Human Herpesviruses in Chronic Fatigue Syndrome

Patient selection.Our patients were both civilians and veterans of the Persian Gulf War. Veterans from 10 states east of the Mississippi River were evaluated by a screening questionnaire designed to identify those with CFS and those who were in good health. After being identified, veterans came to the Veterans Administration Medical Center (VAMC) in East Orange, N.J., where they were evaluated by a physician in the Gulf War Research Center and divided into those fulfilling the 1994 Centers for Disease Control and Prevention CFS case definition (14) (n = 46) and those in good health (n = 32). Veterans with CFS also underwent a computerized psychiatric interview (3, 25) that was administered by trained personnel to identify those with (n = 33) and without (n = 13) concurrent major axis I psychopathology (e.g., major depression, posttraumatic stress disorder, or a generalized anxiety disorder).

Civilians either were self- or physician referred to the Chronic Fatigue Syndrome Research Center (VAMC, East Orange, N.J.) or were screened by the same means used for veterans. Potential research study participants were then evaluated by a neurologist or nurse practitioner and were grouped into those satisfying the 1994 CFS case definition criteria (n = 76) and those in good health (n = 73). Healthy civilians and those with CFS were further evaluated via the computerized psychiatric interview to provide an axis I diagnosis. None of the healthy civilians had an axis I diagnosis, and the CFS group was divided into those with a concurrent axis I diagnosis (n = 32) and those lacking such a diagnosis (n = 44). In both the veteran and civilian research populations, the CFS patients and healthy controls were carefully matched for age, gender, race, and educational level.

Patient demographics.The Persian Gulf War veteran population was 83% male and 73% Caucasian and had a mean age of 35.3 years (range, 26 to 46). The civilian population was 84% female and 93.5% Caucasian and had a mean age of 35.2 years (range, 26 to 44). In addition, the mean educational levels for the veteran and civilian groups, ± the standard deviations, were 14 ± 1.95 and 15.2 ± 2.60 years, respectively.

Clinical specimens.Heparinized-blood samples were collected from veterans and civilians at the Gulf War and CFS Research Centers at the VMAC in East Orange, N.J. PBMCs were isolated from the heparinized blood by using Ficoll-Hypaque gradients (Pharmacia, Uppsala, Sweden). Thereafter, PBMCs were aliquoted, pelleted, and immediately frozen at −70°C until required. When possible, a serum sample was also taken from the patient for analysis. All patient PBMC pellets and sera collected were numerically encrypted prior to shipment to our laboratory for analysis.

Avoidance of contamination.Great care was taken to avoid specimen contamination during the course of this investigation. Sample preparation and analysis of amplified products were conducted in designated physically separated laboratory areas. All reagents were aliquoted and stored in distinct locations. Only dedicated PCR pipetting equipment and aerosol-resistant barrier pipette tips were used. Meticulous laboratory technique and adherence to standard PCR anticontamination procedures were the norm. A number of positive and negative controls were included in each PCR assay.

DNA extraction.PBMC pellets were resuspended in 450 μl of autoclaved distilled-deionized water and subjected to three rounds of freezing and thawing. Fifty microliters of 10× proteinase K reaction buffer (50 mM KCl, 100 mM Tris-HCl, 15 mM MgCl₂; pH 8.3) and 100 μg of proteinase K (Boehringer Mannheim, Indianapolis, Ind.) were added to the cell lysate. Digestion was performed for 90 min at 65°C; this was followed by heat inactivation at 98°C for 20 min. Alternatively, digestion was allowed to proceed overnight at 50°C with gentle shaking, prior heat inactivation. The resultant crude DNA extract was visually examined for the presence of cellular debris. If such debris was present, the extract was centrifuged at 1,000 ×g for 5 min and the supernatant was used as a template DNA for subsequent PCR analysis. As a rule, mock DNA extracts, consisting of autoclaved deionized water or JJhan cells (a continuous human T-cell line [30]), were prepared concurrently with the patient PBMC samples to serve as internal negative controls.

In some instances, the crude DNA extracts were further purified by phenol-chloroform extraction followed by ethanol precipitation. Briefly, the crude DNA extract was thoroughly mixed with 2 volumes of 25:24:1 (vol/vol/vol) Tris-buffered phenol-chloroform-isoamyl alcohol–0.1% (wt/vol) 8-hydroxyquinoline and centrifuged at 14,000 × g for 5 min at 4°C. The top, clear, aqueous phase was transferred to a new tube and combined with 2 μl of Pellet Paint coprecipitant (Novagen, Madison, Wis.) with 0.1 volume of 3 M sodium acetate, pH 5.2. Thereafter, 2 volumes of −20°C absolute ethanol were added. The DNA was precipitated by a 30-min incubation at −70°C. The DNA was then pelleted by a 4°C centrifugation at 14,000 × g for 15 min, the supernatant was removed, and the DNA pellet was washed with ice-cold 70% ethanol. The DNA pellet was allowed to air dry and then was resuspended in 200 μl of autoclaved deionized water.

