ABSTRACT
Sylvilagus floridanus Papillomavirus (SfPV) causes growth of large horn-like tumors on rabbits. SfPV was described in cottontail rabbits (probably Sylvilagus floridanus) from Kansas and Iowa by Richard Shope in 1933, and detected in S. audubonii in 2011. It is known almost exclusively from the US Midwest. We explored the University of Kansas Natural History Museum for historical museum specimens infected with SfPV, using molecular techniques, to assess if additional wild species host SfPV, and whether SfPV occurs throughout the host range, or just in the Midwest. Secondary aims were to detect distinct strains, and evidence for strain spatio-temporal specificity. We found 20 of 1395 rabbits in the KU collection SfPV symptomatic. Three of 17 lagomorph species (S. nuttallii, and the two known hosts) were symptomatic, while Brachylagus, Lepus and eight additional Sylvilagus species were not. 13 symptomatic individuals were positive by molecular testing, including the first S. nuttallii detection. Prevalence of symptomatic individuals was significantly higher in Sylvilagus (1.8%) than Lepus. Half of these specimens came from Kansas, though new molecular detections were obtained from Jalisco-Mexico's first-and Nebraska, Nevada, New Mexico, and Texas, USA. We document the oldest lab-confirmed case (Kansas, 1915), pre-dating Shope's first case. SfPV amplification was possible from 63.2% of symptomatic museum specimens. Using multiple methodologies, rolling circle amplification and, multiple isothermal displacement amplification in addition to PCR, greatly improved detection rates. Short sequences were obtained from six individuals for two genes. L1 gene sequences were identical to all previously detected sequences; E7 gene sequences, were more variable, yielding five distinct SfPV1 strains that differing by less than 2% from strains circulating in the Midwest and Mexico, between 1915 and 2005. Our results do not clarify whether strains are host species specific, though they are consistent with SfPV specificity to genus Sylvilagus.
Subject(s)
Cottontail rabbit papillomavirus/isolation & purification , Papillomavirus Infections/veterinary , Rabbits/virology , Skin Neoplasms/veterinary , Animals , Antigens, Viral/genetics , Base Sequence , Colorado/epidemiology , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/pathogenicity , DNA, Viral/genetics , DNA, Viral/isolation & purification , Genes, Viral , History, 20th Century , History, 21st Century , Host Specificity , Kansas/epidemiology , Mexico/epidemiology , Midwestern United States/epidemiology , Molecular Sequence Data , Museums , Oncogene Proteins, Viral/genetics , Papillomavirus Infections/epidemiology , Papillomavirus Infections/history , Papillomavirus Infections/virology , Phylogeny , Rabbits/classification , Sequence Homology, Nucleic Acid , Skin Neoplasms/epidemiology , Skin Neoplasms/history , Skin Neoplasms/virology , Species Specificity , Tumor Virus Infections/epidemiology , Tumor Virus Infections/history , Tumor Virus Infections/veterinary , Tumor Virus Infections/virology , Viral Structural Proteins/geneticsABSTRACT
A wild-caught desert cottontail rabbit (Sylvilagus audubonii) from Colorado was observed to have large, pedunculated, dark cutaneous lesions on its abdomen and cylindrical masses on its mouth. Morphologically, the masses were consistent with previous reports of virally induced papillomas. Subsequent DNA analysis indicated widespread infection with cottontail rabbit papillomavirus.
