Herpesviruses in Metastatic Lucké Renal Adenocarcinoma

The etiologic agent of the renal adenocarcinoma of leopard frogs, Rana pipiens , is the Lucké tumor herpesvirus (LTHV). The virus is easily detected with thin section electron microscopy in primary tumors of frogs which have been exposed to a cold environment. Several spontaneous metastatic nodules and a large primary tumor were detected at autopsy of a frog which had been maintained at 4°C for 73 days. LTHV was found not only in the primary tumor, as previously reported, but also was present in metastatic tumor cells in the liver, fat body, and bladder. The presence of LTHV in metastatic cells demonstrates that the differentiated state of primary Lucké tumor cells is retained in its metastatic colonies even at the fine structure level revealed by electron microscopy.     

Herpesviruses in metastatic Lucké renal adenocarcinoma

The etiologic agent of the renal adenocarcinoma of leopard frogs, Rana pipiens, is the Lucké tumor herpesvirus (LTHV). The virus is easily detected with thin section electron microscopy in primary tumors of frogs which have been exposed to a cold environment. Several spontaneous metastatic nodules and a large primary tumor were detected at autopsy of a frog which had been maintained at 4 degrees C for 73 days. LTHV was found not only in the primary tumor, as previously reported, but also was present in metastatic tumor cells in the liver, fat body, and bladder. The presence of LTHV in metastatic cells demonstrates that the differentiated state of primary Lucké tumor cells is retained in its metastatic colonies even at the fine structure level revealed by electron microscopy.     

Augmentation of retrovirus-induced lymphoid leukosis by Marek''s disease herpesviruses in White Leghorn chickens.

Our objective was to determine whether the cell-associated herpesvirus vaccines used in chickens to control Marek's disease tumors can augment development of lymphoid leukosis (LL) induced by exogenous avian leukosis virus (ALV). Various single or mixed Marek's disease vaccines were inoculated at day 1, and ALV was injected at 1 to 10 days, with chickens of several experimental or commercial strains. Development of LL was monitored at 16 to 48 weeks in various experiments. In several strains of chickens we repeatedly found that the widely used serotype 3 turkey herpesvirus vaccine did not augment LL in comparison with unvaccinated controls. However, LL development and incidence were prominently augmented in several chicken strains vaccinated with serotype 2 vaccines, used alone or as mixtures with other serotypes. In one chicken strain, augmentation was demonstrated after natural exposure to ALV or serotype 2 Marek's disease virus viremic shedder chickens. Augmentation of LL by virulent or attenuated Marek's disease viruses of serotype 1 was intermediate in effect. Serotype 2 Marek's disease virus augmentation of LL was prominent in three laboratory lines and one commercial strain of White Leghorns, but it was not observed in an LL-resistant laboratory line or four commercial strains susceptible to ALV infection. Chickens developed similar levels of viremia and neutralizing antibodies to ALV regardless of the presence of augmentation of LL, suggesting that the mechanism of enhanced LL did not result from differences in susceptibility or immune response to ALV. We postulate that the serotype 2 herpesviruses may augment LL through one of several possible influences on bursal cells that are subsequently transformed by exogenous ALV.     

禽肿瘤性病毒研究的回顾和展望

反转录病毒及疱诊病毒可以鸡引起肿瘤,近几十年中,通过突变或相互间的基因重组,这类病毒在病原性和抗原性等方面不断发生着演变,一方面,我国饲养量巨大的鸡群将不断面对着这类病毒感染的威胁;另一方面,庞大的鸡群也是这类病毒不断演变的温床,以家禽肿瘤病毒为对象,曾对生物学的发展有里程碑性的成果,因此,对这些病毒的进一步深入研究,不仅对畜牧业有重要经济意义,也具有生物学意义。     

Analysis of the src gene of sarcoma viruses generated by recombination between transformation-defective mutants and quail cellular sequences.