HHV6 PCR protocol.PCR was performed with HHV6 primers, denoted HHV6 A and HHV6 B (Table1), that amplify a 161-bp region of a strain-conserved open reading frame (ORF), U30 (16, 20, 21). The ORF U30 gene exhibits the highest degree of sequence homology to the HCMV UL47 gene, which encodes a capsid assembly myosin (16). This set of primers is specific for both HHV6 strains and does not exhibit any cross-reaction with HHV7, EBV, or HCMV (Fig.1).

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Fig. 1.

HHV6 primer specificity control. Shown is a nondenaturing polyacrylamide gel stained with a 1:20,000 dilution of Sybr Green I nucleic acid stain (Molecular Probes). The first lane (M) represents a 100-bp DNA ladder (Gibco BRL Life Technologies). Serial twofold dilutions of the various HHV DNAs, ranging from 1,000 to 125 pg (lanes A to D), were amplified with HHV6 PCR primers A and B for a total of 40 cycles. A prominent 161-bp band can be seen in each of the HHV6 A strain U1102 and HHV6 B strain Z29 lanes. In contrast, no bands are apparent in the HHV7, EBV, or HCMV genomic-DNA dilution lanes. Therefore, we conclude that this primer set is specific for HHV6 alone and does not cross-react with its genetically proximate viral relatives.

Thirty microliters of the crude DNA extract was used as the template DNA for each PCR. A 50-μl PCR reaction mixture consisted of 0.25 μM each primer and 0.2 mM each of four deoxynucleoside triphosphates (Gibco BRL Life Technologies, Grand Island, N.Y.) in 10 mM Tris-HCl (pH 8.3)–50 mM KCl-1.5 mM MgCl₂, and 1 U of Taq DNA polymerase (Gibco BRL Life Technologies). Before being added to the PCR reaction mixture, the Taq DNA polymerase was incubated for 5 min with Taq Start antibody (Clontech Laboratories, Palo Alto, Calif.). Taq Start inactivatesTaq DNA polymerase until it is denatured at 70°C, reducing the amount of mispriming and increasing the chances of amplifying rare target DNAs. Reaction mixtures were initially denatured by heating to 94°C for 5 min and then subjected to 40 successive rounds of cycling as follows: 94°C for 1 min, 55°C for 1 min, and 72°C for 75 s. These cycling conditions were optimized for use with a DNA Thermo Cycler model 480 (Perkin-Elmer Cetus Corporation, Norwalk, Conn.). For each experiment, a number of positive and negative controls were included. The positive controls consisted of serial dilutions of HHV6 genomic DNA. The negative controls consisted of the mock and JJhan cell DNA extracts coupled with water samples that were interspersed within assays to ensure that no cross-contamination occurred during pipetting. Using this PCR protocol, we could routinely detect ≥25 fg of HHV6 genomic DNA. This level of sensitivity permits detection of less than one viral genome copy per cell.

HHV7 PCR protocol.The HHV7 PCR assay followed the protocol for HHV6 with a few modifications. The primer annealing temperature was increased to 58°C and the optimum MgCl₂concentration was determined to be 4.5 mM. The HHV7 primers used, denoted HHV7 #1 and HHV7 #2 (Table 1), amplify a 124-bp region of a conserved gene named ORF U10 (5, 11). These primers are specific for HHV7 DNA alone and do not cross-react with EBV, HCMV, or either strain of HHV6 (data not shown). Again, this PCR protocol permitted the detection of ≥25 fg of HHV7 genomic DNA.

EBV PCR protocol.The EBV PCR protocol was the same as that used for HHV7 except the optimum MgCl₂ concentration was determined to be 2.5 mM. The EBV primers used, denoted EBV #1 and EBV #2 (Table 1), amplify a conserved region of the EBV nuclear antigen 1 (EBNA1)-encoding BKRF1 gene outside the gly-ala repeat that varies among wild-type EBV strains (26). The resultant PCR product is 296 bp in size. These primers are specific for EBV alone and do not cross-react with HHV7, HCMV, or either strain of HHV6 (data not shown). The sensitivity of this protocol is comparable to those for HHV6 and HHV7.