Subject(s)
Cottontail rabbit papillomavirus/isolation & purification , Papilloma/veterinary , Papillomavirus Infections/veterinary , Animals , Colorado/epidemiology , Male , Papilloma/epidemiology , Papilloma/pathology , Papilloma/virology , Papillomavirus Infections/epidemiology , Papillomavirus Infections/pathology , Papillomavirus Infections/virology , RabbitsABSTRACT
A wildlife sanctuary presented an adult female cottontail rabbit (Sylvilagus spp.), age unknown, to the Colorado State University Pathology service for postmortem examination. Gross examination revealed numerous pigmented wartlike lesions arising from the skin of the head surrounding the ears, eyes, nares, mouth, and dorsum. Masses were firm, friable, and easily detached from the underlying skin. Differential diagnoses included Cottontail rabbit papillomavirus, Rabbit fibroma virus, and Myxoma virus. Histological examination revealed multiple papillary masses lined by stratified squamous epithelial cells with central cores of fibrovascular connective tissue and parakeratotic hyperkeratosis. Cells of the Stratum spinosum were frequently swollen with abundant perinuclear, cytoplasmic, clearing, and occasional intranuclear basophilic, glassy, spherical inclusions up to 3 microm in diameter. The lesions were consistent with Cottontail rabbit papillomavirus infection. Papilloma virus antigens were identified by immunohistochemistry. In addition, papillomavirus particles were identified by transmission electron microscopy within Langerhans cells of the epidermis, suggesting a unique mechanism for systemic dissemination of the virus. The present case report highlights the finding of viral particles within the Langerhans cells and suggests a novel mechanism of pathogenesis.
Subject(s)
Cottontail rabbit papillomavirus/isolation & purification , Langerhans Cells/virology , Papillomavirus Infections/veterinary , Animals , Animals, Wild/virology , Female , Hair/pathology , Hair/virology , Immunohistochemistry , Langerhans Cells/pathology , Papillomavirus Infections/pathology , Rabbits , Skin/pathology , Skin/virologyABSTRACT
Shope papillomavirus or cottontail rabbit papillomavirus (CRPV) is one of the first small DNA tumour viruses to be characterized. Although the natural host for CRPV is the cottontail rabbit (Sylvilagus floridanus), CRPV can infect domestic laboratory rabbits (Oryctolagus cuniculus) and induce tumour outgrowth and cancer development. In previous studies, investigators attempted to passage CRPV in domestic rabbits, but achieved very limited success, leading to the suggestion that CRPV infection in domestic rabbits was abortive. The persistence of specific anti-L1 antibody in sera from rabbits infected with either virus or viral DNA led us to revisit the questions as to whether L1 and infectious CRPV can be produced in domestic rabbit tissues. We detected various levels of L1 protein in most papillomas from CRPV-infected rabbits using recently developed monoclonal antibodies. Sensitive in vitro infectivity assays additionally confirmed that extracts from these papillomas were infectious. These studies demonstrated that the CRPV/New Zealand White rabbit model could be used as an in vivo model to study natural virus infection and viral life cycle of CRPV and not be limited to studies on abortive infections.
Subject(s)
Antibodies, Viral/blood , Antigens, Viral/immunology , Antigens, Viral/isolation & purification , Cottontail rabbit papillomavirus , Papillomavirus Infections/blood , Papillomavirus Infections/virology , Viral Structural Proteins/immunology , Viral Structural Proteins/isolation & purification , Virion/isolation & purification , Animals , Antigens, Viral/metabolism , Cells, Cultured , Cottontail rabbit papillomavirus/immunology , Cottontail rabbit papillomavirus/isolation & purification , DNA, Viral/isolation & purification , Enzyme-Linked Immunosorbent Assay , Immunohistochemistry , Papillomavirus Infections/metabolism , Rabbits , Reverse Transcriptase Polymerase Chain Reaction , Viral Structural Proteins/metabolismABSTRACT
A 3-yr-old female Flemish Giant pet rabbit developed a papilloma on the right nictitating membrane. Although the papilloma was excised surgically, it promptly recurred. Examination of the eye 10 wk after surgery revealed that in addition to the initial mass, 2 smaller papillomas were present on the lower eyelid. All 3 masses were excised, and histology revealed papillomatous hyperplasia of the conjunctival epithelium, koilocytosis, and intranuclear viral inclusions. Polymerase chain reaction amplified papillomaviral DNA from the largest papilloma. Sequencing of the amplicon revealed 99.3% homology with rabbit oral papillomavirus (ROPV). All 3 masses recurred after removal. In addition, the rabbit was noted to be losing weight. Weight loss continued until the rabbit died 3 mo later. All 3 papillomas persisted until death. This article provides the fi rst description of ROPV causing conjunctival papillomas and is the fi rst report of ROPV from the southern hemisphere. The persistence of the papillomas in this case is also unusual and may suggest that ROPV-induced conjunctival papillomas are less likely than oral papillomas to spontaneously regress. Alternatively, the death of this rabbit may indicate a compromised immune system that allowed papillomaviral persistence.
