In vitro transformation and molecular characterization of Colobus monkey venereal papillomavirus DNA.

The DNA of a monkey papillomavirus (CgPV-1), originally isolated from a penile lesion on a Colobus monkey was cloned into the EcoRI site of the pUC18 vector and characterized. Using a variety of restriction enzymes a physical map of the DNA was constructed. Cross-hybridization with a variety of animal and human papillomaviruses under high (Tm-22 degrees C) and low (Tm-40 degrees C) stringency conditions indicated various degrees of homology. CgPV-1 showed higher homology with HPVs than it did with any other animal papillomaviruses tested. DNA similarities with the human papillomaviruses HPV-16 and HPV-18 that are frequently associated with cervical cancer, were manifested by extensive cross-hybridization under stringent conditions. Functional alignment of the genomic map of CgPV-1 with that of HPV-16 was carried out by determination of homology between specific restriction fragments of the two viral genomes in cross-hybridization analyses. This alignment was refined by sequencing two regions of approximately 200 bp of the CgPV-1 DNA, and aligning them by computer with their homologous HPV-16 counterparts. CgPV-1 DNA in its pUC18 vector, transformed NIH 3T3 cells with roughly the same efficiency as BPV-1, as determined by the number of transformed foci generated per ug of DNA. The data presented indicate that the state of the CgPV-1 viral DNA in these transformed cells is integrated and partially deleted, not unlike the genomes of HPV-16 and HPV-18 characterized in cell lines derived from cervical cancers.

Cutaneous fibropapillomas of green turtles (Chelonia mydas).

Six juvenile green turtles (Chelonia mydas) from the Indian River Lagoon System, Florida, U.S.A., with multiple cutaneous fibropapillomas, were kept in isolation and examined over a 6-month period. Histologically, the fibropapillomas consisted of a slightly to moderately hyperplastic epidermis overlying a thickened hypercellular dermis. In the earliest lesions, ballooning degeneration was present predominantly in the stratum basale where rete ridges extended into the dermis; aggregates of mixed inflammatory cells were present around dermal vessels. As the lesions matured, they developed an arborizing, papillary pattern. More mature lesions had a less verrucous, often ulcerated surface, with the dermis composed primarily of large collagenous fascicles and relatively few fibroblasts. While numerous trematode eggs were present within dermal capillaries of a histologically similar biopsy specimen from an Hawaiian green turtle, no trematode eggs were observed in any of 28 biopsies examined from the six Florida green turtles in this study. Low stringency Southern blot hybridization and a reverse Southern blot failed to demonstrate papillomavirus DNA in any of the samples extracted. Ultrastructural evaluation of the earliest lesions demonstrated membrane-bound intracytoplasmic vacuoles within epidermal cells in the stratum basale. Similar vacuoles were also observed in the epidermal intercellular spaces and within the dermis. Occasionally, particles with electron-dense centres and measuring 155 to 190 nm were observed in these vacuoles.

Papillomas and carcinomas associated with a papillomavirus in European harvest mice (Micromys minutus).

Papillomaviruses, group-specific papillomavirus antigens, or extrachromosomal papillomavirus DNA were detected in cutaneous, mucocutaneous, and pulmonary tumors affecting a colony of European harvest mice (Micromys minutus). Skin lesions were classified as acanthomatous hyperplasia, epidermal inclusion cysts, squamous papillomas, inverted papillomas, trichoepitheliomas, and sebaceous carcinomas. Cutaneous horns (hyperkeratotic papillomas) were on mucocutaneous junctions of one animal. One mouse, with a cutaneous sebaceous carcinoma, had multiple pulmonary keratinaceous cysts. Papillomavirus antigens, detected by the avidin-biotin technique, were in 20 of 31 lesions tested. In contrast, by Southern blot hybridization all 28 lesions tested contained papillomavirus DNA. Papillomavirus DNA was demonstrated in two of ten benign cutaneous lesions by in situ hybridization.

Papillary squamous cell carcinoma in three young dogs.

