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What Is Coming Through That Needle? The Problem of Pathogenic Vaccine Contamination
Abstract
In recent times mankind is experiencing a situation never previously encountered, that being the threat of release of pathogens intended to kill or disable large numbers of people. That danger has prompted certain health agencies to prepare for possible mass vaccination of the populace. The purpose of this report is to examine the existing scientific evidence of pathogenic contaminants in vaccines. This summary, while making no claim of being a complete review of the subject, will point out sufficient examples and illustrations of contamination with bacteria, viruses, and their components, so as to enable the reader to make a more informed decision regarding accepting a vaccination (or forcing others to receive one). It is presented in a format intended for the public, their physicians, and their agency or governmental representatives, and may be freely copied in its entirety. If you as an individual are too busy to read this brief summary in one sitting, please be aware there is ample evidence in the scientific literature that serious viruses, bacteria; or components and toxins therefrom; as well as foreign animal or cancer-related proteins and DNA are finding their way into the commercial vaccines intended for humans, pets, and agricultural animals. If you are interested in the short and long-term health of yourself and those you care about, or serve as a public servant or medic al advisor, you do owe it to yourself to be informed. In the production of viral vaccines on a commercial scale, the virus of concern must be reproduced in large quantities. Viruses cannot survive or reproduce without being introduced into cells that nourish them, which enables the viral reproductive activity. In that sense all viruses can be considered parasitic on other cells. Living cell types commonly used to reproduce viruses in the lab include monkey kidney cells, chicken embryos, as well as other animal and human cells. These cells must also be nourished with food, and are most often fed with a nutrient mix containing in large part, bovine (cow) calf serum (usually, serum extracted from fetal calf blood). This product can carry many types of bovine blood-borne viruses, and is one of the primary sources of vaccine contaminants. A journal article states, "a potential risk associated with the production and use of biological products is viral contamination. This contamination may be present in the source material, e.g. human blood, human or animal tissues, cell banks, or introduced in the manufacturing process through the use of animal sera..."(1) Bovine viruses
... known to suffer allergic/inflammatory conditions are at Post exposure vaccination is unlikely to be of value in most risk from live vaccines [55]. animals, as the death usually occurs before appreciable immunity has had time to develop [15]. ...
... However, other factors other than understood as the disturbance of the vital force by generic reaction are also responsible for adverse reaction vaccination that results in mental, emotional and physical after vaccination. changes that can, in some cases be a permanent condition [55,56]. ...
... Economic Challenges: The economic costs of rabies in a Chronic Symptoms after Vaccination: Chronic symptoms animal bite investigations, conferment and quarantine of look very much like the acute illnesses but they are often domestic animals which bite humans or which are not life-threatening unless allowed to continue for years suspected of exposure to rabid animals, salaries of animal and years. According to O'Driscoll [57], McRearden [55] control officers, laboratory diagnosis and treatment and and Blanco [56], symptoms after vaccination include: consultation, public education, staff training and clerical restless nature (suspicion of others, aggression to animals costs [15]. and people); changes in behavior (aloofness, ...
Rabies has one of the highest case-fatality ratios of any infectious disease, almost always fatal, caused by lyssavirus infection. It is associated with dysfunction of the neurons after the entrance of rabies virus to the central nervous system, usually in the spinal cord. The bite route is still regarded as the most important means of transmission. Although wild animals are regarded as a host for rabies, dogs and cats remain the most important sources of human exposure. The disease is worldwide in distribution except in Antarctica. The most affected regions are tropical countries in Africa, Asia and South America, which have limited resources for diagnosis, treatment, control surveillance and vaccine production and improvement. Controlling rabies is challenging, due to wide host range and worldwide distribution, availability of many free roaming/stray dogs and lack of awareness about the disease. Diagnosis of the rabies is one of the most difficult duties because of non-specific clinical symptoms, long incubation period and limited diagnostic techniques. Vaccines are expensive and consequently, out of the reach for many people. Apart from high cost and unavailability, they are associated with serious neurologic complications. The economic costs of rabies in a country are associated with vaccinations, laboratory diagnosis, treatment and public education. So, the main objectives of this paper are to highlight the general characteristics of rabies virus, indicate the overall course of rabies and review possible challenges of rabies.
A comprehensive text-reference which covers major aspects of cell wall deficient microbes (L forms). The book discusses protoplasts as efficient genetic tools, L forms essential to the food chain for their ability to reclaim nitrogen in the air, an L form pathogenic to a flowering plant, and new information on male sterility caused by streptococcal L forms in drosphila. This new edition covers the L forms implicated in sarcoidosis, chronic stages of rheumatoid arthritis, uveitis, Crohn's disease, and Whipple's disease. AIDs-related data covered includes micrographs of an agent found in six Kaposi cases that resembles clinical L forms. The book presents methods for rapid diagnosing of common serious infections such as tuberculosis; describes little-known characteristics of mycobacterium leprae, including optimum temperature and pH. The book is aimed at haematologists, cellular biologists, microbiologists and clinical laboratory personnel.
Twenty samples of commercial vaccines intended for administration to humans were assayed for the presence of bacterial endotoxins by using the Limulus amebocyte lysate test. Sixteen of the vaccines contained more than 0.1 ng of endotoxin per ml (which corresponds to 103 bacterial cell wall equivalents per ml in the undiluted vaccines). These results suggest that at some stage of preparation, the vaccines have contained varying amounts of gram-negative bacteria and may indicate the presence of other bacterial products as well. It might be useful to list the level of endotoxins, phage, and other contaminants on each vaccine lot to facilitate studies on any side effects of these contaminants. Selection of vaccine lots with the least endotoxin might reduce some of the adverse effects of vaccinations.