Subject(s)
Conjunctival Neoplasms/pathology , Cottontail rabbit papillomavirus/isolation & purification , Papilloma/pathology , Papillomavirus Infections/pathology , Animals , Conjunctival Neoplasms/surgery , Conjunctival Neoplasms/virology , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/physiology , DNA, Viral/analysis , Fatal Outcome , Female , Papilloma/surgery , Papilloma/virology , Papillomavirus Infections/surgery , Papillomavirus Infections/virology , RabbitsABSTRACT
Cervical cancer arises from lesions caused by infection with high-risk types of human papillomavirus (HPV). Therefore, vaccination against HPV could prevent carcinogenesis by preventing HPV infection or inducing lesion regression. HPV E2 protein is an attractive candidate for vaccine development because it is required for papilloma formation, is involved in all stages of the virus life cycle, and is expressed in all premalignant lesions as well as some cancers. This study reports vaccination against E2 protein using a rabbit model of papillomavirus infection. A recombinant adenovirus (Ad) vector expressing the E2 protein of cottontail rabbit papillomavirus (CRPV) was tested for therapeutic efficacy in CRPV-infected rabbits. Primary immunization with the Ad-E2 vaccine, compared to immunization with a control Ad vector, reduced the number of papilloma-forming sites from 17 of 45 to 4 of 45. After booster immunization, vaccinated rabbits formed no new papillomas versus an additional 23 papillomas in rabbits that received the control vector. Papillomas in the Ad-E2 vaccinees were significantly smaller than those in the control rabbits, and all four papillomas in the Ad-E2 vaccinated rabbits regressed. No CRPV DNA was detected either in the regression sites or in sites that did not form papillomas, indicating that the vaccination led to clearance of CRPV from all infected sites.
Subject(s)
Adenoviridae/genetics , Cottontail rabbit papillomavirus/immunology , Papilloma/prevention & control , Papillomavirus Infections/prevention & control , Transcription Factors/administration & dosage , Viral Proteins/administration & dosage , Viral Vaccines/administration & dosage , Animals , Antibodies, Viral/blood , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/isolation & purification , Female , Genetic Vectors , Papilloma/virology , Papillomavirus Infections/virology , Rabbits , Transcription Factors/genetics , Transcription Factors/immunology , Transcription Factors/metabolism , Vaccination , Vaccines, DNA/administration & dosage , Vaccines, DNA/immunology , Viral Proteins/genetics , Viral Proteins/immunology , Viral Proteins/metabolism , Viral Vaccines/immunologyABSTRACT
The present study examined topical effects of cidofovir on cutaneous rabbit warts. Based on an inoculum-dependency study, each New Zealand White rabbit was inoculated with a high and low titer of cottontail rabbit papillomavirus (CRPV) at four sites on each dorsolateral area. Inoculation with 50 ID(50) induced papillomas at 100% of the inoculation sites within 16+/-1 days, and the wart growth curve plateaued within approximately 7 weeks. With an inoculum of 5 ID(50), 80% of the inoculated sites developed papillomas within 21+/-1 days and their size plateaued at a later time. Cidofovir was applied topically twice daily on the inoculated sites at a concentration of 1% for 18 days, starting at three different time points. In the first experiment, treatment was initiated 7 days post-inoculation. One of the inoculated sides received cidofovir or the vehicle, PBS, while the other side was left untreated. With this treatment regimen, cidofovir significantly delayed the time of onset and the growth rate of papillomas induced with the high titer of inoculum. It completely prevented papilloma-induction on the sites inoculated with the low titer of CRPV. Reversible side-effects of cidofovir were observed on the directly treated area including erythema, necrosis, and flaking. Both therapeutic and side-effects were limited to the sites of direct exposure. In the second experiment, one of the two sides in each group of rabbits received cidofovir or vehicle starting on day 29 post-inoculation. With this treatment regimen, cidofovir significantly reduced wart growth against the low titer only. Topical treatment initiated on day 49 post-inoculation was not effective on warts initiated with either viral titer. These results demonstrated that topical cidofovir could be very effective against papillomavirus-induced wart growth if it is initiated early during the infection, especially against low titers of inoculum.
