Real time quantitative PCR as a method to evaluate simian virus 40 removal during pharmaceutical protein purification.

Continuous cell lines used for pharmaceutical protein manufacturing have the potential to be contaminated by viruses. To ensure the safety of pharmaceutical proteins derived from continuous cell lines, validation of the ability of the manufacturing process to clear potential contaminating viruses is required for product registration. In this paper, a real time quantitative PCR method has been applied to the evaluation of simian virus 40 (SV40) removal during chromatography and filtration procedures. This method takes advantage of the 5'-3' exonuclease activity of Taq DNA polymerase and utilizes the PRISM 7700 sequence detection system of PE Applied Biosystems for automated SV40 DNA quantification through a dual-labeled fluorogenic probe. This method provides accurate and reproducible quantification of SV40 DNA. The SV40 clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. This method offers significant advantages over cell-based infectivity assays, such as higher sensitivity, greater reliability, higher sample throughput and lower cost. This method can be potentially used to evaluate the clearance of all model viruses during chromatography and filtration procedures. This method can be used to substitute cell-based infectivity assays for process validation of viral removal procedures and the availability of this method should greatly facilitate and reduce the cost of viral clearance evaluations required for new biologic product development.     

High-fidelity PCR amplification of infectious copies of the complete simian virus 40 genome from plasmids and virus-infected cell lysates

We describe here a long-polymerase chain reaction (PCR) method that can be used to amplify complete simian virus 40 (SV40) DNA with high fidelity, and we show that authentic, viable virus can be produced from molecular clones of the PCR-amplified viral DNAs. A commercial long-PCR kit that employed a combination of Taq and GB-D polymerases was used, together with a pair of overlapping primers that recognized a unique EcoRI site in the SV40 genome. Efficient amplification required linearization of the circular SV40 genomic DNAs with EcoRI. Entire SV40 genomes were successfully PCR-amplified from an SV40 plasmid and from two different SV40-infected cell lysates and were cloned into pUC-19. Three separate segments of the cloned viral genomes were DNA sequenced, and no nucleotide changes relative to the parental virus were detected, suggesting that the viral DNAs had been amplified with high fidelity. Each PCR clone was infectious, and no differences were detected in the growth characteristics of viruses derived from these clones as compared to the original viral strain. The procedure we utilized shortens and simplifies the molecular cloning of small double-stranded DNA viruses and will be useful for viral diagnostic tests and for recovery of virus from clinical samples. The results of these experiments have broad implications, as the methodology is applicable to many systems.     

Real time quantitative PCR as a method to evaluate xenotropic murine leukemia virus removal during pharmaceutical protein purification

Chinese hamster ovary cells used for pharmaceutical protein production express noninfectious retrovirus-like particles. To assure the safety of pharmaceutical proteins, validation of the ability of manufacturing processes to clear retrovirus-like particles is required for product registration. Xenotropic murine leukemia virus (X-MuLV) is often used as a model virus for clearance studies. Traditionally, cell-based infectivity assay has been the standard virus quantification method. In this article, a real time quantitative PCR (Q-PCR) method has been developed for X-MuLV detection/quantification. This method provides accurate and reproducible quantification of X-MuLV particle RNA (pRNA) over a linear dynamic range of at least 100,000-fold with a quantification limit of approximately 1.5 pRNA copies microL(-1). It is about 100-fold more sensitive than the cell-based infectivity assay. High concentrations of protein and cellular DNA present in test samples have been demonstrated to have no impact on X-MuLV quantification. The X-MuLV clearance during chromatography and filtration procedures determined by this method is highly comparable with that determined by the cell-based infectivity assay. X-MuLV clearance measured by both methods showed that anion exchange chromatography (QSFF) and DV50 viral filtration are robust retroviral removal steps. In addition, combination of the two methods was able to distinguish the viral removal from inactivation by the Protein A chromatography, and fully recognize the viral clearance capacity of this step. This new method offers significant advantages over cell-based infectivity assays. It could be used to substitute cell-based infectivity assays for process validation of viral removal procedures, but not inactivation steps. Its availability should greatly facilitate and reduce the cost of viral clearance evaluations for new biologic product development.     

