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The association between undifferentiated nasopharyngeal carcinoma (NPC) and Epstein-Barr virus (EBV) is well established. Nevertheless, available evidence suggests that other cofactors are required for the development of undifferentiated NPC. Several investigations reported simian virus 40 (SV40) footprints in human tumors of different histotypes. Serum samples from patients with undifferentiated NPC (n = 64) and healthy subjects (n = 130) were analyzed by an indirect enzyme-linked immunosorbent assay (ELISA) with SV40 synthetic peptides to detect antibodies against viral peptide (VP) capsid proteins VP1, 2, and 3. Immunologic data indicate that in sera from patients with undifferentiated NPC, the prevalence of SV40 antibodies was 25%, whereas in controls it was 16%. This difference is not statistically significant (p > .05). A similar prevalence of SV40 antibodies was detected in undifferentiated NPC and healthy subjects. Our serologic data suggest no association between undifferentiated NPC and SV40 infection. This investigation may stimulate further studies aimed at determining the possible contribution of other risk factors in the pathogenesis of undifferentiated NPC.

BACKGROUND Simian virus 40 (SV40) has been considered to be an oncogenic viral agent in the development of osteosarcoma (OS), which to the authors' knowledge continues to be of unknown etiology. METHODS In the current study, serum samples from patients with OS were investigated with an indirect enzyme-linked immunoadsorbent assay (ELISA) to test for the presence of immunoglobulin G antibodies, which react with SV40 antigens. In ELISA, SV40 antigens were represented by 2 synthetic polypeptides that mimic epitopes of the viral capsid proteins 1 to 3. Additional sera from patients with breast cancer and undifferentiated nasopharyngeal carcinoma as well as healthy subjects were the controls. RESULTS Immunologic results suggested that antibodies that react with SV40 mimotopes were more prevalent (44%) in serum samples from patients with OS compared with healthy subjects (17%). The difference in prevalence between these cohorts was statistically significant (P

Simian Virus 40, experimentally assayed in vitro in different animal and human cells and in vivo in rodents, was classified as a small DNA tumor virus. In previous studies, many groups identified Simian Virus 40 sequences in healthy individuals and cancer patients using PCR techniques, whereas others failed to detect the viral sequences in human specimens. These conflicting results prompted us to develop a novel indirect ELISA with synthetic peptides, mimicking Simian Virus 40 capsid viral protein antigens, named mimotopes. This immunologic assay allowed us to investigate the presence of serum antibodies against Simian Virus 40 and to verify whether Simian Virus 40 is circulating in humans. In this investigation two mimotopes from Simian Virus 40 large T antigen, the viral replication protein and oncoprotein, were employed to analyze for specific reactions to human sera antibodies. This indirect ELISA with synthetic peptides from Simian Virus 40 large T antigen was used to assay a new collection of serum samples from healthy subjects. This novel assay revealed that serum antibodies against Simian Virus 40 large T antigen mimotopes are detectable, at low titer, in healthy subjects aged from 18-65 years old. The overall prevalence of reactivity with the two Simian Virus 40 large T antigen peptides was 20%. This new ELISA with two mimotopes of the early viral regions is able to detect in a specific manner Simian Virus 40 large T antigen-antibody responses.

BK virus (BKV), a human polyomavirus closely related to JC virus and Simian Virus 40, is ubiquitous in human populations worldwide. After primary infection, BKV establishes a lifelong latent infection in many organs. BKV transforms rodent cells to the neoplastic phenotype and is highly oncogenic in rodents. This review considers the oncogenic potential of BKV in humans and its possible involvement in human tumors. BKV sequences and T antigen (Tag) are detected in several types of human neoplasms, although the viral load is generally low, with less than one copy of the viral genome per cell. The possible causative role of BKV in human oncogenesis rests on the ability of BKV Tag to inactivate the functions of tumor suppressor proteins p53 and pRB family as well as on its ability to induce chromosomal aberrations in human cells. A 'hit and run' mechanism and secretion of paracrine growth factors by BKV Tag-positive cells, recruiting into proliferation neighboring and distant cells, are discussed as possible BKV pathogenic elements in human oncogenesis.

Glioblastoma multiforme (GBM) is the most severe form of cancer among gliomas. Indeed, GBM is a World Health Organization grade IV tumor of the CNS, with a mean survival time of 0.5 years.1 Its incidence is ∼1/100 000, which represents 30% of all CNS malignancies.2 According to the recent Eurocare 3 report,3 the mean age of patients affected by glioblastoma is 66 years. The extremely grim prognosis of GBM patients probably explains why such a rare tumor accounts for 2% of all cancer-related deaths in both Europe and the United States.4 GBM is characterized by high genotypic and phenotypic heterogeneities, but it is invariably and homogeneously resistant to conventional chemo/radiotherapy.1 The vast majority (95%) of GBM is represented by primary de novo glioblastomas.5 GBM is considered, like other tumors, a genetic disease of the somatic cell due to an impressive series of alterations of the cellular genome, such as point mutations, gene amplifications, translocations, and deletions, as well as epigenetic modifications, including methylation of specific genes. A limited percentage (5%) of GBM is linked to genetic predispositions, which are mainly associated with rare hereditary syndromes, such as Li–Fraumeni syndrome, Turcot syndrome, Peutz–Jegher syndrome, and neurofibromatosis 1 and 2.2 Exposure to chemical and physical carcinogenic agents, the genetic predisposition, and/or infections by oncogenic viruses, including simian virus 40 (SV40), have been repeatedly advocated in the literature.6,7 SV40 is a nonenveloped small DNA virus of ∼5.2 kb in size. SV40 was recognized in the 1960s as a contaminant of both inactivated (Salk) and live attenuated (Sabin) antipoliomyelitis vaccines. As a consequence, SV40 was inadvertently administered to human populations worldwide mainly by antipoliomyelitis vaccines, produced in SV40 naturally infected monkey kidney cells. Soon after its isolation, SV40 was characterized as a transforming and tumorigenic viral agent.6,7 Cell transformation and tumorigenesis by SV40 is induced by 2 oncoproteins, the large T antigen (Tag) and the small t antigen (tag), which target key cellular proteins, such as the tumor suppressor p53 and retinoblastoma family proteins, inactivating their functions.6,8 This small DNA tumor virus was found to be associated with some human cancers, including GBM. SV40 footprints were detected at high prevalence in GBM specimens, but SV40 sequences have also been revealed, although at lower prevalence, in blood specimens from healthy donors.9–11 In this context it is worth recalling that other investigations did not find SV40 footprints in human brain tumor specimens12,13 or higher SV40 antibodies in serum samples from brain tumor patients than in normal individuals.14 It has been reported that SV40-negative data on human tumor samples obtained by some groups were affected by technical flaws that invalidate the conclusions.15–17 A considerable debate, because of these contrasting reports, has developed in the scientific community.6,7 SV40 antibody detection had been performed in most cases using serologic methods with SV40 virus-like particles, but due to the high protein homology among the 3 main polyomaviruses—SV40, BKV, and JCV—final results were always influenced by some cross-reactivity.18–24 Altogether these reports prompted us to use an indirect ELISA with synthetic peptides employed as specific mimotopes, mimicking SV40 viral capsid antigens. Indeed, recent investigations have reported on the development of a specific and sensitive serologic test for SV40. The test is based on an indirect ELISA employing synthetic peptides as mimotopes/antigens of SV40 viral capsid proteins (VPs), without cross-reactivity with other closely related human polyomaviruses, such as BKV and JCV.25–28 This immunologic assay was used to detect specific serum antibodies against SV40 VPs in normal individuals of different ages and oncologic patients affected by malignant pleural mesothelioma and breast cancer (BC).25–28 In this investigation, serum samples from GBM patients were comparatively analyzed for the presence and titer of antibodies against SV40 infection, with serum samples from healthy individuals and from other reliable controls. No other slow virus infections were investigated.