Subject(s)
Antiviral Agents/administration & dosage , Cottontail rabbit papillomavirus , Cytosine/analogs & derivatives , Organophosphonates , Organophosphorus Compounds/administration & dosage , Warts/drug therapy , Administration, Topical , Animals , Cidofovir , Cottontail rabbit papillomavirus/drug effects , Cottontail rabbit papillomavirus/isolation & purification , Cytosine/administration & dosage , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Rabbits , Time Factors , Warts/pathology , Warts/virologyABSTRACT
We previously observed that warts induced by an isolate of cottontail rabbit papillomavirus (CRPV) showed incomplete instead of systemic regression in some domestic rabbits. We report that the viral isolate contained, as a major component, a CRPV strain (CRPVb) showing an unexpectedly high divergence in the E6 and E7 open reading frames (ORFs), compared to the prototype CRPVa present in the isolate as a minor component. The E6 and E7 oncoproteins of CRPVa and -b disclosed only 87.5% identical amino acids and differed in size by three and two amino acids, respectively. This divergence involved (i) a great number (4.4%) of nucleotide substitutions and a high rate (83.3%) of nonsynonymous mutations; (ii) mutations changing the E6 and E7 stop codons; and (iii) in-frame sequence insertions in the E6 ORF (18 nucleotides) and downstream of the mutated E7 stop codon (6 nucleotides), both likely to result from a duplication of adjacent sequences. These extensive differences could account for distinct biological and antigenic properties. Strikingly, only four (0.8%) amino acids of the L1 major capsid protein were variable. Thus, it seems likely that sequence duplications and mutations affecting stop codons exert a strong selection pressure on the fixation of nonsynonymous mutations and that phylogenetic calculations based only on point mutations may misevaluate the time scale of the evolution of papillomaviruses.
Subject(s)
Cottontail rabbit papillomavirus/genetics , Evolution, Molecular , Genetic Variation , Oncogene Proteins, Viral/genetics , Amino Acid Sequence , Animals , Base Sequence , Capsid/genetics , Cloning, Molecular , Cottontail rabbit papillomavirus/isolation & purification , Molecular Sequence Data , Point Mutation , Polymerase Chain Reaction , RabbitsABSTRACT
Several small, raised lesions from the underside of the tongue of domestic rabbits were isolated, and an extract prepared and tested for the presence of rabbit oral papillomavirus (ROPV). Two weeks after inoculation of this extract into the underside of rabbit tongues, multiple small discrete, grey-white nodules were observed that reached a maximum size of 2 mm in diameter by 5 weeks. These lesions showed typical ROPV pathology, and nuclei stained positive for papillomavirus (PV) group-specific antigen (GSA) by immunocytochemistry. Tissue fragments from rabbit tongues were incubated with a suspension of ROPV and placed subrenally into athymic mice. After 60 days, cysts were removed, sections cut for histology, and a virus stock prepared. GSA staining and in situ hybridization demonstrated that the xenografts were morphologically transformed with areas showing strong nuclear staining for viral capsid antigen and ROPV DNA. Extracts prepared from the pooled xenografts contained infectious ROPV as demonstrated by inoculation into the undersurface of tongues of nonimmune New Zealand White rabbits. The results demonstrated that stocks of infectious ROPV can be prepared in the athymic mouse xenograft system for use in studies on the experimental transmission of a mucosal-targeting animal papillomavirus.