Tumors were produced in quails about 2 months after injection with a transformation-defective mutant of the Schmidt-Ruppin strain of Rous sarcoma virus, subgroup A (SR-A), that retains a small portion of the src gene. Sarcoma viruses were isolated from each of five such tumors. A transformation-defective mutant which has a nearly complete deletion of the src gene was unable to induce tumors. The avian sarcoma viruses recovered from quail tumors (rASV-Q) had biological properties similar to those of the avian sarcoma viruses previously acquired from chicken tumors (rASV-C); these chicken tumors had been induced by the same transformation-defective mutants. Both rASV-Q and rASV-C transformed cells in culture with similar focus morphology and produced tumors within 7 to 14 days after injection into chickens or quails. The size of rASV-Q genomic RNA was indistinguishable from that of SR-A by polyacrylamide gel electrophoresis. The sequences of rASV-Q RNA genomes were analyzed and compared with those of the parental transformation-defective virus, SR-A and of rASV-C by RNase T1 fingerprinting and oligonucleotide mapping. We found that the src sequences of all five isolates of rASV-Q were identical to each other but different from those of SR-A and rASV-C. Of 13 oligonucleotides of rASV-Q identified as src specific, two were not found in either SR-A or rASV-C RNA. Furthermore, some oligonucleotides present in SR-A or rASV-C or both were absent in rASV-Q. No differences were found for the sequences outside the src region in any of the viruses examined. In addition, rASV-Q-infected cells possessed a 60,000-dalton protein specifically precipitable by rabbit serum raised against SR-D-induced tumors. The facts that the src sequences are essentially the same for rASV's recovered from one animal species and different for rASV's obtained from different species provide conclusive evidence that cellular sequences of normal birds were inserted into the viral genome and supplied to the resulting recombinant viruses genetic information for cell transformation.     

Recombinant Murine Gamma Herpesvirus 68 Carrying KSHV G Protein-Coupled Receptor Induces Angiogenic Lesions in Mice

Human gamma herpesviruses, including Kaposi’s sarcoma-associated herpesvirus (KSHV) and Epstein-Barr virus (EBV), are capable of inducing tumors, particularly in in immune-compromised individuals. Due to the stringent host tropism, rodents are resistant to infection by human gamma herpesviruses, creating a significant barrier for the in vivo study of viral genes that contribute to tumorigenesis. The closely-related murine gamma herpesvirus 68 (γHV68) efficiently infects laboratory mouse strains and establishes robust persistent infection without causing apparent disease. Here, we report that a recombinant γHV68 carrying the KSHV G protein-coupled receptor (kGPCR) in place of its murine counterpart induces angiogenic tumors in infected mice. Although viral GPCRs are conserved in all gamma herpesviruses, kGPCR potently activated downstream signaling and induced tumor formation in nude mouse, whereas γHV68 GPCR failed to do so. Recombinant γHV68 carrying kGPCR demonstrated more robust lytic replication ex vivo than wild-type γHV68, although both viruses underwent similar acute and latent infection in vivo. Infection of immunosuppressed mice with γHV68 carrying kGPCR, but not wild-type γHV68, induced tumors in mice that exhibited angiogenic and inflammatory features shared with human Kaposi’s sarcoma. Immunohistochemistry staining identified abundant latently-infected cells and a small number of cells supporting lytic replication in tumor tissue. Thus, mouse infection with a recombinant γHV68 carrying kGPCR provides a useful small animal model for tumorigenesis induced by a human gamma herpesvirus gene in the setting of a natural course of infection.     

Spontaneous and experimental malignancies in non‐human primates

Contrary to earlier established opinion that tumors in monkeys are found rarely, now the large material confirms that monkey tumors are frequent phenomenon. Tumor incidence clearly increases with age. Frequencies of benign and malignant tumors of various locations and histogenesis are slightly different. Tumors of hematopoietic system are the most frequent. Sporadic cases and enzootic outbreaks of lymphomas are described for different kinds of monkeys, including apes, and probably are caused by viruses. Two viruses were isolated by us from sick monkeys – the retrovirus C‐type STLV‐1 and the herpes virus papio HVP. Inoculation of virus cultures into monkeys and rabbits induces neoplasms. Monkey neoplasms can be induced by exposure to various chemical agents, and by oncogenic and non‐oncogenic viruses. There is no strict species specificity of tumor viruses. The role of polyoma viruses in neoplasms etiology is discussed.     