A series of retroviral vectors with potential anti-tat and anti- constructs inhibited virus replication at a high multiplicity of rev activity was developed. Vectors containing a tat trans- infection (MOI). At a low MOI, two cell clones containing dominant negative mutant (tat22/37) and an RRE decoy in tat22/37 and the RRE decoy in 3′ position showed a long different positions, directed by the same promoter or by lasting protection against virus replication, in comparison different promoters, were generated. Retroviral vectors to control cultures expressing tat22/37 or RRE alone. Combicontaining tat 22/37 and the RevM10 transdominant negative nation of tat and rev mutants was ineffective in inhibiting mutant were also constructed. Jurkat cells were trans- HIV-1 replication at both low and high MOIs. At a low MOI, duced with the recombinant retroviruses to produce mono- HIV-1 replication was efficiently blocked in two cell clones clonal and polyclonal cultures. In these cell lines the expressing the RevM10 mutant alone. These results show recombinant proviruses were correctly integrated and a synergic effect of anti-tat and anti-rev molecules when expression of the inserted genes was detected by Northern the RRE sequence is cloned 3′ to tat22/37, suggesting the blot or RT-PCR analysis. However, infection of these cell possibility of using this vector design to control HIV-1 lines with HIV-1 showed that none of these recombinant replication.

At present Simian virus 40 (SV40) infection in humans appears to be transmitted independently from early contaminated vaccines. In order to test the spread of SV40 infection in children, an immunologic assay employing specific SV40 synthetic peptides corresponding to its viral protein (VP) antigens was employed to estimate the seroprevalence of this polyomavirus in Italian infants and adolescents. Serum samples from 328 children and adolescents, up to 17 years, were investigated. Serum antibodies against SV40 VPs were detected by indirect enzyme-linked immunosorbent assays. The seroprevalence of this polyomavirus was calculated after stratifying the subjects by age. Anti-viral capsid protein 1-2-3 SV40 IgG antibodies were detected in 16% of the study participants. The prevalence of antibodies against SV40 VPs tended to increase with age in children, up to 10 year old (21%). Then, in the cohort of individuals aged 11-17 years, the prevalence decreased (16%). A higher prevalence rate (23%) of SV40 VP antibodies was detected in the cohorts of 1-3 year and 7-10 year old children, than in children and adolescents of the other age groups. This age corresponds to children starting nursery and primary school, respectively, in Italy. IgM antibodies against SV40 VP mimotopes were detected in 6-8 month old children suggesting that SV40 seroconversion can occur early in life. SV40 VP antibodies are present at low prevalence in Italian children (16%), suggesting that SV40 infection, although acquired early in life, probably through different routes, is not widespread. The low SV40 seroprevalence suggests that SV40 is less transmissible than other common polyomaviruses, such as BKV and JCV. Alternatively, our immunologic data could be due to another, as yet undiscovered, human polyomavirus closely related to SV40.

We have analyzed by PCR skin lesions from classic, endemic and AIDS-related Kaposi's sarcoma (KS), as well as from KS-derived cell lines, the presence of ubiquitous transforming viruses. BK virus (BKV), a transforming human papovavirus which has been associated with human tumors, was detected in 100% of KS skin lesions and 75% of KS cell lines. KS specimens contained a full-length, intact BKV early region, but minor rearrangements were observed in some tumors. BKV was also detected with a high prevalence (57-67%) in genital tissues and sperm, thus fulfilling the role of a sexually transmitted agent in KS. The closely related JC virus (JCV), which has never been associated with human malignancies, was present in 11-20% of KS specimens and was detected with a low prevalence (0-21%) in genital tissues and sperm. Simian virus 40 (SV40) was not detected in any KS lesions. Herpes simplex virus (HSV) DNA sequences were detected in 20-25% of KS lesions. Malignant human papillomavirus (HPV) types 16 and 18 and benign HPV types 6 and 11 were detected in KS specimens with a similar prevalence of 11-83%, suggesting that the presence of HPV-transforming sequences is not a specific trait of HPV interaction with KS tissue. Furthermore, JCV, SV40, HSV and HPV DNA sequences were not detected in KS cell lines, suggesting that these viruses are not associated to KS neoplastic cells in KS tissue. KS cell lines were also negative for DNA sequences of KS-HV, the novel herpesvirus detected in primary KS lesions. The constant association of BKV DNA with KS lesions and KS cell lines suggests that BKV-transforming functions may participate in the development of KS.

