Author: Redig Mandy
Institution: Biochemistry
Date: September 2005
The lethal members of the various viral families seem to dominate public perceptions of the field of virology. Hepatitis is linked to liver cancer, and Ebola and other hemorrhagic fevers have long been a favorite of novelists and screenwriters. Recent political events have shifted our attention to the specter of smallpox and other putative agents of biological warfare, and the human immunodeficiency virus is tragically reshaping communities in many parts of the world.
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In the midst of such weighty concerns, it is easy to ignore more innocuous viruses, particularly when they don't cause human suffering of any great magnitude. Yet, despite their relatively low profile and the fact that they don't infect humans, viruses such as rodent parvoviruses can cause significant problems, even for those of us who don't have a hamster or gerbil at home. The biological characteristics of rodent parvoviruses and the symptoms they cause in infected rodents combine to make such viruses a serious threat to the validity and efficiency of animal research studies - something upon which human lives do depend. The quest to detect and prevent rodent parvovirus contamination is an untold part of the development of research medicine.
Rodent parvoviruses were first isolated from cell culture stocks in the 1960s, and since that time an ever-increasing number of serologically distinct viruses have been isolated and characterized from both rodent colonies - the virus's natural host - as well as cell culture stocks. With the exception of hamster parvovirus (HaPV) and minute virus of mice immunosuppressive strain (MVMi), nearly all of the rodent parvoviruses result in sub-clinical infections, which means that the infected animals do not show any outward signs of being sick. Instead of causing an observable malaise, the virus localizes to epithelial tissue and silently begins to modulate immune response and alter growth patterns of rapidly dividing cells.
A problem in research rodents
Rodent parvoviruses become a problem when they infect research rodents. Rodents are the most commonly used species of research animal. A range of medical advancements, from prescription medication and vaccinations to developing treatments for diseases and genetic disorders, would not be possible without extensive testing and validation involving rodents. Rodent parvoviruses' destructive potential lies in their ability to alter cellular growth kinetics and immune response, two factors often under scrutiny in animal testing.
Research on immune disorders from diabetes to multiple sclerosis depends upon accurate representation of the immune system, while the issue of cellular growth encompasses an even wider range of research interests. Oncology connections to rodent testing are well known. Cancer is fundamentally a disease of rapidly dividing cells, but many other fields, including the study of neurodegenerative disorders or drug development programs, also require experiments that attempt to answer questions about cellular growth. In addition, since rodent parvoviruses can contaminate animal products (cell lines, blood samples, sera samples) in addition to the animals themselves, even research protocols that do not use animals directly can be affected.
The problems of parvovirus contamination become even more vexing when the biological characteristics of the viruses are considered. Rodent parvoviruses have been identified in the urine, feces, saliva, nasopharyngeal region, and lungs of infected animals, indicating that it can be spread through bodily excretions and possibly even expired air. Parvoviruses can survive almost indefinitely at -80oC and can only be destroyed following extensive exposure to caustic viricides and ultraviolet radiation. If such a virus is introduced into an animal facility, it can silently infect a few animals, even just one, and in a matter of days be transferred throughout the facility and beyond.
Any investigator or technician contact with contaminated cages becomes another entry-exit source for the virus. Just a few virions on the gloves or shoecovers of the person in question and the virus has escaped into the hall, where it can sit on the floor until someone else's shoes pick it up and take it into a lab full of cell culture stocks. With the possibility of multiple laboratory collaboration, it is even possible for the virus to be transported from one facility to another through shipments of cell lines or other such samples.
Preventing parvovirus contamination
The key to preventing parvovirus contamination is effective detection, so that a potential outbreak can be prevented before it begins. Until recently, serology testing was considered the best way to identify parvoviral contamination in rodent colonies. The similar technique of rodent antibody production (RAP) testing serves as a corollary to identify contamination in biological materials such as cell culture or tissue samples. Serology tests are based on the identification of antibodies, a protein produced by the immune system specific to the antigen (in this case parvovirus) causing the response. The presence of parvovirus-specific antibodies in the blood of a research rodent is assumed to be the result of parvovirus infection.
A similar strategy is employed in RAP testing. Test rodents are exposed to samples thought to be contaminated, and after 21 days their blood is examined for the presence of anti-parvovirus antibodies; a positive RAP result is assumed to result from parvovirus in the initial sample triggering an immune response in the test animal.
Unfortunately, both RAP testing and serology are expensive and time-consuming since they require adequate time for an antibody response to be generated, and are not highly sensitive due to their propensity for both false positive and false negative results. Non-specific antibody interactions can signal a false positive while inadequate antibody production can give a negative result when in fact parvovirus contamination is present. The key to the problem with RAP testing is that it relies upon a protein intermediary, the antibody, to identify parvovirus contamination.