Quantification of enterococci and human adenoviruses in environmental samples by real-time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

Quantification of Enterococci and Human Adenoviruses in Environmental Samples by Real-Time PCR

Pathogenic bacteria and enteric viruses can be introduced into the environment via human waste discharge. Methods for rapid detection and quantification of human viruses and fecal indicator bacteria in water are urgently needed to prevent human exposure to pathogens through drinking and recreational waters. Here we describe the development of two real-time PCR methods to detect and quantify human adenoviruses and enterococci in environmental waters. For real-time quantification of enterococci, a set of primers and a probe targeting the 23S rRNA gene were used. The standard curve generated using Enterococcus faecalis genomic DNA was linear over a 7-log-dilution series. Serial dilutions of E. faecalis suspensions resulted in a lower limit of detection (LLD) of 5 CFU/reaction. To develop real-time PCR for adenoviruses, degenerate primers and a Taqman probe targeting a 163-bp region of the adenovirus hexon gene were designed to specifically amplify 14 different serotypes of human adenoviruses, including enteric adenovirus serotype 40 and 41. The standard curve generated was linear over a 5-log-dilution series, and the LLD was 100 PFU/reaction using serial dilutions of purified adenoviral particles of serotype 40. Both methods were optimized to be applicable to environmental samples. The real-time PCR methods showed a greater sensitivity in detection of adenoviruses in sewage samples than the viral plaque assay and in detection of enterococci in coastal waters than the bacterial culture method. However, enterococcus real-time PCR overestimated the number of bacteria in chlorinated sewage in comparison with the bacterial culture method. Overall, the ability via real-time PCR to detect enterococci and adenoviruses rapidly and quantitatively in the various environmental samples represents a considerable advancement and a great potential for environmental applications.     

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.     

Structural organization of chromosomes in interphase nuclei

The in situ molecular hybridization method has been applied to the detection, at the electron microscope level, of SV40 viral DNA in permissively infected monkey kidney cell cultures. The observations suggest an important role of the host cell nucleolus during the lytic infection with SV40.     

Detection of Ostreid Herpesvirus 1 (OsHV-1) DNA in seawater by PCR: influence of water parameters in bioassays

Since 1991, herpesvirus infections have been reported among larvae and juveniles of various bivalves. Most of the studies focused on detection of viral infections of economically important species. However, the persistence of bivalve herpesviruses in the marine environment is poorly documented. The present study concerns the role of seawater parameters in Ostreid Herpesvirus 1 (OsHV-1) detection by polymerase chain reaction (PCR). Viral DNA extracted from purified particles or virions present in infected oyster larvae were detected by PCR after storage in different media at different temperatures. The lowest detection threshold was found using distilled water or Tris EDTA buffer. In seawater, the threshold was higher. The use of sterile media permitted detection of viral DNA stored over a longer period. Storage temperature also had a significant influence on detection, with lower temperatures promoting DNA detection over a longer period. In summary, water parameters such as temperature influenced detection of OsHV-1 DNA by PCR. However, the PCR technique may also be successfully applied to samples in natural seawater. Indeed, the PCR technique permitted detection of naked viral DNA at 100 ng l(-1) in seawater in bioassays.     

Structure of integrated simian virus 40 DNA in transformed mouse cells

The structure of integrated viral DNA sequences in four lines of simian virus 40 (SV40)-transformed Balb/c 3T3 cells has been probed using restriction endonucleases and the Southern (1975) transfer method. By considering data from a large number of restriction digests of DNA from each line, and by using a novel method of handling the data, we have constructed fairly detailed physical maps of the integrated DNA in each line. The most striking of the features of the maps described here is that none is easily explained by the integration of a single SV40 genome into the DNA of the host cell. Three of the lines contain at least two distinct integrated segments and the fourth contains a single segment longer than the viral DNA. Considered individually, only two of the seven segments that we have mapped might be unit length. Of the remaining five, two are longer and three are shorter than the viral genome. It seems likely, therefore, that at least in SV40-transformed Balb/c 3T3 cells simple, single integrations are rare.The endpoints of these seven segments of integrated DNA fall at many positions distributed over the entire genome, confirming earlier studies (Ketner &; Kelly, 1976; Botchan et al., 1976), which indicated that SV40 integration is not absolutely site-specific.Finally, one of the lines mapped here (SVB209) does not possess an intact SV40 early region, an observation that suggests the possibility that a normal viral large T polypeptide is not synthesized by this line.     

Quantitation of herpes simplex virus DNA in cerebrospinal fluid of patients with herpes simplex encephalitis by the polymerase chain reaction