Methods used by the National Animal Disease Center to test fetal calf serum for contamination with bovine viral diarrhea virus (BVDV) and antibodies against BVDV are described. Using those methods, virus was isolated from 332 of 1,608 (20.6%) lots of raw fetal calf serum obtained specifically for the Center and 93 of 190 (49%) lots of commercially available fetal calf serum. Virus neutralization and immunoperoxidase staining tests were used to detect antibodies against BVDV in 224 of the 1,608 (13.9%) lots of raw fetal calf serum. Both BVDV and antibodies against BVDV were detected in 50 lots of raw serum. The molecular specificity of antibodies against BVDV was determined by radioimmunoprecipitation. Lots of fetal calf serum that contained BVDV-specific antibodies that did not neutralize virus were identified.
We have previously shown that avian leukosis virus (ALV) induces erythroblastosis by insertional activation of the c-erbB gene. In 25% of the ALV-induced leukemic samples we have analyzed, acute retroviruses that have captured the activated erbB oncogene were released. The unusually high frequency at which erbB transduction occurs makes this an ideal system for studying the mechanism of oncogene transduction. In addition, these leukemic samples provide a rich source for the isolation of novel erbB-transducing viruses. We report here our characterization of several new erbB-transducing proviruses. The 5' recombination points of all these viruses mapped to the same intron in which proviral insertions cluster, supporting the hypothesis that transduction begins with proviral insertion near the oncogene. The 3' recombination points usually occurred within the 3' untranslated region downstream from the termination codon of the c-erbB gene. Three of the erbB-containing proviruses were molecularly cloned and analyzed in detail. Two of them were capable of releasing acute viruses, and interestingly, both retained poly(A) tracts of erbB messages in their genomes. A stretch of six adenosine residues in the ALV env gene appeared to mediate the 3' recombination events required for the generation of these viruses. These data provide further insight into the mechanism by which oncogenes are transduced into retroviral genomes.
Oncogene transduction, the process by which a cellular gene is captured by a retrovirus was mainly described in vivo. We have developed a biological system allowing stepwise analysis of transduction mechanisms in tissue culture. Avian neuroretina (NR) cells dissected at the 8th day of embryonic development rapidly cease to divide and differentiate in culture. Serial passaging of a retrovirus that does not carry an oncogene on such cultures leads with a high frequency to the emergence of new viruses that have transduced oncogenes from the mil/raf family of serine/threonine kinases. These viruses have been selected by their ability to induce NR cell division. This experimental system allowed the isolation of the following molecular intermediates generated during the successive steps of oncogene transduction: a chimeric transcript containing viral and cellular sequences joined together by an alternative splicing mechanism; then a complete retrovirus with a 5' end identical to that of chimeric RNA; finally, a retrovirus that has acquired additional gag sequences and consequently, an increased replicative capacity. Structural analysis of these molecules led us to propose a general model for oncogene transduction in which the key step is the synthesis of chimeric RNAs. This model also explains generation of the vast majority of acutely transforming retroviruses isolated in vivo.
A reference strain (Movár 33/63) of bovine herpesvirus type 4 (BHV-4) was inoculated into 14 different human cell lines and five primary cell cultures representing various human tissues. BHV-4 replicated in two embryonic lung cell lines, MRC-5 and Wistar-38, and in a giant-cell glioblastoma cell culture. Cytopathic effect and intranuclear inclusion bodies were observed in these cells. PCR detected a 10,000-times-higher level of BHV-4 DNA. Titration of the supernatant indicated a 100-fold increase of infectious particles. Since this is the first bovine (human herpesvirus 8 and Epstein-Barr virus related) herpesvirus which replicates on human cells in vitro, the danger of possible human BHV-4 infection should not be ignored.
Restriction fragment length polymorphism analyses were used to examine endogenous viral genes (ev genes or ALVE genes) of the avian leukosis viral (ALV) family in semi-congenic lines of meat chickens. The Generation 6 lines examined in this study were semi-congenic in that each contained birds with either zero or with one ALVE gene in hemizygous state plus some solitary long terminal repeat (LTR) elements. Using four restriction enzymes on chicken genomic DNA and two probes, one representing the entire ALV retroviral genome and one with only a small part plus the LTR, four ALVE genes were characterized. Each seemed to be complete with no detectable deletions. None appeared to be similar to known ALVE genes of White Leghorns, whereas two of the four may be the same as ALVE genes reported by others in White Plymouth Rock chickens.
On 23 February 1999, the Dutch Animal Health Service advised all Dutch veterinary practices to postpone vaccination against bovine herpesvirus 1 (BHV1) immediately. The day before severe disease problems were diagnosed on four dairy farms after vaccination with the same batch of BHV1 marker vaccine. Using monoclonal antibodies, bovine virus diarrhoea virus (BVDV) type 2 was found in the vaccine batch. This paper describes an outbreak of BVDV type 2 infection caused by the use of a batch of modified live BHV1 marker vaccine contaminated with BDVD. Sources of information used were reports of farm visits, minutes of meetings, laboratory results, and oral communications from the people involved. The first symptoms of disease were observed on average six days after vaccination. Morbidity was high on 11 of the 12 farms. On five farms more than 70% of the animals became ill, while on one farm no symptoms could be detected. During the first week after vaccination, feed intake and milk production decreased. During the second week, some animals became clinically diseased having nasal discharge, fever, and diarrhoea. At the end of the second week and at the start of the third week, the number of diseased animals increased rapidly, the symptoms became more severe, and some animals died. Mortality varied among herds. Necropsy most often revealed erosions and ulcers of the mucosa of the digestive tract. In addition, degeneration of the liver, hyperaemia of the abomasum, and swollen mesenterial lymph nodes and swollen spleen were found. On 11 of the 12 farms all animals were culled between 32 and 68 days after vaccination after an agreement was reached with the manufacturer of the vaccine. This was the third outbreak of BVD in cattle after administration of a contaminated vaccine in the Netherlands. The possibilities to prevent contamination of a vaccine as a consequence of infection of fetal calf serum with BVDV are discussed. Improvement of controls to prevent contamination before and during vaccine production, and improvement of the monitoring of side-effects is necessary.
