Fig. 22.1
Electron photomicrograph showing cell infected with SV40 virus. Depicted is the nucleus of the cell packed with paracrystalline arrays of viruses, seen here as approximately 40 nm dark spherical particles (Image courtesy of Dr. Sara Miller, Professor of Pathology and Director Electron Microscopy Shared Resource, Duke University Medical Center)
In 1960, Sweet and Hilleman identified and added a new member to the list of viruses commonly appearing as contaminants within tissue cultures of monkey origin, particularly the kidney. Owing to its potential to result in distinctive, vacuolar cytopathic effect within infected tissues, these investigators termed the virus the vacuolating virus, simian virus 40 (Sweet and Hilleman 1960). At that time, poliovirus vaccine was prepared using kidney cell cultures from rhesus and cynomolgus monkeys, natural hosts for the virus, wherein the cytopathic effects of SV40 infection are not identifiable within the cultured cells. It is only upon the introduction of SV40 into a heterologous species (C. aethiops, the African green monkey) that the cytopathic effects to indicate infection become evident (Sweet and Hilleman 1960). Subsequent investigations indicated that SV40 was isolatable from both the live attenuated and injectable Sabin poliovirus vaccine, as well as the inactivated and oral Salk poliovirus vaccine. In the latter, the conditions to inactivate the poliovirus were not sufficient to eradicate the contaminating SV40 (Gerber et al. 1961). Following the mass poliovirus immunization programs in the United States which spanned the mid-1950s through early 1960s, tens of millions of Americans were exposed to SV40-contaminated poliovirus vaccine, as well as many millions more worldwide following the distribution of Salk and Sabin vaccines and the cell cultures necessary to produce the vaccine (Poulin and DeCaprio 2006; Shah 2006; Strickler 2001). Despite regulations requiring new stocks of polio vaccine to be SV40-free which were passed in 1961, recalls of older stocks of vaccine were not implemented, and immunization with SV40-contaminated vaccine likely continued until 1963 (Shah and Nathanson 1976). And while the titer of SV40 present in the contaminated stocks varied, as most individuals received multiple vaccines, probably the vast majority of the vaccinated during that time period received exposure of some degree to SV40. SV40-contaminated lots polio vaccines prepared in Eastern Europe and the former Soviet Union may have been used until the late 1970s (Cutrone et al. 2005). Poliovirus vaccine was not the only vaccine to sustain SV40 contamination; the virus also contaminated the adenovirus vaccine, used parenterally to inoculate hundreds of thousands of military recruits during the late 1950s and early 1960s (Rollison et al. 2004).
Following its discovery, intense efforts followed to understand the virus’ molecular biology and potential for inducing disease owing to the perceived public health risk for developing cancer resulting from the inadvertent exposure to SV40 from the contaminated poliovirus vaccines. Following infection of a natural host cell and its integration of SV40 DNA into its genome, the virus completes a lytic replication cycle allowing for high levels of its major oncogenic proteins, large T and small t antigens, to be expressed, followed by synthesis of viral structural proteins, and the creation of progeny virions, typically released following cell lysis (Butel and Lednicky 1999; Garcea and Imperiale 2003). The first substantial purification of SV40 large T antigen was performed by Lazarus et al. from tumor nuclei in hamsters induced by SV40 inoculation (Lazarus et al. 1967). Large T antigen has since been shown to be important for viral replication but also binds to and inactivates cellular p53 and the retinoblastoma protein pRb, tumor suppressor proteins which prevent tumorigenesis through regulation of cellular proliferation and causing apoptosis of injured cells (Poulin and DeCaprio 2006). Highly oncogenic in animal models, SV40 large T antigen alone suffices to transform lines of rodent cells, leading to lymphomas, mesothelioma brain tumors, and sarcomas (Butel and Lednicky 1999; Garcea and Imperiale 2003). Both small and large T antigens, likely in concert with other oncogenic proteins, are required to transform human cell lines in vitro (Bikel et al. 1987; Butel and Lednicky 1999; Hahn et al. 1999, 2002). With the progression of knowledge of the oncogenic properties and capabilities of SV40, both within human and animal models, investigations commenced to assess the role of SV40 in human carcinogenesis at the molecular and epidemiologic level. The results of these investigations will be discussed in the following section.
22.3 Putative Role for SVO in Human Disease, SV40 Oncogenicity, and Malignant Mesothelioma in Humans
SV40 DNA has been detected in human tumors using polymerase chain reaction (PCR) detection assays. The types of tumors demonstrating SV40 have included ependymoma, choroid plexus tumors, non-Hodgkin’s lymphomas, osteosarcomas, and mesothelioma (Poulin and DeCaprio 2006). A number of SV40 DNA-positive tumors have been identified in children too young to have received contaminated poliovirus vaccine (Strickler 2001), which would indicate an active SV40 infection among the population in the community at large, and the potential for person-to-person transmission of the virus for which no strong evidence exists (Strickler 2001). Other studies failed to detect SV40 DNA sequences in CNS tumors, and in one study where SV40 DNA was detected in a few CNS tumors, none of the SV40 (+) malignancies were shown to express large T antigen, suggesting the virus played no role in the tumorigenesis in these cases (Weggen et al. 2002).