Background: Previous studies have shown the diagnostic utility of qualitative detection of herpes simplex virus (HSV) DNA by the polymerase chain reaction (PCR) in cerebrospinal fluid samples (CSF) from patients with herpes simplex encephalitis (HSE).Objectives: To determine whether quantitation of HSV DNA in CSF could be useful for monitoring efficacy of antiviral therapy and provide prognostic indications.Study design: A quantitative PCR assay using an internal control for evaluation of PCR efficiency and detection of PCR inhibitors was developed and used for retrospective testing of 98 CSF samples from 26 patients with serologically diagnosed HSE during the period 1980–1995.Results: HSV DNA was detected in 36 CSF samples from 23 patients. PCR positivity was 100% for CSF samples collected within 10 days after onset, and 30.4 and 18.7% for samples collected 11–20 and 21–40 days later, respectively. The 3 PCR-negative patients had their first CSF collected 14, 16, and 28 days after onset, respectively. Three of 98 (3.1%) CSF samples were completely or partially inhibitory to PCR. Initial DNA levels were not significantly different in patients with HSE due to either primary or recurrent HSV infection. In addition, they were not related to severity of clinical symptoms nor were predictive of the outcome. A progressive decrease in viral DNA levels was observed both in patients who received acyclovir therapy and in a small number of untreated patients.Conclusions: This study: (i) confirms the high sensitivity of PCR for the diagnosis of HSE; (ii) emphasizes the need for an internal control of amplification to achieve maximal sensitivity and perform reliable quantitation of viral DNA; and (iii) suggests that CSF might not be the best specimen to investigate in studies of the natural history of HSE.     

Development,characterization, and validation of a sensitive primate-specific quantification assay for forensic analysis

Accurate human-specific DNA quantification is essential for forensic casework analysis. In this work, we describe a microplate-based quantification assay that utilizes the PCR amplification of human-specific TH01 primers. This method enables the reliable quantification of human DNA samples from 0.2 to 40 ng, even in mixtures with nonhuman DNA. Analysis of samples can be semi-automated using 96-well microplates and a spreadsheet-based concentration calculator for high-throughput demands. We have used this quantification method with more than 15,000 forensic samples.     

Exploring the prevalence of ten polyomaviruses and two herpes viruses in breast cancer

Several different viruses have been proposed to play a role in breast carcinogenesis. The aim of this study was to investigate the prevalence of a subset of viruses in breast cancer tissue. We investigated the prevalence of 12 DNA viruses: EBV and CMV from the Herpesviridae family and SV40, BKV, JCV, MCV, WUV, KIV, LPV, HPyV6, HPyV7, and TSV from the Polyomaviridae family in 54 fresh frozen breast tumour specimens. Relevant clinical data and basic lifestyle data were available for all patients. The tissue samples were DNA extracted and real-time PCR assays were used for viral detection.The highest prevalence, 10% (5/54), was found for EBV. MCV, HPyV6, and HPyV7 were detected in single patient samples (2% each), while WUV, KIV, JCV, BKV, LPV, SV40, TSV and CMV were not detected in the 54 breast cancer specimens analysed here. Further investigations are needed to elucidate the potential role of viruses, and particularly EBV, in breast carcinogenesis.     

Real-time PCR test for detection and quantification of human polyomavirus BK (BKV) DNA

The human polyomavirus BK (BKV) is wide-spread pathogen, associated with urogenital tract disorders or even nephropathy in immunosuppressed patients. Nowadays molecular detection by real-time PCR (qPCR) is recognized as a method-of-choice for detecting human polyomaviruses in clinical samples. The aim of the study was development of real-time PCR assay for detection and quantification of polyomavirus BK DNA in clinical samples, using specific primers targeting a viral DNA VP3 gene and a TaqMan hydrolyzing probe. The analytical sensitivity of assay was tested using serial dilutions of BKV DNA in range between 13500 and 15 copies/ml. 27 urine samples and 23 plasma samples taken from a group of 22 adult recipients of allogeneic HSCT were tested for the presence of polyomavirus BK in the LightCycler system. Described in-house real-time PCR assay detected BKV DNA in 8 specimens (6 urine and 2 plasma). Detected average viral load was 170 copies/ml for plasma and 1250 copies/ml for urine samples, respectively. The results of this study show that developed TaqMan-based probe qPCR assay is very reliable and valuable for detection and quantification of BKV DNA, both in urine and plasma samples. These data, combined with its rapid turnaround time for results and decreased hands-on time, make the LightCycler PCR assay highly suitable for the rapid diagnostics of polyomavirus BK infections in the clinical laboratory.     

Does simian virus 40 DNA integrate into cellular DNA during productive infection?