Subject(s)
Cottontail rabbit papillomavirus/growth & development , Papillomavirus Infections/veterinary , Tumor Virus Infections/veterinary , Animals , Antigens, Viral/analysis , Base Sequence , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/isolation & purification , Cottontail rabbit papillomavirus/pathogenicity , DNA, Viral , Immunohistochemistry , In Situ Hybridization , Mice , Mice, Nude , Molecular Sequence Data , Papillomavirus Infections/virology , Polymerase Chain Reaction , Rabbits , Tissue Transplantation , Tongue/virology , Transplantation, Heterologous , Tumor Virus Infections/virology , Virus CultivationABSTRACT
Empirical scanning of natural or engineered peptide sequences for functional residues is inherently dependent upon efficient expression of large numbers of individual sequence variants to assay their relative functional potency. The insect baculovirus system has been widely used for expression of viral coat proteins, but it generally requires prior isolation and expansion of a plaque-purified recombinant viral stock to generate useful quantities of self-assembled virus-like particles. In search of a more rapid means of expression of analytical levels of the L1 coat protein of cottontail rabbit and human type 11 papilloma-viruses, we found that even brief transient cotransfection of insect cells with baculovirus plasmid transfer vectors and viral DNA yielded assembled particles that were immunologically indistinguishable from particles obtained with plaque-purified viral stocks. Within six days of plasmid/viral DNA cotransfection of Sf9 cells, at least 1-2 micrograms of assembled L1 particles/100-mm plate could be demonstrated, which proved more than sufficient to assay functionality. Transient cotransfection of insect cells should provide general utility for rapid high-level expression of sets of protein sequence variants, as well as other sequence-scanning applications such as sequence optimization in protein engineering.
Subject(s)
Baculoviridae/genetics , Capsid Proteins , Cottontail rabbit papillomavirus/isolation & purification , Gene Expression Regulation, Viral/genetics , Spodoptera/virology , Animals , Antibodies, Viral/analysis , Cell Line , Cottontail rabbit papillomavirus/genetics , DNA, Viral/analysis , Epitopes , Oncogene Proteins, Viral/biosynthesis , Rabbits , TransfectionABSTRACT
Papillomaviruses are small DNA tumor viruses with a life cycle inseparably linked to the differentiation of the pluristratified epithelium. The infection of epithelial layers of the skin may remain latent or may result in the development of benign tumors. A certain number of distinct papillomavirus types, however, cause lesions which have a high risk of progression into carcinomas, and extensive efforts have been made to understand this process. comparatively little is known about the initial events during the establishment of a persistent infection and papilloma development. Although it is generally accepted that the growth of a papilloma requires the infection of cells in the basal layer of the epithelium, it remains unknown which cells perform this task. We have analyzed by in situ hybridization biopsy samples taken at various time points after infection of domestic rabbits with cottontail rabbit papillomavirus. The positive cells detected at a low frequency in biopsy samples taken after 11 days predominantly expressed high levels of E6 and E7 mRNA and were localized in the outer epithelial root sheath and in the bulbs of hair follicles. A clonal analysis of keratinocytes isolated from different subfragments of individual rabbit hair follicles demonstrated a clear colocalization of cottontail rabbit papillomavirus mRNA-positive cells with clonogenic cells in hair follicles. These data suggest that the cells competent to establish papillomatous growth represent a subpopulation of keratinocytes in hair follicles with properties expected of epithelial stem cells.