SV40 bears a special relationship to the pleura. Mesothelioma, an epithelial malignancy arising in the pleura, peritoneum, pericardium, and tunica vaginalis testis, is highly associated with malignancies related to exposure to amphibole asbestos species (Sporn and Roggli 2004). Despite the myriad usages for asbestos in the industries of shipbuilding, insulation, and construction, with large numbers of workers sustaining significant exposure to asbestos over the past century, mesothelioma remains a rare albeit highly lethal disease with only several thousand new cases diagnosed per year (Sporn and Roggli 2004). Although gender and anatomic site specific, a large number of mesothelioma cases have no known exposure to asbestos, and a distinct minority (5–10 %) of highly asbestos-exposed workers will develop the disease (Bochetta et al. 2000). Asbestos exposure remains the only established cause of mesothelioma, although there are sporadic reports of mesothelioma following exposure to therapeutic irradiation for solid tumors or to certain radiologic contrast materials (Thorotrast) (Sporn and Roggli 2004), and efforts to identify cocarcinogens in the causation of mesothelioma have been largely unsuccessful given the capability of SV40 to cause mesothelioma in newborn hamsters (Cicala et al. 1993), and with the potential for massive exposure of the population through contaminated poliovirus vaccine, the virus seemed to warrant scrutiny as a possible “missing link” or cocarcinogen. Following identification of SV40 gene sequences, including those encoding the large T antigen in some mesotheliomas, some reports attributed SV40 in the etiology of 40–60 % of mesotheliomas (Carbone et al. 1994; Garcea and Imperiale 2003; Gazdar et al 2002; Jasani et al 2001). Galateau-Salle detected using PCR sequences related to SV40 large T antigen in cases of lung cancer, mesothelioma, as well as nonneoplastic pleuropulmonary disorders, albeit at significantly lesser occurrence in the latter conditions (Galateau-Salle et al. 1998). Interestingly, in this study immunohistochemistry using monoclonal antibodies to large T antigen did not show the expected nuclear immunoreactivity either in paraffin-embedded archival tumoral material or in cell lines shown to exhibit SV40-like sequences. Positive controls reacted appropriately, but the lack of nuclear immunoreactivity in the tumors and cell lines constituted a discrepancy the authors found problematic (Galateau-Salle et al. 1998).
In addition to its detection in mesothelioma, SV40 was reported to induce telomerase activity in human mesothelial cell lines (Foddis et al. 2002) and to serve as a cocarcinogen in the induction of mesothelioma in human mesothelial cell lines exposed in vitro to crocidolite asbestos (Bochetta 2000). Carbone et al. reported that SV40 large T antigen binds and inactivates p53 in human mesotheliomas, a cellular protein whose physiologic expression is involved in tumor suppression (Carbone et al. 1997).
Concern for the public health risk posed by a suspected massive exposure to SV40 and new laboratory evidence suggesting demonstrable presence of the SV40 DNA sequences in certain human tumors prompted the Institute of Medicine to report that there was in fact evidence for SV40 contamination of poliovirus vaccine and that SV40 was a likely human carcinogen and recommended additional investigation (Stratton et al. 2003). But in the wake following the initial reporting of the identification of SV40 in human mesotheliomas, and a possible cocarcinogen in the induction of the disease, and as a result of subsequent investigations, considerable doubt has in fact emerged as to the true significance of such reports. Such doubt, as well as skepticism, has evolved due to concerns regarding laboratory methodology for the identification of the viral DNA sequences, as well as epidemiologic studies reporting the incidence of disease, or lack thereof, in cohorts exposed to contaminated vaccine. These concerns will be addressed below.
At the level of laboratory methodology, potential exists for cross-reactivity on ELISA immunoassays between viruslike particles from SV40, JC, and BKV (Viscidi et al. 2003). In human models, Carter et al. have shown that the low level of immunoreactivity of human sera to SV40 antibodies that has been demonstrated is highly associated with the common antibodies to JCV and BKV, and such immunoreactivity may be neutralized by both JCV and BKV antibody-positive sera. These authors conclude that such cross-reactivity does not support the hypothesis that SV40 is a prevalent human pathogen (Carter et al. 2003).
The International SV40 Working Group of nine independent laboratories failed to demonstrate reproducibly positive detection of the SV40 DNA in either selected mesotheliomas or human lung tissue (Strickler et al. 2001). Lopez-Rios et al. raised the possibility of artifactual detection of SV40 within mesothelioma, owing to contamination by plasmids. In a study of 71 mesotheliomas, SV40 sequences identified by PCR contained deletions found only in plasmids, not the sequences required for viral replication and transformation. All mesotheliomas studied were negative for T antigen using RT-PCR and did not demonstrate T-antigen-positive cells using immunoperoxidase stains (Lopez-Rios et al 2004). The issue of false-positive SV40 detection due to plasmid contamination is critical owing to the ubiquity of SV40 DNA sequences in the majority of molecular biology plasmids to facilitate gene expression (Poulin and DeCaprio 2006). A similar study by Manfredi et al. failed to detect SV40 large T antigen in 69 mesotheliomas (Manfredi et al. 2005). This study included an immunohistochemical evaluation of large T antigen using a highly specific SV40 antibody, including some other cases reported to contain SV40 T antigen. These immunohistochemical studies were also negative. An earlier study by Dhaene et al. had also identified SV40 large T-antigen-like DNA sequences in 46 % of a series of Belgian mesotheliomas, but failed to detect nuclear expression of the viral oncoprotein in tumor cells using immunohistochemistry (Dhaene et al. 1999). Brousset et al. similarly failed to detect SV40 T antigen using histochemistry in a series of mesotheliomas a proportion of which had previously been reported to contain SV40 DNA sequences (Brousset et al. 2005).
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