A BK virus (BKV) episomal vector (pRPneoCMV) was constructed for expression of cDNAs under control of the cytomegalovirus (CMV) immediate-early promoter. Transfection of pRPneoCMV for expression of the chloramphenicol acetyltransferase (CAT) gene in several human cell lines showed that the CMV promoter is more efficient than the HIV-1 and RSV LTRs in directing gene expression from episomal vectors. In 293 human cells pRPneoCMV/CAT is twenty times more active in CAT expression than the well known pSV2CAT vector in COS7 cells. Stable expression of the gene of the herpes simplex virus type 1 and type 2 glycoprotein G, cloned into pRPneoCMV, was obtained in 293 cells. This vector will allow direct cloning of newly synthesized cDNAs whose expression can be monitored in human cells.

Human malignant pleural mesothelioma (MPM) is considered a rare tumor, but recent estimations indicate that one-quarter million people will die of this neoplasm in Europe in the next three decades. The mineral asbestos is considered the main causative agent of this neoplasm. MPM is largely unresponsive to conventional chemotherapy/radiotherapy. In addition to asbestos exposure, genetic predisposition to asbestos carcinogenesis and to simian virus (SV)40 infection has also been suggested. SV40 is a DNA tumor virus found in some studies to be associated at high prevalence with MPM. SV40 sequences have also been detected, although at a lower prevalence than in MPM, in blood specimens from healthy donors. However, some studies have failed to reveal SV40 footprints in MPM and its association with this neoplasm. These conflicting results indicate the need for further investigations with new approaches. We report on the presence of antibodies in serum samples from patients affected by MPM that specifically react with two different SV40 mimotopes. The two SV40 peptides used in indirect ELISAs correspond to viral capsid proteins. ELISA with the two SV40 mimotopes gave overlapping results. Our data indicate that in serum samples from MPM-affected patients ( n = 97), the prevalence of antibodies against SV40 viral capsid protein antigens is significantly higher (26%, P = 0.043) than in the control group (15%) represented by healthy subjects ( n = 168) with the same median age (66 y) and sex. Our results suggest that SV40 is associated with a subset of MPM and circulates in humans.

Simian virus 40 (SV40), a small DNA tumor virus, was inadvertently administered to human populations with the use of contaminated vaccines. SV40 sequences have mainly been detected in healthy individuals and cancer patients using polymerase chain reaction techniques. However, some studies have failed to reveal the presence of SV40 in human specimens. These conflicting results indicate the need for new research to verify whether SV40 is circulating in humans. Mimotopes from SV40 structural peptides were tested to investigate for specific reactions to human sera antibodies. An indirect enzyme-linked immunosorbent assay with synthetic peptides from SV40 viral capsid proteins 1-2-3 (VPs 1-2-3) was set up and employed to test 855 serum samples from healthy blood donors. Data from immunologic assays indicate that serum antibodies against SV40 VP mimotopes are detectable, although with a low titer, in blood donors 18 to 65 years old. The overall prevalence of serum samples that reacted with the 2 SV40 VP peptides was 18%. The strong points for this novel method include the simplicity of its approach and the potential to discriminate between SV40-specific antibody responses and to draw correlations between responses to the 2 independent SV40 peptides. These data suggest that SV40, or a yet undetected closely related polyomavirus, is circulating in human populations, but with lower prevalence than that of the ubiquitous BK and JC human polyomaviruses.

BK virus (BKV), JC virus (JCV) and Simian Virus 40 (SV40) are polyomaviruses, highly homologous at the DNA and protein levels. While the human polyomaviruses BKV and JCV are ubiquitous in humans, SV40 is a simian virus which was introduced in the human population, between 1955 and 1963, by contaminated poliovaccines produced in SV40-infected monkey cells. Alternatively, SV40 or an SV40-like virus may have entered the human population before anti-poliovirus vaccination. Epidemiological evidence suggests that SV40 is now contagiously transmitted in the human population by horizontal infection, independently from the earlier contaminated poliovaccines. All three polyomaviruses transform rodent and human cells and are oncogenic in rodents. JCV induces tumors also in experimentally inoculated monkeys. Transformation and oncogenicity induced by BKV, JCV and SV40 are due to the two viral oncoproteins, the large T antigen (Tag) and the small t antigen (tag), encoded in the early region of the viral genome. Both proteins display several functions. The large Tag acts mainly by blocking the functions of p53 and pRB family tumor suppressor proteins and by inducing in host cells chromosomal aberrations and instability. The principal effect of small tag is to bind the catalytic and regulatory subunits of the protein phosphatase PP2A, thereby constitutively activating the β-catenin pathway which drives cells into proliferation. All three polyomaviruses are associated with specific human tumor types which correspond to the tumors induced by experimental inoculation of the three viruses in rodents and to the neoplasms arising in mice transgenic for the polyomavirus early region gene directed by the native viral early promoter-enhancer. Human tumors associated with BKV. JCV and SV40 contain viral DNA, generally episomic, express viral RNA and are positive for large Tag by immunohistochemistry. The low copy number of viral genomes in human tumors suggests that polyomaviruses may transform human cells by a “hit and run” mechanism. An autocrine-paracrine effect, involving secretion of growth factors by cells expressing polyomavirus Tag, may be responsible for recruiting to proliferation Tag-negative cells in polyomavirus-associated human tumors.

In this study, 82 DNA samples of simian virus 40 (SV40)-positive human tumors and normal tissues were transfected into SV40-permissive monkey cells. SV40 wild-type strain 776 was reactivated from two DNA samples, derived from peripheral blood mononuclear cells (PBMCs) of a blood donor and from a vulvar tissue. SV40 reactivation was confirmed by obtaining rescue of SV40 from the DNA of the vulvar tissue in a second transfection experiment. This investigation indicates that infectious SV40 is present in normal human tissues and suggests that (i) PBMCs are probably vectors of SV40 to different tissues of the host and (ii) blood and sexual transmission may be routes of SV40 infection in humans, leading to (iii) virus spread in the human population.

