College of Pharmacy, Chungbuk National University, 12 Gaeshindong, Cheongju,
Chungbuk, Republic of Korea.
In this study, we describe our newly-developed sensitive two-stage PCR procedure
for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M.
bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum,
M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum).
For primary amplification, the DNA regions encompassing the 16S and 23S rRNA
genes of 13 species were targeted using general mycoplasma primers. The primary
PCR products were then subjected to secondary nested PCR, using two different
primer pair sets, designed via the multiple alignment of nucleotide sequences
obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA
fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target
DNA, and evidenced no cross-reactivity with the genomic DNA of related
microorganisms or of human cell lines, thereby confirming the sensitivity and
specificity of the primers used. The identification of contaminated species was
achieved via the performance of restriction fragment length polymorphism (RFLP)
coupled with Sau3AI digestion. The results obtained in this study furnish
evidence suggesting that the employed assay system constitutes an effective tool
for the diagnosis of mycoplasmal contamination in cell culture systems.
Cell and Gene Bank, Biotechnology Department of Razi Vaccine and Serum Research
Institute (CGBRI), Hesarrak, Karaj, Iran. ali_mirjalili@yahoo.com
Cell line contamination is a major
drawback of main cell banks of the world and it has cost of losing important
biological products or valuable research. The causative agents are different
chemicals, invertebrates, bacteria, fungi, parasites, viral species and even
other cell lines. In this retrospective study, cell lines from various
species such as human, fish, insect, animals either offered or accessed through
usual official accession in CGBRI were studied during 2 years (2002-2004) to
detect their microbial contaminations and the causative organisms. Samples were
taken for sterility test upon cell lines receipt and upon each cell line
sub-culture. Samples were examined for bacterial (including mycoplasmas) and
fungal contamination using conventional microbiological techniques. The study
excluded parasites, viruses and other contaminating agents.
This study revealed 39% of specimens
were contaminated. The major contaminating agents were mycoplasmas
(19%) followed by mixed infection (8%), fungi (8%) and bacteria (4%).
Among various bacterial species (except mycoplasmas) Bacillus sp., Enterococcus
sp. and Staphylococcus sp. are main bacterial agents and among various fungi
Aspergillus sp. followed by Penicillium sp., Sepedonium sp. and Botrytis sp.
were main fungal causative agents of CGBRI cell line contamination. Our study
also delineates each cell line contamination rate and its causative agents. This
is the first report of cell culture contamination from cell banks of Middle-East
countries like Iran.
Stem Cell Bank of Andalucia (Spanish Central Node), Hospital Universitario
Virgen de las Nieves, Avda Fuerzas Armadas, 2, 18014, Granada, Spain. fernancobo@fundacionhvn.org
The transplant of cells of human origin is an increasingly complex sector of
medicine which entails great opportunities for the treatment of a range of
diseases. Stem cell banks should assure the quality, traceability and safety of
cultures for transplantation and must implement an effective programme to
prevent contamination of the final product. In donors, the presence of
infectious micro-organisms, like human immunodeficiency virus, hepatitis B
virus, hepatitis C virus and human T cell lymphotrophic virus, should be
evaluated in addition to the possibility of other new infectious agents (e.g.
transmissible spongiform encephalopathies and severe acute respiratory
syndrome). The introduction of the nucleic acid amplification can avoid the
window period of these viral infections. Contamination from the laboratory
environment can be achieved by routine screening for bacteria, fungi, yeast and
mycoplasma by European pharmacopoeia tests. Fastidious micro-organisms, and an
adventitious or endogenous virus, is a well-known fact that will also have to be
considered for processes involving in vitro culture of stem cells. It is also a
standard part of current good practice in stem cell banks to carry out routine
environmental microbiological monitoring of the cleanrooms where the cell
cultures and their products are prepared. The risk of viral contamination from
products of animal origin, like bovine serum and mouse fibroblasts as a "feeder
layer" for the development of embryonic cell lines, should also be considered.
Stem cell lines should be tested for prion particles and a virus of animal
origin that assure an acceptable quality.
Biopharmaceutical Services, Charles River Laboratories, Ijamsville, Maryland
21754, USA. lschiff@bps.criver.com
A critical component in controlling the production of biological products
derived from human and animal cell lines is the characterization and testing of
banked cell substrates. The objective is to confirm the identity, purity, and
suitability of these cells for manufacturing use. Quality concerns for
biological products derived from cell lines originate from the presence of
cellular and adventitious contaminants. Well-characterized cell banks not only
permit a consistent source of production cells throughout the life of a product
but also decrease the likelihood of contamination by other cell lines and
adventitious agents. An important part
of qualifying a cell line is choosing the appropriate testing for the presence
of adventitious contaminants. The qualification of cell banks includes tests for
cell identity and endogenous and adventitious microbial contaminants (bacteria,
fungi, mycoplasmas,
and viruses). For cells producing recombinant deoxyribonucleic
acid-derived products, analysis of the expression construct at the nucleic acid
level (genetic stability) is also a primary concern. The strategy for designing
a safety-testing program for banked cells should be based on sound scientific
principles and current regulatory guidance.
DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig,
Germany.
Mycoplasma contamination of cell lines
is one of the major problems in cell culture technology. The specific,
sensitive, and reliable detection of mycoplasma contamination is an important
part of mycoplasma control and should be an established method in every cell
culture laboratory. New cell lines as well as cell lines in continuous culture
must be tested in regular intervals. The polymerase chain reaction (PCR)
methodology offers a fast and sensitive technique to monitor all cultures in a
laboratory. The technique can also be used to determine the contaminating
mycoplasma species.The described assay can be performed within 3 h, including
sample preparation, DNA extraction, performing the PCR reaction, and analysis of
the PCR products. Special precautions necessary to avoid false-negative results
resulting from inhibitors of the Taq polymerase present in the crude samples and
the interpretation of the results are also described.
Rapid detection and differentiation of the major
mycoplasma contaminants in cell
cultures using real-time PCR with SYBR Green I and melting curve analysis.
Veterinary Microbiology, Department of Veterinary Medicine, Iwate University,
Morioka, Iwate 020-8550, Japan. harasawa-tky@umin.ac.jp
A quantitative real-time polymerase chain reaction (PCR) procedure followed by
melting curve analysis, using the green fluorescence dye SYBR Green I, was
developed for rapid detection and differentiation of mycoplasma contaminants in
cell cultures. This method showed that the detection of the target sequence was
linear over a range from 10(4) to 10 colony-forming units (CFU) of the
mycoplasma cells. Analysis of the melting temperature of the PCR products
allowed differentiation of the major mycoplasma contaminants. These results
demonstrate that the protocol described in the present study can decrease the
time to obtain reproducible results by simultaneous detection and
differentiation of the Mycoplasma species contaminating cell cultures.
Centre for Infectious Diseases and Microbiology Laboratory Services, Institute
of Clinical Pathology and Medical Research, Westmead, New South Wales,
Australia.
We have developed a reverse line blot (RLB)
hybridization assay to detect and identify the commonest mollicutes
causing cell line contamination (Mycoplasma arginini,
Mycoplasma fermentans,
Mycoplasma hyorhinis,
Mycoplasma orale,
and Acholeplasmalaidlawii)
and human infection (Mycoplasma
pneumoniae, Mycoplasma hominis,
Mycoplasma genitalium,
Ureaplasmaparvum,
and Ureaplasmaurealyticum).
We developed a nested PCR assay with "universal" primers targeting the
mollicute 16S-23S rRNA intergenic spacer region. Amplified biotin-labeled PCR
products were hybridized to membrane-bound species-specific oligonucleotide
probes. The assay correctly identified reference strains of 10 mollicute
species. Cell cultures submitted for detection of mollicute contamination,
clinical specimens, and clinical isolates were initially tested by PCR assay
targeting a presumed mollicute-specific sequence of the 16S rRNA gene. Any that
were positive were assessed by the RLB assay, with species-specific PCR assay as
the reference method. Initially, 100 clinical and 88 of 92 cell culture
specimens gave concordant results, including 18 in which two or more mollicute
species were detected by both methods. PCR and sequencing of the 16S-23S rRNA
intergenic spacer region and subsequent retesting by species-specific PCR assay
of the four cell culture specimens for which results were initially discrepant
confirmed the original RLB results. Sequencing of amplicons from 12 cell culture
specimens that were positive in the 16S rRNA PCR assay but negative by both the
RLB and species-specific PCR assays failed to identify any mollicute species.
