SV40 and human mesothelioma.

Simian virus 40 (SV40) is a DNA tumor virus capable of infecting and transforming human mesothelial (HM) cells in vitro. Hamsters injected intracardially to expose most tissue types to SV40 preferentially develop mesotheliomas. In humans, asbestos is the main cause of mesothelioma, and asbestos and SV40 are co-carcinogens in transforming HM cells in tissue culture and in causing mesothelioma in hamsters. Laser microdissection experiments conducted in the laboratory of Adi Gazdar demonstrated that SV40 was present specifically in the malignant mesothelioma cells and not in nearby stromal cells. Further experiments demonstrated that SV40 remains episomal in HM cells and astrocytes because of the production of a long antisense RNA that represses viral capsid protein production. Thus, the potent SV40 oncoprotein, T-antigen (Tag), is expressed, but because the capsid proteins are not produced, the cells are not lysed and, instead, become transformed. Together this evidence suggests that SV40 may contribute to the development of mesotheliomas in humans. However, epidemiological evidence to support this hypothesis is lacking. This chapter also summarizes the introduction of SV40, a monkey virus, into the human population as an unrecognized contaminant of early poliovaccines. In addition to mesotheliomas, SV40 now is linked with brain cancers, osteosarcomas, and lymphomas in humans. Explanations are provided for the apparent geographic variations in SV40 prevalence and for controversies about the role of SV40 in human cancer.

SV40 seroprevalence in two Latin American countries involved in field trials of candidate oral poliovaccines.

OBJECTIVES: This study sought to determine SV40 seroprevalence in residents of two Latin American countries, Colombia and Nicaragua, which were sites of prelicensure oral poliovaccine (OPV) trials. METHODS: Archival sera were tested for SV40 neutralizing antibody using a virus-specific plaque-reduction assay. Samples included 517 sera from Colombia and 149 sera from Nicaragua. RESULTS: Overall SV40 seroprevalence was 22.8% for Colombian subjects and 12.8% for Nicaraguans. Subgroups of Colombian subjects ranged in frequency of SV40 seropositivity from 10.0% to 38.6%. Birth cohorts both older and younger than the age cohort that contained potential OPV vaccinees from both countries had SV40 antibodies. Gender and ethnicity had no significant effects on SV40 seropositivity. CONCLUSIONS: Inhabitants of both Colombia and Nicaragua had detectable SV40 neutralizing antibody, including those of ages presumably not recipients of potentially SV40-contaminated OPV. This observation provides support for the concept that transmission of SV40 human infections can occur. Frequency of SV40 antibody positivity was elevated over that reported for the US where there was limited use of contaminated OPV. This investigation indicates also that study results of SV40 infections in humans will reflect whether subject populations had probable exposures to contaminated poliovaccines and to environmental conditions favoring cycles of viral transmission.

Viral microRNA effects on persistent infection of human lymphoid cells by polyomavirus SV40.

BACKGROUND: Polyomaviruses, including simian virus 40 (SV40), display evidence of lymphotropic properties. This study analyzed the nature of SV40-human lymphocyte interactions in established cell lines and in primary lymphocytes. The effects of viral microRNA and the structure of the viral regulatory region on SV40 persistence were examined. RESULTS: SV40 DNA was maintained in infected B cell and myeloid cell lines during cell growth for at least 28 days. Limiting dilution analysis showed that low amounts of SV40 DNA (~2 copies per cell) were retained over time. Infected B cells remained viable and able to proliferate. Genome copies of the SV40 microRNA-null mutant persisted at higher levels than the DNA of wild-type viruses. Complex viral regulatory regions produced modestly higher DNA levels than simple regulatory regions. Viral large T-antigen protein was detected at low frequency and at low levels in infected B cells. Following infection of primary lymphocytes, SV40 DNA was detected in CD19+ B cells and CD14+ monocytes, but not in CD3+ T cells. Rescue attempts using either lysates of SV40-infected B lymphocytes, coculture of live cells, or infectious center assays all showed that replication-competent SV40 could be recovered on rare occasions. SV40 infections altered the expression of several B cell surface markers, with more pronounced changes following infections with the microRNA-null mutant. CONCLUSION: These findings indicate that SV40 can establish persistent infections in human B lymphocytes. The cells retain low copy numbers of viral DNA; the infections are nonproductive and noncytolytic but can occasionally produce infectious virus. SV40 microRNA negatively regulates the degree of viral effects on B cells. SIGNIFICANCE: Lymphocytes may serve as viral reservoirs and may function to disseminate polyomaviruses to different tissues in a host. To our knowledge, this report is the first extensive analysis of viral microRNA effects on SV40 infection of human lymphocytes.

