Copyright, The Nightingale Research Foundation
Detection of Viral Related Sequences in CFS Patients Using the
Polymerase Chain Reaction
W. John Martin M.D., Ph.D., Professor of Pathology, USC School of Medicine,
Los Angeles, California 90033
The chronic fatigue syndrome (CFS) refers to an illness characterized by unexplained fatigue lasting beyond 6 months which results in greater than 50% reduction in an individual's normal level of activity(1). The pathophysiology of this illness is poorly understood with many conflicting theories and few substantiated facts. An exciting new technology, termed the polymerase chain reaction (PCR) has recently become available to investigate whether some forms of this disease are associated with a persistent viral infection. This paper will provide a brief overview of the PCR technique and will describe some of the preliminary findings using PCR assays in CFS patients.
Polymerase Chain Reaction (PCR)
PCR refers to enzymatic amplification of a defined DNA sequence (2,3). It requires the following reactants: target DNA containing the sequence to be amplified; oligonucleotide primers complimentary to the flanking regions, on opposing DNA strands, of the particular segment of double stranded DNA to be replicated, DNA polymerase enzyme, deoxynucleotide triphosphates (dNTP) and buffer. The PCR is performed in a thermal cycling machine. PCR proceeds by denaturing the double stranded DNA molecule by heat; and cooling in the presence of the oligonucleotide primers. Because of their high concentration and greater mobility in solution, the primers bind more rapidly to the target DNA than the slower reannealing process exhibited by the larger complimentary DNA strands. The primer DNA complex provides a substrate for DNA polymerase. In the presence of dNTP, the polymerase will extend the primers in a DNA sythesis reaction. Each newly synthesized strand will be complimentary to the template DNA and will acquire at its 3' end, the sequence complimentary to the other primer used in the PCR. On reheating, the newly formed hybrids will denature, thereby providing two additional template molecules during the next primer annealing step. With each successive cycle of heating, primer annealing and primer extension, there will be an exponential increase in the targeted segment of DNA. Eventually, the reaction will become rate limiting due mainly to competition between primer binding and reannealing of the greatly amplified single DNA molecules synthesized during the PCR. In a typical reaction, however, amplification in the order of 106 fold can be achieved in from 25-30 cycles. The specifically amplified PCR product will be of uniform size corresponding to the distance separating the 5' ends of the two primer binding sites on the opposing strands of the target segment of DNA. It can be identified by electrophoresis in agarose gels and further characterized by a hybridization reaction using a labeled probe reactive with the amplified sequence.
PCR has been applied to the detection of an ever increasing number of human pathogens. Examples include human papillomavirus (4), human immunodeficiency virus-1 (5) and -2 (6), human T lymphotropic virus type I and type II (7), cytomegalovirus (CMV) (8), herpes simplex virus (HSV) (9), Epstein-Barr virus (10), human herpesvirus-6 (HHV-6) (11), hepatitis B virus (12), B16 parvovirus (13), JC and BK viruses (14), rubella virus (15), mycobacteria (16), Toxoplasmosis gondii (17), Trypanosoma cruzi (18), and malaria (19). PCR can be applied to the detection of virtually any pathogen for which even limited DNA (or RNA) sequence information is known and in which a specimen of infected tissue can be readily obtained.
PCR assays can be positive in the absence of detectable antibody responses (20,21) PCR assays can also be more discriminative than conventional serology. Thus, most clinical serological reactions are directed against a mixture of antigenic epitopes which can be variously shared by closely related pathogens. Thus, for example, it is difficult to distinguish HIV-1 from HIV-2 or HTLV-1 from HTLV-II by serology, yet such distinctions can be readily made on the basis of PCR amplification of type specific genetic sequences (6,7).Recently, PCR analysis has led to the detection of different strains of EBV (22), HBV (23) and of Plasmodium vivax malaria (19).
Conventional culture techniques have been established to more of less provide a broad screen for a range of microorganisms. In principle, PCR can also be used as an initial screen for major categories of pathogens. The primer sets required would need to be reactive with conserved regions of bacterial, viral or fungal genomes. Moreover, the PCR and subsequent hybridization reactions may need to be run at a lower than normal stringency. This approach is expecially suited for the detection of new types of pathogens (24).