The RLB hybridization assay is sensitive and specific and able to rapidly detect
and identify mollicute species from clinical and cell line specimens.
Cancer Research UK Oncology Unit, Western General Hospital, Edinburgh, UK.
For the cell culturist, two types
of contamination require careful monitoring and constant vigilance: the
contamination of cell cultures with microbiological organisms and the
contamination of one cell line with another. Both forms of contamination are
extremely prevalent and cannot be underestimated. Neither type can be
eliminated, only controlled and managed to minimize the possibility of
occurrence. Contamination consequences can range from minor
inconvenience (a flask of cells becoming contaminated with bacteria) to a
major disaster (published results that may be invalid owing to
cross-contamination of one cell line with another). Other types of
contaminants, such as chemical contamination, may also cause problems (e.g.,
deposits of disinfectants or detergents on glassware; residues, impurities,
and toxins in water, media or sera), but the common recurring problems are
likely to be biological in origin.
Comment: Is the reason there is no mention of
possible disease transmission that it is assumed that such contamination
cannot cause disease, or has it been thoroughly tested and been shown to
(almost) never occur? (Examples
of cross-species disease transmission, and in particular that of SV40, suggest that the former
may be the case.)
Wohl Virion Centre, Division of Infection and Immunity, University College
London, W1T 4JF, UK. rweiss@ucl.ac.uk
Fifty years ago, the age-old scourge of infectious disease was receding in the
developed world in response to improved public health measures, while the
advent of antibiotics, better vaccines, insecticides and improved surveillance
held the promise of eradicating residual problems. By the late twentieth
century, however, an increase in the emergence and re-emergence of infectious
diseases was evident in many parts of the world. This upturn looms as the
fourth major transition in human-microbe relationships since the advent of
agriculture around 10,000 years ago. About 30 new diseases have been
identified, including Legionnaires' disease, human immunodeficiency virus
(HIV)/acquired immune deficiency syndrome (AIDS), hepatitis C, bovine
spongiform encephalopathy (BSE)/variant Creutzfeldt-Jakob disease (vCJD),
Nipah virus, several viral hemorrhagic fevers and, most recently, severe acute
respiratory syndrome (SARS) and avian influenza. The emergence of these
diseases, and resurgence of old ones like tuberculosis and cholera, reflects
various changes in human ecology: rural-to-urban migration resulting in
high-density peri-urban slums; increasing long-distance mobility and trade;
the social disruption of war and conflict; changes in personal behavior; and,
increasingly, human-induced global changes, including widespread forest
clearance and climate change. Political ignorance, denial and obduracy (as
with HIV/AIDS) further compound the risks.
The use and misuse of medical
technology also pose risks, such as drug-resistant microbes and contaminated
equipment or biological medicines. A better understanding of the
evolving social dynamics of emerging infectious diseases ought to help us to
anticipate and hopefully ameliorate current and future risks.
Division of Infectious Diseases and Food Chain Quality, Animal Sciences Group,
Wageningen University and Research Centre (WUR), P.O. Box 65, Lelystad AB8200,
The Netherlands. adriaan.antonis@wur.nl
Veterinary vaccines are usually tested
for the absence of contaminants. However, the quality control does not always
imply that vaccines are not contaminated as, for example, illustrated by the
bovine herpes virus 1 (BHV1) vaccine used in The Netherlands in 1999 that
contained a small amount of bovine viral diarrhoea
virus (BVDV1).
Thousands of cows were vaccinated with BHV1 vaccine
batches, and the question arose as to whether these small amounts of BVDV1, most
likely not detected with in vitro tests, could have infected cattle. More in
general, the question was whether the outcome of the in vitro tests, i.e. the in
vitro infectivity, was indicative for the infectivity for cattle, i.e. the in
vivo infectivity. We therefore carried out in vitro experiments to determine the
sensitivity of a BVDV1 isolation assay. In addition, we performed two animal
experiments, in which we estimated the lowest dose needed to infect calves with
BVDV1. We extrapolated the experimental in vitro and in vivo results from a
tissue culture infectious dose (TCID50) to a cattle infectious dose (CID50). We
observed a partial response in the calves inoculated with this dose: four out of
six calves turned out to be infected. In the tissue culture test, all 20 samples
tested negative. The response in vivo, however, was not significantly higher
than the in vitro response, which implies that no difference in susceptibility
was observed between the animal test and the tissue culture test. Based on the
results in our experiments, some cattle may have been infected with BVDV1 after
the application of the contaminated BHV1 vaccine during the vaccination
campaign. The question remains that how
many cattle received contaminated vaccine, and became infected with BVDV1.
Department of Clinical Immunology and Allergology, Faculty of Medicine in
Hradec Kralove, University Hospital, Charles University in Prague, Sokolska
tr. 408, 500 05 Hradec Kralove, Czech Republic. mloudova@post.cz
The authors describe a rapid, simple and inexpensive method for the routine
testing of mycoplasma contamination of the continuous mouse macrophage-like
cell line J774.2 using specific anti-mouse monoclonal antibodies (antiCD14,
antiCD80) and flow cytometry.
School of Humanities, Faculty of Arts, Education, and Social Sciences,
University of Western Sydney, Locked Bag 1797, South Penrith Distribution
Centre, NSW 1797, Australia. chris.wilson@uws.edu.au
Q-One Biotech Limited, Todd Campus, West of Scotland Science Park, Glasgow,
G20 0XA, UK. cnairn@q-one.co.uk
Bovine polyomavirus
(BPyV)
is a member of the
Polyomaviridae, a virus that
was originally thought to be of simian origin but was later shown to be of
bovine origin, the primate cultures having been contaminated through the use
of foetal
bovine serum. The significance of this agent to the biotechnology industry
cannot be underestimated. The presence of BPyV
in serum batches poses a serious risk for the contamination of human
therapeutic products. The current PCR
based assays provide a means of detecting virus sequences but give no
indication as to the infectious nature of the virus. The communication
reports the successful development of an assay to detect infectious BPyV using
an in vitro amplification system followed by PCR. A lengthy culture period on
bovine cells was required before replicating BPyV could be detected and
distinguished from non-replicating virus in the cell culture supernatant. A
mock-test assay using foetal bovine serum positive for BPyV showed that there
was no evidence of replicating BPyV in the serum sample. The BPyV spiked serum
control showed that replicating virus was present thus confirming that the
serum itself did not inhibit replication of the virus. Cells harvested during
the culture period were subjected to fixation, embedding and sectioning and
examined by electron microscopy. Intact virus-like particles of approximately
40-50nm were observed in the nucleus of the bovine kidney cells, the site of
polyomavirus replication.
Bovine viral diarrhoea virus antigen in foetal calf serum
batches and consequences of such contamination for vaccine production.
Makoschey B, van Gelder PT, Keijsers V, Goovaerts D.
Virological R&D Department, Intervet International b.v., Wim de Korverstraat
35, NL-5831 AN, Boxmeer, The Netherlands. Birgit.Makoschey@Intervet.com
A protocol to test foetal calf serum (FCS) for contamination with bovine viral
diarrhoea virus (BVDV) is described. Following this protocol, which combines
cell culture methods and detection of pestivirus RNA,
seven batches of FCS
were tested. Infectious BVDV
was detected in four of those batches. One of the remaining batches
contained a relatively high number of non-infectious BVDV particles. A sample
of this batch was formulated with aluminium hydroxide and aluminium phosphate
as adjuvant into an experimental vaccine preparation. This product was
injected twice into BVDV seronegative cattle with a 4 week interval. Blood
samples taken 4 weeks after the second application were negative for BVDV
specific antibodies. Our data stress
that detection of BVDV
RNA is not sufficient for a complete risk assessment on FCS.
Discrimination between infectious and non-infectious BVDV
is essential. This can only be achieved by cell culture methods.
Department of Obstetrics and Gynecology, Chang Gung Memorial Hospital, Taipei,
ROC.
BACKGROUND: Mycoplasmas, the smallest
and simplest prokaryotes that reside in endosomes
of mammalian cells, are widespread contaminants found in cell cultures. About
30% of all cell cultures, varying from 15 to 80%, are reportedly contaminated
with mycoplasmas.