Publisher Summary Several viruses are ubiquitous in the human population and produce a persistent or latent infection in humans. Some of them—such as human polyomaviruses BK (BKV) and JC (JCV)—are oncogenic in experimental systems and are associated with human tumors. This chapter considers the general properties of the human polyomaviruses BK and JC, the characteristics of the latent infection and of the ubiquitous state of these viruses in humans, their transforming capacity in vitro , their oncogenicity in experimental animals, and their possible etiologic role in human tumors are also evaluated. It is noted that the classic Koch's postulates cannot be applied to latent viruses. New rules are considered for these viruses to establish their oncogenic role. It is reported that BKV fulfills these criteria, suggesting that BKV may cooperate, under peculiar conditions, as a cofactor in the development or progression of human tumors. BKV oncogenicity is revealed by cooperating events, such as oncogene activation, loss of tumor suppressor genes, or rearrangements of the viral genome, inducing proliferation of clonal neoplastic cells in a population of latently BKV-infected cells.

T-antigen (TAg) coding sequences specific for BK virus (BKV) were detected, by PCR amplification followed by Southern-blot hybridization, in a high percentage of human tumors and tumor-cell lines, as well as in normal tissues, by analysis of 189 specimens. Specifically, the BKV early region was detected in 85% of brain tumors and in all normal brain tissues, in 78% of osteosarcomas, in 38% of Ewing's tumors, in 40% of normal bone specimens and in 71% of normal peripheral blood cell samples. Wilms' tumor tissues used as a control were all negative for BKV sequences. RT-PCR analysis indicated that TAg coding sequences were expressed in specimens carrying BKV early region, ranging from 64% of the osteosarcomas to 100% of glioblastomas, Ewing's tumors, peripheral blood cells and normal bone. Moreover, DNA sequencing performed in 12 different positive samples revealed that the amplified PCR products are identical to the early-region sequence of wild-type BKV. The role of BKV TAg and its possible mechanism of action in human tumorigenesis are discussed.

Cells from BKV/tat transgenic mice were characterized for their tumorigenic phenotype in nude and syngeneic BDF mice. The results indicate that the BKV/tat recombinant transgene has a weak tumorigenic potential, mostly predisposing to oncogenesis, and that second events are required for the development of tumorigenicity. Tat is endogenously produced and released by tumor cells. It is taken up by recipient cells directly from the culture medium, without need of cell to cell contact. Extracellular Tat stimulates proliferation of cells from BKV/tat transgenic mice and protects them from apoptosis under conditions of serum starvation. Our results are in agreement with a model in which Tat induces its effects on target cells in two different ways. Growth promotion may require interaction of extracellular Tat with surface receptors eliciting a signal for cell proliferation, whereas intranuclear localization of Tat is necessary for transactivation of viral and cellular genes.

The human polyomaviruses BK (BKV) and JC (JCV) are ubiquitous in human populations and have a worldwide distribution.(1) They are oncogenic in rodents and monkeys and transform cells in vitro to a neoplastic phenotype. For all these reasons, BKV and JCV have been considered possible candidates in the etiology of human tumors. Association of BKV, but not of JCV, with human tumors has been described, although a formal proof for an etiological role of BKV in human oncogenesis is still lacking. SV40, which is not a ubiquitous human virus, has also been sporadically detected in human tumors. In this chapter, we consider their general characteristics, the natural history of infection, experimental transformation and oncogenicity by BKV and JCV, as well as the evidence linking BKV, JCV, and SV40 to human neoplasia.

BK virus (BKV) is a human papovavirus that readily transforms rodent cells, but not human cells, to a neoplastic phenotype, suggesting that tumor-suppressor functions expressed in human cells control BKV oncogenicity. Transfer of a normal human chromosome 11 to BKV-transformed mouse cells suppresses the malignant phenotype. In this report we map the regions of chromosome involved in tumor suppression. Transfer of chromosome 11 to the BKV-transformed hamster cell line HKBK produces monochromosomic hybrids retaining only portions of the transferred human chromosome. We have compared the tumorigenicity of the hybrids with the molecular mapping of chromosome 11 retained regions. This analysis indicated that 3 regions of human chromosome 11, 11p15.5, 11p13 and 11q13, cooperate in tumor suppression. However, 11q13 seems the most important, since all the HKBK/H11-induced tumors analysed had lost this region, whereas 11p15.5 and 11p13 were sometimes retained. The chromosomal regions identified in this study are deleted in several types of human tumors, suggesting that the BKV transformation system specifically detects tumor-suppressor genes on chromosome 11 that are involved in human oncogenesis. This model may be of use in isolating and cloning such genes. The results of this report raise the possibility that BKV may have a synergistic tumorigenic effect in human cells where tumor-suppressor genes controlling its oncogenk potential are inactivated. © 1994 Wiley-Liss, Inc.

Episomal BK virus (BKV) DNA was detected in primary human brain tumours, in Kaposi's sarcoma and in cell lines from brain tumours. Ewing sarcoma and osteogenic sarcoma. Infectious BKV was rescued from several tumours and tumour cell lines by transfection of total cellular DNA into human embryonic fibroblasts. Restriction endonuclease and nucleotide sequence analysis showed that all the rescued viruses are similar to BKV-IR, a BK variant previously isolated from a human tumour of pancreatic islets, indicating that a specific BKV strain may be associated with certain types of human tumours. All the variants contain a putative transposable elements in the regulatory region of the viral genome. This region has mutagenic properties and enhancing activity in transformation, suggesting a possible role of these variants in tumour induction or progression.

The herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) gene, deleted of 639 nucleotides that encode the transmembrane anchor sequence and reconstructed with the extramembrane and intracytoplasmic domains, was cloned under control of the Rous sarcoma virus long terminal repeat in the episomal replicating vector pRP-RSV, which contains the origin of replication and early region of the human papovavirus BK as well as a cDNA for a mutant mouse dihydrofolate reductase that is resistant to methotrexate. gB-1 (0.15 to 0.25 pg per cell per 24 h) was constitutively secreted into the culture medium of pRP-RSV-gBs-transformed human 293 cells. Treatment of transformed cells with methotrexate at high concentrations (0.6 to 6 microM) increased gB-1 production 10- to 100-fold, because of an amplification of the episomal recombinant. Mice immunized with secreted gB-1 produced HSV-1- and HSV-2-neutralizing antibodies and were protected against HSV-1 lethal, latent, and recurrent infections. Constitutive expression of secreted gB-1 in human cells may establish a system to develop diagnostic material and a subunit vaccine for HSV infections.

BK virus (BKV)-induced tumors in hamsters were investigated for the presence of viral DNA by the blot-transfer hybridization technique. Several viral genomes per cell were found in tumor tissues and in their derived cell lines and clones. Most of the detected viral genomes were integrated into the cellular DNA, but some tumors also contained free viral DNA sequences. Integration patterns were different from each other, and many different integration sites were available on the cellular or on the viral DNA or on both. Typical features of integration patterns were found in ependymomas, which were the most frequent (72%) among BKV-induced tumors. Readily detectable viral DNA sequences were only found in neoplastic tissues, but traces of BKV DNA were also present in the apparently normal portion of the brain of an animal that had developed an ependymoma and in the brain (but not in the liver) of another animal 15 days after virus inoculation. A cell line and a single-cell clone derived from a tumor had hybridization patterns considerably simpler that the pattern of the original tumor, lacking several integrated viral genomes and all free viral sequences.

Analysis of factors determining replication of BK virus (BKV) episomal vectors in human cells showed that vector copy number was related to the level of BKV T antigen expression. T antigen was synthesized efficiently, as assessed by indirect immunofluorescence, in vector-transfected primary embryonic fibroblasts undergoing neoplastic transformation. Surprisingly, transfected continuous cell lines (143B, HeLa and KB), kept under biochemical selection or tested in transient assays, produced negligible amounts or no T antigen, revealed only by a sensitive ELISA test, suggesting that in these cells vector amplification was under the control of cellular factors. Presence or absence of BKV late region sequences, BKV strain, orientation of the inserted genes and presence or absence of selection were not relevant for vector replication. Type of biochemical selection, however, was important, since BKV vectors containing the thymidine kinase gene replicated better than those containing theneo gene. Despite great variability, vector copy number increased in transfected clones of adenovirus 5-transformed 293 cells, in the absence of immunofluorescence detectable T antigen. These cells express adenovirus immediate early proteins E 1 A and E 1 B which may directly or indirectly activate BKV origin of replication.

Hamster kidney cells were transformed, with comparable efficiency, by circular or linear molecules of complete BK virus (BKV) genome and by agarose gel-purified fragments of BKV DNA obtained by single or double digestions with various restriction endonucleases. Only fragments containing the complete early region of BKV DNA displayed transforming activity. Analysis by blot hybridization of the arrangement of viral DNA sequences in a cloned cell line transformed by a 3.8-kilobase fragment, obtained after sequential digestion of BKV DNA with HhaI and BamHI, showed the presence of seven viral integrations into the cellular DNA. Apparently all of the integrated viral molecules contained the entire early region of BKV DNA. Large T antigen, small t antigen, and the 56,000-dalton nonviral Tau antigen were detected in transformed cells by immunoprecipitation. The pattern of integration of viral sequences in transformed cells was constant over many generations. Likewise, large T antigen was always detected in transformed cells at various passage levels. These results may suggest that all of the sequences of the early region coding for large T antigen are required for transformation by BKV. Alternatively, subgenomic segments of the BKV DNA early region may be unable to transform because the appropriate polyadenylation site, necessary to obtain a complete functional transcriptional unit, is removed by the restriction enzyme cleavage.

Tandem integrations of complete viral genomes, which are not usually found in hamster cells transformed by BK virus (BKV) or circular BKV DNA can be established when hamster cells are transfected with linear BKV DNA with cohesive ends. Treatment of linear BKV DNA with S1 nuclease or use of linear BKV DNA with staggered but non-complementary ends prevents tandem integration. These results support a model in which formation of tandems of free viral DNA is a preliminary step for tandem integration.

BK virus (BKV) DNA sequences were not detected in 142 human tumors analyzed by DNA-DNA reassociation kinetics and blot hybridization. The investigation was focused mainly on those rare types of human tumors (ependymomas, choroid plexus papillomas, tumors of pancreatic islets and osteosarcomas) that are induced with highest frequency by BKV in experimental animals. In addition, other tumors of the urinary apparatus and of the central nervous system were analyzed. BKV tumor (T) antigen was not detected in neoplastic tissues, and BKV T antibodies were not found in sera and cerebrospinal fluids from patients with neoplasms. Sequences homologous to BKV DNA were found in normal tissue from a kidney carrying a carcinoma. The neoplastic tissue from the same organ, however, had no sequences homologous to BKV DNA. Such DNA does not belong to BKV but probably to another papovavirus related to BKV.

SummaryA measles-related virus was isolated from two out of three brain biopsies of patients with SSPE by the fusion of brain cells with susceptible cells. β-propiolactone-inactivated Sendai virus was used in the fusion experiments. The mechanism of activation and the characterization of the isolated agents are discussed. Their association with another virus is reported in light of the etiology of SSPE.

The early events in the interaction of simian virus 40 (SV40) with permissive cells were investigated. Evidence is presented that 30 min after infection intact virions penetrate the nuclei of infected cells. The uncoating of the virus is carried out in the nuclei with a complete dissociation of the viral genome from the protein coat. Opening of the circular parental deoxyribonucleic acid (DNA), i.e., conversion of component I to component II of SV40 DNA, takes place after uncoating, followed by the appearance of a new component sedimenting faster than component I at alkaline p H.