Papillary squamous cell carcinomas were located on the gingiva of 3 young dogs. The tumors locally invaded the soft tissues of each dog, and invaded bone in 2 dogs. Surgical excision was unsuccessful in eliminating 2 of the tumors. Surgery and radiotherapy were effective, and recurrence has not been observed in 39 months in 1 dog, 32 months in a second, and 10 months in a third. Superficially, the oral masses resembled papillomas, which are known to be caused by viruses. Cytopathologic indication of productive infection was not evident, and papillomavirus antigens could not be detected by immunohistochemical methods. Electron microscopy failed to identify viral particles in 2 of the tumors. High and low molecular weight DNA extracts from 2 of the tumors contained no detectable papillomavirus genome when probed under conditions of either high or low stringency by Southern blot hybridization with a cloned canine oral papillomavirus genome.

Papillomavirus in the vapor of carbon dioxide laser-treated verrucae.

Vapor produced by the carbon dioxide laser during the vaporization of papillomavirus-infected verrucae was analyzed for viral DNA content. Two models were used for evaluation: an in vitro cutaneous bovine fibropapilloma and an in vivo human verruca model. Four bovine fibropapillomas were exposed to various laser parameters with power densities of 38,200 to 130 W/cm2 and energy fluences of 3820 to 130 J/cm2. The generated vapor was collected in a chamber in line with a vacuum system. Hybridization with bovine papillomavirus DNA probes revealed intact bovine papillomavirus DNA for all power densities and energy fluences used. The laser vapor from seven patients undergoing carbon dioxide laser therapy for plantar or mosaic verrucae was also collected. Laser parameter settings were similar to those usually chosen for clinical tissue vaporization. Intact human papillomavirus DNA was present in the vapor from two of seven patients. These studies indicate that intact viral DNA is liberated into the air with the vapor of laser-treated verrucae. It would be prudent for all practitioners who use the laser in treating patients with viral infections or conditions associated with viruses to practice extreme care and safety throughout the laser procedure.

Cutaneous inverted papillomas in dogs.

Inverted papillomas of the skin occurred in five dogs. Lesions were 1-2 cm, circumscribed, flask-like structures below the level of the surrounding normal skin. Walls of the structures consisted of hyperplastic epidermis, forming thin papillary projections on thin fibrovascular stalks. Cells in the stratum granulosum had clear cytoplasm, numerous keratohyalin-like granules of various sizes, and poorly defined intranuclear inclusions. These cells stained positively for papillomavirus group-specific antigens by both the peroxidase-antiperoxidase and avidin-biotin methods. Virions with a mean diameter of 35.7 nm were present within nuclei in cells of the stratum granulosum when examined by electron microscopy. In situ DNA hybridization, using a canine oral papillomavirus probe, localized papillomavirus DNA in canine oral papillomas, but not in canine cutaneous squamous or inverted papillomas, suggesting that a different papillomavirus type was present in the latter lesions. Although these lesions resembled intracutaneous cornifying epitheliomas (keratoacanthomas), they appear to be a distinct lesion, probably with a different etiology.

Cloning and molecular characterization of an oral papillomavirus of domestic rabbits.

DNA obtained from New Zealand white rabbit oral papillomas was analyzed for the presence of papillomavirus DNA. The viral genome was cloned as three separate subclones, which were each mapped and oriented with respect to one another. Comparisons with other papillomavirus DNAs by Southern blot hybridization under various conditions of stringency revealed a strong area of conservation among the DNAs of the rabbit oral papillomavirus (ROPV) and CRPV, HPV-1a, HPV-16, and BPV-5, but not with 12 other papillomavirus DNAs. This region, which spans the junction of the presumptive E2 and L2 open reading frames of ROPV, was sequenced and compared to other known papillomavirus sequences. These analyses revealed a high degree of DNA homology in the C-terminal E2 and N-terminal L2 regions between ROPV and both HPV-1a and CRPV. The homology with HPV-16 was limited to the L2 open reading frame. The predicted amino acid sequences of each region were also compared and bore out the same conclusions. In addition, no E5 open reading frame was detected in the ROPV sequence.

