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Article

Human Herpesviruses in Chronic Fatigue Syndrome

Howard L. Wallace II, Benjamin Natelson, William Gause, John Hay
Howard L. Wallace II
Department of Microbiology, State University of New York at Buffalo, Buffalo, New York;
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Benjamin Natelson
Veterans Administration Medical Center, East Orange, New Jersey; and
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William Gause
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, Maryland
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John Hay
Department of Microbiology, State University of New York at Buffalo, Buffalo, New York;
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DOI: 10.1128/CDLI.6.2.216-223.1999
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  • 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.

  • Fig. 2.
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    Fig. 2.

    Detection of HHV6 DNA from human PBMCs. PCR products were resolved on a 2% agarose gel and stained with ethidium bromide. The first lane (M) represents a 100-bp DNA ladder (Gibco BRL Life Technologies). Lane 4 is the reagent control, and lanes 3, 21, 38, and 44 are empty. The HHV6 A strain U1102 DNA titration controls are in lanes 2 and 39 to 43 and represent template DNA quantities of 800, 400, 200, 100, 50, and 25 fg, respectively. Amplified products generated from various patient PBMC DNA extracts are in the remaining lanes. HHV6 DNA was detected in patient sample lanes 6, 8, 12, 14 to 18, 23 to 26, and 28 to 36.

  • Fig. 3.
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    Fig. 3.

    Detection of HHV7 DNA from human PBMCs. PCR products were resolved on a 2% agarose gel and stained with ethidium bromide. The first lane (M) represents a 100-bp DNA ladder (Gibco BRL Life Technologies). Lane 4 is the mock DNA extract control, and lanes 3, 38, and 44 are empty. The HHV7 strain JI genomic-DNA titration controls are in lanes 2 and 39 to 43 and represent template DNA quantities of 800, 400, 200, 100, 50, and 25 fg, respectively. Amplified products generated from various patient PBMC DNA extracts are in the remaining lanes. A prominent positive 124-bp band can be seen in patient lanes 5 to 8, 10, 11, 13 to 18, 22, 27, 28, 30, 33, 35, and 36. Faint positive bands can be seen in lanes 12, 19, 20, 25, 29, 31, and 32.

  • Fig. 4.
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    Fig. 4.

    Detection of EBV DNA from human PBMCs. PCR products were resolved on a 2% agarose gel and stained with ethidium bromide. The first lane (M) represents a 100-bp DNA ladder (Gibco BRL Life Technologies). Lanes 2 and 13 are empty. The EBV strain B95-8 DNA titration controls are in lanes 1 and 14 to 17 and represent template DNA quantities of 800, 400, 200, 100, and 50 fg, respectively. Amplified products generated from various patient PBMC DNA extracts are in the remaining lanes. A prominent positive 296-bp band can be seen in patient lane 6. The remaining patient samples on this gel are negative.

  • Fig. 5.
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    Fig. 5.

    Detection of HCMV DNA from human PBMCs. PCR products were resolved on a 2% agarose gel and stained with ethidium bromide. The first lane (M) represents a 100-bp DNA ladder (Gibco BRL Life Technologies). Lanes 2, 6, and 14 are empty. The HCMV DNA titration controls are in lanes 1 and 15 to 18 and represent template DNA quantities of 800, 400, 200, 100, and 50 fg, respectively. Amplified products generated from various patient PBMC DNA extracts are in the remaining lanes. All of the patient DNA extracts represented on this gel are negative and fail to exhibit a band at 419 bp.

  • Fig. 6.
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    Fig. 6.

    HHV6 serology determined by indirect IFA. (A) The indirect IFA results for 51 healthy civilians and 58 civilian CFS patients are graphically represented. The mean serum anti-HHV6 antibody titers ± the standard deviations were 286 ± 62.9 and 295 ± 62.6 for the healthy and CFS groups, respectively. (B) Shown are the same data as were represented in panel A but after stratification of the CFS patient population with regard to the presence (CFS Axis I) or absence (CFS No Axis I) of concurrent psychological symptoms. The CFS axis I diagnosis group (n = 22) had a mean serum anti-HHV6 antibody titer of 155 ± 59.8, compared to the CFS no-axis I diagnosis group (n = 36) mean titer of 381 ± 91.8.

  • Fig. 7.
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    Fig. 7.

    EBV serology determined by indirect IFA. The indirect-IFA results generated from sera of 42 healthy civilians and 47 civilian CFS patients are graphically represented. The error bars indicate 1 standard deviation from the mean serum antibody titer. The CFS patient population shown was stratified by the presence (CFS Axis I; n = 17) or absence (CFS No Axis I; n = 30) of concurrent psychological symptoms. The mean serum anti-EBV VCA (both IgG [A] and IgM [B]), EBNA1 (C), and IE (D) antibody titers are depicted.