Fecal Polyomavirus Excretion in Infancy.

Qualitative polymerase chain reaction (PCR) was used to determine the prevalence of fecal excretion of BK virus, JC virus, and simian virus 40 in 1-year-old infants. Overall, 17.8% of 321 specimens from 64.1% of 39 infants were polyomavirus positive. These data suggest that the gastrointestinal tract may be a site of polyomavirus persistence in humans.

Specific Antibodies Reacting with SV40 Large T Antigen Mimotopes in Serum Samples of Healthy Subjects.

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.

Neutralizing and IgG antibodies against simian virus 40 in healthy pregnant women in Italy.

OBJECTIVE: Polyomavirus simian virus 40 (SV40) sequences have been detected in various human specimens and SV40 antibodies have been found in human sera from both healthy individuals and cancer patients. This study analyzed serum samples from healthy pregnant women as well as cord blood samples to determine the prevalence of SV40 antibodies in pregnancy. METHODS: Serum samples were collected at the time of delivery from two groups of pregnant women as well as cord bloods from one group. The women were born between 1967 and 1993. Samples were assayed by two different serological methods, one group by neutralization of viral infectivity and the other by indirect ELISA employing specific SV40 mimotopes as antigens. Viral DNA assays by real-time polymerase chain reaction were carried out on blood samples. RESULTS: Neutralization and ELISA tests indicated that the pregnant women were SV40 antibody-positive with overall prevalences of 10.6% (13/123) and 12.7% (14/110), respectively. SV40 neutralizing antibodies were detected in a low number of cord blood samples. Antibody titers were generally low. No viral DNA was detected in either maternal or cord bloods. CONCLUSIONS: SV40-specific serum antibodies were detected in pregnant women at the time of delivery and in cord bloods. There was no evidence of transplacental transmission of SV40. These data indicate that SV40 is circulating at a low prevalence in the northern Italian population long after the use of contaminated vaccines.

Complete genomic sequence of a new Human polyomavirus 9 strain with an altered noncoding control region.

A complete Human polyomavirus 9 (HPyV9) genome, designated HPyV9 UF-1, was amplified by rolling circle DNA amplification from DNA extracted from the peripheral blood mononuclear cells (PBMC) of an AIDS patient. The noncoding control (enhancer/promoter) region (NCCR) of HPyV9 UF-1 has one less AML-1a binding site and three more potential Sp1/GC box binding sites than the NCCRs of two previously described HPyV9 genomes. Nucleotide polymorphisms within the coding regions result in two amino acid differences in the deduced VP2 and VP3 proteins of HPyV9 UF-1 relative to those of the two previously described HPyV9 genomes. Exhaustive attempts to detect HPyV9 in DNA samples extracted from the PBMC of 40 healthy humans and 9 other AIDS patients were unsuccessful, highlighting the need for improved search strategies and optimal specimens for the detection of HPyV9 in humans.

Naturally arising strains of polyomaviruses with severely attenuated microRNA expression.

UNLABELLED: Several different polyomaviruses (PyVs) encode microRNAs (miRNAs) that regulate viral as well as host gene expression. However, the functions of polyomaviral miRNAs, particularly during in vivo infection, remain poorly understood. Here we identify rare naturally arising PyVs that are severely attenuated or null for miRNA expression. We identify hypomorphic or null strains for miRNA expression from rhesus macaque simian virus 40 (SV40) and human JC virus. These strains were isolated from immunocompromised hosts and derive from insertions or deletions in the viral DNA that preserve the amino acid reading frame of opposing-strand large T antigen gene. Characterization of the SV40 miRNA hypomorph, K661, shows that it is inhibited at the early miRNA biogenesis step of Drosha-mediated processing. Despite having a nonrearranged enhancer, which a previous study has shown renders some PyVs more susceptible to the autoregulatory activities of the miRNA, restoring miRNA expression to K661 has little effect on virus growth in either immortalized or primary monkey kidney cells. Thus, in addition to any effect of accompanying genomic elements, these results suggest that the cellular context also determines susceptibility to PyV miRNA-mediated effects. Combined, these results demonstrate that polyomaviruses lacking miRNAs can arise infrequently and that the functional importance of polyomaviral miRNAs is context dependent, consistent with an activity connected to the immune status of the host. IMPORTANCE: Diverse virus families encode miRNAs, yet much remains unknown about viral miRNA function and contribution to the infectious cycle. Polyomaviruses (PyVs) are small DNA viruses, long known to be important as etiological agents of rare diseases and valuable models of DNA virus infection. Here, in immunosuppressed hosts, we uncover rare naturally arising variants of different PyVs that have lost the ability to express miRNAs. This represents some of the only known natural viruses to have lost miRNA expression. By probing the biogenesis pathways of these variants, we uncover that miRNA expression is lost via small insertions or deletions that render the transcripts resistant to early steps of miRNA biogenesis while preserving the reading frame of the opposing T antigen transcripts. Overall, our study informs how miRNA genes evolve/devolve in viruses and suggests that miRNA function is exquisitely dependent not only on viral genomic context but also on the cellular and host environment.