Comparison of a cheetah herpesvirus isolate to feline herpesvirus type 1.

A cytopathogenic virus with size and structural characteristics of a Herpesviridae was isolated from a cheetah with severe ulcerative dermatitis. Restriction endonuclease analysis and cross-hybridization studies revealed that the isolate was related to feline herpesvirus type 1 (FHV-1). Antigenic comparison studies using anti-FHV-1 serum demonstrated the presence of common antigens in the FHV-1 and the isolate from the cheetah.

Cloning and characterization of a papillomavirus associated with papillomas and carcinomas in the European harvest mouse (Micromys minutus).

Individuals in a colony of European harvest mice (Micromys minutus) were diagnosed with a variety of skin tumors including papillomas, trichoepitheliomas, and sebaceous carcinomas. Papillomavirus group-specific antigens and viruslike particles were detected in the papillomas. A 7.6-kilobase supercoiled circular DNA, which was cleaved once by EcoRI, was visualized in papilloma extracts by low-stringency Southern blot hybridization with a bovine papillomavirus type 2 probe. The molecule was cloned in pUC18, and a restriction map was generated. The molecule was shown to be colinear with the genome of human papillomavirus type 1a by partial sequence analysis. The DNA hybridized to human papillomavirus type 1a, rabbit oral papillomavirus, and the genome of Mastomys natalensis papillomavirus at Tm - 33 degrees C but not to the DNAs of 13 other papillomaviruses. Transformation of NIH 3T3 or C127I cells by tail papilloma extracts or transfected viral DNA was not observed. All 17 tumors examined contained large amounts of viral DNA in a supercoiled, unintegrated form as revealed by Southern blot hybridization. Furthermore, many extracts (25 of 35) from normal organs and skin of individuals with lesions elsewhere on their bodies contained viral DNA. This represents the first reported molecular cloning of a papillomavirus genome from a mouse species.

Venereal papilloma and papillomavirus in a colobus monkey (Colobus guereza).

A papilloma on the penis of a colobus monkey was found to contain papilloma-virus group specific antigens by immunohistochemical analysis and virus-like particles in the nuclei of epithelial cells by transmission electron microscopy. In low-stringency Southern blot hybridizations, DNA from the lesion annealed with human papillomavirus 11 DNA, but not with the DNAs of 13 other papillomaviruses. Using human papillomavirus 11 DNA as a probe in Southern blot hybridizations, DNA from the penile papilloma was shown to contain a supercoiled DNA approximately 8 kilobases in size. This represents the first demonstration of a papillomavirus-associated venereal lesion in a nonhuman primate.

Cross-hybridization and relationships of various papillomavirus DNAs at different degrees of stringency.

Cloned DNAs of 21 different papillomaviruses which naturally infect mammals and one bird papillomavirus were compared for relative homology by Southern blot hybridization. Blots were carried out under low (Tm-40 degrees), medium (Tm-33 degrees), and high (Tm-22 degrees) stringency conditions. At higher stringency, human papillomaviruses cross-hybridized with each other reflecting species-specific similarities. Bovine papillomavirus types 1, 2, 5, European elk papillomavirus, and deer papillomavirus also cross-hybridized at higher stringencies probably reflecting the association of these viruses with fibroblast-prolific lesions. The hybridization data presented here may be useful in future classification attempts. They are also useful as a guide in the selection of papillomavirus DNA probes for analysis of extracts from warts and tumors.

Cloning and characterization of an equine cutaneous papillomavirus.