One of the ‘early’ effects of cellular infection by DNA-containing viruses is an increase of various enzymic activities concerned in DNA synthesis, which in most instances seems dependent upon the synthesis of virus-coded enzymes (Keir, 1968). As far as poxviruses are concerned, relevant information relates almost exclusively to the vaccinia subgroup and very little is known about other poxviruses (Joklik, 1966). The Shope fibroma virus is an oncogenic pox virus of the myxoma subgroup (Joklik, 1966) and has only recently been subjected to systematic study in tissue cultures. It has been shown to replicate in primary cell cultures of human amnion (Hodes et al. 1966; La Placa et al. 1967; Hodes & Chang, 1968) where it causes a peculiar cytopathic effect characterized by the appearance of pock-like lesions (La Placa et al. 1967). The time-course of nucleic acid synthesis (Ewton & Hodes, 1967) and the induction of DNA nucleotidyltransferase (Chang & Hodes, 1967) in cells infected with Shope fibroma virus have also been reported.

Human fibroblasts, transfected with a recombinant DNA containing the neo gene and BK Virus (BKV) early region, which expresses BKV large T antigen (TAg), show cytogenetic alterations characterized by dicentric chromosomes and other structural aberrations such as deletions, duplications, translocations, and ring chromosomes. Such alterations were absent or significantly less frequent in human fibroblasts transfected with a plasmid expressing only the neo gene. The chromosome damage in BKV-transfected cells was evident before the appearance of the morphologically transformed phenotype and therefore seems to be a primary effect of TAg expression in human cells. The specific pattern of chromosome aberrations suggests the prevalence of an indirect clastogenic effect, determined by the inhibition of p53 regulatory functions on genome stability by BKV TAg. Due to the widespread distribution of BKV in the human population and to the latent state of BKV DNA in many human organs, the clastogenic activity of BKV TAg may potentially participate in an oncogenic process involving BKV latently infected cells.

Simian virus 40 (SV40) is a monkey virus that was introduced in the human population by contaminated poliovaccines, produced in SV40-infected monkey cells, between 1955 and 1963. Epidemiological evidence now suggests that SV40 may be contagiously transmitted in humans by horizontal infection, independent of the earlier administration of SV40-contaminated poliovaccines. This evidence includes detection of SV40 DNA sequences in human tissues and of SV40 antibodies in human sera, as well as rescue of infectious SV40 from a human tumor. Detection of SV40 DNA sequences in blood and sperm and of SV40 virions in sewage points to the hematic, sexual, and orofecal routes as means of virus transmission in humans. The site of latent infection in humans is not known, but the presence of SV40 in urine suggests the kidney as a possible site of latency, as it occurs in the natural monkey host. SV40 in humans is associated with inflammatory kidney diseases and with specific tumor types: mesothelioma, lymphoma, brain, and bone. These human tumors correspond to the neoplasms that are induced by SV40 experimental inoculation in rodents and by generation of transgenic mice with the SV40 early region gene directed by its own early promoter–enhancer. The mechanisms of SV40 tumorigenesis in humans are related to the properties of the two viral oncoproteins, the large T antigen (Tag) and the small t antigen (tag). Tag acts mainly by blocking the functions of p53 and RB tumor suppressor proteins, as well as by inducing chromosomal aberrations in the host cell. These chromosome alterations may hit genes important in oncogenesis and generate genetic instability in tumor cells. The clastogenic activity of Tag, which fixes the chromosome damage in the infected cells, may explain the low viral load in SV40-positive human tumors and the observation that Tag is expressed only in a fraction of tumor cells. “Hit and run” seems the most plausible mechanism to support this situation. The small tag, like large Tag, displays several functions, but its principal role in transformation is to bind the protein phosphatase PP2A. This leads to constitutive activation of the Wnt pathway, resulting in continuous cell proliferation. The possibility that SV40 is implicated as a cofactor in the etiology of some human tumors has stimulated the preparation of a vaccine against the large Tag. Such a vaccine may represent in the future a useful immunoprophylactic and immunotherapeutic intervention against human tumors associated with SV40.

SUBACUTE sclerosing panencephalitis (SSPE), a progressive neurological disease of children, is characterized by intracytoplasmic and intranuclear inclusions in the neurones and glial cells. Although no infectious agent was isolated directly from the diseased brain tissue, a paramyxovirus, related serologically to the measles–rinderpest virus group, was recovered from SSPE brain cells maintained in tissue culture in serial passages1. These cultures were characterized by the formation of syncytia and by their capacity to undergo transformation2. Only by fusion with African green monkey kidney cells (AGMK) could an infectious syncytiogenic paramyxovirus be recovered from the brain cells.

We describe the molecular and biological properties of BK virus (BKV)-IR, a new BKV variant isolated from a human tumor of pancreatic islets. BKV-IR bears a 253-base-pair (bp) deletion and an 80-bp insertion in the early region of the genome. The deletion abolishes the expression of small-t antigen. The inserted sequences, grouped in four clusters, produce rearrangements in the first and second enhancer elements. They are bound by 12-bp direct repeats and could form a 217-base stem-loop structure suggestive of an insertion sequence. As compared with wild-type BKV, BKV-IR transformed hamster cells with a reduced efficiency and induced ependymomas in hamsters at a lower frequency and with a longer latency period. Tumors induced by BKV-IR, however, showed features of higher malignancy. The possible role of the insertion sequence-like element in transformation by BKV-IR is discussed.

BK virus DNA can be extracted from virions as a nucleoprotein complex containing about 20 nucleosomes. Transcription of this "minichromosome" with Escherichia coli RNA polymerase indicates that both initiation and elongation of RNA chains are reduced by the presence of nucleosomes. Hybridization analysis of RNA made on the complex shows preferential transcription of one region of BK virus genome. No increase in strand selection is observed with respect to transcription of purified superhelical BK virus DNA.

Semipermissive rabbit liver, brain, and kidney cells were transformed by BK virus (BKV). All cells of the three resulting cell lines produced BKV T antigen. Viral DNA was detected by DNA-DNA reassociation kinetics and by blot-transfer hybridization. Hybridization patterns were different for the three lines, indicating a different mode of integration for each line. In addition to integrated viral DNA, two of the lines contained also free viral DNA sequences, which in one case were defective viral genomes. Absence or splitting of particular regions of the viral genome was not a necessary condition for the maintenance of the transformed state. HindIII-generated segment A, which contains all the sequences coding for the late viral proteins, was absent (in an intact form) in the only line from which virus could not be rescued.