HCMV PCR protocol.The HCMV primers used, denoted HCMV #1 and HCMV #2 (Table 1), specifically amplify a 419-bp region of the highly conserved CMV lower-matrix phosphoprotein pp56 gene (13). These primers were designed by Mary Jo Evans (Children’s Hospital, Buffalo, N.Y.), who graciously provided us with their sequences. Currently, these primer sequences are protected by patent applications (13a). The cycling parameters were essentially the same as for HHV6, but the cycle number was 35. These primers are specific for HCMV alone and do not cross-react with HHV7, EBV, or either strain of HHV6 (data not shown). Once more, we were able to routinely obtain sensitivity levels comparable to those of the other viral PCR assays.

TCR-β PCR protocol.To verify that each patient PBMC DNA extract prepared did indeed contain cellular DNA and to show that inhibitors of PCR amplification were absent, we employed a PCR assay specific for human T-cell receptor beta chain (TCR-β). The human TCR-β gene locus is located on chromosome 7 and is present at a copy number of two per cell genome. The TCR-β primers were designed by Mary Jo Evans, who kindly provided us with their sequences. These primers, denoted TCR-β #1 and TCR-β #2 (Table 1), amplify a 124-bp portion of the TCR-β constant region (13). Each TCR-β primer was used at a final concentration of 0.2 μM. Amplification was carried out as previously described except that the cycling elongation time was shortened to 1 min and the cycle number was reduced to 35. A positive TCR-β result was considered proof that the DNA extraction procedure was successful and that overt PCR inhibitors were absent.

Control DNAs.Positive-control HHV genomic DNAs, including HHV6 strain A (U1102), HHV6 strain B (Z29), HHV7 strain JI, EBV strain B95-8, and HCMV DNAs, were purchased from Advanced Biotechnologies Inc. (Columbia, Md.). The DNA concentrations of these viral DNA stocks were determined spectrophotometrically. The stocks were serially diluted in the presence of 10 ng of purified JJhan cell DNA/μl, and titrations of them near the limits of the PCR protocol’s detection capabilities were included in each PCR assay. These controls ensured that assay conditions were up to par and also allowed us to gauge the relative amount of PCR product amplified in positive patient PBMC samples. Negative controls consisted of autoclaved deionized water or JJhan cell DNA.

Fluorescence-based detection of amplified products.During the course of this study, two slightly different methods were employed to evaluate the amplified PCR products. The first involved mixing 20 μl of the amplified PCR product with 5 μl of a gel loading buffer containing 25 μg of bromphenol blue/ml, 25 μg of xylene cyanol FF, 50% (wt/vol) glycerol, and 1:4,000-diluted Sybr Green I nucleic acid gel stain (Molecular Probes, Eugene, Oreg.) and loading it onto a nondenaturing 5% polyacrylamide gel. The products were electrophoresed for 2 h and 15 min at 125 V, with 0.5× TBE (44.5 mM Tris base, 44.5 mM boric acid, 1 mM EDTA; pH 8.0) as the running buffer. After electrophoresis, without the gel being removed from the glass plates, it was inserted into a Fluor Imager model 575 fluorimeter (Molecular Dynamics, Sunnyvale, Calif.) and scanned. The gel image was projected onto a computer screen, and amplified PCR product bands were directly quantitated by using Image QuanT software (Molecular Dynamics).

Alternatively, 20 μl of the PCR product was mixed with 4 μl of a gel loading buffer containing 20% (wt/vol) Ficoll 400 (Pharmacia Biotech, Piscataway, N.J.), 10 mM Na₂ EDTA (pH 8.0), and 2.5 μg of xylene cyanol FF/ml and loaded into a 2% Ultra Pure Aqua Por low-electroendosmosis agarose gel (National Diagnostics, Atlanta, Ga.). Electrophoresis was allowed to proceed at 125 V for 2 h and 15 min, with 1× TBE as the running buffer. Afterward, the gel was stained for 20 min in a 1-μg/ml ethidium bromide solution. The gel was then destained for 5 min in distilled-deionized water. The PCR products were visualized by using Molecular Analyst Gel Doc 1000 (Bio-Rad Laboratories, Hercules, Calif.). The amplified PCR product bands were directly quantitated by using Molecular Analyst software (Bio-Rad Laboratories). Using either method, the relative amounts of PCR product from the patients’ PBMC samples were determined by linear-regression analysis. For each PCR assay, a standard curve was generated by using known concentrations of viral genomic DNA, with limiting DNA dilutions being run on each gel. Standard curves were deemed acceptable only if they were composed of at least four data points and had a correlation coefficient of at least 0.87. Each patient PBMC sample was tested for the presence of each virus in at least three separate experiments. Only patient samples yielding consistently positive results with both virus and TCR-β primers were considered positive.

Additional controls.To test for the presence of subtler PCR inhibitors, we selected patient PBMC DNA extracts that had tested negative for each of the four HHV DNAs evaluated. We spiked PCR tubes containing 30 μl of these respective patient PBMC template DNAs with known concentrations of the DNAs. The relative amounts of amplified PCR product generated were compared to those generated using the same viral DNA concentrations in the absence of the patient template DNA. We found that the relative amounts of product generated were virtually identical (data not shown). This implies that the likelihood of a false-negative result due to the presence of a Taq DNA polymerase inhibitor in the extract is small.