Equine papillomaviruses (EqPV) from naturally occurring cases of cutaneous papillomatosis in several ponies and one horse were isolated, cloned, and characterized. Group specific papillomavirus structural antigens were detected in sections of the papillomas by the peroxidase-antiperoxidase technique, and virions were observed in the in the nuclei of cells in the stratum granulosum and corneum. Negatively stained virions purified from papilloma homogenates by isopycnic CsCl centrifugation were 55 nm in diameter and had typical papillomavirus morphology. The entire viral genomes of two separate isolates were cloned at a single BamHI site into pBR322. A detailed restriction map of the viral genome is presented. Using nick-translated subgenomic fragments of BPV-1 as probes in Southern blot hybridizations, the organization of the EqPV genome was established. Southern blot analysis under various conditions of stringency revealed that EqPV shares relatively more homology with the late region of the BPV-1 genome and with the E2 region of the HPV-1 genome than with other parts of the same viral DNAs. Papillomavirus-specific sequences were found in papillomas from other anatomic sites using the EqPV DNA as a probe in Southern blot hybridizations. Genomes detected in DNA from penile papillomas had a different restriction pattern and hybridized to the EqPV probe only under nonstringent conditions.

Cloning and characterization of a canine oral papillomavirus.

A papillomavirus, isolated from oral papillomas in young Beagles, was used to produce a live-virus vaccine. After the IM use of this vaccine, some dogs developed squamous cell carcinomas at the inoculation site. The virus was isolated from the original vaccine and was cloned into pBR322. A detailed restriction map of the viral genome was generated.

Characterization of New Jersey vesicular stomatitis virus isolates from horses and black flies during the 1982 outbreak in Colorado.

Vesicular stomatitis viruses isolated from horses, afflicted during the recent outbreak in the western United States, and from black flies (Simuliidae) were characterized with respect to the homology of their genomic RNAs and the mobility of their proteins in polyacrylamide gels. All the isolates were very similar, if not identical, with respect to these two parameters. When the black fly isolate was compared to other VSV isolates, this virus appeared to belong in the Hazelhurst subgroup of the New Jersey serotype of VSV. Since the other viruses in this division were obtained from infected swine, the natural host range of this subgroup has been extended to horses.

The requirement of protein synthesis for VSV inhibition of host cell RNA synthesis.

Published ultraviolet (uv) inactivation data and in vitro transcription studies have suggested that vesicular stomatitis virus (VSV) leader RNA was solely responsible for the inhibition of host cell RNA synthesis by this virus. Since no protein product is encoded in leader RNA, this conclusion implied that no protein synthesis should be required for this effect. Therefore, the inhibitory activity of VSV was examined in the presence of the protein synthesis inhibitors, cycloheximide, pactamycin, and emetine. Protein synthesis inhibitors are known not to interfere with VSV primary transcription, but in their presence viral replication and amplification of transcription do not take place. Although at 39 degrees the VSV mutant tsG22 could undergo only primary transcription, maximum inhibition of host cell RNA synthesis took place. However, in the presence of the protein synthesis inhibitors the VSV mutant was no longer able to interfere with host cell RNA synthesis. These results could not be explained by a change in the concentration of intracellular leader RNA which remained unaltered by the drugs. Similar results were also obtained with wild-type VSV in the presence of cycloheximide. Upon removal of the drug, inhibition of host cell RNA synthesis was reestablished in parallel with the restoration of protein synthesis. It is concluded that protein synthesis is required for the inhibitory activity of VSV, presumably because the active inhibitory complex is a nucleoprotein containing leader RNA and either a cellular protein or the viral N protein. The cellular protein would have to be in limiting supply since de novo protein synthesis was required for the inhibition to take place.

Inhibition of SV40 DNA synthesis by vesicular stomatitis virus in doubly infected monkey kidney cells.

Vesicular stomatitis virus (VSV) inhibited SV40 DNA synthesis in doubly infected synchronized Vero cells. Gel-electrophoretic profiles demonstrated that SV40 DNA monomers accumulated in all stages of supercoiling, regardless of whether cells were superinfected with VSV early or late in the S phase. These gel profiles were indistinguishable from ones obtained from SV40-infected, cycloheximide-treated cells in the absence of VSV infection. Radiolabel in the partial supercoils could be chased into supercoils, but only by restoring protein synthesis. The relative rates of SV40 DNA chain elongation were determined in VSV-superinfected and nonsuperinfected cells. The gradients of 3H incorporation as a function of distance from the origin of replication in pulse-labeled form I DNA were unaffected by VSV. It is concluded that VSV inhibition of SV40 DNA synthesis is an indirect result of inhibition of host cell protein synthesis and it is suggested that incompletely supercoiled SV40 chromatin is not a good template for DNA synthesis.

