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Fetal equine fibroblasts exposed to bovine papillomavirus became transformed by the criteria of morphological alterations and the acquisition of an increased life span, although they failed to grow in soft agar. Papillomavirus genome persisted in the transformed fibroblasts and was apparently not integrated with the cellular genome. These findings support the notion that bovine papillomaviruses are involved in the production of equine sarcoids.

A hypothesis linking the concept of mucosal immune surveillance (Langerhans' cells and intra-epithelial lymphocytes) with recent evidence of human papillomavirus (HPV) infection of the oesophagus in a sequence of events which leads to squamous dysplasia and invasive carcinoma is presented. It is believed that the essential pathway to squamous carcinoma involves an aberration of the Langerhans' cell/lymphocyte network and its symbiotic relationship with squamous cells as a result of persistent HPV infection in the epithelium. Neoplastic transformation may occur when this 'at-risk' mucosa is exposed to one or several co-carcinogenic factors present in the environment. This hypothesis is supported by morphological changes present in acanthotic lesions of the oesophagus and by immunocytochemical evidence of HPV infection of the mucosa. In addition, this viewpoint offers a possible explanation for the regional variation of oesophageal carcinoma, the rising incidence of the disease, and the association with multifocal squamous carcinomas of the upper aerodigestive tract.

In these experiments a technique for enhancing the virus replication in tissue culture (RK13 cells) has been used. The method consisted in growing the cells in presence of mug 0.4-0.8 of 6-Azauridine/ml of cellular suspension until the monolayers were formed. This pretreatment enhances the replication of several animal viruses with increased infectious titers (vesicular stomatitis, equine arteritis, equine rhinopneumonitis, Aujeszky disease and myxomatosis virus) and with increased yield of total virus in the culture (myxomatosis virus). In other experiment the 6-Azauridine pretreatment of the cells has shown to render the cells more susceptible to interferon preparation action with subsequent high rate of vesicular stomatitis virus plaques reduction.

A papovavirus, CCL 33 PV, isolated from a porcine cell line, CCL 33 (GT), was characterized. Based on a comparison of four isoenzyme systems, CCL 33 (GT) and CCL 33 (ATCC), obtained directly from the American Type Culture Collection, appeared to be the same. In addition to the previously characterized C-type virus of CCL 33 cultures, CCL 33 (GT) produced CCL 33 PV in high quantity, but CCL 33 (ATCC) produces a papovalike virus, presumably the same as CCL 33 PV, in barely detectable amounts. Serological results showed that CCL 33 PV is apparently identical to a papovavirus (SPV) isolated by Newman and Smith after inoculation of CCL 33 with concentrated porcine trypsin. These studies extend the characterization of this papovavirus, demonstrating that CCL 33 PV is weakly hemagglutinating after neuraminidase treatment, has a high affinity for a component of fetal bovine serum and is highly infectious in appropriate porcine cell systems rather than very defective as reported previously. Moreover, it was concluded from the data that CCL 33 PV is probably indigenous to the CCL 33 porcine cell line.

Studies were initiated to determine the practicality of using various tissue cultures for the propagation of murine viruses isolated from laboratory animals. The cytopathogenic effects of 10 murine viruses known to cause disease in laboratory rodents were compared in monolayer cultures of L929, BHK-21, WI-38, BSC-1, and Vero cells. The susceptibility of primary hamster embryo, hamster kidney, mouse embryo, mouse kidney, and rat embryo cell cultures was also tested. Seven of the viruses produced effects in at least 1 of the cell substrates. The remaining 3 viruses, namely H-1, K, and mouse hepatitis, produced no effects in the cell cultures tested.

We have studied the presence and state of human papillomavirus (HPV) genomes in condylomata acuminata of the vulva, vagina, and cervix and in flat condylomas of the cervix from Japanese females by DNA hybridization with HPV 6 and 11 DNAs. Unintegrated HPV 6-related DNA (HPV 6 or 11 DNA) was detected in 55 out of 60 condylomata acuminata and in 1 out of 4 flat condylomas under stringent hybridization conditions. The HPV DNAs were identified as HPV 6 DNA in 29 condylomata acuminata (20 vulvar, 3 vaginal, and 6 cervical samples) and 1 flat condyloma and as HPV 11 DNA in 6 condylomata acuminata (6 vulvar samples). The data indicate close association of HPV types 6 and 11 with condylomata.

Monkey B-lymphotropic papovavirus (LPV) replicates only in B-lymphoblastoid cells, whereas the LPV mutant 76 (LPV-76) can grow in either B- or T-lymphoblastoid cells. The nucleotide sequence of the wild-type LPV PstI B segment was compared with that of LPV-76 PstI-B, within which the mutation responsible for the extended host range had been located. The VP-1 coding region of LPV-76 was found to have mutations, single-base substitutions causing three amino acid substitutions.

