Infectious SV40 Research Articles

Some Oral Poliovirus Vaccines Were Contaminated With Infectious SV40 After 1961

Some Oral Poliovirus Vaccines Were Contaminated With Infectious SV40 After 1961

Some Oral Poliovirus Vaccines Were Contaminated with Infectious SV40 after 1961

Some polio vaccines prepared from 1954 to 1961 were contaminated with infectious SV40. It has been assumed that all polio vaccines were SV40 free in the United States after 1961 and in other countries after 1962. Following a WHO requirement that was prompted by the detection of SV40 in some human tumors, we conducted a multilaboratory study to test for SV40 polio vaccines prepared after 1961. Vaccine samples from 13 countries and the WHO seed were initially tested by PCR. The possible presence of intact and/or infectious SV40 DNA in PCR-positive samples was tested by transfection and infection of permissive CV-1 cells. All results were verified by immunohistochemistry, cloning, and sequencing. All the vaccines were SV40 free, except for vaccines from a major eastern European manufacturer that contained infectious SV40. We determined that the procedure used by this manufacturer to inactivate SV40 in oral poliovirus vaccine seed stocks based on heat inactivation in the presence of MgCl2 did not completely inactivate SV40. These SV40-contaminated vaccines were produced from early 1960s to about 1978 and were used throughout the world. Our findings underscore the potential risks of using primary monkey cells for preparing poliovirus vaccines, because of the possible contamination with SV40 or other monkey viruses, and emphasize the importance of using well-characterized cell substrates that are free from adventitious agents. Moreover, our results indicate possible geographic differences in SV40 exposure and offer a possible explanation for the different percentage of SV40-positive tumors detected in some laboratories.

Isolation of SV40 from the environment of a colony of cynomolgus monkeys naturally infected with the virus

The presence of SV40 viral particles in the environment of cynomolgus monkeys naturally infected with this virus has been analyzed by testing waste of the cage samples. SV40 was detected in 2/4 cages tested where mixed infection of SV40 and adenoviruses was observed after inoculation of virions concentrated from cage waste in CV-1 cells. The detected SV40 strains were identical in the regions studied to strain W17, isolated at National Institute for Biological Standards and Control, UK (NIBSC) from a (1/19) monkey kidney biopsy and contains an archetypal regulatory region. The recovery of infectious SV40 virions from the cages provides information about the potential mechanism of transmission of this virus.

The simian virus 40 minor structural protein Vp3, but not Vp2, is essential for infectious virion formation.

The SV40 capsid is composed of pentameric capsomeres of the major structural protein Vp1. The two minor structural proteins, Vp2 and Vp3, interact with the capsid. Here, the roles of Vp2 and Vp3 were explored during the course of SV40 infection. Start codons of Vp2, Vp3, or both Vp2 and Vp3, were destroyed by site-directed mutagenesis, and mutant genomes were transfected into CV-1 cells. SV40DeltaVp2 produced plaques and infectious virion particles with titres indistinguishable from wild-type. SV40DeltaVp3 and SV40 DeltaVp2/Vp3 were defective in plaque formation and rendered no infectious particles. All three mutants showed normal nuclear localization of T-Ag and Vp1; they also showed packaging of SV40 DNA by nuclease digestion assays. Thus, Vp3 is essential for formation of infectious SV40 particles, whereas Vp2 is not. One critical role of full-length Vp3 appears to be in virus-cell interactions at post-packaging steps of a permissive infection.

BK virus and SV40 co-infection in polyomavirus nephropathy.