Tables

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  • Table 1.

    Primers used in this study

    Primer pairRefer-ence(s)SenseSequenceGenBank locus (accession no.)Bases spannedPCR product size (bp)Target gene
    TCR-β#113Sense5′GTGTTCCCACCCGAGGTCGCTGTGTTTGAGCC3′C2CDNA16–47124TCR-β
    TCR-β #2Antisense5′GTGCTGACCCCACTGTGCACCTCCTTCCCATT 3′139–110
    HHV6 A16, 20, 21Sense5′TCTCACAGCCCAGGACAATGGATTATATA 3′HHV6AGNM (X83413 )43155–43183161ORF U30 (putative capsid myosin)
    HHV6 BAntisense5′TGAGATCATTCTCCCGTTCTTCTTGAGGG 3′43315–43295
    HHV7 #15, 11Sense5′CAGAAATGATAGACAGATGTTGG 3′HHU43400 (U43400 )15678–15700124ORF U10
    HHV7#2Antisense5′TAGATTTTTTGAAAAAGATTTAATAAC 3′15801–15775
    EBV #126Sense5′AAGGAGGGTGGTTTGGAAAG 3′EBV (V01555 )109331–109350296EBNA1
    EBV#2Antisense5′ AGACAATGGACTCCCTTAGA3′109628–109609
    HCMV#113Sense5′CACGCAACTTTTGGCCGCCACACCTGTCAC 3′HS5PPBC (M15120 )2081–2110419Phosphorylated ma-trix protein pp65
    HCMV #2Antisense5′CACCACGCAGCGGCCCTTGATGTTT 3′2500–2476
  • Table 2.

    Prevalence of HHV6 and HHV7 in the civilian population: healthy adults versus the stratified CFS subsets

    PBMC donorsNo. with virus/no. tested (%)
    HHV6aHHV7bEither HHV6 or HHV7Both HHV6 and HHV7
    Healthy adults19/71 (26.7%)51/73 (69.9%)56/73 (76.7%)14/71 (19.7%)
    CFS patients26/74 (35.1%)58/75 (77.3%)61/76 (80.3%)23/73 (31.5%)
      P valuec0.290.350.690.13
    CFS patients with axis I diagnosis11/32 (34.4%)24/31 (77.4%)27/32 (84.4%)8/31 (25.8%)
    CFS patients without axis I diagnosis15/42 (35.7%)34/44 (77.3%)34/44 (77.3%)15/42 (35.7%)
      P valuec0.550.650.700.17
    CFS patients reporting acute onset17/52 (32.7%)41/53 (77.4%)42/53 (79.3%)42/53 (79.3%)
    CFS patients reporting gradual onset9/22 (40.9%)17/22 (77.3%)19/23 (82.6%)19/23 (82.6%)
      P valuec0.430.600.890.26
    • ↵a Two CFS and healthy adult control donor samples were omitted from the HHV6 PCR analysis because a definitive determination as to the presence or absence of viral DNA could not be made.

    • ↵b One CFS patient sample was omitted from the HHV7 PCR analysis because a definitive determination as to the presence or absence of viral DNA could not be made.

    • ↵c Determined by Fisher’s exact test.

  • Table 3.

    Prevalence of HHV7 in the Persian Gulf War veteran population

    PBMC donor population No. with HHV7/no. tested (%)
    Healthy veteran controls14/32 (43.8%)
    Veterans with CFS22/46 (47.8%)
      P valuea0.82
    Veteran CFS patients with axis I diagnosis13/33 (39.4%)
    Veteran CFS patients without axis I diagnosis9/13 (69.2%)
      P valuea0.19
    Veteran CFS patients reporting acute onset1/5 (20.0%)
    Veteran CFS patients reporting gradual onset21/41 (51.2%)
      P valuea0.48
    • ↵a Determined by Fisher’s exact test.

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Human Herpesviruses in Chronic Fatigue Syndrome
Howard L. Wallace II, Benjamin Natelson, William Gause, John Hay
Clinical and Diagnostic Laboratory Immunology Mar 1999, 6 (2) 216-223; DOI: 10.1128/CDLI.6.2.216-223.1999

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Human Herpesviruses in Chronic Fatigue Syndrome
Howard L. Wallace II, Benjamin Natelson, William Gause, John Hay
Clinical and Diagnostic Laboratory Immunology Mar 1999, 6 (2) 216-223; DOI: 10.1128/CDLI.6.2.216-223.1999
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