This paper summarizes the history of and information on bovine herpesvirus type 4 (BoHV-4) from the first isolation to the most recent results. For almost twenty years BoHV-4 has been considered a typical herpes 'orphan' virus, which infects several species but causes no illness. The latest experiments revealed the close relationship of this virus with the immune system and other tissues. The virus was even considered as a possible candidate for a vector vaccine. BoHV-4 as a strange herpesvirus has several features which are not characteristic of other herpesviruses, such as several latency sites, persistence in serum, dividing cells necessary for virus replication, and the wide host range. In addition to describing the main features of the virion, replication, clinical signs, nomenclature problems, this review intends to concentrate on the new and strange results coming out from several laboratories worldwide. It is also suggested that because the virus combines several properties of various herpesvirus subfamilies and because of its close relationship with the immune system, it may deserve further attention as a representative of a potentially new genus within the Gammaherpesvirinae subfamily.
This paper describes some current work pertaining to transformation of cells by oncogenic viruses.
Part I includes: (1) the effect of a deoxyribonucleic acid (DNA) tumor virus (SV40) on the antigenic characteristics of transformed
cells; (2) in vitro and in vivo methods of detecting virus-specific surface antigens; (3) the role that the host cell may
play in the expression of virus-coded antigens; and (4) the presence of virus-induced antigens as a possible mechanism of
the apparent nononcogenicity of certain virus variants.
Part II discusses (1) the physicochemical properties of the nucleic acid of a ribonucleic acid (RNA) tumor virus-the Moloney
sarcoma-leukemia virus (MSV-MLV) complex —(2) a preliminary analysis of viral RNA replication in cells transformed by MSV-MLV,
and (3) application to human tumors.
A total of 25 lots of bovine serum samples were pelleted in Beem capsules for thin sectioning and were examined by electron microscopy. These included 17 lots of fetal bovine serum pools and five lots of calf serum pools obtained from commercial sources, and three lots of adult bovine serum from local dairy farms. Virus-like particles, 50 to 300 nm in diameter, were detected in 17 of 25 (68%) of the sera. Five of 25 serum samples showed the presence of mycoplasma-like agents. Incubation of bovine serum at 35 C for 1 or 2 weeks appeared to destroy some of these agents, but in certain instances it enhanced bacteria and bacteriophage contaminants. The advantages of electron microscopy using the thin-sectioning technique for detection of microbial contamination in bovine sera are illustrated.
The A.A. have examined by the Limulus assay the possible endotoxin contamination in some biological products (ribosomal vaccines, viral vaccines, interferon). While the preparations of influenza vaccines and a partially purified fraction of ribosomal vaccine from Pseudomonas aeruginosa exhibit a gelation of lysate with high levels of endotoxin, rubeola vaccines, interferon and a purified fraction of ribosomal vaccine, presented a negligible amount of endotoxin. The results are discussed with the aim to examine the possible role of contaminating endotoxin in the mediation of some adverse effects and of the unsuspected extrinsic adjuvant activities developed in clinical and experimental use of these preparations.
IBR virus was found to replicate in WI-38 cells. At a high input multiplicity the virus yield was comparable to that obtained in bovine cells, but comparable degree of CPE took longer to achieve. At a low input multiplicity of IBR virus, such as may be encountered in virus contaminated bovine serum, virus yield was only about 1% of that in bovine cells, with 50% of the cells showing CPE, followed by cell regrowth. Infectious virus was not recoverable from the regrown cells by 5 weeks after initial infection, and these regrown cells were susceptible to reinfection with IBR virus. Aging of WI-38 cells in cultures for as little as 1 week reduced IBR virus yield to 90% less than the yield from the same lot of cells inoculated 7 days earlier.
Two batches of foetal calf serum, free from detectable bacterial, mycotic, mycoplasmal and bovine viral conta mination and possessing good growth-promoting properties induced an abnormally high number of chromatid breaks in WI-38 cells to the extent that the cells were unacceptable as a vaccine substrate. This phenomenon, immediately reversible on changing to a different batch of serum, was unaffected by pasteurisation, suggesting that it was caused by some unidentified toxic factor(s). It was found that the same effect could be brought about by the addition of subcytotoxic concentrations of some bacterial toxins to the culture media. These findings re-emphasise the importance of improved methods for the collection and the maintenance of sterility throughout the processing of such sera, especially when they are intended to be incorporated in media used to establish vaccine substrates.
To estimate the amount of endotoxin in sera and vaccines relatively high quantities of limulus lysate are necessary. Because this makes the control rather expensive, a micromethod was developed in which the amount of limulus lysate was reduced fivefold. This method was used to estimate endotoxin in typhoid vaccines. The relation between the reactions in man and the amount of endotoxin in the vaccines was examined. In these experiments it appeared that the limulus amebocyte lysate (LAL) test demonstrates predominantly the endotoxin in solution. The test was also used as a control test for calf sera. The culture quality of these calf sera as estimated by the lymphocyte stimulation test appeared to be influenced by previous bacterial contamination as could be demonstrated with the LAL test.