Expression of cloned viral and cellular genes in eukaryotic cells is relevant to production of vaccines (Melnick, 1986; Moss and Flexner, 1987) and gene therapy (Anderson, 1984). To this purpose numerous eukaryotic vectors have been developed. Since the level of expression of exogenous genes is directly related to the degree of gene amplification (Rigby, 1983) episomal eukaryotic vectors have been constructed where a viral origin of replication directs vector amplification in suitable cells (DiMaio, 1987; Milanesi et al., 1984). However, only two eukaryotic vectors (Milanesi et al., 1984; Sugden et al., 1985) are specific for human cells and they allow expression of genes endowed with their own regulatory sequences. We have therefore devised a viral vector (pBK-1) that replicates and persists episomally in human cells; The structure and general characteristics of this vector have been described in detail (Milanesi et al., 1984). It contains DNA sequences from the plasmid pML, a deletion derivative of pBR322, as well as the origin of replication and the early region of BK virus (BKV), a human papovavirus that efficently replicates in human cells.

Tumors were induced by BK virus (BKV) inoculated intravenously in 3-week-old Syrian golden hamsters immunosuppressed with anti-lymphocyte serum or methylprednisolone acetate alone or in association with gamma-radiation (60Co). The induced neoplasms were ependymoma, carcinoma of pancreatic islets, lymphoma, osteosarcoma, undifferentiated sarcoma, kidney and renal pelvis carcinoma, pheochromocytoma and hemangiosarcoma. High levels of insulin and glucagon and altered concentrations of glucose were detected in blood of animals with tumors of pancreatic islets. No antibodies to BKV tumor antigen (TAg) and low levels of hemagglutination-inhibition antibodies to BKV viral coat protein Ag were detected in hamster sera. BKV TAg was found in tumors by complement fixation. Blot hybridization analysis of tumor DNA showed the presence of both free and integrated BKV genomes in tumor cells. BKV DNA inoculated intravenously and subcutaneously in immunosuppressed or immunocompetent hamsters was not oncogenic, whereas it was weakly oncogenic when inoculated intracerebrally.

Summary After exposure of human embryonic fibroblasts to BK virus, virus particles adsorbed to the plasma membrane were engulfed by pinocytosis or captured by vesicles, possibly originating from the endoplasmic reticulum, within 2 h after infection. Most of the virus particles were then transported into lysosomes or into the nucleus, while a small amount of virus was found free in the cytoplasm. Virus particles entered the nucleus between 2 and 12 h after infection, were still detectable in the nucleus at 24 h after infection and became morphologically undiscernible at 30 h after infection, suggesting that a nuclear uncoating mechanism was active between 24 and 30 h after infection. Virus progeny started to appear in the nucleus of infected cells at 4 days after infection, but not until 7 to 8 days after infection did the virus escape into the cytoplasm and cell degeneration became evident. The possible explanations for the long replicative cycle of BK virus are discussed.

Sera from 1,279 patients with various diseases were examined for the presence of antibodies to BK virus (BKV) capsid antigens. The percentage of positive sera was comparable in all the diseases except rheumatoid arthritis and chronic nephropathies, where a slightly higher prevalence was found. Sera from 952 patients with tumors were examined for the presence of antibodies to BKV tumor and capsid antigens in comparison with a matched control group of 501 blood donors. Sera from 11 tumor patients (1.15%) and from 4 normal controls (0.80%) had antibodies to BKV tumor antigen. No higher prevalence of antibodies to BKV capsid antigens was found in any cancer type except in carcinomas of the urinary bladder, where the percentage of positive sera and of sera with high titers was higher than in other groups. BKV infection is discussed in relation to its possible connection with human non-neoplastic diseases as well as with human tumors and to its activation under conditions of immunosuppressive therapy.

Early-passage hamster embryo cells were transformed by recombinant DNA molecules containing BK virus (BKV) early-region gene and either the activated c-Ha-ras oncogene (pBK/c-rasA) or the normal c-Ha-ras proto-oncogene (pBK/c-rasN). The recombinant DNAs had a greater transforming ability and converted hamster cells to a more malignant phenotype than the single genes transfected separately. pBK/c-rasA was significantly more powerful than pBK/c-rasN in conferring to cells all the characteristics of transformation. Transfected DNA sequences were integrated mostly as single insertions into cellular DNA. Specific c-Ha-ras and BKV transcripts as well as c-Ha-ras p21 and BKV T antigen were detected in transformed cells. Although stimulation of c-Ha-ras expression by BKV enhancers cannot be excluded in recombinants, super-transfection and co-transfection experiments in hamster embryo cells and pre-neoplastic cell lines showed that BKV early-region and c-Ha-ras co-operate in transformation by contributing separate and independent functions.

The early promoters of BKV and SV40 plasmids were transactivated in both BKV and SV40-transformed cells which failed to support replication of these plasmids. This suggests that the T-antigen of either virus can transactivate BKV and SV40 early promoters by either increasing the availability of cellular transcription factors or by directly interacting with specific sequences which comprise the transcriptional control region of the early promoters. We also observed that removal of 8-bp on the early side of T-antigen binding site I of BKV does not alter viral-plasmid replication.