Murine K-papovavirus was serially passaged 10 times in primary cultures of mouse embryo cells, using cell suspensions and media from infected cultures to inoculate fresh flasks at each passage level. Titers of K virus infectivity in cell suspensions and media rose during serial passage, but viral hemagglutinating activity was not detected in cells or media before the 10th passage. Although the infectivity of K virus for mouse embryo cultures was enhanced by serial passage, lethality of the virus for suckling mice was lost. The present study represents the first successful serial transmission of K virus infection in vitro.

A lymphotropic papovavirus was isolated from a lymphoblastoid cell line of African green monkey (AGM) cells which also contained a herpesvirus and a paramyxovirus-like agent. The papovavirus was analyzed by restriction endonuclease cleavage; its biochemical and serological crossreactivity with SV40 and host range have been determined. Thus far, only B-lymphoblasts of primate and human origin have been found to be susceptible to infection. Although more than 50% of the tested monkey sera were reactive with antigens of this virus, all human sera tested failed to react. Cleavage patterns and hybridization studies with the viral DNA indicate that the virus represents a novel member of the papovavirus group that is characterized by its lymphotropic host range. Papovavirus particles were also demonstrated in a human lymphoblastoid cell line (CCRF-SB) originally derived from a leukemic child. These cells revealed nuclear fluorescence when tested with human sera, but failed to react with AGM sera. Although characterization of this agent has not yet been completed, available evidence suggests that it represents another lymphotropic papovavirus which seems to be spread within the human population.

Purified vesicular stomatitis virus grown in the human cervical carcinoma HeLa cell line, VSV(HeLa), contains a 75 kDa tumor-specific antigen, detectable by immunoblotting of electrophoretically separated proteins with rabbit antiserum made against whole HeLa cells. Nearly identical results were obtained with VSV grown in the tumorigenic human hybrid ESH-5L cells, but not with the matched non-tumorigenic ESH-5E cells. Growth of VSV in 4 other independently isolated human cervical carcinoma cell lines led to the concentration of the same 75 kDa tumor-specific antigen by VSV. Infection of 2 other human cervical carcinoma cell lines did not lead to the detection of this antigen. The expression of the tumor-specific antigen correlated directly with the amount of RNA expression from human papillomavirus integrated in the DNA of these cells, irrespective of whether the papillomavirus was type 16 or 18.

Primary cultures of mouse embryo cells were inoculated with K virus, a murine papovavirus, and were examined for cytopathic effect (CPE) of for the development of fluorescent antibody staining specific for K virus V antigen. CPE was not observed. However, numerous cells in infected cultures exhibited positive nuclear fluorescence, and the presence of papovavirus virions was demonstrated by electron microscopy. Extracts from infected cultures produced typical K virus pneumonia in newborn mice. Inoculation of cultures with serial dilutions of virus demonstrated that these cells provide a fluorescent antibody assay for K virus equal in sensitivity to animal inoculation methods. Although specific K virus fluorescence was also detected in cultures of fetal mouse endocardial cells, livers, placentas, and brains, positive cells were much less abundant in these cultures than in cultures of mouse embryo cells. The mouse embryo culture assay described in the present paper represents the first method of measuring K virus infectivity in vitro.

JC human papovavirus was found to replicate in primary human amnion cells. The virus has undergone eight passages in amnion cells and was identified by serological methods as JC virus. By restriction endonuclease analysis of the viral DNA, the fragments observed were identical to those previously reported for the prototype strain.

Clinical and epidemiological data have implicated the human papillomaviruses (HPVs) as having an etiologic role in some anogenital malignancies, with HPV-16 being most frequently (greater than 60%) detected in cervical carcinoma. HPV-16 is actively transcribed in the cancers; the most abundant transcripts map to the E6 and E7 early open reading frames. Evidence is presented that the HPV-16 E7 open reading frame encodes transcriptional transactivation and cellular transformation functions analogous to those of adenovirus E1A proteins. Specifically, the HPV-16 E7 gene product could transactivate the adenovirus E2 promoter and cooperate with an activated ras oncogene to transform primary baby rat kidney cells. The E7 transforming function differed somewhat from that of adenovirus E1A in that E7 was also able to transform established mouse cells. Examination of the amino acid sequence of HPV-16 E7 revealed striking similarities with conserved domains 1 and 2 of adenovirus E1A proteins.