Some human and animal continuous cell lines as well as primary cell cultures were examined by karyological, electron microscopial, virological and molecular biological methods and also by the electrophoretic motility of glucose-6-phosphate dehydrogenase (G-6-PDG) in polyacrylamide gel. All human and animal continuous cell lines were shown to contain mycoplasma, 17-to contain intracytoplasmic particles of type A oncornaviruses, 5 -- type B oncornaviruses similar to Mason-Pfizer virus, 8 -- paramyxoviruses, 2 --oncornaviruses type C. A high molecular RNA with sedimentation constant 64--70 S was found in oncornaviruses isolated from cell cultures. Intracellular virus or subviral structures were detected by association of the reverse transcriptase activity with high molecular RNA. The presence in the cell cultures of marker chromosomes of HeLa cells, the absence in these cultures of Y chromosomes, the presence of the G-6-PDG enzyme with type A motility indicate the possibility of contamination of human continuous cell lines with HeLa cells.
In the Federal Republic of Germany inactivated vaccines against poliomyelitis and measles are still produced in monkey kidney cell cultures which may be contaminated by simian viruses. One of these viruses is the oncogenic adenovirus SV20. A control of monkey sera from the institute's monkey house showed a high incidence of hemagglutination-inhibiting and neutralizing antibodies against this virus (table 1). Experimentally could be demonstrated that inactivated virus vaccines may be contaminated with SV20 antigen. Vero cells were infected at the time of their seeding with small doses of SV20. After 7 and 14 days CPE and hemagglutinin were frequently undetectable although infectivity could be shown by passages (table 2). Vaccines experimentally contaminated with SV20 were injected into guinea-pigs or rabbits (tables 3-6). In this way small amounts of the contaminating virus antigen could be detected by demonstrating neutralizing and hemagglutination-inhibiting antibodies. It is suggested to include passages of control cultures and of the virus harvest, after neutralization of the vaccine virus, for the control of virulent extraneous viruses. Alternatively to the last suggestion high amounts already inactivated virus pools could be inoculated into animals for the detection of antibodies against extraneous virus antigens. These controls are necessary until inactivated virus vaccines will be produced in safer substrates.
Bovine viral diarrhea virus (BVDV) infection is common in the bovine population. Infection in utero leads to virus and antibody contamination of the fetal bovine serum used in cell cultures. These contaminants can interfere with diagnosis of viral infection. The high frequency of virus and antibody detection in individual animal or small pool samples suggests that any large pool of unscreened sera will be contaminated. Infection of cell cultures with BVDV can lead to interference with the growth of other viruses. Vaccine produced on contaminated cells may in turn be contaminated, leading to seroconversion or disease in the vaccine. The safety, purity, and efficacy of viral vaccines require BVDV testing of ingredients, cell substrates and final product. Methods for detection of BVDV in nutrient serum, cell cultures, seed viruses, and viral vaccines, and the frequency of their detection at the National Veterinary Services Laboratories are discussed.
Four viral contaminants have been routinely detected in unprocessed and commercial lots of fetal bovine serum: bacteriophage, infectious bovine rhinotracheitis, parainfluenza-3 and bovine viral diarrhea virus (BVDV). Of those, BVDV is consistently present in a majority of commercial lots of fetal bovine serum. Methods for BVDV detection and removal are reviewed. The tentative role of an unclassified pestivirus in microcephaly of infants has been reported. Its significance remains uncertain.
Since the 1920's, a small number of researchers have been quite regularly isolating highly pleomorphic bacteria from the blood and tumors of humans and animals with cancer. These bacteria show characteristics of cell wall deficient bacteria, and can apparently be observed regularly in the blood of cancer patients by darkfield microscopy. Cancer appears to be induced by injecting these bacteria into experimental animals, and some forms of cancer can be prevented by prevaccination with killed bacteria, isolated from experimental animals affected with the specific cancer. The bacteria regularly produce a protein ressembling chorionic gonadotropin hormone, a substance which appears to protect trophoblastic and cancer cells from immune recognition. There is some evidence that a plasmid may be responsible for this property, and even that these bacteria may in some manner be intimately associated with retroviruses. Further studies are proposed to clarify the role these bacteria play in cancer.
The National Veterinary Services Laboratories (NVSL) routinely monitors continuous cell lines (CCL's) used for veterinary biologicals and diagnostic virology. All veterinary biologicals produced in CCL's must follow the master seed concept which limits the use of the master seed CCL to up to 20 passages beyond the passage level characterized and deposited at NVSL. All CLL's are evaluated for the presence of adventitious agents such as mycoplasma, bovine viral diarrhea virus, and other bacteria and viruses. Previously, CCLs were evaluated for tumorigenicity by the Syrian hamster cheek pouch method; however, this procedure has now been eliminated. The adventitious agents most frequently detected in CCL's have been bovine viral diarrhea virus and mycoplasma. Our laboratory has consistently found that the source of bovine viral diarrhea contamination of CCLs has been the use of contaminated fetal bovine cell culture enrichment serum. Gamma irradiation at 2.5-3.5 megarads at -40 degrees C of carefully screened fetal bovine serum has been used in the Diagnostic Virology Laboratory for over 10 years. If the irradiated serum is used at a final concentration of 10 percent, there is no untoward effect on cell susceptibility for virus propagation or cell culture growth. Gamma irradiation has also been demonstrated to be a very efficient inactivator of mycoplasma. Specific conditions utilized by our laboratory to preserve fetal bovine serum cell culture growth factors while eliminating adventitious bovine viral diarrhea virus will be presented.