The absence of p53 promotes metastasis in a novel somatic mouse model for hepatocellular carcinoma

We have generated a mouse model for hepatocellular carcinoma using somatic delivery of oncogene-bearing avian retroviral vectors to the liver cells of mice expressing the viral receptor TVA under the control of the albumin gene promoter (Alb-TVA mice). Viruses encoding mouse polyoma virus middle T antigen (PyMT) induced tumors, which can be visualized with magnetic resonance imaging, in 65% of TVA-positive animals. While these tumors can exceed 10 mm in diameter, they do not invade locally or metastasize to the lungs. Delivery of PyMT-expressing viruses to Alb-TVA mice lacking an intact p53 gene does not increase tumor incidence. However, the resulting tumors are poorly differentiated, invasive, and metastatic to the lungs. Gene expression microarrays identified over 100 genes that are differentially expressed between tumors found in p53 wild-type and p53 null mice. Some of these genes, such as cathepsin E and Igf2, have been previously implicated in tumor cell migration and invasion. Tumors induced in p53 null, TVA transgenic mice by PyMT mutants with changes in specific tyrosine residues fail to form metastases, indicating that metastasis is dependent on both the oncogene and the absence of p53.     

Kaposi's sarcoma-like tumors in a human herpesvirus 8 ORF74 transgenic mouse

The product of human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is related structurally and functionally to cellular chemokine receptors. ORF74 activates several cellular signaling pathways in the absence of added ligands, and NIH 3T3 cells expressing ORF74 are tumorigenic in nude mice. We have generated a line of transgenic (Tg) mice with ORF74 driven by the simian virus 40 early promoter. A minority (approximately 30%) of the Tg mice, including the founder, developed tumors resembling Kaposi's sarcoma (KS) lesions, which occurred most typically on the tail or legs. The tumors were highly vascularized, had a spindle cell component, expressed VEGF-C mRNA, and contained a majority of CD31(+) cells. CD31 and VEGF-C are typically expressed in KS. Tumors generally (but not always) occurred at single sites and most were relatively indolent, although several mice developed large visceral tumors. ORF74 was expressed in a minority of cells in the Tg tumors and in a few other tissues of mice with tumors; mice without tumors did not express detectable ORF74 in any tissues tested. Cell lines established from tumors expressed ORF74 in a majority of cells, expressed VEGF-C mRNA, and were tumorigenic in nude mice. The resultant tumors grew rapidly, metastasized, and continued to express ORF74. Cell lines established from these secondary tumors also expressed ORF74 and were tumorigenic. These data strongly suggest that ORF74 plays a role in the pathology of KS and confirm and extend previous findings on the tumorigenic potential of ORF74.     

IL-12 treatment of endogenously arising murine brain tumors

A number of recent studies have indicated that T cells can be stimulated to attack transplanted brain tumors in rodent models. As IL-12 has been shown to activate cytotoxic T cell responses, we tested the idea that it might stimulate a T cell response against endogenous brain tumors that arise in SV40 large T Ag transgenic mice (SV11). SV11 mice develop tumors of the choroid plexus, a specialization of the ependymal lining of the brain ventricles. They are a particularly relevant model of human disease, because they are immunocompetent but immunologically tolerant of the tumors. SV11 mice were treated with recombinant murine IL-12 for 10 days. Tumors grew more slowly than in control treated mice, and in some cases were reduced in size, as assessed by magnetic resonance imaging before and after treatment. At the end of treatment, tumors, but not brain parenchyma, exhibited extensive infiltration of activated CD8(+) and CD4(+) T cells. Tumors also showed a reduction in vascular density. Mice treated with IL-12 lived significantly longer than control mice. Tumors that progressed were nearly devoid of T cells, indicating that the T cell response was not sustained. In addition, some mice that had a substantial tumor burden at the beginning of treatment displayed evidence of immunosuppression, which might be related to TGF-ss2 detected in tumors. We conclude that IL-12 treatment can initiate an anti-tumor response even against endogenously arising brain tumors, but factors that will allow a sustained and more effective anti-tumor response need to be determined.     