Monkey B-lymphotropic papovavirus (LPV) DNA present as free copies in LPV-transformed hamster embryo cells was molecularly cloned in Escherichia coli. Twenty-two of 24 cloned DNAs were 4.9 kilobases long and shorter than the wild-type LPV DNA (5.1 kilobases). The shorter DNA was nondefective and generated infectious virus (designated LPV-76) upon transfection of human B-lymphoblastoid BJA-B cells. LPV-76 DNA had a small deletion in the early region and a deletion and an insertion in the control region for transcription. LPV-76 VP-1 was apparently larger than that of the wild-type LPV. LPV-76 could grow in human T-lymphoblastoid MOLT-4 cells, whereas the wild-type LPV replicated only in B-lymphoblastoid cells. Characterization of constructed recombinant viruses between wild-type LPV and LPV-76 showed that the mutation responsible for the extended host range of LPV-76 was within the PstI B fragment, which includes the VP-1 coding region. These data strongly suggest that the mutation of VP-1 altered the host range of LPV.

Foetal mouse glial cultures were inoculated with murine K papovavirus and subjected to serial subcultivation. Two cell lines were developed. The first of these, KVBCG2A, remained positive for viral infectivity and K virus capsid (V) antigen for over 30 subcultivations. Productive infection was not abolished by serial subcultivation in the presence of antiviral antibody. The second cell line, KVBCG1B, became negative for infectious virus and K virus V antigen, could be cloned from single cells and produced tumours in mice. Sera from tumour-bearing animals produced nuclear fluorescence of KVBCG1B cells and K virus-infected mouse embryo cells but did not react with uninfected mouse embryo cells or with cells infected by polyoma virus. DNA hybridization studies confirmed the presence of K virus DNA in KVBCG1B cells and suggested integration of the viral genome into host chromosomal DNA. K virus produces both persistent infection and cell transformation in glial cultures derived from its natural host.

Open reading frame (ORF) fragments (putative gene fragments) from human papillomavirus type 6b (HPV-6b) were inserted into the bacterial expression vector pHK413 to provide viral antigenic determinants. Approximately 86% of the entire L1 ORF, 82% of the E2 ORF, and 52% of the L2 ORF were expressed in Escherichia coli. The E1 ORF was cloned as two fragments. The constructions containing E1n (coding for the N-terminal region) and E1c (coding for the C-terminal region) expressed 27% and 16% of the E1 ORF, respectively. Protein encoded by the L1 ORF, but not that encoded by the L2 ORF, reacted with antibodies elicited by disrupted bovine papillomavirus. These reagents will be extremely useful in unravelling the HPV-6b replication cycle.

A virus suspected of causing high death rates in fledgling budgerigars in Georgia and Texas aviaries was isolated in budgerigar embryo fibroblasts inoculated with tissue homogenates from affected birds. Virus was most easily recovered from tissues containing many intranuclear inclusion bodies. Cytopathic effect on fibroblasts of all four isolates was characterized by a swollen nucleus followed by rounding and detachment of the affected cell from the monolayer. Properties suggesting the B-931 isolate belongs to the papovaviridae family are (1) presence of DNA; (2) insensitivity to treatment with CHCl3; and (3) presence of cubic viral particles 42 to 49 nm in diameter in the nucleus of infected chicken embryo fibroblasts. The isolate did not hemagglutinate erythrocytes of chickens, turkeys, budgerigars, guinea pigs, or type O humans and was basically stable against heating and freeze-thawing. An examination of fledgling budgerigars from infected aviaries demonstrated that sick birds carried more virus than healthy birds.

Adult human brain (AHB) cells infected with JC virus (JCV) developed a cytopathic effect (CPE) beginning 12--14 days after infection. Ultrastructurally, 37--40 nm papova virions were seen in the nuclei of infected cells, and both T and V antigen were demonstrated by indirect immunofluorescence. The hemagglutinating titer of JCV in infected AHB cells was 10--40 times higher than the amount of JCV used to initiate infection. AHB cells are more readily available than primary human fetal brain cells, they can be subcultured 15--25 times in vitro and they support JCV replication after multiple subcultivations. These properties make the AHB cell line useful for propagating JCV.

Papillomaviruses induce tumors of keratinocytes. Vegetative viral DNA replication and virion assembly are seen in those cells which are in the process of keratinizing or are keratinized. To date, no cell culture system has been developed that permits expression of the complete viral life cycle. Keratinocytes infected in culture may harbor the virus as a stable, replicating episome, but they do not support vegetative viral growth, nor do they become immortalized or transformed. The major obstacle in using keratinocyte cultures may be related to a dual need for transformation and full differentiation. Some animal papillomaviruses have been shown to be capable of transforming cultured murine fibroblasts. The fibroblast model is useful for identifying the viral-transforming gene(s) and their products.