Application of PCR to CFS Patients
We were interested in applying the PCR to study patients diagnosed as having CFS. We initially chose primer sets reactive with a sequence contained in a recently described herpesvirus termed human herpesvirus-6. Two sets of primers were synthesized based on data kindly provided to us by Dr. S. Josephs working in Dr. Gallo's laboratory. Blood samples obtained from patients seen by Dr. Jay Goldstein were processed by simple lysis and proteinase K digestion. Following the PCR, the products were dot blotted onto nylon membrane and probed with oligonucleotide representing a region of HHV-6 genome that would have been amplified. Blood from healthy donors rarely (<1%) gave a discernable positive hybridization reaction following PCR amplification. With a known isolate of HHV-6, strong 3+ to 4+ responses were obtained. Furthermore, the HHV-6 amplified products were easily detectable on agarose electrophoresis and had the expected sizes as predicted from the regions of homology with primer sets. We next examined over 100 blood samples from CFS patients. Detectable 1+ to 2+ responses were seen in only 3-5% of the patients tested in different assays. The PCR products from CFS patients were not easily visualized on agarose electrophoresis. The variability between assays reflected on the critical importance of the temperatures at which the PCR and subsequent hybridization steps were performed. By lowering the temperatures of primer annealing and primer extension and by performing hybridization at lower than normal stringency, we were able to obtain weak but detectable responses in up to 20% of CFS patients tested. Unfortunately, the reactivity of the positive samples appeared to deteriorate with storage, even at 20C. The data were suggestive, but certainly not conclusive, that CFS patients may harbor a virus related to, but distinguishable from, HHV-6.
We next tested a set of PCR primers reactive with the late antigen of CMV (8) for potential cross-reactivity with all known human herpesviruses. Again under conditions of reduced stringency, CMV, EBV, HHV-6 and HSV gave a clearly discernable hybridization signal. Using similar PCR condition, clinical testing was performed on normal individuals, CMV infected individuals, and CFS patients. While no responses were seen with the normal individuals, positive, but weak, responses were seen with a significant number of CFS patients (approximately 20-30% in different groups tested). The responses seen in CFS patients gave positvie PCR when tested with a second set of primers reactive with sequences within the (I-E) gene of CMV (8). There was also a lack of reactivity with a set of EBV specific primers or with HHV-6 primers run under highly stringent conditions.
Although, the PCR findings distinguished CFS patients a a group, from normal individuals, and possibly distinguished some CFS patients from others, the data were difficult to interpret. If the detectable sequences are of CMV, EBV or HHV6 origin, the data would suggest that the virus is incomplete and only partially represented. More likely, the data reflect a new virus with partial DNA sequence homology with herpesviruses. The low stringency conditions used would allow cross-priming to distantly related viral and even normal cellular DNA sequences. Analyses of agarose gels confirmed that cross-priming was occurring since multiple diffuse bands were seen with DNA from both normal individuals and CFS patients. Even though there was no hybridization to amplified products from normal DNA, the results in the CFS patients could not be confidently ascribed to a specific type of virus.
An ongoing study has provided supprot for the view that some CFS patients may have a persistent neurological infection with an atypical virus. A patient with dysphasia, dyspraxia and MRI scan showing periventricular lesion, recently underwent a stereotactic brain biopsy to exclude lymphoma. Instead, the biopsy showed mild gliosis associated with demyelination. On electron microscopy, enveloped viral particles were seen . The findings with PCR have closely approximated those seen with the CFS patients. Thus, a positive but weak response occurred with the HHV-6 and the cross-reactive CMV reactive primer sets when tested on the brain biopsy sample and on the CSF from this patient. Interestingly, all of the routine chemistries on the CSF, including testing for myelin basic protein, were normal. Attempts to further characterize this neurotropic virus are in progress.
The PCR technology represents a major breakthrough in efforts to detect persistent viral infections. Highly specific assays can be performed providing the exact DNA or RNA sequence is known. The stringency, and therefore, the specificity of the assay has to be compromised when one is searching for an unknown virus using primer sets matched for a known virus. The logical progression is to amplify a product using the cross-reactive primer set; sequence the amplified product; and synthesize perfectly matched primer sets based upon the sequence data.
The preliminary results suggest that the first goal has been achieved. The PCR assay has also provided evidence for neurological disease associated with a persistent atypical viral infection. Clinically, one can distinguish two major sub-groups of CFS patients on the basis of whether the patient has neurological evidence for a thought-processing (cognitive) disorder. Patients with this form of CFS often complain of dysphasia and dyspraxia and have an impediment of short term memory. They appear to have a defect in processing and in expressing higher intellectual functions. Fatigue in these patients may be less prominent and, certainly is less distressing, than the neurological defect. This type of illness may often have an acute onset and follow a fluctuating clinical course. The other broad category of diseas manifestation is primarily one of "rapid draining of energy". It may have features in common with fibromyalgia. Although there may be an acute origin, some of these patients have a life-long pattern of illness. As the PCR technology becomes increasingly applied to CFS patients, it should be possible to test the validity of this distinction and determine if it is based on the type and location of a persistent viral infection.
I wish to thank Dr. Jay Goldstein for allowing me to test some of his patients. I also wish to thank Dr. Rick George, Peymon Javaherbin, Jeanette Santiago, Victor Ramirez, Keith Callahan and Anton Mayr for helping to process blood samples and in establishing the PCR assay for HHV-6. The work was supported by a grant from DiaTech (Cooperative Agreement DPE-5935-A00-5065-00 between the Program for Appropriate Technology in Health and the U.S. Agency for International Development).
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