Subject(s)
Cottontail rabbit papillomavirus/physiology , Hair Follicle/virology , Papillomavirus Infections/virology , Stem Cells/virology , Tumor Virus Infections/virology , 3T3 Cells , Animals , Cottontail rabbit papillomavirus/genetics , Cottontail rabbit papillomavirus/isolation & purification , Hair Follicle/cytology , Hair Follicle/metabolism , Mice , Papillomavirus Infections/pathology , Papillomavirus Infections/veterinary , RNA, Viral , Rabbits , Skin/pathology , Skin/virology , Stem Cells/cytology , Stem Cells/metabolism , Time Factors , Tumor Virus Infections/pathology , Tumor Virus Infections/veterinary , Viral Proteins/geneticsABSTRACT
Los virus del papilloma humano estan asociados estrechamente con el cancer de cuello de utero. Se ha demostrado que los virus 6 y 11 estan asociados con las lesiones premalignas, mientras que los 16 y 18 a las lesiones malignas del cancer cervical. En el presente trabajo se describe la obtencion de un anticuerpo monoclonal murino contra el virus 16 del pailloma humano, utilizando el metodo convencional de fusion somatica, el cual reconoce por inmunohistoquimica sobre cortes de tejido de biopsias de cervix uterino las celulas coilociticas y disqueratosicas ubicadas en las capas mas superficiales del epitelio infectado. La presencia del virus tipo 16 de las muestras analizadas se comprobo por estudios de hibridacion in situ realizadas con anterioridad. El uso de este anticuerpo monoclonal en Anatomia Patologica permitira el diagnostico de la presencia del virus 16 del papiloma humano en aquellas muestras que presenten las atipias celulares caracteristicas de esta enfermedad
Subject(s)
Animals , Mice , Antibodies, Monoclonal , Antibodies, Monoclonal/isolation & purification , Cell Fusion , Cottontail rabbit papillomavirus/isolation & purification , Immunohistochemistry/methods , Uterine Cervical Neoplasms/diagnosisABSTRACT
Los virus del papilloma humano están asociados estrechamente con el cáncer de cuello de útero. Se ha demostrado que los virus 6 y 11 están asociados con las lesiones premalignas, mientras que los 16 y 18 a las lesiones malignas del cáncer cervical. En el presente trabajo se describe la obtención de un anticuerpo monoclonal murino contra el virus 16 del pailloma humano, utilizando el método convencional de fusión somática, el cual reconoce por inmunohistoquímica sobre cortes de tejido de biopsias de cérvix uterino las células coilocíticas y disqueratósicas ubicadas en las capas más superficiales del epitelio infectado. La presencia del virus tipo 16 de las muestras analizadas se comprobó por estudios de hibridación in situ realizadas con anterioridad. El uso de este anticuerpo monoclonal en Anatomía Patológica permitirá el diagnóstico de la presencia del virus 16 del papiloma humano en aquellas muestras que presenten las atipias celulares características de esta enfermedad (AU)
Subject(s)
Animals , Mice , Uterine Cervical Neoplasms/diagnosis , Cottontail rabbit papillomavirus/isolation & purification , Antibodies, Monoclonal/isolation & purification , Antibodies, Monoclonal , Cell Fusion , Immunohistochemistry/methodsABSTRACT
The ability to obtain infectious papillomavirus virions from molecularly cloned DNA has not been previously reported. We demonstrate here that viral genomes isolated from a recombinant++ DNA clone of cottontail rabbit papillomavirus (CRPV) gave rise to infectious virus when inoculated into cottontail rabbit skin. Replication occurred in papillomas that formed at inoculation sites. Extract of a DNA-induced papilloma was serially passaged to naive rabbits with high efficiency. Complete virus was fractionated on cesium chloride density gradients, and papillomavirus particles were visualized by electron microscopy. CRPV DNA isolated from virions contained DNA sequence polymorphisms that are characteristic of the input CRPV-WA strain of virus, thereby proving that the newly generated virus originated from the molecularly cloned viral genome. These findings indicate that this will be a useful system in which to perform genetic analysis of viral gene functions involved in replication.
Subject(s)
Cottontail rabbit papillomavirus/growth & development , DNA, Viral/metabolism , Papilloma/microbiology , Skin Neoplasms/microbiology , Virion/pathogenicity , Animals , Cloning, Molecular , Cottontail rabbit papillomavirus/isolation & purification , Cottontail rabbit papillomavirus/pathogenicity , Cottontail rabbit papillomavirus/ultrastructure , DNA Replication , DNA, Viral/genetics , Polymorphism, Genetic , Rabbits , Sequence Analysis, DNA , Serial Passage , Skin/microbiology , Virion/isolation & purification , Virion/ultrastructure , Virulence , Virus ReplicationABSTRACT
In situ hybridization (ISH) for human papillomavirus (HPV)-6, -11, -16, -18, and -31 DNA was performed on 615 formalin-fixed paraffin-embedded cervical biopsies using biotinylated DNA probes. Results were obtained from 584 samples with 266 (45.5%) containing HPV-DNA sequences. Ninety percent of condyloma acuminatum specimens were positive for HPV-DNA with 18 of 19 positive cases containing HPV-6 or -11 DNA. The detection rate of HPV in cervical intraepithelial neoplasia (CIN) lesions was 50.6% (239 of 472), while only 8 of 91 (8.9%) cervical biopsies considered to be histologically normal or with minimal dysplasia contained HPV-DNA as demonstrated by ISH. The prevalence of HPV-16, -18, and/or -31 DNA increased with the severity of the lesions, with 20 of 20 (100%) positive CIN-III lesions containing these viral types compared with 102 of 157 (65.0%) positive CIN-I lesions. ISH with biotinylated DNA probes appears helpful in identifying lesions containing higher risk viral strains.