Viral microRNA effects on pathogenesis of polyomavirus SV40 infections in syrian golden hamsters.

Effects of polyomavirus SV40 microRNA on pathogenesis of viral infections in vivo are not known. Syrian golden hamsters are the small animal model for studies of SV40. We report here effects of SV40 microRNA and influence of the structure of the regulatory region on dynamics of SV40 DNA levels in vivo. Outbred young adult hamsters were inoculated by the intracardiac route with 1×107 plaque-forming units of four different variants of SV40. Infected animals were sacrificed from 3 to 270 days postinfection and viral DNA loads in different tissues determined by quantitative real-time polymerase chain reaction assays. All SV40 strains displayed frequent establishment of persistent infections and slow viral clearance. SV40 had a broad tissue tropism, with infected tissues including liver, kidney, spleen, lung, and brain. Liver and kidney contained higher viral DNA loads than other tissues; kidneys were the preferred site for long-term persistent infection although detectable virus was also retained in livers. Expression of SV40 microRNA was demonstrated in wild-type SV40-infected tissues. MicroRNA-negative mutant viruses consistently produced higher viral DNA loads than wild-type SV40 in both liver and kidney. Viruses with complex regulatory regions displayed modestly higher viral DNA loads in the kidney than those with simple regulatory regions. Early viral transcripts were detected at higher levels than late transcripts in liver and kidney. Infectious virus was detected infrequently. There was limited evidence of increased clearance of microRNA-deficient viruses. Wild-type and microRNA-negative mutants of SV40 showed similar rates of transformation of mouse cells in vitro and tumor induction in weanling hamsters in vivo. This report identified broad tissue tropism for SV40 in vivo in hamsters and provides the first evidence of expression and function of SV40 microRNA in vivo. Viral microRNA dampened viral DNA levels in tissues infected by SV40 strains with simple or complex regulatory regions.

Ethnic differences in polyomavirus simian virus 40 seroprevalence among women in Houston, Texas.

OBJECTIVE: To examine the prevalence and distribution among racial/ethnic groups of polyomavirus SV40 antibodies in women in Houston, Texas. METHODS: Women in three different cohorts reflecting the evolving demographics of Houston were evaluated for frequency of SV40 antibodies using a plaque-reduction neutralization assay. RESULTS: Women in cohort A (enrolled 1972-1973) were 68% (145/212) African-American and 32% Caucasian; the overall frequency of SV40 neutralizing antibodies was 7%. Women in cohort B (enrolled 1975-1977) were Caucasian with an overall frequency of SV40 neutralizing antibodies of 18% (37/211). Women in cohort C (enrolled 1993-1995) were 50% (199/400) African-American, 25% Caucasian, and 25% Hispanic; the overall frequency of SV40 neutralizing antibodies was 10%. Logistic regression analysis for cohort A showed no difference in SV40 neutralizing antibodies with respect to race/ethnicity, pregnancy status, number of previous pregnancies, or history of sexually transmitted diseases. For cohort C, race/ethnicity was identified as a significant factor associated with SV40 neutralizing antibodies, with Hispanics having a seroprevalence of 23% compared to 5-6% in the other two groups (p = 0.01). CONCLUSIONS: A significantly higher SV40 seroprevalence was found among Hispanics than other racial/ethnic groups in the city of Houston. Findings are compatible with a model that certain population groups potentially exposed to SV40-contaminated oral poliovaccines have maintained cycles of SV40 infections.

Effects of route of inoculation and viral genetic variation on antibody responses to polyomavirus SV40 in Syrian golden hamsters.