A two-part study was done to assess the value of human fibroblast interferon (IFN-beta) in the treatment of condylomata acuminata. The first part was an open study of different IFN-beta preparations, which showed that intramuscular injection was the most suitable mode of administration of IFN-beta. In the double-blind placebo section 22 patients were given injections of 2 X 10(6) units IFN-beta or placebo for 10 consecutive days and followed up for 3 months. In 9 of the 11 in the IFN-beta group and 2 in the placebo group lesions disappeared from about 5 weeks after completion of the course of injections. After 3 months 8 of the non-responders were given a course of IFN-beta and all responded to treatment. None of those who had responded has had a recurrence, the disease-free period now being 12 months. Changes in (2'-5')oligo A synthetase levels in white blood cells confirm that intramuscular injections of IFN-beta produce a systemic response.

A transient assay has been used to study bovine papilloma virus type 1 (BPV-1) replication. We show that BPV-1 early replication occurs faster than cellular DNA synthesis. Initial replication events are dependent on a gene product(s), encoded by the BPV-1 E1 open reading frame. Mutational analysis of the viral upstream regulatory region shows the requirement of two domains in cis for replication. Domain one, located outside of the viral 69% transforming fragment, is an enhancer-like activity and can be replaced by other known viral enhancers. Domain two lies within sequences previously defined as plasmid maintenance sequence 1. The apparent requirement for a proximal enhancer function for replication may explain why certain BPV-1 constructions, when linked to bacterial plasmid sequences, can be maintained extrachromosomally while others cannot.

The growth of African green monkey lymphotropic papovavirus (LPV) in human lymphoblastoid cell line BJA-B was found to be slow and inefficient due to the accumulation of defective particles. An analysis of molecularly cloned LPV DNAs showed that 3 of 19 clones had DNAs that were longer (5.1 kilobases) than the DNAs of the other clones. The 5.1-kilobase DNA was infectious for BJA-B cells, whereas the shorter (4.8-kilobase) molecules were defective. Unlike the wild-type virus, stocks of LPV made from cloned, infectious DNAs were homogeneous and had higher titers. Using stocks of nondefective LPV, we investigated other biological properties. LPV replication in another human B-lymphoblastoid cell line was observed. The virus did not cause tumors when it was inoculated into newborn hamsters. Serological surveys of human and nonhuman primate sera indicated that virtually all primates, including humans, show evidence of infection by viruses antigenically related to LPV.

BK virus, a widespread human papovavirus, is shown to cause malignant transformation of BHK(21) clone 13 cells. The transformed cells grow as tumors upon inoculation in adult hamsters. The question of the possible role of this virus in human neoplasia is raised.

The occurrence and localization of vegetative viral DNA replication was studied in sections of tumors induced by the rabbit Shope papilloma virus, in cottontail and domestic rabbit papillomas, in primary domestic rabbit carcinoma, and in transplantable VX2 carcinoma, by in situ hybridization of radioactive RNA complementary to viral DNA. Vegetative viral DNA replication and viral protein synthesis were compared by means of cytological hybridization and immunofluorescence techniques on adjacent frozen sections. Vegetative viral DNA replication is completely repressed in the proliferating cellular layers of these tumors, which suggests a provirus state of the viral genome, as in other cells transformed by oncogenic DNA viruses. Vegetative viral DNA replication is induced, after initiation of the keratinization, in cells of cottonail rabbit papillomas, where it is usually followed by viral protein synthesis; this illustrates the influence of the physiological state of the host cell on the control of viral functions. Vegetative viral DNA replication is deteced only in a few cells of domestic rabbit papillomas, at the end of the keratinization process; this observation provides indirect evidence that the DNA synthesis specifically induced in these tumors after the onset of keratinization reflects mostly the induction of cellular DNA synthesis.

Some of the properties of a new human papovavirus, BK, have been examined. Host range studies of BK virus (BKV) showed human cells to be more sensitive to infection than monkey cells; human fetal brain cells appear to be highly sensitive to BKV, with the production of extensive cytopathology characterized by cytoplasmic vacuolization. The hemagglutinin of BKV is associated with the virion and is resistant to ether or heating at 56 C for 30 min. Fluorescent antibody as well as neutralization tests indicated antigenic similarities between simian virus 40 (SV40) and BKV. Cells undergoing lytic infection with BKV synthesized intranuclear T antigen(s) which reacted with SV40 T antibody demonstrable by immunofluorescence. However, BKV did not appear to induce SV40 transplantation antigens in transplantation-resistance tests. Evidence was obtained that BKV was present in humans prior to the widespread use of polio vaccines, thus ruling out the possibility that BKV is an SV40-related monkey virus, introduced into the human population by accidental contamination of poliovirus vaccines.

A new member of the papovavirus group has been isolated and appears to infect swine. The new agent, tentatively named swine papovavirus, appears to be very defective and replicates only within a very narrow host cell range. The original source of the isolate is under investigation. Preliminary evidence suggests that the origin of swine papovavirus is either a stable pig kidney cell line or pancreas-derived trypsin