Here, we present our experience in successfully detecting and treating
mycoplasmal infection in various cell lines. METHODS: The nested polymerase
chain reaction (PCR) detection and microscopic examination, including
phase-contrast, fluorescent, as well as differential interference contrast, were
used for detecting potential mycoplasma contamination of cell lines used in our
laboratory. As soon as mycoplasma was identified, antibiotic treatment was
initiated. RESULTS: Mycoplasmal contamination was detected in six of 15 cell
lines using the nested PCR amplification of mycoplasma DNA, which was further
demonstrated using 4, 6-Diamidino-2-phenylindole (DAPI) staining and fluorescent
microscopy. Alternate treatment with two antibiotics, macrolide (tiamulin) and
tetracycline (minocycline), effectively eliminated mycoplasma, which was
validated by both PCR and microscopic studies. CONCLUSIONS: The nested PCR using
genomic DNA extracted from cultured cells as templates is a rapid and sensitive
method for detecting mycoplasma contamination. Treatment with combined
antibiotics can completely eradicate mycoplasmal infection from cultured cells.
For the ease of use, PCR and/or DAPI staining appear suitable for detecting
potential mycoplasmal contamination in laboratories that rely heavily on the
cell culture system.
Detection and characterization of pestivirus contaminations
in human live viral vaccines.
Studer E, Bertoni G, Candrian U.
Official Medicines Control Laboratory Biologika and R&D Unit, Division of
Biologicals, Swiss Federal Office of Public Health, P.O. Box 3003, Bern,
Switzerland.
In view of the use of potentially
contaminated foetal
calf serum (FCS)
in cell cultures pestiviruses
may be present in live viral vaccines. Thirty-six lots of human live viral
vaccines produced by three manufacturers were tested for the presence of
pestiviruses.
Bovine viral diarrhoea
virus (BVDV)
RNA was detected in 33% of the vaccine lots. All positive results were caused
by the mumps component of a single manufacturer. Partial sequences of
the 5' untranslated region of BVD viral RNA were determined. The sequences
were closely related to that of the NADL strain of BVDV. The amount of BVDV
RNA in the vaccines was determined by real-time RT-PCR using the LightCycler.
Between 3.3*10(2) and 6.2*10(5) RNA copies per dose were found to be present
in the vaccine samples. Additionally, culture tests were done with FCS and
human diploid cells used in the vaccine production of the manufacturer whose
vaccines were positive by PCR. All attempts to detect virus antigen in MRC-5
human diploid cells or to infect these cells with BVDV failed.
This suggests that BVDV
RNA detected in human live viral vaccines represents passive carry over of
BVDV
from contaminated FCS
rather than active virus replication in human diploid cells. Our results
indicate that contamination with BVDV
of FCS
used in vaccine production does not appear to be of immediate concern to human
health. Furthermore, our results indicate that gamma-irradiation of FCS
destroys BVDV particles and is also effective in preventing the presence of
BVDV RNA in the vaccines. Copyright 2002 The International Association for
Biologicals. Published by Elsevier Science Ltd. All rights reserved.
Comment: Let's hope they are right about the
consequences re: human health..
Comparative PCR analysis for detection of mycoplasma
infections in continuous cell lines.
Uphoff CC, Drexler HG.
Department of Human and Animal Cell Cultures, DSMZ-German Collection of
Microorganisms & Cell Cultures, Braunschweig. cup@dsmz.de
Mycoplasma
contamination of cell lines is one of the major problems in cell culturing.
About 15-35% of all cell lines are infected with a limited number of mycoplasma
species of predominantly human, swine, or bovine origin. We examined
the mycoplasma contamination status in 495 cell cultures by polymerase chain
reaction (PCR) assay, microbiological culture method, and deoxyribonucleic
acid-ribonucleic acid (DNA-RNA) hybridization, and in 103 cell cultures by PCR
and DNA-RNA hybridization, in order to determine the sensitivity and
specificity of the PCR assay in routine cell culture. For those two cohorts,
results for the three or two assays were concordant in 92 and 91% of the
cases, respectively. The sensitivity (detection of true positives) of this PCR
detection assay was 86%, and the specificity (detection of true negatives) was
93%, with positive and negative predictive values (probability of correct
results) of 73 and 97%, respectively. PCR defined the mycoplasma status with
92% accuracy (detection of true positives and true negatives). The mycoplasma
contaminants were speciated by analyzing the PCR amplification fragment using
several restriction enzymes. Most of the cultures (47%) were infected with
Mycoplasma fermentans, followed by M. hyorhinis (19%), M. orale (10%), M.
arginini (9%), Acholeplasma laidlawii (6%), and M. hominis (3%). To sum up,
PCR represents a sensitive, specific, accurate, inexpensive, and quick
mycoplasma detection assay that is suitable for the routine screening of cell
cultures.
No evidence of infectious retroviruses in measles virus
vaccines produced in chicken embryo cell cultures.
Shahabuddin M, Sears JF, Khan AS.
Laboratory of Retrovirus Research, Division of Viral Products, Center for
Biologics Evaluation and Research, U.S. Food and Drug Administration,
Bethesda, Maryland 20892, USA.
All vaccines that are prepared in
chicken embryo fibroblasts (CEFs)
contain a low level of particle-associated reverse transcriptase (RT)
activity, which is produced from the avian cell substrate. The RNAs
present in the particles have sequence homology to viral DNAs belonging to the
ancient endogenous avian virus (EAV) family or to the avian sarcoma-leukosis
virus (ALV)-related subgroup E endogenous virus loci. Although no
replication-competent retrovirus has been associated with the RT activity
produced from CEFs, there have been some theoretical safety concerns regarding
potential consequences of integration of EAV and ALV sequences in human DNA,
which may result from nonproductive infection with replication-defective
particles or infection with EAV and ALV pseudotypes bearing measles virus
envelopes. To address these possibilities, we have analyzed EAV and ALV
particles in a measles virus vaccine equivalent (MVVE) preparation, obtained
from a U.S. manufacturer, for integration and for replication in human
peripheral blood mononuclear cells (PBMCs). The results show the absence of
EAV and ALV integrants in DNA prepared from MVVE-inoculated human cells by
direct DNA PCR and Alu PCR assays and no propagation of retrovirus in 18-day
cultures of MVVE-inoculated human PBMCs by a highly sensitive PCR-based RT
assay. These results provide further
confidence regarding the safety of chicken RT
activity in live viral vaccines and support the continued use of
chick-cell-derived vaccines in humans.
For over half a century, cell cultures
derived from animals and humans have served researchers in various fields. To
this day, cross-contamination of cultures has plagued many researchers, often
leading to mistaken results, retractions of results, cover-ups and some
out-and-out falsification of data and results following inadvertent use of the
wrong cells. Also, during years of examining cultures for purity we
learned that many virologists were not too concerned about the specificity of
the cultures they used to propagate the particular virus under study as long
as the substrate (whatever it might have been) gave optimal virus yield. Polio
virus propagates in primate cells, and much research has involved cells from
man and various species of primates. In the 1950s a large number of
chimpanzees were held in captivity in Africa for extensive studies of the
efficacy of polio vaccine in production at the Wistar Institute in
Philadelphia and elsewhere. Chimpanzee tissues, particularly kidneys, were
thus readily available and could have also provided substrates for polio virus
production, since little was known about the purity of substrates and little
attention was paid to their specificity at that time.
Publication Types:
Review
Review, Tutorial
Comment: How much concern has there been about
possible cross-species transfer of disease?
Experimental infection of calves with bovine viral
diarrhoea virus type-2 (BVDV-2) isolated from a contaminated vaccine.
Falcone E, Cordioli P, Tarantino M, Muscillo M, Sala G, La Rosa G, Archetti
IL, Marianelli C, Lombardi G, Tollis M.
Istituto Superiore di Sanita, Laboratorio di Medicina Veterinaria, Viale
Regina Elena 299, 00161 Rome, Italy.