Genetic variants of polyomavirus SV40 are powerful agents with which to define viral effects on cells and carcinogenesis pathways. We hypothesized that differences in biologic variation among viral strains affect the process of viral infection and are reflected in antibody responses to the viral nonstructural large T-antigen (TAg) protein but not in neutralizing antibody responses against the inoculated viral particles. We analyzed the production of TAg antibody and neutralizing antibody in Syrian golden hamsters that were inoculated with SV40 viral strains by intracardiac, intravenous, or intraperitoneal routes and remained tumor free. Compared with the intraperitoneal route, intravascular (that is, intravenous, intracardiac) inoculation resulted in increased frequency of responsiveness to TAg but not in higher TAg antibody titers. The intravascular route was superior both for eliciting neutralizing antibody responses and for higher titers of those responses. Viruses with complex regulatory regions induced TAg antibody more often than did viruses with simple regulatory regions after intraperitoneal but not intravascular injections, with no differences in antibody titers. This viral genetic variation had no effect on neutralizing antibody production after intraperitoneal or intravascular inoculations or on neutralizing antibody titers achieved. These findings confirm that SV40 variants differ in their biologic properties. Route of inoculation combined with viral genetic variation significantly influence the development of serum antibodies to SV40 TAg in tumor-free hamsters. Route of inoculation-but not viral genetic variation-is an important factor in production of neutralizing antibody to SV40.

Patterns of polyomavirus SV40 infections and associated cancers in humans: a model.

A model is described that predicts patterns of polyomavirus SV40 infections and associated cancers in humans. The model proposes that SV40 infections were established in humans primarily by exposure to contaminated oral poliovaccines and that infections persist today in geographic regions where poor sanitation or living conditions allow maintenance of infections transmitted by a fecal/urine-oral route. Predictions from the model include that SV40 infections and virus-associated malignancies will be restricted geographically and demographically and that in developed countries, such as the US, SV40 prevalence rates will be generally very low. The model highlights the importance of selection of populations for investigations of SV40 human infections. This model can explain inconsistencies in the published literature of SV40 infections in humans and can guide the design of future studies.

Polyomavirus JC urinary shedding in kidney and liver transplant recipients associated with reduced creatinine clearance.

BACKGROUND: Polyomavirus reactivation can cause significant morbidity in solid organ transplant recipients, particularly BK virus (BKV) in kidney transplant patients. Less is known about dynamics of John Cunningham virus (JCV) in nonkidney organ transplant patients. METHODS: We examined the frequency of urinary shedding of polyomaviruses BKV and JCV and their relationship to creatinine clearance (CrCl) in a longitudinal study of 41 kidney and 33 liver transplant recipients. RESULTS: Any polyomavirus urinary shedding was more frequent in liver than kidney recipients (64% vs 39%; P= .03). JCV was excreted more frequently by liver than kidney recipients (71% vs 38%), whereas BKV was shed more often by kidney than liver patients (69% vs 52%). Mean JCV loads were significantly higher than those of BKV in both patient groups (P< .0001). Lower mean CrCl values were significantly associated with JCV shedding in both kidney and liver recipients (P< .001). CONCLUSIONS: These findings suggest that BKV and JCV display different patterns of reactivation and shedding in kidney and liver transplant patients and that JCV may have a role in renal dysfunction in some solid organ transplant recipients.

Lymphotropism of Merkel cell polyomavirus infection, Nova Scotia, Canada.

To test the hypothesis that Merkel cell polyomavirus (MCPyV) can infect cells of the lymphoid system, we analyzed 353 specimens, including 152 non-Hodgkin lymphomas, 44 Hodgkin lymphomas, 110 benign lymph nodes, 27 lymph nodes with metastasis, and 20 extranodal tissue samples. MCPyV DNA was detected by quantitative PCR in 13 (6.6%) of 196 lymphomas, including 5 (20.8%) of 24 chronic lymphocytic leukemia specimens, and in 11 (10%) of 110 benign lymph nodes, including 8 (13.1%) of 61 samples of reactive hyperplasia and 3 (10.3%) of 29 normal lymph nodes. Other samples were MCPyV negative. Sequence analysis of 9 virus-positive samples confirmed the identity of MCPyV; 3 viral strains were represented. Immunohistochemical testing showed that 1 T-cell lymphoma expressed MCPyV T-antigen. These findings suggest that the lymphoid system plays a role in MCPyV infection and may be a site for MCPyV persistence.

A system for the analysis of BKV non-coding control regions: application to clinical isolates from an HIV/AIDS patient.

The human polyomavirus BK virus (BKV) is an important opportunistic pathogen whose disease prevalence continues to increase with the growing immunocompromised population. To date, the major determinant of replication in cell culture has not been formally proven. BKV exists as archetype virus and rearranged variants, which are classified based on the DNA sequence of their non-coding control regions (NCCRs). The archetype BKV NCCR is divided into five blocks of sequence and rearranged variants contain deletions and duplications of these blocks. In this study, a genetic system was developed and used to identify the major determinant of replication ability in primary renal proximal tubule epithelial cells, the natural host cell of BKV. This system was also used to analyze NCCR variants isolated from an immunocompromised patient which contain assorted rearrangement patterns and functional differences. This study solidifies the NCCR as the major genetic determinant of BKV replication ability in vitro.