Three Novel Herpesviruses of Endangered Clemmys and Glyptemys Turtles

The rich diversity of the world’s reptiles is at risk due to significant population declines of broad taxonomic and geographic scope. Significant factors attributed to these declines include habitat loss, pollution, unsustainable collection and infectious disease. To investigate the presence and significance of a potential pathogen on populations of critically endangered bog turtles (Glyptemys muhlenbergii) as well sympatric endangered wood (G. insculpta) and endangered spotted (Clemmys guttata) turtles in the northeastern United States, choanal and cloacal swabs collected from 230 turtles from 19 sites in 5 states were screened for herpesvirus by polymerase chain reaction. We found a high incidence of herpesvirus infection in bog turtles (51.5%; 105/204) and smaller numbers of positive wood (5) and spotted (1) turtles. Sequence and phylogenetic analysis revealed three previously uncharacterized alphaherpesviruses. Glyptemys herpesvirus 1 was the predominant herpesvirus detected and was found exclusively in bog turtles in all states sampled. Glyptemys herpesvirus 2 was found only in wood turtles. Emydid herpesvirus 2 was found in a small number of bog turtles and a single spotted turtle from one state. Based on these findings, Glyptemys herpesvirus 1 appears to be a common infection in the study population, whereas Glyptemys herpesvirus 2 and Emydid herpesvirus 2 were not as frequently detected. Emydid herpesvirus 2 was the only virus detected in more than one species. Herpesviruses are most often associated with subclinical or mild infections in their natural hosts, and no sampled turtles showed overt signs of disease at sampling. However, infection of host-adapted viruses in closely related species can result in significant disease. The pathogenic potential of these viruses, particularly Emydid herpesvirus 2, in sympatric chelonians warrants additional study in order to better understand the relationship of these viruses with their endangered hosts.     

What Makes Tumors Multidrug Resistant?

Tumors arising “spontaneously” in genetically modified mice now make it possible to study mechanisms of drug resistance in animal tumors resembling their human counterparts. We have studied mouse mammary tumors induced by conditional deletion of Brca1 and p53. These tumors respond to monotherapy with the maximal tolerable dose of doxorubicin, or docetaxel, but eventually always become resistant to the drugs. Resistance in most tumors is caused by upregulation of drug transporters and not by interference with apoptosis/senescence. The tumors also respond to cisplatin, but do not become resistant, even after repeated treatments at the maximum tolerable dose. We conclude that resistance due to interference with cell death effector pathways (apoptosis/senescence) is not an option in these tumors, re-emphasizing doubts that such mechanisms play a role in epithelial tumors. Tumors responding to drug may shrink to less than 5% of their volume before relapsing. We argue that this resistant remnant fraction may provide a test for the tumor stem cell hypothesis and, more generally, that “spontaneous” mouse tumors resembling their human counterparts provide a useful new tool for drug development and for improving treatment regimens.     

Serological diagnosis of infection with human herpesvirus type 6.

OBJECTIVE--To identify clinical consequences of acute human herpesvirus type 6 infection by hypothesising that the virus will induce similar clinical syndromes to cytomegalovirus. DESIGN--Examination of consecutive serum samples from patients with illnesses compatible with acute cytomegalovirus infection or exanthem subitum by indirect immunofluorescence for the presence of antibodies to human herpesvirus type 6. An IgG absorption step was included to avoid false positive and negative results for IgM. The criterion standard for diagnosis of human herpesvirus type 6 infection was the presence of IgM human herpesvirus type 6 antibody (titre greater than 20) and a rising titre of IgG human herpesvirus type 6 antibody without serological evidence of alternative infection. SETTING--Routine viral diagnostic and reference laboratory in the largest teaching hospital in Sydney. PATIENTS--341 Consecutive serum samples were analysed from patients with hepatitis (147 samples); infectious mononucleosis-like illness (106); screens for toxoplasma, other viruses, rubella, cytomegalovirus, and herpesvirus (38); fever in an immunocompromised patient (eight); unusual neurological (nine) or haematological syndromes (14); splenomegaly (six); and rash in a child (13). RESULTS--Three cases of acute human herpesvirus type 6 infection were identified: in one patient aged 65 with a previous diagnosis of acute non-A non-B hepatitis, one aged 25 with a glandular fever-like illness, and one aged 6 with a glandular fever-like illness. All three illnesses resolved completely. 15 Further serum samples were positive for human herpesvirus type 6 antibody but were also diagnostic for acute infection with other viruses (cytomegalovirus (nine), Epstein-Barr virus (three), and HIV (one] or had a titre of IgM human herpesvirus type 6 antibody less than 20 (two). CONCLUSIONS--Acute human herpesvirus type 6 infection in immunocompetent patients may result in a mononucleosis-like illness or an acute but self limiting hepatitis.     