Polyomavirus (PV) nephropathy has been attributed to reactivation of BK virus (BKV) or more rarely JC virus (JCV). The simian virus (SV) 40 is PV that was likely introduced into the human population through contaminated vaccines. The purpose of this study was to identify and characterize the PV that is associated with PV nephropathy. The clinical diagnosis of PV nephropathy (PVN) was made in patients with acute deterioration in renal function whose renal biopsies showed typical viral cytopathic changes in tubular epithelial cells and staining for PV T antigen. Polymerase chain reaction (PCR) amplification of DNA from peripheral blood mononuclear cells (PBMC), urinary cells, and renal biopsy tissue was performed using specific primers for the transcription control regions of BKV, JCV, and SV40, respectively. Six cases of PV nephropathy were identified in 91 renal transplant recipients (7%). Immunosuppressive therapy was modified in all patients. Renal function stabilized or improved in four patients and deteriorated in two patients, and one patient has lost his allograft, after follow-up from 2 to 25 months. PCR detection demonstrated BKV genome in three of five PBMC samples, six of six urinary cell samples, and two of four renal biopsies. SV40 genome was detected in two of five PBMC samples, one of six urinary cell samples, and two of four renal biopsies. Infectious SV40 and BKV was demonstrated in CV-1 co-cultures using urine from one patient. JCV was not detected in any PVN sample. Co-infection with BKV and SV40 was found in two PVN patients. Urine samples obtained 12 months after transplant from 26 transplant recipients without PVN on simultaneous protocol renal biopsy were analyzed by PCR; BKV genome was demonstrated in 5 of 25 samples, JCV genome was demonstrated in 3 of 25 samples, and SV40 genome was demonstrated in 0 of 25 samples. The authors report molecular evidence that co-infection with BKV and SV40 occurs in renal transplant patients with PVN, suggesting that SV40 may contribute to PVN after renal transplant.

Examination of Poliovirus Vaccine Preparations for SV40 Sequences

Oral poliovaccines derived from the strains developed by Sabin have been the basis of vaccination against poliomyelitis in the U.K. since 1962. Contamination of earlier materials with the monkey virus SV40, particularly inactivated Salk type poliovaccines, is well documented. Precautions have been in place for more than 30 years to prevent SV40 contamination of oral poliovaccines based on screening of donor animals and tests for SV40 infectivity. PCR was applied to examine all archived samples of oral poliovaccines available to us dating from 1966 to the present, including all vaccines used in the U.K. since 1980, for the presence of SV40 sequences. Of 132 materials examined, 118 were negative on initial testing and fourteen gave reactions which on further examination were attributed either to cross contamination during handling in the laboratory at National Institute for Biological Standards and Control (NIBSC) or to non-specific amplification. It was concluded that none of the samples contained SV40 sequences. The materials included 69 separate monovalent bulks of poliovirus type 1, 2 or 3 grown on monkey kidney cells from four different manufacturers and 74 bulks grown on human diploid cells from two manufacturers.One additional seed material from 1962 contained low levels of unique and characteristic SV40 sequences. The seed had been treated by the manufacturer to inactivate DNA viruses and tests by the manufacturer and at NIBSC failed to demonstrate the presence of infectious SV40 virus. Monovalent bulks prepared by the manufacturer from this seed were negative for SV40 sequences by PCR.The PCR studies provide no evidence of contamination of oral poliovaccines used in the UK with infectious SV40 and suggest that the steps taken to ensure the absence of infectious SV40 are satisfactory.

A Packaging System for SV40 Vectors without Viral Coding Sequences

SV40 vectors have been used as expression vectors for mammalian cells since the early 1980s. More recently, they have been used as gene transfer vectors in mice and in human peripheral blood cells. Here we described a system for packaging SV40 vectors without viral coding sequences. Recombinant adenovirus-expressing SV40 capsids can effectively package plasmids that contain the SV40 replication origin. The final yield of infectious SV40 vector is about 3 × 105, with a SV40 to adenoviral vector ratio of about 1000:1. Helper adenoviruses can be effectively heat-inactivated with no effect on the infectivity of SV40 vectors.

Excess antisense RNA from infectious recombinant SV40 fails to inhibit expression of a transfected, interferon‐inducible gene

SV40-based infectious virus constructs were used to produce a high copy number of full-length antisense RNA in essentially every cell in a population. Chloramphenicol acetyltransferase (CAT) cDNA was placed in either the sense or antisense orientation relative to the SV40 early promoter in helper-free recombinant virus. RNA synthesized at high levels from the antisense virus was without effect on the expression of a stably-transfected CAT mini-gene controlled by an interferon-inducible promoter in monkey CV1 and large T antigen-expressing tsCOS cells. In double infection experiments the antisense RNA was similarly without effect on expression from CAT cDNA placed in the sense orientation in a second virus vector. No activation of the ppp(A2'p)nA(n greater than or equal to 2) system was observed after interferon treatment in either type of experiment. There was no evidence, therefore, for the formation of double-stranded (ds)RNA. It can be concluded that a large excess of a full-length antisense RNA is not necessarily sufficient to cause inhibition of gene expression even when interferon treatment is used to enhance any effect of dsRNA.