The adenovirus type 12 (Ad12) early region 1A (E1A) gene is thought to play a major role in repressing class I major histocompatibility complex expression in transformed rodent cells. However, since transformation by adenovirus requires both E1A and E1B genes, it has not been demonstrated whether the Ad12 E1A gene acts alone or synergistically with the E1B gene to accomplish this effect. Moreover, it is not known whether the repression of class I antigen synthesis by Ad12-transforming gene products occurs only in rodent cells. We show that the Ad12 E1A gene, in the absence of the E1B gene, is capable of greatly reducing the levels of class I HLA antigens and mRNAs in primary human cells transformed by the E1A gene of Ad12 and the large tumor antigen (T-antigen) gene of BK virus; control cells transformed by BK virus T-antigen gene alone or the highly related simian virus 40 T-antigen gene showed no apparent alteration in class I HLA expression. Human recombinant interferon gamma was able to restore synthesis of class I HLA antigens in transformed cells that produced Ad12 E1A proteins, indicating that these cells were not deficient for class I genes. These results strongly indicate that the Ad12 E1A proteins modulate class I gene expression by similar mechanisms in both transformed rodent and human cells.

BK virus (BKV)-transformed cells and BKV-induced tumors as well as in vitro derived tumor cell lines were all found to contain BKV DNA sequences when analysed by DNA-DNA reassociation kinetics. In transformed cells the number of viral genome equivalents per diploid cell genome (CG divided by VG) decreased with increasing generations. Likewise, single cell clones had a lower CG divided by VG than parental transformed cells. BKV-induced tumors had a high CG divided by VG. Tumor cells cultivated in vitro and their clones had a lower CG divided by VG than BKV-induced tumors from which they were derived, suggesting a multiclonal origin of tumors. Hamster tumors induced by subcutaneous inoculation of BKV-transformed cells or tumor cell lines had a higher CG divided by VG than cells producing them. Variation in CG divided by VG is discussed in terms of cell selection depending on different in vitro or in vivo conditions of cell growth. In some cases, however, the decrease in CG divided by VG most likely depends on loss on free viral sequences from transformed cells or tumors.

Expression of herpes simplex virus type 1 (HSV-1) glycoprotein B (gB-1) was obtained in human cells from the gB-1 gene cloned in the episomal replicating vector pBK-1, which contains the origin of replication and early region of the human papovavirus BK. Selective systems for the TK+ phenotype in TK-143B cells and for resistance to G418 in adenovirus 5-transformed 293 cells were used to obtain stable transformants that produced gB-1. While gB-1 expression in 143B cells required induction by HSV-1 early proteins, constitutive gB1 production was observed in 293 cells, where endogenous trans-acting factors probably replace the need for early viral products in the activation of the cloned gB-1 gene. The amount of recombinant gB-1 was comparable to that produced during HSV-1 lytic infection in human cells, due to amplification of the inserted gene in the replicating episomal vector. Expression of gB-1 was induced by cadmium and zinc when the promoter of the mouse metallothionein-I gene was placed upstream of gB1 structural sequences. The inducible system where the gB-1 gene is under the control of its own promoter could be employed to clarify the role of early viral products in induction of gB-1 synthesis. Constitutive expression of gB-1 in human cells could provide useful material for diagnostic purposes and for the preparation of a subunit vaccine against HSV infections.

Secondary mouse and hamster cells were transformed with BK virus (BKV) or with purified BKV DNA. Restriction analysis of viral sequences present in transformed cell lines yielded complex patterns, indicating the presence in most lines of more than one viral intergration/cell and the existence of a large number of available integration sites. In spite of this variability, however, the arrangement of integrated viral sequences had specific features in the two types of transformed cells. Tandem insertions of full-size BKV genomes, which were a constant feature of transformed mouse lines, were not found in hamster lines. These appeared to contain no complete, intact BKV genomes, but only genomes interrupted by the insertion event or tandem integrations of incomplete viral sequences. Traces of free, full-size viral DNA were observed in all transformed mouse lines, but not in hamster lines. One transformed hamster line, after 84 passages in culture, showed the presence of tandemly integrated, full-size BKV DNA, which was not found in cells at 14 passages after the appearance of the transformed phenotype. This was the only BKV transformed line from which virus could be rescued by fusion with permissive cells. These results suggest that BKV DNA integration occurs differently in hamster (nonpermissive) and mouse (semipermissive) cells, and support the idea that excision of integrated viral DNA occurs usually via homologous recombination.

Summary Semi-permissive mouse, rabbit and monkey cells were stably transformed by BK virus (BKV). The specificity of transformation was demonstrated by the presence of BKV tumour (T) antigen in nuclei of transformed cells and by virus rescue with Sendai virus-mediated fusion or transfection. Two out of seven BKV-transformed cell lines were oncogenic. Permissive human cells were only abortively transformed by BKV, since morphologically modified cells persisted in culture for a few passages and eventually died.

A preliminary report [1] described encephalitis in ferrets injected intracerebrally with suspensions of brain tissue obtained at biopsy of 3 patients with subacute sclerosing panencephalitis (SSPE). This encephalitis, characterized by abnormal electroencephalographic (EEG) patterns and histologic lesions, was successfully transmitted serially in ferrets by intracerebral subinoculation of suspensions of ferret brain tissue. Subsequent to this report, several laboratories, including our own, succeeded in establishing cultures of brain cells from biopsies of patients with SSPE. These tissue cultures, after serial cell transfers, showed the presence of syncytia and giant cells. Cells in these cultures contained intranuclear

Evidence that the resistance of simian virus (SV40)-transformed permissive cells to superinfection with SV40 is due to lack of virus uptake is presented. When virus uptake is enhanced, the events of infection proceed as in normal permissive cells, resulting in production of infectious virus.

ALTHOUGH a viral etiology for subacute sclerosing panencephalitis (SSPE) was first postulated by Dawson 1 in 1933, the isolation of measles-like viruses from brain cell cultures established from biopsies of SSPE patients has been a recent accomplishment. 2-4 The SSPE agents, either present in brain biopsies of patients or isolated from their brain tissue maintained in culture, have been found to be pathogenic for ferrets, 5,6 with encephalitis becoming manifest after a prolonged incubation period. Because of the difficulties involved in handling ferrets and the excessive costs of their maintenance, a search became necessary for another experimental host—an animal at least as susceptible as the ferret to SSPE infection, but easier to handle and less costly to maintain. The present study shows that the hamster meets these criteria; intracerebral inoculation of hamsters with human brain culture cells carrying the SSPE agents, or with cell-free SSPE virus, produces encephalitis. Materials and