A non-cytopathic strain of BVDV-2 was isolated from a batch of live infectious
bovine rhinotracheitis (IBR) vaccine, and inoculated intranasally into four
3-month-old calves. Severe signs of disease developed by days 4 and 6 in three
of the calves, free of BVDV and antibodies to BVDV, that had been exposed to
the virus. These calves survived the acute phase of the infection and
progressively recovered. BVDV was consistently isolated, or the respective
viral RNA was detected, in the buffy coats from blood samples collected
starting from days 2 or 4 up to days 11 or 14 after the experimental
infection. Viral RNA was also detected in sera from these infected calves
until the presence in the serum of virus neutralizing antibodies was
demonstrated. By contrast, the only calf having pre-existing neutralizing
antibodies to BVDV at the start of the study was protected from the disease.
No virus was detected at any time after experimental inoculation of this calf.
Genomic characterization of the BVDV-2 isolated in cell cultures, or detected
in sera from the experimentally infected animals, revealed 100%, homology in
the nucleotide sequence with the BVDV-2 detected as a contaminant of the live
IBR virus vaccine. These findings
provided evidence of the infective nature of the contaminant BVDV-2 and of its
potential to generate disease outbreaks when inoculated into susceptible
animals.
Official Medicines Control Laboratory Biologika and R&D Unit, Division of
Biologicals, Swiss Federal Office of Public Health, P.O. Box 3003, Bern,
Switzerland.
In view of the use of potentially
contaminated foetal
calf serum (FCS)
in cell cultures pestiviruses
may be present in live viral vaccines. Thirty-six lots of human live viral
vaccines produced by three manufacturers were tested for the presence of pestiviruses.
Bovine viral diarrhoea
virus (BVDV)
RNA was detected in 33% of the vaccine lots. All positive results were
caused by the mumps component of a single manufacturer. Partial sequences of the
5' untranslated region of BVD viral RNA were determined. The sequences were
closely related to that of the NADL strain of BVDV. The amount of BVDV RNA in
the vaccines was determined by real-time RT-PCR using the LightCycler. Between
3.3*10(2) and 6.2*10(5) RNA copies per dose were found to be present in the
vaccine samples.Additionally, culture tests were done with FCS and human diploid
cells used in the vaccine production of the manufacturer whose vaccines were
positive by PCR. All attempts to detect virus antigen in MRC-5 human diploid
cells or to infect these cells with BVDV failed. This suggests that BVDV RNA
detected in human live viral vaccines represents passive carry over of BVDV from
contaminated FCS rather than active virus replication in human diploid cells.
Our results indicate that contamination with BVDV of FCS used in vaccine
production does not appear to be of immediate concern to human health.
Furthermore, our results indicate that gamma-irradiation of FCS destroys BVDV
particles and is also effective in preventing the presence of BVDV RNA in the
vaccines. Copyright 2002 The International Association for Biologicals.
Published by Elsevier Science Ltd. All rights reserved.
Division of Neurology, Department of Medicine, Chiang Mai University, Chiang Mai
50200, Thailand. spiyasir@mail.med.cmu.ac.th
Public tolerance to adverse reactions is minimal. Several reporting systems have
been established to monitor adverse events following immunization. The present
review summarizes data on neurologic complications following vaccination, and
provides evidence that indicates whether they were directly associated with the
vaccines. These complications include autism (measles vaccine), multiple
sclerosis (hepatitis B vaccine), meningoencephalitis (Japanese encephalitis
vaccine), Guillain-Barre syndrome and giant cell arteritis (influenza vaccine),
and reactions after exposure to animal rabies vaccine. Seizures and hypotonic/hyporesponsive
episodes following pertussis vaccination and potential risks associated with
varicella vaccination, as well as vaccine-associated paralytic poliomyelitis
following oral poliovirus vaccination, are also described.
In addition, claims that complications
are caused by adjuvants,
preservatives and contaminants [i.e. macrophagic myofasciitis
(aluminium), neurotoxicity
(thimerosal), and new variant Creutzfeldt-Jakob disease (bovine-derived
materials)] are discussed.
Department of Pharmacy Practice and Science, College of Pharmacy, University of
Arizona, 1703 E. Mabel Street, Tucson, AZ 85721-0207, USA. erstad@pharmacy.arizona.edu
The implications of prion-induced diseases for the use of medications that
theoretically could harbor the infectious pathogens are discussed. Prions have
been identified as protein particles that lack nucleic acids. There is evidence
that prions cause the transmissible neurodegenerative diseases known as
transmissible spongiform encephalopathies.
Of these diseases, bovine spongiform
encephalopathy (BSE)
and the human spongiform encephalopathy to which it has been linked, new variant
Creutzfeldt-Jakob disease (CJD),
have generated the most attention. The first cases of new variant CJD
appeared in Britain in the mid-1990s. Ingestion of prion-infected beef remains
the only known cause of new variant CJD. No cases of BSE or new variant CJD have
been documented in the United States. The time from exposure to the development
of clinical sequelae appears to be about 10 years. The median duration of
illness is 14 months, and the outcome is invariably death. There is no
treatment; currently the only available approach is prevention. There is no
reliable method of predicting the number of new cases that might occur because
of lack of definitive information on the efficiency of transmission from animals
to humans and the number of people currently infected and at risk for infection.
The infectivity of medications and plasma fractionation products containing
material from cattle with BSE is unknown, but the risk is believed to be very
low. No cases of such transmission have been identified. Guidelines to keep the
risk of transmission via medications low have been promulgated by FDA, and
further research is warranted. There
have been no reports of medications or plasma fractionation products being
contaminated with the prions
that cause new variant CJD.
Ongoing vigilance and research are appropriate, however.
Ten commandments for preventing contamination of primary
cell cultures.
Vierck JL, Byrne K, Mir PS, Dodson MV.
Department of Animal Sciences, Washington State University, Pullman,
Washington, USA.
Procedures for preventing contamination in primary cell cultures must be
carefully defined and strictly followed in order to obtain healthy cells.
Protocols have been developed and refined in our laboratory for establishing
primary cultures of muscle and fat stem cells without contamination from a
variety of animals. Contamination of cell cultures is not only frustrating,
but is also very expensive both in time and loss of materials. Through the
consistent use of proper aseptic techniques, most instances of contamination
may be avoided. We suggest that the basic principles detailed here will find
wide applicability in the culturing of primary cells without contamination
from many different types of animals and tissues.
Comment: Is it merely "frustrating" and
"expensive", or are there negative health consequences associated with their
use as well?
[Contamination of bovine fetal serum with bovine viral
diarrhea virus]
[Article in Spanish]
Zabal O, Kobrak AL, Lager IA, Schudel AA, Weber EL.
Instituto de Virologia, CICVyA, INTA Castelar, Buenos Aires, Argentina.
Fetal bovine serum (FBS) used in cell culture may be contaminated with
viruses, among them bovine viral diarrhea virus (BVDV) affecting the
production of biological reagents and the results of diagnosis. The filtration
process used in the preparation of commercial FBS abrogates most viral agents
that may be present in raw FBS, but BVDV may pass through the filters because
of its small size and its pleomorphism.
While detection of bovine herpes
virus-1 and parainfluenza-3 (PI-3) is determined by observation of the cytopathic
effect, and also by
hemadsorption in the case of
PI-3, the most frequently isolated BVDV
is non cytopathic,
and infects cells without morphological alterations, inducing problems that
arise after several cell generations. Batches of raw and processed FBS
were analyzed. Frequencies of BVDV detection in raw serum in Argentina were
similar to those published for USA. By conventional methods for BVDV
detection, only 2 of 20 commercial batches of FBS had BVDV. Using cell
cultures maintained with high concentrations of the serum under study for at
least 2 weeks, with detection of viral antigen by indirect immunofluorescence,
the percentage of BVDV detection was 80%. This method shows that most lots of
commercial FBS contain BVDV. RT-PCR allows faster detection of the viral
genome, but it must be validated, as it does not show viral replication. To
eliminate the problem of BVDV contamination in FBS, only gamma irradiated FBS
is used in our laboratory.
Benefits and risks due to animal serum used in cell culture
production.
Wessman SJ, Levings RL.
USDA, APHIS, VS, Center for Veterinary Biologics-Laboratory, Ames, Iowa, USA.