Characterization of mutant human fibroblast cultures transformed with simian virus 40.

Fibroblast cell strains derived from a normal individual and from eight patients with various genetic mutations were transformed by a small-plaque variant of simian virus 40 (SV40, strain 776), cloned and studied after long-term in vitro maintenance. Seven of the cultures continued to express the mutant phenotype. Cultures derived from a patient with phosphoglycerate kinase I deficiency exhibited reappearance of normal enzyme activity after transformation. Compared to untransformed controls, all transformed cultures displayed decreased population doubling times, an increase in the relative number of cycling cells and increased saturation density on solid substrates, and did not show evidence of cellular senescence after long-term cultivation. Unlike previous studies on wild-type SV40-transformed human fibroblasts, the majority of cultures transformed by the small-plaque variant of SV40 did not exhibit signs of crisis. The cells also exhibited a decreased dependence on serum and were able to grow in semi-solid medium. The different transformed cultures expressed variable levels of SV40 large T-antigen, synthesized some infectious SV40 virus, and contained both unique arrangements and quantities of covalently integrated and episomal SV40 DNA. No correlation was observed between the rate of growth and synthesis of infectious virus in the different transformed clones. These studies indicate that this small-plaque variant of SV40 can be used effectively to generate long-lived human cultures, which generally retain their mutant phenotype. Transformation with this SV40 variant permits the generation of large quantities of clonal cell cultures for the biochemical and molecular analysis of their genetic defects.

Rescue of SV40 following transfection of TC7 cells with cellular DNAs containing complete and partial SV40 genomes.

Infectious SV40 virions could be rescued from permissive TC7 cells within one to three subcultures following cotransfection with two cellular DNAs, each containing a complementary portion of the SV40 genome. SV40 virions could also be rescued by transfection of TC7 cells with cellular DNAs from a variety of SV40 transformed cells containing complete genome equivalents but not from cells containing subgenomes alone or defective genomes. Infectious virus was not rescued if the transfecting DNA species was treated with DNAase or if the DEAE-dextran pretreatment of the recipient cells was omitted.

Inhibition of nuclear migration of wild-type SV40 tumor antigen by a transport-defective mutant of SV40-adenovirus 7 hybrid virus

A mutant of the defective SV40-adenovirus 7 hybrid, PARA, induces the synthesis of SV40 large T-antigen (T-ag) that is not transported to the nucleus and accumulates in the cytoplasm of infected or transformed cells. The effect of this cT mutation on the transport of wild-type (WT) T-ag was examined by coinfection experiments. Immunofluorescence staining for T-ag revealed that when SV40-transformed green monkey kidney cells were infected with the cT mutant of PARA [PARA(2cT)] there was a total loss of nuclear T-ag (nT-ag) reactivity concomitant with the appearance of cytoplasmic T-ag (CT-ag) reactivity. The dominance of the cT-ag phenotype was also manifest in cells coinfected with PARA(2cT) and either SV40 or WT PARA, both of which induce nT-ag during single infection. Two lines of evidence indicate that the absence of nuclear T-ag reactivity in coinfected cells is due to a failure of migration of WT T-ag into the nucleus, rather than an inhibition of its synthesis. First, coinfection of cells with SV40 and either helper adenovirus or PARA-adenovirus populations, at the multiplicities of infection employed in these studies, did not reduce the yields of infectious SV40. Second, cells coinfected with PARA(2cT) and deletion mutants of SV40 which encode T-ag polypeptides of reduced molecular weight expressed the cT-ag phenotype, while the presence of the deleted forms of T-ag was confirmed by immunoprecipitation and SDS-polyacrylamide gel electrophoresis. The observation that the replication of infectious SV40 is not inhibited in cells coinfected with PARA(2cT) and expressing the cT phenotype indicates that levels of nuclear T-ag below the limits of detection by immunofluorescence are sufficient to promote SV40 DNA synthesis. The dominant effect of the cT mutation was specific for transport of SV40 T-ag, since normal migration of adenovirus tumor and virion antigens as well as SV40 virion antigen occurred. Several possible mechanisms for the dominance of the CT-ag phenotype are presented.