The advent of polymerase chain reaction (PCR), a molecular technique that identifies specific regions of DNA, represented a great improvement over serology because of the technique's ability to detect the virus directly rather than relying on an antibody intermediate. PCR reactions can also be completed in a matter of hours rather than weeks. In the mid-1990s, PCR assays to detect rodent parvoviruses were developed and incorporated into existing testing protocols.
Yet even with PCR, problems still exist. The PCR assay itself cannot distinguish between target DNA legitimately present in the sample being tested and target DNA introduced on the gloves of the person running the assay. Animal facilities need to perform comprehensive, continual screening for parvoviruses; while the cost of PCR is reasonable in an experimental setting, it can be prohibitive in light of a never-ending stream of samples requiring testing.
A recent detection techique
For these reasons, a recent technique known as fluorogenic nuclease PCR appears particularly promising because of its ability to retain the positive benefits of conventional solution PCR while eliminating some of the drawbacks. Fluorogenic PCR is still based upon direct targeting of DNA, but in addition to primers, the assay also incorporates a fluorogenic probe that must anneal between the two primers in order for a positive signal to be generated. This additional step increases both the specificity and sensitivity of the assay with some published results indicating that specific assays can detect down to a single copy of the assay target in a testing sample.
In addition, the direct relationship between the assay's fluorogenic signal and the amount of assay target present in a given sample means that the assay can be quantitative. Finally, the ability to scale down the total volume of reagents used for each sample means that fluorogenic PCR can be suited for the high-throughput testing needed to continually screen a large volume of samples for parvovirus.
Beginning in 1999, efforts in the laboratory of Dr. David Besselsen at the University of Arizona centered around using fluorogenic nuclease PCR to detect viral contaminants, including parvoviruses, known to be problematic in medical research. Bioinformatic techniques were used to compare all known rodent parvoviral sequences in order to identify both conserved and unique regions. Three distinct fluorogenic nuclease assays were developed, one targeting all rodent parvoviruses, one targeting mouse parvovirus strains (MPV), and one targeting MVM strains.
Analysis of all three assays showed them to be specific for their intended targets and remarkably sensitive, able to detect a mere 10 copies of virus in a spiked sample. The MPV assay was the most sensitive, able to detect a single copy of its target virus. Animal infectivity studies demonstrated the in vivo utility of such assays, and with the publication of the primer and probe sequences, it is hoped the assays will become a part of the repertoire of techniques available for animal screening.
With the continual improvement of techniques used to identify parvoviruses, it is possible for investigators to remain just far enough ahead of the viruses to prevent a widespread outbreak. In some ways then, the fact that most people are unaware of parvoviruses can actually be construed as a positive sign; despite the dangers posed to animal-based research, rodent parvoviruses have not yet been able to permanently impede the progress of medical science.
Suggested Reading
Besselsen D.G., C.L. Besch-Williford, D.J. Pintel et al. "Detection of Newly Recognized Rodent Parvoviruses by PCR." Journal of Clinical Microbiology 33(11):2859-2863, 1995.
Besselsen DG, Pintel DJ, Besch-Williford CL, Purdy GA, Franklin CL, Hook RR Jr, Riley LK. "Molecular Characterization of Newly Recognized Rodent Parvoviruses." Journal of General Virology 77(5):899-911, 1996.
Besselsen D.G., A.M. Wagner and J.K. Loganbill. "Effect of mouse strain and age on detection of mouse parvovirus 1 by use of serologic testing and polymerase chain reaction analysis." Comparative Medicine 50(5):498-502 2000.
Crawford L.V., E.A. Follett, M.G. Burdon et al. "The DNA of a minute virus of mice." Journal of General Virology 4(1): 37-46, 1969.
Jacoby R.O., L.J. Ball-Goodrich, D.G. Besselsen et al. "Rodent Parvovirus Infections." Laboratory Animal. Science 46(4):370-380, 1996.
Kendall L.V., D.G. Besselsen, L.K. Riley. "Technology Update: Fluorogenic 5 Nuclease PCR." Contemporary Topics in Laboratory Animal Science 39(5):41, 2000.
McKisic M.D. et al. "Identification and propagation of a putative immunosuppressive orphan parvovirus in cloned T cells." Journal of Immunology 150(2):419-428, 1993.
McKisic M.D. et al. "Mouse parvovirus infection potentiates allogeneic skin graft rejection and induces syngeneic graft." Transplantation 65(11): 1436-1446 1998.
Radich, J.P. "The detection and significance of minimal residual disease in chronic myeloid leukemia." Medicina 60 Suppl 2:66-70, 2000.
Redig A.J. and D.G. Besselsen. "Detection of Rodent Parvoviruses by Fluorogenic Nuclease Polymerase Chain Reaction." Comparative Medicine 51:326-331, 2001.