Herpesvirus-associated lymphomas: Investigations in humans and animal models

Lymphomas are solid tumors consisting of lymphoid cells; they form a heterogeneous group of less or more malignant disorders. A portion of lymphomas develop due to latent herpesvirus infections established in B and/or T-lymphocytes. The basis for latency is a lifelong presence of the circularized covalently linked viral genome within nuclei of carrier lymphocytes. In certain cases, however, the essential event leading to tumor formation is the integration of a portion(s) of viral DNA into the host cell DNA. This leads to rearrangements within the host cell genome on one hand, and, on other hand, to unregulated expression of oncoproteins encoded by the integrated fragment. Our review deals with mechanisms of lymphoma formation regarding to the role of non-structural herpesvirus oncoproteins interfering with the regulation of cell division and/or exerting anti-apoptotic effects. In addition, the authors wish to highlight the common procedures, which allowed isolation and/or identification of lymphoma-associated viruses in cell cultures derived from tumors and/or proliferating lymphatic tissues.     

Effect of temperature on host response to Batrachochytrium dendrobatidis infection in the mountain yellow-legged frog (Rana muscosa)

The pathogenic chytrid fungus Batrachochytrium dendrobatidis, which causes the disease chytridiomycosis, has been implicated in declines of amphibian populations throughout the world, including declines and extinctions of local populations of mountain yellow-legged frogs, Rana muscosa, in the California Sierra Nevada. Previous studies have shown B. dendrobatidis achieves its maximum growth rate in culture in the temperature range of 17-25 C, and exposure to very high temperatures can clear frogs of B. dendrobatidis infection. Here we present the results of a laboratory experiment in which experimentally infected R. muscosa tadpoles were followed through metamorphosis at temperatures of 17 and 22 C. All infected animals developed clinical disease within a similar time frame. However, frogs housed at 22 C exhibited a significantly lower mortality than those housed at 17 C. Within 35 days after metamorphosis, 50% of the frogs housed at 22 C died, while 95% of the frogs housed at 17 C died. Clinical signs subsided in the surviving frogs at 22 C, despite persistent infection. Because both temperatures are within the optimal thermal range for growth of B. dendrobatidis, we propose that the difference in outcome indicates the effect of temperature on the host's resistance to chytridiomycosis, rather than an effect on the fungus alone.     

New molecular mechanisms of virus-mediated carcinogenesis: oncogenic transformation of cells by retroviral structural protein Envelope

RNA tumor viruses as classified in Retroviruses have been isolated and identified to induce tumors in a variety of animals including chickens, mice, and rats, or even in human in the last 100 years, since the first one has been reported in 1908. The RNA tumor viruses have been historically classified into two groups, acute transforming RNA tumor viruses and nonacute RNA tumor viruses. Acute transforming RNA tumor viruses are basically replication-defective and rapidly induce tumors by expressing the viral oncogenes captured from cellular genome in host cells. The first oncogene derived from Rous sarcoma virus was the src non-receptor tyrosine kinase, which has been identified to play the significant roles for signal transduction. On the other hand, nonacute RNA tumor viruses, which consist of only gag, pro, pol, and env regions but do not carry oncogenes, are replication-competent and could activate the cellular proto-oncogenes by inserting the viral long terminal repeat close to the proto-oncogenes to induce tumors with a long incubation period, as is termed a promoter insertion. These molecular mechanisms have been thought to induce tumors. However, very recently several reports have described that the retroviral structural protein Envelope could directly induce tumors in vivo and transform cells in vitro. These are very unusual examples of native retroviral structural proteins with transformation potential. In this review we look back over the history of oncogenic retrovirus research and summarize recent progress for our understanding of the molecular mechanisms of oncogenic transformation by retrovirus Envelope proteins.     

Association of herpesvirus with fibropapillomatosis of the green turtle Chelonia mydas and the loggerhead turtle Caretta caretta in Florida.