Structure of the components of the adenovirus-simian virus 40 hybrid population Ad2 ++HEY and reciprocal molecular interactions

The heterogeneous adenovirus-simian virus 40 hybrid population Ad2 ++HEY consists of Ad2 † † Abbreviations used: Ad, adenovirus; SV40, simian virus 40; WT, wild-type. Hybrid particles in the Ad2 ++HEY population containing 0.45, 1.43 and 2.39 integrated SV40 genomes are referred to as HEY-0.4, HEY-1.4 and HEY-2.4, respectively. One unit of DNA length(Ad unit, SV40 unit, HEY unit) is equal to the complete length of the corresponding genome (WT Ad2, SV40, HEY-1.4). The nomenclature for restriction enzymes was proposed by Smith & Nathans (1973). p.f.u., plaque-forming units. helper virions, non-hybrid SV40 virions, and three defective hybrid virions (HEY-0.4, HEY-1.4 and HEY-2.4) whose genomes differ in SV40 DNA content (0.45, 1.43 and 2.39 SV40 genomes, respectively). These components were purified by centrifugation in a CsCl gradient. The gradient fractions were identified and the structure of the hybrid DNA molecules was examined by using electron microscope heteroduplex methods. Furthermore, structural information was obtained by analyzing the DNA fragments resulting from digestion of hybrid DNA with restriction endonucleases. In HEY-1.4 and HEY-2.4 hybrids, which contain more than one complete SV40 genome, the excess SV40 DNA is organized as a head-to-tail tandem repetition. It was found that purified HEY-1.4 and HEY-2.4 hybrids interconvert readily in African green monkey kidney cells in the presence of Ad2 helper. Thus, the original composition of the population is restored by recombinational events. Also, HEY-2.4 could be cloned with Ad5 as helper, but similar experiments with HEY-1.4 and Ad5 only gave progeny in which part of the Ad2 information in. the hybrid genome had been exchanged for Ad5 sequences. Apparently, encapsidation by proteins, which are largely Ad5-specific, requires adapted structural features of the hybrid genomes. Infectious, non-hybrid SV40 particles are efficiently generated by the Ad2 ++HEY population. It was shown that HEY-2.4 hybrids yielded infectious non-hybrid SV40 virions much more efficiently than did HEY-1.4 hybrids. Excision of non-hybrid SV40 DNA is a very accurate process resulting in a homogeneous population of DNA molecules. No evidence was found for the presence of Ad or cellular DNA sequences in the excised nonhybrid SV40 genomes. Although the genome of the non-hybrid SV40 progeny differed slightly from that of SV40 strain 776, there is no reason to believe that these changes are causally related to the integration and excision phenomena.

Induction of Infectious Virus DNA and Virus Particles by Mitomycin C in SV40-Transformed Mouse Cells

A line of SV40-transformed mouse cells (SV/3T3-4E) was isolated and four clones were derived from this line. Spontaneous production of small amounts of infectious SV40 DNA was detected in the parental line and in three out of the four clones tested, although no synthesis of virions could be demonstrated. The yields of SV40 DNA were significantly enhanced following treatment of these cells with mitomycin C and infectious virus particles became detectable occasionally.

Simian Virus 40 -- Chinese Hamster Kidney Cell Interaction. III. Characteristics of Chemical Induction in a Clone of Virogenic Transformed Cells

A number of chemical and physical agents were screened to determine their effectiveness in inducing simian virus (SV40) production in a virogenic clone of SV40-transformed Chinese hamster cells. Mitomycin C (MC) was the most effective inducing agent, and MC induction was further characterized. It was found that levels of infectious SV40 DNA were increased above control levels as early as 6 h after addition of MC to the culture medium and reached maximum levels by 48 h. Virus capsid (V) antigen and virions followed with a lag of about 24 h. V antigen production was sensitive to hydroxyurea, suggesting a dependence on virus DNA synthesis. The proportion of virus-producing cells (infectious centres) and the virus burst per cell were both stimulated by MC. Studies of 3H-thmidine incorporation demonstrated that the rate of SV40 DNA synthesis was maximal at 48 h post-induction, at which time cellular DNA synthesis was almost abolished. Caffeine, at doses not toxic to non-induced cells, strongly inhibited SV40production in both non-induced and induced cells, suggesting some role for DNA repair mechanisms.