Subject(s)
Cottontail rabbit papillomavirus/isolation & purification , DNA Probes, HPV , DNA, Viral/analysis , Tumor Virus Infections/microbiology , Uterine Cervical Neoplasms/microbiology , Biopsy , Biotin , Female , Humans , Nucleic Acid Hybridization , Tumor Virus Infections/pathology , Uterine Cervical Neoplasms/pathologyABSTRACT
We have compared warts and carcinomas from cottontail and domestic rabbits for the presence of cottontail rabbit papillomavirus (CRPV) and the status of the viral DNA genome. Our studies indicate that benign warts from cottontail rabbits, whether found naturally or induced in the laboratory, contain large amounts of virus and on the average 1000 copies of the virus genome per cell. Both benign warts and carcinomas from domestic rabbits contain significantly reduced levels of virus relative to cottontail rabbit warts and an average of 100 copies of the virus genome per cell. A single sample of a naturally occurring cottontail rabbit carcinoma contained approximately 80 copies of the viral genome per cell. None of the tumors that we have analyzed thus far appear to have integrated viral genomes by Southern blot analysis of undigested and restriction endonuclease-digested DNA samples. Furthermore, the CRPV genome present in domestic rabbit carcinomas and a cottontail rabbit carcinoma appears identical by restriction endonuclease mapping to that present in papillomas of cottontail and domestic rabbits indicating that no major deletions or rearrangements of the CRPV genome had occurred during the progression of benign to malignant tumors nor was a variant of wild-type CRPV responsible for this phenomenon. Finally, we have demonstrated morphological transformation in vitro of NIH 3T3 and C127 cells upon infection with purified CRPV and upon transfection with purified CRPV DNA. Furthermore, single cell clones derived from transformed foci contain free forms of CRPV DNA that persist through continued passage in culture. Cells transformed by CRPV grow in soft agar in vitro and produce tumors in athymic nude mice.
Subject(s)
Carcinoma/microbiology , Cottontail rabbit papillomavirus/genetics , DNA, Viral/analysis , Papillomaviridae/genetics , Tumor Virus Infections/microbiology , Warts/microbiology , Animals , Cell Line , Cell Transformation, Viral , Cottontail rabbit papillomavirus/isolation & purification , DNA, Viral/physiology , Genes, Viral , Nucleic Acid Hybridization , RabbitsABSTRACT
Four methods for preparation of Shope papilloma virus have been compared. These comprise: (1) alternate centrifugation at low and high speed after grinding with alundum and saline, (2) purification with a fluorocarbon after similar extraction, (3) extraction with the aid of 2-mercaptoethanol and purification by alternate low- and high-speed centrifugation, and (4) extraction with 2-mercaptoethanol followed immediately by centrifugation to equilibrium in CsCI. It is necessary to remove CsCI before chemical analysis, and this has been accomplished by chromatography on Sephadex G-75. After banding, the virus from preparation (4) appears homogeneous in the electron microscope. The deoxyribonucleic acid (DNA) contents of the preparations were (1) 7.8, (2) 8.3, (3) 8.0, and (4) 11.9 percent of total nucleoprotein. At equal inputs of DNA, there was no significant difference in infectivity of any of the preparations. Method (4) is simpler than the others and results in a higher yield of a homogeneous preparation.