Infection with bovine viral diarrhoea
virus (BVDV)
and other viruses is frequent in the bovine population. In utero
infection leads to virus and antibody contamination of foetal
and other serum used in cell culture production. The use of contaminated cells
for vaccine production may result in contaminated vaccines, which may lead to
seroconversion
or disease in the vaccinated animal. Contaminated serum or cell cultures may
also interfere with the diagnosis of viral infections. Methods for the
detection of BVDV and other viruses in serum, cell cultures, seed viruses and
vaccines at the CVB-L, and the frequency of detection are described. Reasons
for continued use of serum in cell culture production, and the risks of using
serum, are discussed.
Cancer risk associated with simian virus 40 contaminated
polio vaccine.
Fisher SG, Weber L, Carbone M.
Cancer Cause and Prevention Program, Loyola University Medical Center,
Maywood, Illinois 60153, USA.
BACKGROUND: The presence of SV40 in monkey cell cultures used in the
preparation of the polio vaccine from 1955 through 1961 is well documented.
Investigations have consistently demonstrated the oncogenic behavior of SV40
in animal models. Early epidemiologic studies were inadequate in demonstrating
an increase in cancer incidence associated with contaminated vaccine.
Recently, investigators have provided persuasive evidence that SV40 is present
in human ependymomas, choroid plexus tumors, bone tumors, and mesotheliomas,
however, the etiologic role of the virus in tumorigenesis has not been
established. MATERIALS AND METHODS: Using data from SEER, we analyzed the
incidence of brain tumors, bone tumors, and mesotheliomas from 1973-1993 and
the possible relationship of these tumors with the administration of the SV40
contaminated vaccine. RESULTS: Our
analysis indicates increased rates of ependymomas
(37%), osteogenic
sarcomas (26%), other bone tumors (34%) and mesothelioma
(90%) among those in the exposed as compared to the unexposed birth cohort.
CONCLUSIONS: These data suggest that there may be an increased incidence of
certain cancers among the 98 million persons exposed to contaminated polio
vaccine in the U.S.; further investigations are clearly justified.
Cell cross-contamination in cell cultures: the silent and
neglected danger.
Markovic O, Markovic N.
BioSciCon, Inc., Rockville, MD 20852, USA.
Cell cross-contamination in cell
cultures is a common problem during cell culturing and use. Contamination
invalidates research results, compromises the comparison of results between
laboratories, reduces reproducibility required in industrial production of
cell lines, and may lead to unusable therapeutic products. The problem
can be solved by increasing the awareness of its seriousness and by
introducing regular quality control of cell cross-contamination in every
laboratory where cells are grown and used.
[Presence of Mycoplasma in laboratory cell cultures from
Cordoba, Argentina]
[Article in Spanish]
Cumino AC, Cordoba P, Zapata TM.
Instituto de Virologia J.M. Vanella, Facultad de Medicina, Universidad
Nacional de Cordoba, Argentina.
In this paper we determined the prevalence of mycoplasma contamination in 17
cell lines. Eighty per cent of the laboratories that currently use cell
culture techniques participated in this study. Hoechst 33258 dye was used to
detect mycoplasma contamination. The relationship between culture maintenance
conditions and the presence of mycoplasma were analyzed, considering the use
of antibiotics in the culture media,
fetal calf serum (FCS)
quality, culture media processing, use of disponsable labware, type of
laminar flow cabinet, quantity of operators, and cell culture system.
Thirty-five per cent of the analyzed
cell lines showed mycoplasma contamination. Those lines belonged to 2
of the 8 surveyed laboratories. When confronting the working conditions versus
mycoplasma contamination, 66% of the laboratories that employ non-certified
FCS or reuse their labware, show mycoplasma contamination.
Mycoplasma presence was found in 50%
of the laboratories that use closed culture system, or more than one operator.
Laboratories that process their culture media or that include antibiotic in
the growing media, show a 40% contamination. The results obtained help
to establish working conditions necessary to avoid introducing or spreading
the microorganism.
Division of Viral and Rickettsial Diseases, National Center for Infectious
Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30333, USA.
Virus zoonoses causing haemorrhagic fever have been recognized in three major
families: Arenaviridae, Bunyaviridae and Filoviridae. All are
negative-stranded RNA viruses, with genomes in two segments, three segments,
or non-segmented, respectively. Acquisition of haemorrhagic fever in man
generally requires close contact with a vertebrate vector species, usually
rodents, for the arenaviruses and bunyaviruses. In the case of filoviruses,
the vector is currently unknown, but these viruses may infect monkeys, and may
contaminate cell cultures prepared from them. Both bunyavirus and arenavirus
haemorrhagic fevers have arisen in humans following exposure to rodents, and
in the case of Hantaan, a virus causing haemorrhagic fever with renal syndrome
(HFRS), there have been numerous laboratory-acquired infections among animal
care workers. As the technology to differentiate virus species has improved,
it has become clear that there are numerous potentially hazardous viruses
capable of causing HFRS or hantavirus pulmonary syndrome (HPS) within the
feral rodent population. In many cases
it would be desirable to introduce screening methods for such viruses before
preparing cell cultures from these rodent or simian species that will be used
to prepare biological products for human use.
Contamination of commercially available fetal bovine sera
with bovine viral diarrhea virus genomes: implications for the study of
hepatitis C virus in cell cultures.
Yanagi M, Bukh J, Emerson SU, Purcell RH.
Laboratory of Infectious Diseases, National Institute of Allergy and
Infectious Diseases, National Institutes of Health, Bethesda, Maryland
20892-0740, USA.
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 HCV, 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.
Experience with viral contamination in cell culture.
Garnick RL.
Genentech, Inc., South San Francisco, CA, USA.
Genentech has had direct experience with two contaminations of large-scale
cell cultures by Minute Virus of Mice (MVM). No definitive source of either
contamination has been identified, although the conclusions of the
investigation were consistent with a raw material used in cell culture as the
source. Effective analytical barriers were developed following the first
contamination using polymerase chain reaction (PCR) and cell culture-based
methodology for MVM, and these were employed on a routine basis. This approach
was effectively used to detect and contain the second contamination, thus
dramatically minimizing its impact. Close interactions with regulatory
authorities were critical in the positive management of these situations.
Detection of bovine viral diarrhoea virus antigen and RNA
in oviduct and granulosa cells of persistently infected cattle.
Booth PJ, Stevens DA, Collins ME, Brownlie J.
Institute for Animal Health, Compton, Newbury, Berkshire, UK.
Large-scale in vitro bovine embryo production systems commonly use genital
tracts obtained from an abattoir as a source of both cumulus-oocyte complexes
and co-culture feeder cells. Tissues
derived from this source may be contaminated with non-cytopathogenic
bovine viral diarrhoea
virus (BVDV)
since, in several countries surveyed, approximately 1% of animals tested are
persistently infected with this pathogen. Therefore, the use of such material
in in vitro fertilization systems presents a potential risk for the
transmission of BVDV
to bovine embryos and via embryo transfer. This potential was
investigated by obtaining oviduct epithelial cells and granulosa cells, which
are commonly used as feeder cells, from cattle persistently infected with BVDV
and examining them for the presence of BVD viral antigen (p80 non-structural
protein and gp53 envelope glycoprotein) by indirect immunofluorescent
histochemistry, and also viral RNA (encoding the p80 region) by in situ
hybridization. In addition, titres of virus present in oviduct, ovary and
blood were assayed by immunodetection on calf testis cell cultures. Luminal
epithelial cells from the oviduct and primary cultures of granulosa cells and
oviduct epithelial cells from such cattle were shown to contain both viral
antigen and RNA. The susceptibility of both cell types to BVDV infection was
further established by inoculating primary cell cultures of cells derived from
cattle not infected with BVDV with a cloned isolate of non-cytopathogenic BVDV
(Pe515). RNA encoding BVDV and the antigen were detected 12 h after
inoculation.(ABSTRACT TRUNCATED AT 250 WORDS)
The possibility of air-droplet cell transmission during cell transfer was
experimentally confirmed, this appearing to be the most probable route of
cross contamination of cell cultures. The consequences of such contamination
are assessed and measures reducing the risk of intercellular contamination
proposed.
Department of Membrane and Ultrastructure Research, Hebrew University-Hadassah
Medical School, Jerusalem, Israel.
Mycoplasma infection of cell cultures
is widespread and has major detrimental effects on cellular physiology and
metabolism. Since cell culture is used extensively, both in research and in
industrial production processes, questions of primary concern arise, such as:
how can mycoplasma contamination be detected; what are the effects of such
contamination on cellular functions; what methods are available for
eliminating contamination?