Characterization of Simian Virus 40 Transformed African Green Monkey Cells (CV‐1)

Several clones of SV40 transformed CV-1 cells have been characterized for the production of T- and V-antigens and for the state of viral genome. The transformed CV-1 cells failed to produce infectious virions as assayed after sonication or cocultivation and fusion with normal CV-1 cells, and were resistant to super-infection by SV40. Some clones of the transformed cells contained V-antigens. The population of V-antigen positive cells varied from 0 to 100% depending on the passage number while the T-antigen positive cells were always 100%. The virions isolated from the transformed cells were similar in morphology to complete SV40, but lighter in density than complete SV40. In one clone, a small amount of SV40 DNA was detectable in a free state while a large proportion of the DNA hybridizable with SV40 3H cRNA was linearly integrated into the cell DNA. The free SV40 DNA was noninfectious, closed circular DNA with a size smaller than infectious SV40 DNA component I. Since the cell extracts of the transformed cells contained an agent(s) which induced T- and V-antigens in normal CV-1 cells, it was suggested that the SV40 transformed CV-1 cells contained free as well as integrated defective SV40 genomes responsible for the synthesis of T- and V-antigens.

Biology of SV40 transplantation antigen (TrAg): I. Demonstration of SV40 TrAg on glutaraldehyde-fixed SV40-infected African green monkey kidney cells

The synthesis of papovavirus SV40 transplantation antigen (TrAg) in SV40-infected permissive African green monkey kidney cells (TC7) was demonstrated by using the in vivo transplantation-rejection test. Adult BALB c mice immunized with SV40-infected cells, which had been fixed with 0.25% glutaraldehyde for 5 min at room temperature, rejected a challenge of syngeneic virus-free SV40-transformed mouse cells (VLM). Fixed uninfected TC-7 cells failed to induce transplantation immunity against VLM cells in mice. The induction of SV40 tumor immunity in mice after immunization with glutaraldehyde-fixed infected TC-7 cells was mediated by the TrAg synthesized in permissive monkey cells as a result of virus infection, rather than by the residual infectious virus in the glutaraldehyde-fixed infected cells. The treatment of infectious SV40 with 0.25% glutaraldehyde drastically reduced both viral infectivity and tumor immunity-inducing ability of the virus. Glutaraldehyde-inactivated virus also failed to induce SV40 T antigen in monkey cells, thus ruling out any possibility that the inactivated cell-associated virus was responsible for the induction of tumor immunity in vivo. The amount of infectious SV40 required to induce detectable tumor immunity was found to be 2 × 10 5 PFU of virus administered to adult mice, and is far greater than the amount of virus (1 × 10 4 PFU) which remained associated with SV40-infected monkey cells after fixation of cells with glutaraldehyde. The synthesis of SV40 TrAg in SV40-infected TC-7 cells was inhibited by 5 μg/ml of actinomycin D, but not by 15 μg/ml of cytosine arabinoside, indicating that transcription in the infected cells, but not viral-DNA synthesis, is required for the expression of TrAg. By using 0.25% glutaraldehyde to fix infected cells, it should be possible to test available temperature-sensitive mutants of SV40 for their ability to synthesize TrAg at the nonpermissive temperature, as the glutaraldehyde fixation reduces infectivity of intracellular and extracellular virus, stops the infectious cycle at a particular stage, and preserves immunogenicity of TrAg.