The data presented in this paper confirm earlier findings that the antithymocyte globulin (ATG) treated newborn Wistar rat is more sensitive than the nude mouse in allowing the metastatic potential of a cell substrate to be displayed. Several human neoplastic cell lines such as HeLa, FL, Hep-2, KB and WiDr cells as well as monkey kidney cell lines such as Vero and BSC-1 cells, which failed to metastasize in nude mice, formed metastases in ATG-treated newborn rats. Earlier studies on the tumorigenicity of Vero monkey kidney cells were extended to various passage levels of BSC-1 aneuploid and CV-1 diploid monkey kidney cells in order to establish the effect of prolonged in vitro culture on the oncogenic potential of African green monkey kidney cells. It was found that BSC-1 and CV-1 cells--like Vero cells--showed increased tumorigenicity in the rat model with increasing cell passage. Cells of all three cell lines passaged 146 to 257 times formed invasive adenocarcinomas in the muscle (inoculation site) of the animals. Considering the passage levels, at which the adenocarcinomas appeared and lung metastases were detected, the malignant potential was most pronounced in CV-1 cells, followed by Vero cells and was lowest in BSC-1 cells. Results obtained from the in vitro tumorigenicity assay of cell colony formation in soft agar confirmed the rat test results: increased cell passage of all three monkey kidney cell lines resulted in higher cell colony formation. The in vitro chick embryo skin culture assay was found unsuitable for the differentiation of various passage levels of these cell lines.
Endotoxins liberated by gram-negative bacteria are frequent contaminants of aqueous and physiological solutions. Because of their potent biological effects in vivo and in vitro, their detection and removal are essential for the safe parenteral administration of products produced from natural sources, as well as those produced by recombinant DNA technology. Traditional methods of endotoxin detection include the U.S. Pharmacopeia rabbit test and the Limulus amebocyte lysate test. Elimination of endotoxins, however, continues to be a problem. Standard methods of sterilization, such as autoclaving or sterile filtration, have little effect on endotoxin levels. Various techniques for the prevention of endotoxin contamination and endotoxin removal have been discussed. The overall role of endotoxin prevention, detection, and elimination in biotechnology is emphasized.
There has been increasing interest over the past decade in exploring the possibility of using continuous cell lines as substrates for the production of both old and new biological products. The recent introduction of hybridoma technology to produce monoclonal antibodies, and the use of lymphoblastoid cells to produce interferon has broadened the discussion even further because both of those cell substrates are frankly tumorigenic. More recently, recombinant DNA technology has expanded beyond bacterial cells to mammalian cells, some of which may also be tumorigenic. One of the major safety concerns in biologicals produced from other than normal cells relates to the possibility that one or more of the biological characteristics of abnormal cells might be transmitted through residual DNA in the biological product to the human recipients. The assessment of risk for a given product must ultimately be based on the totality of evidence available including generic data on DNA as well as specific pieces of information on the product itself. These issues are discussed in the context of attempting to identify those data elements for the product which are essential for making an assessment of risk, plus generic information which may help to put the risk of DNA into an overall biological perspective.
INTERSPECIFIC cell culture contamination has been detected several times by karyotypic and immunological procedures1-3. These same measurements are of little value as detectors of intraspecific contamination, but polymorphic variants detectable at the cell culture level can be very useful for this purpose, A survey of twenty heteroploid human cell lines for glucose-6-phosphate de-hydrogenase (G6PD) and phosphoglucomutase (PGM) electrophoretic polymorphisms revealed that all had the same G6PD and PGM phenotypes. These results were interpreted as reflecting widespread contamination of cultures by the first established human cell line HeLa. These findings were presented at the Second Decennial Review Conference on Cell Tissue and Organ Culture4 and have since been extended to include other American Type Culture Collection lines. Further evidence on the stability of these markers in various conditions has been obtained, and in view of the widespread use of cell lines in a variety of investigations it seems important to make these findings readily accessible to other workers.
The mechanism by which neurotropic arboviruses gain access to the central nervous system remains uncertain, although it is generally assumed that viremic infection results in growth across or passive diffusion through brain capillaries. In contrast to the natural reservoir hosts of these arboviruses, clinical hosts (e.g., horses, humans) have viremias of very brief duration and low magnitude. We investigated the question of neuroinvasion in 5- to 6-week-old Syrian hamsters infected with St. Louis encephalitis virus (strain TBH-28). This model shares with the human disease low or undetectable viremia and many clinical and pathoanatomical features. The mortality rate after intraperitoneal inoculation of a moderate viral dose was 88%. No viremia was detectable by a sensitive assay in 31% of the animals. In the remaining hamsters, the mean peak viremia was 1.0 log10 plaque-forming units/0.05 ml and the mean duration 1 to 2 days. There was no correlation between viremia and outcome of infection, length of incubation period, or brain virus titer. Tissue infectivity studies showed a rise in titer in the olfactory neuroepithelium on day 4 postinoculation, then in the olfactory bulbs (day 5 postinoculation), and finally in the remainder of the brain (day 6 postinoculation). Specific immunofluorescence was demonstrated in the bipolar neurons of the olfactory epithelium, their dendrites, and in axon bundles of the olfactory nerves in the submucosa. By electron microscopy, virus particles and associated tubular structures were demonstrated within dendrites, perikarya, and axons of olfactory neurons, and to a lesser extent in macrophages and Bowman's gland cells in the lamina propria. In cells of Bowman's glands large numbers of virions were sequestered within secretory granules. Virus was recovered from nasal washings on day 4 postinoculation. Similar findings were obtained in weanling mice inoculated intraperitoneally with another (mouse-virulent) St. Louis encephalitis viral strain (77V-12908). These data taken together indicate that the olfactory pathway is the principal route of viral entry into the central nervous system. After peripheral inoculation a low-level viremia results in infection of highly susceptible cells in the olfactory neuroepithelium, allowing centripetal axonal transport of virus to the olfactory bulb, whence spread is unimpeded throughout the neuropil of the central nervous system. Infection of Bowman's gland cells in the olfactory mucosa and shedding of virus in nasal mucus may be an adaptation for nonarthropod-borne transmission, a feature of many flaviviruses.