The benefits of using animal or human cell cultures have been clearly
demonstrated in diagnostic and therapeutic research and in their application
for manufacturing. Cell cultures serve
as a tools for the production of vaccines, receptors, enzymes, monoclonal
antibodies and recombinant DNA-derived proteins. They represent an
integral part of drug development for which corresponding facilities,
equipment and manufacturing processes are required.
Although the cells themselves offer no
particular risk to workers in laboratories and production areas or to the
environment, the cell cultures may be contaminated with viruses, mycoplasma,
bacteria, yeast and fungi or might contain endogenous viruses. The
containment level for animal and human cells is therefore determined by the
risk class of these agents. The history of animal and human cell cultures has
proved that they can be handled safely. The recommendations in this
publication concern the safe handling of cell cultures (tissue explants,
primary cell cultures) and permanent cell lines of animal and human origin. A
classification system of safety precautions has been elaborated according to
the potential for contamination with the pathogenic agents involved.
Comparison of methods used for detection of mycoplasma
contamination in cell cultures, sera, and live-virus vaccines.
Benisheva T, Sovova V, Ivanov I, Opalchenova G.
National Drug Institute, Sofia.
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.
Bovine polyomavirus, a cell-transforming virus with
tumorigenic potential.
Schuurman R, van Strien A, van Steenis B, van der Noordaa J, Sol C.
Department of Virology, University of Amsterdam, Academic Medical Centre,
The Netherlands.
The early region of bovine polyomavirus (BPyV) was tested for its cell
transformation potential employing an assay of dense focus formation. Dense
foci of morphologically transformed cells were observed upon transfection of
primary rodent cells with a plasmid construct encoding the complete early
region of BPyV under the transcriptional control of the long terminal repeat
of Rous sarcoma virus. No transformation of primary rodent cells was
observed upon transfection of these cells with a plasmid encoding the
complete early region of BPyV under the control of its own transcriptional
regulatory sequences. In BPyV-transformed cells, the viral sequences had
become integrated into the cellular genome, and expression of large T
antigen could be detected in a high percentage of cells.
The transformed cells were
demonstrated to be capable of anchorage-independent growth and to be oncogenic
in immunocompromised
newborn rats. Therefore BPyV
should be considered as a potentially tumorigenicpolyomavirus.
Since many commercial batches of calf serum have been shown to be
contaminated with BPyV,
our observations may have implications for the use of calf serum in cell
culture.
Detection of Mycoplasma contamination in primary calf
kidney cell cultures.
Pfutzner H, Otto P.
Research Institute for Bacterial Animal Diseases, Jena, Germany.
A total of 31 primary cell culture preparations of calf kidney including their
processing stages (kidney, washing fluid, cell suspension, cell culture
monolayer) were investigated for mycoplasmas by cultural methods. Mycoplasma
(M.) arginini, was isolated from 8 out of 27 investigated samples of calf
kidney from 3 out of 26 washing fluids, 5 out of 20 cell suspensions, and from
9 out of 21 cell culture monolayers. Furthermore, M. arginini was repeatedly
found in throat swabs of the cell laboratory technicians. The results give
indications as to the source and route of mycoplasma infections of primary
cell cultures.
Peters CJ, Jahrling PB, Ksiazek TG, Johnson ED, Lupton HW.
Disease Assessment Division, U.S. Army Medical Research Institute of
Infectious Diseases, Fort Detrick, Frederick, MD 21701-5011.
The filoviruses Marburg and Ebola comprise a newly recognized family of
viruses. The first filovirus to be isolated was Marburg virus in 1967. This
virus was imported in shipments of African green monkeys from Uganda and
infected several cell-culture technicians, with serious illness resulting. The
rarity of Marburg and Ebola virus transmission, decreasing use of imported
African monkeys, and quarantine efforts have presumably been responsible for
the lack of additional episodes until 1989, when a new filovirus related to
Ebola was isolated from quarantined monkeys in Reston, Virginia. This virus
was imported on multiple occasions from a Philippine supplier of cynomolgus
macaques as a consequence of an epidemic of acute infections in the foreign
holding facility. While quarantine procedures prevented the use of any of
these animals in research and the three human infections that occurred were
asymptomatic, this episode emphasizes that these little understood viruses
have considerable potential for mischief. The finding of antibodies reacting
with Ebola viruses in many biomedically important Old World primates,
including colonized monkeys in the U.S., emphasizes the need for more research
to understand the specificity of the antibodies, spectrum of filovirus strains
in nature, potential hosts, and true distribution of the family. The
filoviruses grow well in primary and established cell strains and cell lines,
and cytopathogenic effects may be absent or require several days to be
manifest, leading to the possibility of occult contamination. The known
viruses are readily detected by polyclonal and monoclonal antibody staining of
cells and by electron microscopy; nucleic acid probes exist to develop more
sensitive techniques if warranted.
Latent parvoviral infection of continuous cell lines.
Fikrig MK, Tattersall P.
Department of Medicine, Yale University School of Medicine, New Haven, CT
06510.
The parvoviruses are a family of single-stranded DNA-containing viruses which
are known to establish inapparent infections of continuous, and in some cases,
primary cell cultures. Their small
size and great stability suggest that they would be difficult to eliminate
from a biological component purified from a contaminated cell line. Thus,
precautions should be taken to exclude such agents from initial cell cultures,
and from the reagents used to maintain them.
Cell line issues: historical and future perspectives.
Petricciani JC.
Pharmaceutical Manufacturers Association, Washington, DC 20005.
The initial decision to use only primary cell cultures for the production of
human biological products was challenged in the late 1960s by the introduction
of human diploid cells (HDCs), and again in the 1980s by continuous cell lines
(CCLs). The history of the HDC controversy is reviewed and lessons from that
era that are relevant to the use of CCLs are pointed out. With the
introduction of recombinant DNA technology in the 1980s, and the potential
usefulness of CCLs in product development, the issue of cell acceptability
became more urgent, and several attempts were made to reach a consensus on
regulatory issues. In 1986, the World Health Organization convened a Study
Group to review the safety issues related to products derived from CCLs. The
Study Group made a clear recommendation to pursue CCLs in product development
because of the demonstrated capability of modern manufacturing processes to
cope with contaminants. Issues such as acceptable levels of cellular DNA in
products, the relationship of purity to safety, and the relevance of the
genetic stability of recombinant cells to product consistency are current
examples of areas in need of discussion and agreement. A system in which
regulatory authorities, industry, and the general biomedical community
cooperate in finding solutions is ultimately in everyone's best interest.
Frequent detection of bovine polyomavirus in commercial
batches of calf serum by using the polymerase chain reaction.
Schuurman R, van Steenis B, van Strien A, van der Noordaa J, Sol C.
Department of Virology, University of Amsterdam, The Netherlands.
Twenty commercial batches of calf serum, obtained from several suppliers, were
tested for the presence of bovine polyomavirus (BPyV) DNA and antibodies
against the virus. Using polymerase
chain reaction (PCR)
technology, BPyV
DNA was detected in 70% of the batches; no BPyV
was detected in any of the negative control samples. The specificity of
the amplification reactions was proven by hybridization. PCR results were
confirmed by virus isolation experiments performed with five PCR-positive and
five PCR-negative serum batches. The
results indicate that the use of calf serum to supplement tissue culture media
involves a serious risk of contaminating cell cultures with BPyV.
No correlation was observed between the presence or absence of anti-BPyV
immunoglobulins and the detection of BPyV-specific DNA sequences in the serum
batches.
Virus zoonoses and their potential for contamination of
cell cultures.
Mahy BW, Dykewicz C, Fisher-Hoch S, Ostroff S, Tipple M, Sanchez A.
Division of Viral and Rickettsial Diseases, Centers for Disease Control,
Atlanta, GA 30333.