Late after infection of permissive monkey cells by simian virus 40 (SV40), large amounts of SV40 DNA (30,000 to 220,000 viral genome equivalents per cell) can be isolated with the high-molecular-weight fraction of cellular DNA. Hirai and Defendi (J. Virol.9:705-707, 1972) and H?lzel and Sokol (J. Mol. Biol. 84:423-444, 1974) suggested that this SV40 DNA is covalently integrated into the cellular DNA. However, our data indicate that the high-molecular-weight viral DNA is composed of tandem, "head-to-tail" repeats of SV40 DNA and that very little, if any, of this viral DNA is covalently joined to the cellular DNA. This was deduced from the following experimental findings. The size of the SV40 DNA associated with the high-molecular-weight cellular DNA fraction is greater than 45 kilobases, based on its electrophoretic mobility in agarose gels. In this form the SV40 DNA did not produce heteroduplex structures with a marker viral DNA (an SV40 genome with a characteristic deletion and duplication). After the high-molecular-weight DNA was digested with EcoRI or HpaII endonucleases, enzymes which cleave SV40 DNA once, more than 95% of the SV40 DNA migrated as unit-length linear molecules and, after hybridization with the marker viral DNA, the expected heteroduplex structures were easily detected. Digestion of the high-molecular-weight DNA fraction with restriction endonucleases that cleave cellular, but not SV40. DNA did not alter the electrophoretic mobility of the polymeric SV40 DNA, nor did it give rise to molecules that form heteroduplex structures with the marker viral DNA. Polymeric SV40 DNA molecules produced after coinfection by two physically distinguishable SV40 genomes contain only a single type of genome, suggesting that they arise by replication rather than by recombination. The polymeric form of SV40 DNA is highly infectious for CV-1P monolayers (6.5 X 10(4) PFU per microgram of SV40 DNA), yielding virtually exclusively normal, covalently closed circular, monomer-length DNA. Quite clearly these cells have an efficient mechanism for generating monomeric viral DNA from the SV40 DNA polymers.     

Isolation of viral specific RNA from SV40 infected cells by viral DNA chemically linked to a cellulose matrix.

SV40 DNA fragments chemically attached to neutral cellulose powder with a water-soluble carbodiimide have been used to isolate late lytic viral specific RNA from virus infected cells. Exhaustive hybridization to SV40 DNA reveals that virtually all of the isolated RNA molecules contain SV40 specific sequences. Comparison with SV40 cRNA prepared with purified Escherichia coli RNA polymerase and a SV40 DNA I template suggests that the purity of the isolated SV40 specific RNA is very close to 100%. The background level for the nonspecific binding of RNA to a purified cellulose matrix is very low. Retention of nonspecific RNA by SV40 DNA-cellulose is only 1.5% of the viral specific RNA isolated under saturating conditions for the column. Sedimentation in neutral sucrose suggests that the major 16S viral specific RNA has been isolated largely intact.     

Tumor induction by simian virus 40 in mice is controlled by long-term persistence of the viral genome and the immune response of the host.

Simian virus 40 (SV40), which transforms mouse cells in vitro, has not been previously observed to cause tumors when injected in immunocompetent mice. We have investigated both the fate of the injected virion in mice and several immunological parameters as potential factors controlling tumorigenicity. We find that although SV40 does not replicate in mouse cells, the viral DNA can persist for many months postinjection; the majority of the viral DNA is found in the cytoplasm, but a small amount of the viral DNA is integrated at multiple sites in the host nuclear DNA. The persistence of the viral genome is independent of the ability of the mouse to mount an SV40 TSTA specific cytotoxic T-cell response and may be attributed to the cytoplasmic location of the majority of the viral genome. However, in long-term studies of SV40-injected mice, genetically identical except for the major histocompatibility complex, we find that tumors were induced in some mice of the H-2d (low cytotoxic T-lymphocyte responder to SV40 TSTA) but not of the H-2k (high responder to SV40 TSTA) haplotype. Thus, a combination of inefficient disposal of the injected virion and inefficient immunological surveillance and elimination of cells containing nuclear SV40 DNA can eventually result in SV40-induced tumors at multiple sites in mice.     

A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNA

AIMS: The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. METHODS AND RESULTS: A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 10(1) DNA copies per 10 microl of plasmid template and 6.5x10(0) colour changing units of reference strain Ba/2. CONCLUSIONS: The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2-3 h compared with at least 1 week). SIGNIFICANCE AND IMPACT OF THE STUDY: The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies.     

Protein blotting: principles and applications

Extensive studies on the DNA tumor virus Simian Virus 40 (SV40) have provided a wealth of information regarding the genome organization, regulation of viral gene expression, and the mechanism of DNA replication. SV40 can grow lytically in permissive monkey cells or the viral DNA can integrate into the host genome of nonpermissive rodent cells causing morphological transformation. The viral DNA exists as a minichromosome within the nuclei of lytically infected cells and, as a consequence of DNA replication, there is a significant amplification of the viral genome during infection. These properties suggested that SV40 could be developed as a transducing vector to introduce exogenous DNA into mammalian cells and to express this foreign DNA during the SV40 infectious cycle. In this article the properties of SV40 virus vectors and SV40 hybrid plasmid vectors are described and contrasted.