Sea turtle fibropapillomatosis (FP) is a disease marked by proliferation of benign but debilitating cutaneous fibropapillomas and occasional visceral fibromas. Transmission experiments have implicated a chloroform-sensitive transforming agent present in filtered cell-free tumor homogenates in the etiology of FP. In this study, consensus primer PCR methodology was used to test the association of a chelonian herpesvirus with fibropapillomatosis. Fibropapilloma and skin samples were obtained from 17 green and 2 loggerhead turtles affected with FP stranded along the Florida coastline. Ninety-three cutaneous and visceral tumors from the 19 turtles, and 33 skin samples from 16 of the turtles, were tested. All turtles affected with FP had herpesvirus associated with their tumors as detected by PCR. Ninety-six percent (89/93) of the tumors, but only 9% (3/33) of the skin samples, from affected turtles contained detectable herpesvirus. The skin samples that contained herpesvirus were all within 2 cm of a fibropapilloma. Also, 1 of 11 scar tissue samples from sites where fibropapillomas had been removed 2 to 51 wk earlier from 5 green turtles contained detectable herpesvirus. None of 18 normal skin samples from 2 green and 2 loggerhead turtles stranded without FP contained herpesvirus. The data indicated that herpesvirus was detectable only within or close to tumors. To determine if the same virus infected both turtle species, partial nucleotide sequences of the herpesvirus DNA polymerase gene were determined from 6 loggerhead and 2 green turtle samples. The sequences predicted that herpesvirus of loggerhead turtles differed from those of green turtles by only 1 of 60 amino acids in the sequence examined, indicating that a chelonian herpesvirus exhibiting minor intratypic variation was the only herpesvirus present in tumors of both green and loggerhead turtles. The FP-associated herpesvirus resisted cultivation on chelonian cell lines which support the replication of other chelonian herpesviruses. These results lead to the conclusion that a chelonian herpesvirus is regularly associated with fibropapillomatosis and is not merely an incidental finding in affected turtles.     

The URNA genes of herpesvirus saimiri (strain C488) are dispensable for transformation of human T cells in vitro

The herpesvirus saimiri strain C488 genome contains five genes for small nuclear RNAs, termed herpesvirus saimiri URNAs (or HSURs). Using a cosmid-based approach, all HSURs were precisely deleted from the genome. The mutant virus replicated at levels that were similar to those of wild-type viruses in OMK cells. Although the HSURs are expressed in wild-type virus-transformed human T-cell lines, the deletion does not affect viral transformation in cell culture.     

Susceptibility of laboratory mice to intranasal and contact infection with coronaviruses of other species

The susceptibility of laboratory mice to intranasal and contact infection with mouse hepatitis virus (MHV)-related coronaviruses was tested in infant CD1 mice. One day old mouse pups were inoculated intranasally with respiratory MHV-S, enteric MHV-Y, rat sialodacryoadenitis virus (SDAV), human coronavirus OC43 (HCV-OC43) or bovine coronavirus (BCV). Twenty-four hours later, they were placed in direct contact with age matched sham inoculated pups. Indices of infection in virus inoculated mice included lesions by histopathology and viral antigen by immunoperoxidase histochemistry in brain, lung, liver and intestine at 3 days after inoculation. Indices of infection in contact mice included mortality or seroconversion by 21 days after exposure. Infant mice were susceptible to infection with all five viruses. Transmission by direct contact exposure occurred with MHV and SDAV, but not HCV or BCV. Furthermore, adult mice were not susceptible to infection with HCV. Tissue distribution of lesions and antigen varied markedly among viruses, indicating that they do not induce the same disease as MHV. This study demonstrates that although these coronaviruses are antigenically closely related, they are biologically different viruses and disease patterns in susceptible infant mice can be used to differentiate viruses.     

Are type C viruses transforming agents?

Type C RNA viruses have been considered oncogenic because they are found associated with animal tumors and can induce cancers in several animal species. Those viruses that rapidly cause cancer appear to contain an oncogenic gene which resembles genetic sequences present in normal cells. This gene codes for a transforming protein which may be a normal cellular enzyme or a slightly altered cellular product. Its mechanism for transforming a cell is not yet known. Other oncogenic viruses, such as the chronic leukemia viruses, may not produce an oncogenic protein but may affect, by other means, specific target cells so they become malignant. Recent evidence now suggests that the majority of endogenous type C viruses are not transforming agents but inherited in the host to function in other biologic processes. These viruses do not contain transduced cellular genes which are responsible for cancer. Their role probably depends on their expression of other gene products which aid in normal development. These observations suggest that the ultimate control of human cancer may result from the identification of the oncogenic cellular-like genes transduced by some type C viruses even if a true human oncogenic virus is not isolated.