The extensive use of cell cultures for diverse research purposes is one of the truly great international growth industries. With the proliferation of cells comes a responsibility for monitoring them for inter- and intraspecies characteristics. We use multiple genetic markers for cell identification, i.e. species specific antigens, isozymic phenotypes, chromosomal complement, and HL-A haplotypes. The methodologies employed are briefly described, and various examples cited to show how these markers can be utilized for cell line monitoring. Data are summarized from 275 cultures sent to our laboratory for analysis during the past eighteen months. The data show that, overall, 35% of the cultures received were contaminated. The majority of cell cultures submitted were human cell lines. We found that 36% of these cultures were cross contaminated; 25% by cells of another species and 11% by another human cell line. This high incidence of inter- and intraspecies contamination underscores the importance of frequent monitoring of cell cultures.
Viruses of the avian leukosis/sarcoma group (ALSV) and reticuloendotheliosis viruses (REV) are highly prevalent in chickens and turkeys and naturally cause tumors in them. Commercial chickens are positive for antibodies, and a proportion actually carry infectious virus. Virus may be present in chicken products and in eggs, thus human exposure is virtually universal. The viruses show little potential for producing infectious viral particles in mammalian cells; nevertheless, they have the capacity to infect and transform mammalian cells (including human cells) in vitro, and to induce tumors in a variety of mammals, including primates. Most, but not all, of the serological studies in humans have been negative. Given the known behavior of these viruses in mammals, this was not unexpected. Moreover, there were methodological problems with most of the studies. There is some epidemiological evidence associating putative poultry exposure with cancer in humans. However, this has not been rigorously investigated. This paper is a comprehensive review of the extent of the carcinogenic potential these viruses show for humans. It is concluded, virological evidence indicates, that these viruses could conceivably have a carcinogenic potential for humans, but if so, at a level much less than in chickens. Whether this is insignificant, or translates to a real risk, is not known at the moment. Therefore, there is a need for definitive studies to completely rule out this possibility.
Two methods for detection of mycoplasma contamination in cell cultures, sera, and live-virus vaccines were compared: the direct culture test and the DNA staining method employing bisBenzimide (Hoechst No. 33258). Contamination by different species of mycoplasma was found in 39% samples tested. It is recommended to use both techniques for a reliable detection of mycoplasma contamination.
We examined 20 cell lines of various animal origins for the presence of pestivirus contamination by the reverse transcription-polymerase chain reaction (RT-PCR), and found 15 (75%) cell lines were positive. The RT-PCR products of the 5' untranslated region (UTR) of pestivirus genome were sequenced and subjected to genotyping. Stem-loop structures at three variable regions in the 5' UTR render genotyping of the contaminated pestiviruses. Bovine cell lines tested were all contaminated with genotypes I, II, or III of bovine diarrhea virus (BVDV). Cell lines of canine, feline, and primate origin were contaminated with genotype II of BVDV. Cell line Ch1Es of caprine origin was contaminated with border disease virus (BDV).
Serologic evidence of antibodies in humans to avian leukosis/sarcoma viruses (ALSV) and reticuloendotheliosis viruses (REV) has in general been negative. Because of the difficulty in infecting mammalian cells in vitro with these viruses, it is generally held that they do not infect humans. We first provided presumptive evidence of serologic response to these viruses in human sera of workers in poultry slaughtering plants, using an ELISA. We now provide confirmatory evidence using Western blot assay. Our results show that exposed poultry workers and subjects with no occupational exposure to these viruses have antibodies in their sera specifically directed against ALSV p27, p19, p15, and p12 antigens. In addition, we demonstrate evidence of serologic response to REV. This is the first time definitive evidence of exposure to ALSV or REV has been demonstrated in human sera. The significance of this is not known. Further investigation into whether these findings mean that virus has been integrated into the human genome is needed, to assess the public health implications of these results.
The establishment of cell cultures for hepatitis C virus (HCV) is important for its study as a human pathogen. However, in
reported cell lines, HCV demonstrates low levels of replication detected primarily by reverse transcription-polymerase chain
reaction (RT-PCR) assays. In attempts to culture HeV, an additional complication was observed. From mock-infected cultures,
cDNA of appropriate size was obtained by RT-PCR with primers deduced from conserved domains of the 5' noncoding region of
HCV. However, sequence analysis revealed that the cDNA was
amplified from bovine viral diarrhea virus (BVDV). All of 7 bovine sera tested were contaminated with BVDV. In conclusion,
most commercially available bovine sera are contaminated with BVDV and, although there is no evidence that the virus is infectious,
bovine sera should be screened for this virus by RT-PCR when used in conjunction with HCV or for the development or production
of vaccine.
The 5'-untranslated genomic region of the pestivirus strain Europa, originated in human leucocytes and previously identified as bovine diarrhea virus (BVDV), was amplified by reverse transcription-PCR and sequenced. Analyses based on primary nucleotide sequence homology and on secondary palindromic sequence structures characteristic to genotypes revealed that this human isolate should be assigned to a novel genotype of pestivirus, type Ic. This newly emerged genotype was related to, but distinguishable from the three known BVDV genotypes, Ia, Ib and II. Three other bovine field isolates of BVDV originated from Germany were also found to belong to this new genotype Ic. Within pestivirus genotype Ic strains, the overall nucleotide sequence homology was 95-96%, and 88-92%, 88-90% and 77-79% with the other BVDV genotypes Ia, Ib and II, respectively. With the strains from border disease virus (genotype III) and hog cholera virus (genotype IV), homologies were less than 75%.