Silent virus infections of laboratory
animals present a human health hazard, from direct exposure and from
contamination of biological products for human use. Here we report two
recent examples. In 1989, an outbreak of lymphocytic choriomeningitis virus (LCMV)
infections was recognized among workers at a cancer research center after an
animal caretaker developed viral meningitis. Investigation revealed that
multiple tumor cell lines at the facility were infected with LCMV, as were
research animals injected with these cell lines. Of 82 workers tested, eight
(10%) were found to have been infected. The infected workers were more likely
than other animal handlers to report handling athymic (nude) mice (p less than
.0.007). The number of nude mice used in this facilty had increased five-fold
in the previous year, possibly explaining the timing of the outbreak. This is
the first reported LCMV outbreak since 1975, and the first to implicate nude
mice as a source of human LCMV infections. In November 1989 and January 1990,
infections caused by two distinct Ebola-like filoviruses were discovered in
non-human primates at quarantine facilities in Virginia and Pennsylvania.
Although 22 persons were considered to have high- or medium-risk exposures for
Ebola infection, no Ebola-compatible illnesses occurred. One of the
medium-risk persons had Ebola IgG antibodies confirmed by IFA and Western
blot. Rigorous use of barrier precautions may have limited exposure and
infection with these filoviruses. In February 1990, new groups of filovirus-infected
monkeys were identified in Virginia and in Texas. Seroconversion occurred in
four animal handlers, including one to very high titer, but again no illness
was observed.(ABSTRACT TRUNCATED AT 250 WORDS)
Correlations between virus infection and pathogenicity in
humans.
Agut H, Huraux JM.
Laboratoire de Virologie, CERVI, Groupe Hospitalier Pitie-Salpetriere, Paris.
Infection is not synonymous with disease. Infection refers to the
multiplication or the persistence of a virus in tissues while pathogenicity
refers to the emergence of disease in the infected host. Pathogenicity is the
result of a competition between the growth of the virus and the host response
to infection, and its genesis involves many intricate factors. Consequently,
most viruses of medical interest exhibit a wide spectrum of pathogenicity
ranging from asymptomatic infection to lethal disease. The study of
pathogenicity is far more complex than the recognition of infection. Cell
cultures, animal models and molecular biology investigations have provided
substantial insights both into the virulence of viruses and the susceptibility
of the host. However, the prediction of disease often remains hazardous
whereas the detection of a virus can now be obtained in most cases by the
combination of classical methods with recent molecular techniques. Therefore
the prevention of virus diseases transmitted by biologicals is logically
founded on the prevention of virus infections, which implies a constant
adaptation of safety control procedures to the rapid evolution of knowledge in
medical virology.
Bovine viral diarrhea virus contamination of nutrient
serum, cell cultures and viral vaccines.
Levings RL, Wessman SJ.
National Veterinary Services Laboratories, Animal and Plant Health
Inspection Service, USDA, Ames, IA 50010.
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.
Detection and elimination of adventitious agents in
continuous cell lines.
Erickson GA, Landgraf JG, Wessman SJ, Koski TA, Moss LM.
National Veterinary Services Laboratories, Ames, IA 50010.
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.
National AIDS Program, U.S. Public Health Service, Washington, D.C.
The initial discussions and decisions in 1954 on the use of various types of
mammalian cell substrates for the production of human biological products set
the stage for controversy in the late 1960's and again in the 1980's when
"abnormal" cell substrates were proposed as alternates for primary cell
cultures. In the 1960's the issue was human diploid cells, and in the 1980's
it has been the use of continuous cell lines. The parallels between the two
issues have been obvious to all who have been interested in the subject. The
history of the human diploid cell controversy is reviewed from a personal
point of view, and lessons from that era that are relevant to the use of
continuous cell lines are pointed out. Pragmatism based on the perceived need
for human interferon in the late 1970's led to the exploration of human
lymphoblastoid cells as substrates. That bold venture into a prohibited zone
set the stage for a reconsideration of the acceptability of continuous cell
lines as substrates for the production of a wide range of human biologicals.
With the introduction of recombinant DNA technology in the 1980's and the
potential usefulness of continuous cell lines in product development, the
issue of acceptability took on a more acute aspect and several attempts were
made to reach a consensus and a resolution of regulatory issues.
In 1986 the World Health Organization
identified the use of continuous cell lines as of sufficient global importance
to convene a Study Group to review the safety issues and to make
recommendations on their use, especially for vaccines. The result of that
international effort was a clear recommendation to pursue the use of
continuous cell lines in product development because of the demonstrated
capability of modern manufacturing processes to cope with even theoretically
worrying contaminants.(ABSTRACT
TRUNCATED AT 250 WORDS)
[Mycoplasma as a contaminant of cell cultures maintained in
laboratories of private and official institutions]
[Article in Portuguese]
Miyaki C, Pral MM, Gallina NM, de Rizzo E.
Mycoplasma is one of the most serious
contaminants of cell cultures. Its detection is very important in virology, as
well as its eradication. The aim of this study was to verify the
incidence of mycoplasma in cell lines maintained in seven laboratories of
private, government and college institutions of the State of Sao Paulo,
Brazil, for the purposes of research, production of reagents for diagnosis and
production of biologicals for human and animal use. Of the 29 cell lines,
eight were derived from human tissues and 21 from other animal species (dog,
rabbit, mouse, hamster, monkey, pig, chicken and ox).
Using the direct method with specific
liquid and solid media for detection of mycoplasma, 48 out of the 106 cell
samples tested were positive, corresponding to a contamination index of
45.28%. The incidence of contamination among the 35 cell samples of
human origin was 51.43% (18 positive).
Of the 71 samples originated from other species, 30 were positive (42.25%).
The high incidence of contamination found calls for the adoption of measures
for the prevention of this hazard: the elimination of mouth pipetting, the use
of aseptic techniques and a rigid control of trypsin, serum and other
components of cell culture media. The
substitution of mycoplasma-free cultures for all contaminated ones and the
performance of periodical tests for mycoplasma detection must also be carried
out to prevent and avoid the dissemination of these organisms. Data
obtained showed that contamination appeared in the 2nd (72.92%), in the 3rd
(20.83%) and in the 4th passage (6.25%).(ABSTRACT TRUNCATED AT 250 WORDS)
Examinations of 1653 batches of green monkey kidney cell cultures revealed
contamination with foamy virus (FV) in 243 (16%) batches. From some of these
cultures 65 strains of virus belonging to two serotypes were isolated. Tests
on sera from 1122 monkeys revealed antibody to FV in 985 (87.8%) animals. No
correlation between the presence of antibody in the serum and virus recovery
from monkey kidney cell cultures was observed, however. This might be due
either to the lack of the virus in the kidneys or to the failure in its
isolation from primary cultures. Passages of cell cultures were shown to
facilitate additional recovery of FV. These experimental results may be used
for screening and selection of FV-free cultures and for control purposes in
the course of vaccine manufacture.
Two different viruses were isolated from bovine embryonic cell cultures after
two subcultures from the primary cells. One virus was identified as
parainfluenza type 2 simian virus 5 (SV-5), and the other was identified as
infectious bovine rhinotracheitis virus. Six months later, stock cultures of
pig kidney (PK-15) cells were found to be contaminated with SV-5 virus. We
believe that the source of the SV-5 virus in the bovine cells was a
cross-contamination from monkey kidneys during preparation of the cell
cultures. The infectious bovine rhinotracheitis contamination was probably of
endogenous origin. The bovine embryonic cell cultures were the probable source
of contamination of the PK-15 cells with SV-5 virus.
Inter- and intraspecies contamination of human breast tumor
cell lines HBC and BrCa5 and other cell cultures.
Nelson-Rees WA, Flandermeyer RR.
It is shown that the two most recently
reported cell lines derived from malignant human breast tissue, HBC
and BrCa5 are, respectively, rat and HeLa
cell contaminants. The incidence of inter- and intraspecies
contamination among 279 cell cultures from 45 laboratories in an 18-month
survey is also presented.
Viral contamination of bovine foetal serum and cell
cultures.
Nuttall PA, Luther PD, Stott EJ.
From the article: The presence of
adventitious viruses in cell cultures is well recognized, and when the
cultures are of primate origin there are serious hazards for the production of
human viral vaccines. This is one reason for the increasing use of
bovine cell cultures. These cultures, however, are not free from viral
contamination. We found that calf kidney (CK) and calt
testis (CT) cells were often infected by non-cytopathic
mucosal disease virus (MDV):
the cells seemed morphologically healthy but nearly all showed fluorescence
with MDV
antiserum and rabbit-anti-bovine conjugate. We report here that both the
cells and foetal
calf serum, an essential growth factor of cell culture medium, are sources of
the virus...These results indicate a much higher incidence of contamination
than reported previously, and illustrate the inadequacy of commercial
screening methods...Infection of bovine cell cultures by non-cytopathicMDV
has particular significance for the production of viral vaccines...The
importance of contamination by
MDV with regard to human viral
vaccines is unknown, but measles virus vaccine and a potential respiratory
syncytial
virus vaccine are produce in bovine kidney cells grown in the presence of
unheated commercial FCS...Regular
screening of bovine cell cultures is essential if stocks are to be kept free
from MDV.