Effect of intracellular vesicular stomatitis virus mRNA concentration on the inhibition of host cell protein synthesis.

Inhibition of host cellular protein synthesis by vesicular stomatitis virus (VSV) has been suggested to be primarily the result of competition for ribosomes between cellular and viral mRNAs (H. F. Lodish and M. Porter, J. Virol., 36:719-733, 1980; Lodish and Porter, J. Virol. 38:504-517, 1981). This hypothesis was investigated by regulating the extent of VSV mRNA synthesis through the use of defective interfering particles. Although intracellular VSV mRNA concentrations decreased by as much as a factor of 14 at high multiplicities of infection of defective interfering particles, the inhibition of host cell protein synthesis by VSV decreased by a maximum of only 10%. The data also indicated that under these conditions the protein-synthesizing capacity of the cells was not exhausted. We concluded that competition for cellular ribosomes could not have been the major factor in the inhibition of host cell protein synthesis by VSV. This conclusion was further supported by inhibition data obtained with VSV mutants. The ts G22 mutant, defective in replication but not in primary transcription, inhibited host protein synthesis at the nonpermissive temperature (39 degrees C) to the same extent as did wild-type virus, even though it generated only 30 to 50% of the amount of viral mRNA as did wild-type virus. Conversely, in infections with the R1 mutant, which did not inhibit host cell protein synthesis, the amount of total and polysome-bound viral mRNA was indistinguishable from that obtained in infections by wild-type virus.

In vitro and in vivo inhibition of primary transcription of vesicular stomatitis virus by a defective interfering particle.

A unique defective interfering (DI) particle, generated by a heat-resistant (HR) mutant of Indiana serotype vesicular stomatitis virus, was capable of inhibiting primary transcription by heterologous New Jersey serotype virions. The correlation between this phenomenon and the lowering of viral yields from doubly infected cells was investigated by the construction of chimeric DI particles containing the HR DI particle genome with a thermolabile polymerase. At the nonpermissive temperature, these DI particles were unable to self-transcribe, inhibit virion primary transcription, or reduce virion yield, but were able to be replicated. These results suggested that self-transcription of the HR DI particle genome was a prerequisite for heterotypic interference, but not for its own replication. Inhibition of virion primary transcription by HR DI ribonucleocapsids was also observed in vitro. At low HR DI to virion ribonucleocapsid ratios, the extent of inhibition was concentration dependent, whereas at high ratios, the amount of inhibition was concentration independent, approaching a limiting maximum value. A speculative mathematical model, which quantitatively accounts for these data, is presented. According to this model, the higher affinity for polymerase molecules by the HR DI ribonucleocapsids is explained in terms of dissociation events during transcription, which are more frequent in the longer virion ribonucleocapsids.

Rous sarcoma virus infection of synchronized cells establishes provirus integration during S-phase DNA synthesis prior to cellular division.

Synchronized chicken embryo fibroblasts, prepared by addition of serum to stationary cells arrested in Go, were exposed to the Prague strain of Rous sarcoma virus. At different times during the cell cycle, high molecular weight DNA was prepared from infected cells and examined for the presence of newly integrated viral DNA sequences. The results demonstrate that newly integrated viral sequences were first detected during S-phase DNA synthesis 9 hr after infection. The presence of colchicine prevented cellular division and delayed the appearance of progeny virus but it did not affect the appearance of viral specific DNA in the high molecular weight fraction of cellular DNA. Our results indicate that provirus integration, occurring during S-phase DNA synthesis, does not require cell division. Previous experiments have demonstrated that Rous sarcoma virus infection of chicken embryo fibroblasts requires cell division to initiate viral RNA synthesis and the production of progeny virus. The findings presented in this report support the hypothesis that division of the infected cells is required for an event that controls viral expression at the level of the integrated provirus.