There are two possibilities for virus contamination of foodstuff and bioproducts of animal origin: i) the presence of endogenous virus as a result of an acute or subclinical infection of animal raw material used for food processing or ii) contamination of food in the course of processing or thereafter. The latter must be considered as the highest risk for human consumers since the viral contamination mostly is caused by virus shedding people and the transmitted viruses are obligate human pathogens. Food from animals consumed as raw material (e.g. oysters) is listed in a high risk category concerning viral contamination (e.g. hepatovirus). Virus contamination of bioproducts such as vaccines, blood products or biological material used in surgery and for transplantations also is more hazardous because the application of contaminating virus usually occurs by circumvention of the natural barrier systems of the body. Moreover, in many cases immunosuppressed people are treated with bioproducts. Due to an enclosing shield of high protein and lipid content in food and bioproducts viruses are well protected against physical and chemical influences, however most preparation procedures for food are destructive for viruses. The detection of pseudorabies virus and pestivirus in biological fluids was tested using polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR and cell culture propagation. PCR is a powerful method to detect viral nucleic acid whereas the detection of infectious virus in cell cultures is more limited, e.g. due to protein and lipid destroying conditions. Virus contamination of bioproducts should be considered as a hazard no matter which method has been used for its detection. Examples are given about the contamination of cell lines and vaccines.
Large-scale cell culture operations for biotechnology products use millions of litres of complex media and gases as well as huge quantities of organic and inorganic raw materials. These raw materials must always be assumed to contain contamination by adventitious agents such as Minute Virus of Mice (MVM). Genentech has had experience in dealing with two such contaminations. Although the source of these contaminations was not positively identified, there was strong evidence to suggest that the virus entered through a raw material used in cell culture. Analytical methods that can be used to detect the presence of viruses can be used as an early warning system and as part of a strategy to devise barriers against such contamination. Methods such as a polymerase chain reaction (PCR) assay and a cell culture-based infectivity assay have been found to be efficacious in providing early detection of MVM infection. In any contamination, timely interactions with regulatory authorities are vital in minimizing delays in product manufacture.
Biological materials are variable by their very nature. Serum in particular is a complex mix of substances and may contain adventitious agents. Serum for pharmaceutical use is controlled on a batch to batch basis given that each batch may have differing properties. Control points for slaughterhouse obtained bovine and foetal bovine serum exist with the disease status and disease surveillance of the country of origin, veterinary inspection for clinical disease, collection methods and post collection quality control. With these control points many batches of commercially available serum are contaminated with viruses such as BVD. The use of donor serum provides a mechanism to extend Good Manufacturing Practice principles back to the farm environment giving the manufacturer the ability to increase control on production parameters in the donor animal such as disease and specific antibody status, genetic type and history, age, diet, treatments and origin of feed. As well as providing a secure supply of serum for manufacture, donor herds provide added traceability and give the opportunity for in vivo manipulation of the serum and specialised selection of the animals for specific applications with more consistent reproducibility of performance. The BSE crisis of the last 10 years has highlighted the added control that donor serum producers can exert: Many producers of donor serum applied ruminant feed bans on their animals ahead of nation-wide bans in their own countries. SPF donor herds can be set up in several ways and the purchaser should make themselves familiar with the methods used. SPF herds with accompanying seronegative status allow quality control testing of product free from the interference of specific antibodies. Such specific antibody freedom may also allow better growth of viruses for vaccine manufacture.
Several subgroup J-like avian leukosis viruses (ALV-Js) were isolated from broiler breeder (BB) and commercial broiler flocks experiencing myeloid leukosis (ML) at 4 wk of age or older. In all cases, diagnosis of ML was based on the presence of typical gross and microscopic lesions in affected tissues. The isolates were classified as ALV-J by 1) their ability to propagate in chicken embryo fibroblasts (CEF) that are resistant to avian leukosis virus (ALV) subgroups A and E (C/AE) and 2) positive reaction in a polymerase chain reaction with primers specific for ALV-J. The prototype strain of these isolates, an isolate termed ADOL-Hc1, was obtained from an adult BB flock that had a history of ML. The ADOL-Hc1 was isolated and propagated on C/AE CEF and was distinct antigenically from ALV of subgroups A, B, C, D, and E, as determined by virus neutralization tests. Antibody to ADOL-Hc1 neutralized strain HPRS-103, the prototype of ALV-J isolated from meat-type chickens in the United Kingdom, but antibody to HPRS-103 did not neutralize strain ADOL-Hc1. On the basis of both viremia and antibody, prevalence of ALV-J infection in affected flocks was as high as 87%. Viremia in day-old chicks of three different hatches from a BB flock naturally infected with ALV-J varied from 4% to 25%; in two of the three hatches, 100% of chicks that tested negative for virus at hatch had evidence of viremia by 8 wk of age. The data document the isolation of ALV-J from meat-type chickens experiencing ML as young as 4 wk of age. The data also suggest that strain ADOL-Hc1 is antigenically related, but not identical, to strain HPRS-103 and that contact transmission of ALV-J is efficient and can lead to tolerant infection.
Prolonged exposure to asbestos, a potent carcinogen, has been the generally accepted factor responsible for the development of human mesotheliomas. Recent reports documenting the detection of SV40 DNA in human mesotheliomas suggest the possibility that this known tumor virus may be an additional factor involved in the development of some tumors.
A detailed analysis was performed by polymerase chain reaction and DNA sequencing of the genetic characteristics of SV40 viral DNA detected in samples taken from multiple sites of a human mesothelioma.
A single virus variant was detected within the tumor that encoded a novel variable region at the C-terminus of the large T-antigen oncoprotein. The viral regulatory region was predominantly archetypal in sequence (lacking duplications of the enhancer), typical of natural isolates.
These data confirm previous reports from several laboratories showing an association of SV40 DNA with human mesotheliomas and provide the first evidence of a novel virus variant present in separated regions of a mesothelioma.
