained from an infected xenograft can be observed on the plate in f.

Figure 4

Histological features of the bovine xenografts. a shows an H&E-stained section of adult bovine skin. c, e, and f show an H&E-stained section from a bovine fibropapilloma. The sections shown in b, d, f, and h are from bovine organotypic raft/xenografts. The control graft shown in b was generated with uninfected bovine keratinocytes and BALB fibroblasts. The graft shown in d, f, and h was generated with BPV-1-infected keratinocytes, and the dermal equivalent of this graft contained BALB/BPV fibroblasts that resulted in a fibroma. Characteristic features of papillomavirus infection are indicated with arrows: acanthosis (a); koilocytosis (ky); prominent keratohyalin granules (g); parakeratosis (pk); and hyperkeratosis (hk). Flanking mouse skin (ms) is shown in b. Magnifications are indicated.

Figure 5

In situ hybridization for amplified BPV-1 DNA. Examples of grafts with (e and f) and without (a–c) fibroma (because of BALB/BPV fibroblasts) and either uninfected (a), transfected with viral DNA (b and e), or infected with a wart extract (c and f). d shows a natural bovine fibropapilloma. Positive cells containing viral DNA are stained with 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium and counterstained with eosin.

Figure 6

Immunohistochemistry of BPV-1 L1 antigen in xenografts. Examples of grafts with (e and f) and without (a–c) fibroma (because of BALB/BPV fibroblasts) and either uninfected (a), transfected with viral DNA (b and e), or infected with a wart extract (c and f). d shows a bovine fibropapilloma. Positive cells were detected with 3,3′-diaminobenzidine and counterstained with hematoxylin. The stratum corneum (sc), stratum granulosum (sg), and stratum spinosum (ss) are indicated by arrows.