Structure of two adenovirus-simian virus 40 hybrids which contain the entire SV40 genome

Two defective adenovirus-simian virus 40 hybrids which contain the entire SV40 genome (Ad2 + +HEY and Ad2 + +LEY) † † Abbreviations used: Ad, edenovirus; SV, simian virus. have been isolated. Upon infection of cells permissive for SV40 both hybrids give rise to infectious SV40 virions, but with markedly different efficiencies. In the case of Ad2 + +HEY nearly all cells infected with a hybrid particle yield SV40 progeny, whereas in the case of Ad2 + +LEY infectious SV40 is produced in only about one in 10 4 cells infected with hybrid particles. The structures of the DNA molecules in the Ad2 + +HEY and Ad2 + +LEY populations were examined using electron microscope heteroduplex methods. Both populations were found to be heterogeneous. Ad2 + +HEY contained three hybrids (HEY-I, HEY-II, and HEY-III) whose genomes differed only in their content of SV40 DNA (0.45 ± 0.02, 1.43 ± 0.04, and 2.39 ± 0.09 SV40 genomes, respectively). Ad2 + +LEY contained two hybrids (LEY-I and LEY-II), which also differed only in their content of SV40 DNA (0.03 ± 0.01 and 1.05 ± 0.01 SV40 genomes, respectively). In those hybrids which contained more than one complete SV40 genome (HEY-II, HEY-III, LEY-II) the excess SV40 DNA was shown to be organized as a tandem repetition. These data suggest that the various hybrid genomes within each population are interconvertible by recombination events, which insert or excise an SV40 genome. It is proposed that HEY-II and HEY-III yield infectious SV40 with higher efficiency than LEY-II because their SV40 DNA segments contain longer tandem repetitions; thus, the probability of an intramolecular recombination event which results in excision of an SV40 genome is greater.

Further studies on SV40 permissive cell protein factor(s) allowing rescue of infectious virus and viral DNA from non-shedder SV40-transformed cells.

AbstractThe factor (s) responsible for rescue of the SV40 genome from transformed cells (activator) is essentially detected in nuclear extracts from permissive cells. Removal of nucleic acids from these extracts improves their activating capacity on transformed cells. By increasing the yield of infectious viral DNA, virions become frequently detectable. For a given experiment, a correlation is obtained between the protein concentration of the extract and the yield of infectious SV40 DNA. Unlike nuclear extracts from normal or SV40‐transformed permissive cells, those from normal nonpermissive cells are ineffective in rescuing the viral genome. The biological activity of the factor(s) is preserved at −70° C for at least 3 months.

Recombination between temperature-sensitive mutants of simian virus 40

Seven temperature-sensitive (ts) SV40 mutants have been isolated and characterized. All of the mutants were defective in a late function. Four of the mutants were assigned to complementation group B, one to a new group designated C and two could not be assigned to a complementation group. Recombination occurred between mutants in the B and C complementation groups and between mutants in the B group. The recombination frequency (RF) between tsB302 and tsB306 was about 2.0 × 10 −4 when virus was used for infection, but was 10-fold higher when infectious ts SV40 DNAs were used. Treatment of doubly infected cells with 1-β- d-arabinofuranosylcytosine (ara-C) to interrupt DNA synthesis increased the RF 3- to 14-fold. Ultraviolet irradiation of viral inocula to 1–5% survival resulted in a 25- to 40-fold increase in RF. However, only a 2-fold increase in RF was obtained when uv-irradiated ts SV40 DNAs were used for infection. Ultraviolet irradiation of host CV-1 cells or pretreatment of host CV-1 cells with nonirradiated or uv-irradiated CV-1 DNA prior to infection failed to increase the RF between tsB302 and tsB306.

The transformation of BHK 21 hamster cells by simian virus 40.

AbstractAlthough BHK 21 cells remain refractory to direct transformation by SV40 virus even at multiplicities of infection as high as 104 PFU per cell, transformed derivatives may be produced by cocultivation of the untransformed cells with monkey cells infected with SV40 virus.The characteristics of a cloned SV40‐transformed line (C13/SV) have been studied. The cells showed several of the properties of BHK 21 cells transformed by other oncogenic viruses. High‐passage variants of the line (C13/SV‐M) metastasized from the primary tumour that they induced in hamsters. Metastatic foci were the result of dissemination of the cells, and not due to the release of infectious SV40 virus from implanted cells.Comparisons between the in vitro and in vivo growth properties of C13/SV and C13/SV‐M cells did not reveal a correlation with metastasizing ability. The C13/SV‐M cells also retained those properties of transformed cells that are probably associated with surface alterations, although the SV40‐specific transplantation antigen may be partially absent or masked in these cells.