Most laboratories using cells cultured in vitro maintain multiple cell
lines. Such lines should be monitored for species and intraspecies
characteristics to prevent invalidation of research work due to incidents
of cell line cross-contamination. This report describes the results
obtained when 246 cell cultures were examined for evidence of
cross-contamination or mislabeling.
Using species-specific antigens,
isoenzyme
electrophoresis, and chromosomes as markers of identity, 14% of the
cultures submitted were found to be contaminated by cells of another
species. Of human cell lines submitted 25% were of HeLa cell
origin, as determined by 2 intraspecies markers, glucose-6-phosphate
dehydrogenase and chromosome analyses. The fact that, overall, nearly 30%
of the cell lines examined were incorrectly designated makes the
importance of cell line monitoring self-evident.
[Several methodologic problems in the control of cell
cultures]
[Article in Russian]
Demidova SA, Tsareva AA, mikhailova GR, Perekrest VV, Gushchin BV.
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.
Mycoplasmas are common contaminants
of cell cultures and are of great importance because of the deleterious
effects they have upon the infected host cells. Routine detection
testing usually relies upon cultural methods and demonstration of
characteristic colonies on agar. Questions as to the efficacy of this method
have been raised because of increasing realization of the presence of
"non-cultivable" mycoplasmas. Several non-cultural detection methods have
been developed to aid in the detection of these fastidious microorganisms.
These methods include morphology by light and electron microscopy,
immunofluorescence, enzyme assays, autoradiography, sucrose gradient
separation, altered transport of nucleic acid precursors and altered nucleic
acid profiles. A comparison of the relative sensitivities of these methods
will be outlined. The need for, and the feasibility of non-cultural
detection methods as quality control tests in human virus vaccine production
will be discussed.
Spread and control of mycoplasmal infection of cell
cultures.
McGarrity GJ.
Environmental sampling was performed during trypsinization and passage of
3T-6 cell cultures that contained a mean of 4.3 X 10(7) colony forming units
(CFU) per ml supernatant of A. laidlawii. The lip of the culture flask and
the outside of the used pipet were always heavily contaminated. The outside
of the culture flask (3/7), the work surface (8/12) and the outside of a pan
of disinfectant (4/5) were regularly contaminated with mycoplasmas. Airborne
mycoplasmas were detected eight of 32 times (25%) by settling plates;
simultaneous forced-air samplers by two different methods were always
negative. The technician's hands were contaminated two of 15 samples. When
hands were contaminated, more contamination was detected in the environment.
Droplets of A. laidlawii and M. orale inoculated onto work surfaces survived
drying for a minimum of 3 days, even in laminar airflow cabinets.
Twenty-five of 31 (80.6%) cell culture technicians carried M. salivarium in
their throats; only two carried M. orale. It is concluded that
mycoplasma-infected clltures are the most common source of further
infection. Recommendations for prevention and control of mycoplasmal
infection are listed.
Detection of bovine viruses in fetal bovine serum used
in cell culture.
Kniazeff AJ, Wopschall LJ, Hopps HE, Morris CS.
This investigation employed a viral screening method detect endogenous
bovine virus contaminants in commercially supplied fetal bovine serum.
Fifty-one lots of fetal bovine serum from 14 suppliers were examined.
Over 30% of the lots tested were
found to contain bovine viruses; they included bovine virus diarrhea
virus, parainfluenza
type3-like virus, bovine herpesvirus-1, bovine enterovirus
type 4, and an unidentified cytopathogenic
agent. Of the 51 lots, 20 had been pretested
by the suppliers and were considered to be free of known viral
contaminants. Our viral screening methods revealed that five of these
pretested
lots, or 25%, contained endogenous bovine viruses.
Bacteriophages and endotoxin in licensed live-virus
vaccines.
Moody EE, Trousdale MD, Jorgensen JH, Shelokov A.
In confirmation of recent reports,
coliphages
were found in seven of 19 unselected samples of the currently licensed
live-virus vaccines.
Coliphages and pseudomonas
phage were found in 11 and 14, respectively, of the 20 bovine sera
commonly used in the cell culture phase of virus vaccine production. The
same lots of vaccine and serum were examined by the limulus assay for
endotoxin,
another product of bacterial contamination. Eighteen of 20 sera had
detectable endotoxin-like
activity. Our preliminary results suggest that endotoxin
activity may serve as a sensitive indicator of residual products of
previous bacterial contamination, including bacteriophages.
Porcine parvovirus: frequency of naturally occurring
transplacental infection and viral contamination of fetal porcine kidney
cell cultures.
Mengeling WL.
The frequency of naturally occurring transplacental infection of swine with
porcine parvovirus (PPV) and one of the possible consequences of such
infection--the presence of PPV in cell cultures prepared from fetal
tissues--were investigated. Transplacental infection was indicated by the
presence of high titers of hemagglutination inhibiting (HI) antibody for PPV
in serums of 0-day-old, hysterectomy-derived, colostrum-deprived pigs of 3
of 82 litters. All letters were farm-raised dams.
Moreover, cell cultures prepared
from 3 of 49 lots of fetal porcine kidneys (FPK)
collected from an abattoir during an interval of 14 months were found
contaminated with PPV.
Because each lot was usually comprised of kidneys from 2 litters, the
latter finding suggests that 3 of approximately 98 litters were infected.
Prior infection of FPK cell cultures with PPV resulted in only slight
interference of replication of other selected viruses; i.e., porcine
enterovirus (PEV), pseudorabies virus (PRV), vesicular stomatitis virus (VSV),
and hemagglutinating encephalomyelitis virus (HEV). Moreover, PPV and HEV
were propagated in the same cell cultures during 5 serial passages of the
viruses. In contrast, when copropagation of PPV and VSV was attempted, PPV
was not detected after the 2nd serial passage.
[Viral contamination in laboratories and hospital units]
[Article in French]
Chastel C.
Viral contamination is at least as important in hospital laboratories and
wards as contamination by bacteria or microscopic fungi, but it is much more
insidious and sometimes unrecognized. There are two main types: The first
has a purely technical effect and only interest the virologist. This is
contamination of reagents, reference strains, cell cultures, etc.., by
foreign viral agents. It may be the cause of errors on diagnosis or
regrettable errors of interpretation of certain experimental data. It is
most difficult to detect, if not to avoid. The second is much more worrying
as it is liable to cause disease in man, which may induce severe, and even
fatal infections in patients or in the medical, para-medical and technical
personnel. This is the case with type B hepatitis virus which tends to
invade surgical units using extra-corporeal circulation, hemodialysis units
and transplantation units, blood transfusion centres, dental units and even
causes victims in routine laboratories. However, type B hepatitis is not the
only virus which may lead to severe infections; other viruses include:
poxvirus, cytomegalovirus, arbovirus, etc. Finally, other often severe
accidents may occur in research laboratories and in the pharmaceutical
industry, owing to manipulation of dangerous viruses or by contact with
experimental animals, e.g. rodents, or monkeys, which contain the virus in a
latent state, e.g. lymphocytic choriomeningitis, Sabin virus, Marburg virus,
type A hepatitis virus, etc. With regard to such accidents, we are almost
completely powerless from the therapeutic point of view and, even poorly
equipped, from the point of view of prophylaxis.
[Studies on simian viruses as possible contaminants of
inactivated virus vaccines. I. Direct and serologic detection of simian
adenovirus SV20 (author's transl)]
[Article in German]
von Mettenheim AE.
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.
DISCLAIMER: All
information, data, and material contained, presented, or provided here is for
general information purposes only and is not to be construed as reflecting the
knowledge or opinions of the publisher, and is not to be construed or intended
as providing medical or legal advice. The decision whether or not to vaccinate
is an important and complex issue and should be made by you, and you alone, in
consultation with your health care provider.