Vaccines composed of gram-negative bacteria contain endotoxin in considerable amounts. This may result in adverse effects after vaccination of sensitive animals. For reasons of safety and animal welfare the endotoxin level of veterinary vaccines should be limited. The limulus amebocyte lysate test is suitable to check the endotoxin content in most vaccines. Safety tests in animals should only be performed if the results of the limulus test are satisfactory.
Three pestiviruses in fetal calf serum (FCS), a pestivirus in porcine ST cell line, and two pestiviruses in two of five bovine cell lines were detected by RT-PCR method employing panpestivirus primers selected from the 5'-non-coding region (5'-NCR). The 288 bp products were sequenced in both directions. To identify these pestiviruses, their nucleotide sequences and those of reference pestiviruses were used for construction of a dendrogram. Three viruses present in FCS and a virus present in the bovine RP-15 cell line were identified as bovine viral diarrhea virus I (BVDV-1). Bovine viral diarrhea virus 2 (BVDV-2) was identified in a batch of the bovine MDBK cell line. A pestivirus contaminating the porcine ST cell line was identified as a Border disease virus (BDV).
Advances in technology, emerging infectious diseases and accumulating knowledge of the mechanisms of neoplastic development are stimulating the need to develop a regulatory management plan that can be used to evaluate different types of neoplastic cells as possible substrates for vaccine development. To address this challenge, CBER is developing an approach, based on issue identification, issue-based model development and validation, and, where possible, a quantitative risk evaluation, as a basis for regulatory guidance on the use of neoplastic cell substrates for the manufacture of viral vaccines. The issues or concerns that have been identified with the use of neoplastic cell substrates include vaccine contamination with viable human tumour cells or adventitious agents, the possible risks associated with residual cell-substrate DNA or proteins in the vaccine, possible vaccine virus interactions with the cell substrate, and possible uncertainties associated with the instability of the neoplastic cell genome.
T-cell lymphomas developed in three of 10 immunosuppressed rhesus macaques during early experiments using retroviral vectors to transfer marker genes into CD34+ bone marrow cells for subsequent transplantation in the animals. Direct PCR analyses of RNA obtained from tumour tissues from these macaques revealed the presence of several different recombinant murine leukaemia viruses (MuLV). Most prominent was a recombinant designated Mo(LTR)Ampho(env) in which the amphotropic env of the helper packaging virus was joined to a modified form of long terminal repeat (LTR) of the Moloney MuLV-derived vector that contained an additional copy of the core enhancer. This new LTR afforded enhanced replication upon the Mo(LTR)Ampho(env) MuLV in several different rhesus cell types compared with the prototype amphotropic MuLV4070A. Unexpectedly, at least two types of a mink cell focus-forming (MCF) MuLV element, arising from endogenous retroviral sequences expressed in the murine packaging cell line, were also transmitted and highly expressed in one of the macaques. Furthermore, murine virus-like VL-30 sequences were detected in the rhesus lymphomas, but these were not transcribed into RNA. The unanticipated presence of this array of MuLV-related structures in a primate gene transfer recipient highlighted the generation of recombinant retroviruses when the vector producer line produced replication competent viruses. These recombinants had an enhanced tropism and pathogenicity in the primate gene transfer recipients and frequently caused lymphomas. This primate experiment highlights the potential risk from contamination of a vaccine cell substrate with a replicating retrovirus.
Using neoplastic cell lines as substrates for vaccine development could inadvertently result in viral-viral or viral-cellular interactions whose biological consequences are unclear. In this review, the generation of mink cell focus-inducing (MCF) retroviruses in the mouse is discussed as a model for understanding how viral-viral and viral-cellular interactions can result in the generation of new retroviruses with pathological consequences.
The contamination of cell lines with mycoplasmas is certainly one of the major problems occurring in cultured cells. Analyzing more than 460 human leukemia-lymphoma (LL) cell lines, we found that 28% of the cultures were mycoplasma-positive. Mycoplasmas can produce extensive changes, growth arrest and cell death in the infected cultures. While mycoplasma-infected cell lines can be truly cleansed from the contaminants, all the efforts would be in vain when the cells return to a mycoplasma-infested environment or are handled with unsuitable culture practices. Hence, the main focus of mycoplasma control should be on preventing cell culture contamination. Mycoplasmas can be introduced through several routes including culture reagents and laboratory personnel. Cross-contamination from infected cell cultures within one laboratory continues to be the major source for the spread of mycoplasma. Specific technical protocols and cell culturing guidelines may be followed in order to minimize the risk of mycoplasma contamination of cell lines. This "good culture practice" is of utmost importance as faulty cell culture techniques appear to be also the main reason for the high incidence of cross-contaminated LL cell lines which according to our experience using DNA fingerprinting of some 500 LL cell lines is about 15%.
To study the status of simian virus 40 (SV40) infection and its origin in human brain tumors.
Polymerase chain reaction (PCR) and Dot blot hybridization were used to detect SV40 DNA sequences in 516 human brain tumor tissues, 80 peripheral blood cells and 50 sperm fluids from healthy individuals, 100 human embryo tissues from artificial abortion, 30 normal brain tissues and two human glioma cell lines: SHG44 and BT325.
SV40 DNA sequences were found in 36.4% of human brain tumors (188/516), 16.3% of healthy peripheral blood cells (13/80), 22.0% of healthy semen (11/50), 8.0% of human embryo tissues (8/100) and 6.7% of normal brain tissues (2/30). SV40 DNA sequences were also detected in SHG44 and BT325 cell lines. The positive rates of SV40 DNA in human brain tumors, peripheral blood cells and semen from healthy donors were significantly higher than those in human normal brain and embryo tissues (P < 0.05).
(1) SV40 has a higher infection rate in human brain tumors, (2) SV40 is closely related to the etiopathogenesis of human brain tumors, (3) the ways of SV40 spread may due to both horizontal and diaplacental infections in human population.