We also randomly selected crude patient DNA extracts and further purified them as described above. The purified patient DNA extracts were then reevaluated for the presence or absence of viral genomic DNAs (data not shown). We observed similar results whether the sample tested was in the form of a crude or a purified DNA extract.

Statistical analysis.The statistical significance of the results generated from this study was determined by a two-tailed Fisher’s exact test. A P value of ≤0.05 was considered statistically significant.

Virus culture procedure.HHV6 A strain U1102 (12) was propagated in a continuous human CD4⁺-T-cell line known as JJhan (30). Both the cells and virus were kindly donated by Frank J. Jenkins (Uniformed Services University of the Health Sciences, Bethesda, Md.). The cells were grown in RPMI 1640 medium (Gibco BRL Life Technologies) supplemented with 10% heat-inactivated fetal bovine serum, 1 mM l-glutamine, 50 μg of gentamicin/ml, 100 U of penicillin/ml, and 100 μg of streptomycin/ml. Cultures were incubated at 37°C in a humidified 5% CO₂ incubator. For routine propagation of virus, uninfected JJhan cells were cocultured with U1102-infected JJhan cells at a ratio of 5:1 (2). Infected cells were harvested at the peak of infection, usually between 5 and 7 days postinfection, for use in the subsequent experiments. The peak of infection was assessed by the appearance of large “ballooning” refractile cells in culture, indirect immunofluorescence assay (IFA), and HHV6-specific PCR.

Indirect IFA.U1102-infected JJhan cells harvested at the peak of infection and uninfected JJhan cells cultured for the same time period were used as the antigen in indirect IFAs. After being collected, the cells were washed with sterile phosphate-buffered saline (PBS; pH 7.4), air dried onto 10-mm-diameter circular glass slides (Fluoroslides; Erie Scientific Company, Portsmouth, N.H.), and fixed in ice-cold 80% acetone for 10 min. The fixed cells were then washed three times in PBS and stored at −70°C until needed. The primary antibody in this assay was either a dilution (in sterile PBS) of a patient serum or mouse anti-HHV6 p41 monoclonal antibody (designated 9A5D12). This monoclonal antibody, which was used as a positive-control serum, was a gift from N. Balachandran of the University of Kansas Medical Center, Kansas City. It reacts with HHV6 41- and 110-kDa proteins (4). HHV6 P41 is a viral phosphoprotein that is expressed both early and late during infection (4, 9, 30).

Briefly, the fixed cells were incubated at room temperature for 1 h with a 1:100 dilution of antibody. Then the cells were washed three times with sterile PBS prior to incubation for 1 h at room temperature, in the dark, with either a 1:32 dilution of a fluorescein isothiocyanate (FITC)-labeled goat anti-human immunoglobulin G (IgG) (gamma-chain specific) F(ab′)₂ fragment (Sigma Immunochemicals, St. Louis, Mo.) or, if the monoclonal antibody was used, a 1:320 dilution of FITC-labeled goat anti-mouse IgG (Fab specific) F(ab′)₂ fragment (Sigma Immunochemicals). Subsequently, the cells were washed three more times with PBS and then counterstained for 20 s with a 1:100,000 dilution of Evans blue. This was followed by two washings with PBS. Finally, the stained cells were mounted, using Johnsons’ medium (0.1% [wt/vol]p-phenylenediamine, 10% PBS, 90% anhydrous glycerol; pH 8.0), and were observed under a UV microscope.

A positive reaction, indicating the presence of anti-HHV6 antibody, was defined as extensive fluorescent staining of the infected cells compared to that of uninfected JJhan cells. Initially, patient sera were screened for anti-HHV6 antibodies via indirect IFA, using a dilution of 1:100 in PBS. If a positive reaction was observed, the serum was serially diluted twofold to determine the fluorescence end point. If a sample exhibited fluorescent staining in control JJhan cells, it was adsorbed and reevaluated. Adsorption was performed by incubating a 1:100 dilution of the cross-reactive human serum with 3 × 10⁶ JJhan cells overnight at 4°C. The next day, the cells were pelleted by centrifugation at 1,000 × g for 5 min and the adsorbed serum was harvested for subsequent use.

EBV serological analysis.Serological analyses of selected civilian sera for EBV were commercially performed by SmithKline Beecham Laboratories (Philadelphia, Pa.). Titers to EBV viral capsid antigen (VCA) (both IgG and IgM), EBNA1, and immediate-early antigen (IE) were obtained by indirect IFA.