Polyomavirus infection and its impact on renal function and long-term outcomes after lung transplantation.

BACKGROUND: Polyomavirus infection causes nephropathy after kidney transplantation but has not been thoroughly investigated in nonrenal organ transplantation. METHODS: Ninety lung transplant recipients were enrolled, and they provided urine samples for over 4.5 years. Samples were analyzed for BK virus (BKV), JC virus (JCV), and simian virus 40 (SV40) by conventional and quantitative real-time polymerase chain reaction. RESULTS: Fifty-nine (66%) patients had polyomavirus detected at least once, including 38 patients (42%) for BKV, 25 patients (28%) for JCV, and six patients (7%) for SV40. Frequency of virus shedding in serial urine samples by patients positive at least once varied significantly among viruses: JCV, 64%; BKV, 48%; and SV40, 14%. Urinary viral loads for BKV (10 copies/mL) and JCV (10 copies/mL) were higher than for SV40 (10 copies/mL; P=0.001 and 0.0003, respectively). Polyomavirus infection was associated with a pretransplant diagnosis of chronic obstructive pulmonary disease (odds ratio 6.0; P=0.016) but was less common in patients with a history of acute rejection (odds ratio 0.28; P=0.016). SV40 infection was associated with sirolimus-based immunosuppression (P=0.037). Reduced survival was noted for patients with BKV infection (P=0.03). Patients with polyomavirus infection did not have worse renal function than those without infection, but in patients with BKV infection, creatinine clearances were lower at times when viral shedding was detected (P=0.038). CONCLUSIONS: BKV and JCV were commonly detected in the urine of lung transplant recipients; SV40 was found at low frequency. No definite impact of polyomavirus infection on renal function was documented. BKV infection was associated with poorer survival.

Variable frequency of polyomavirus SV40 and herpesvirus EBV in lymphomas from two different urban population groups in Houston, TX.

BACKGROUND: Studies have reported differing frequencies of detection of polyomavirus simian virus 40 (SV40) in association with human lymphomas. OBJECTIVE: We addressed the hypothesis that SV40 positivity in lymphomas can vary among sampled populations. STUDY DESIGN: Archival paraffin-embedded lymphoma specimens (n=171) from patients at two urban hospitals in Houston, TX, USA, were analyzed following a cross-sectional study design. Extracted DNAs were characterized by quantitative polymerase chain reaction for the cellular RNase P gene and for SV40 and herpesvirus Epstein-Barr virus (EBV) sequences. RESULTS: Patient characteristics of the two study populations differed significantly whereas the classification of tumor types studied did not. SV40 DNA was detected more frequently in lymphomas from the public hospital population (10/44, 23%) than in lymphomas from the veterans' hospital (VAMC) (4/127, 3%; P<0.0001). EBV detection in lymphomas also differed between the two groups (17/44, 39% vs. 23/127, 18%; P=0.01). SV40 positivity was associated with a younger age category of VAMC lymphoma patients (P=0.02). Expression of T-antigen was detected by immunohistochemistry in half of lymphomas that contained SV40 DNA. Variation was observed in the quality and quantity of DNA recovered from paraffin-embedded specimens, but there was no difference in recoveries of DNA from samples from the two hospitals. CONCLUSIONS: This study demonstrated that, in a direct comparison, the prevalence of SV40 DNA in lymphomas can differ significantly between groups with different demographic distributions.

Polyomavirus shedding in the stool of healthy adults.

We recently reported the frequent detection of polyomaviruses (BK virus [BKV] or simian virus 40 [SV40]) in 46% of stool samples from hospitalized children. In order to determine if adults exhibit fecal shedding of polyomavirus, single stool specimens from healthy adults were evaluated by PCR. Overall, 20 (18.2%) of 110 specimens were positive for human polyomaviruses: 9 with BKV, 9 with JC virus (JCV), 1 with SV40, and 1 with both JCV and SV40. Among the 94 subjects without immune compromise, 17 (18.1%) were excreting polyomaviruses. This shedding frequency in adults was significantly lower than that observed in children (P < 0.001). These findings support the hypothesis that the gastrointestinal tract may be a site of polyomavirus persistence, and they suggest a fecal-oral route of viral transmission.