Robert J. Suhadolnik, Daniel L Peterson, Paul R Cheney, Susan E. Horvath, Nancy L Reichenbach, Karen O'Brien, Vincent Lombardi, Suzanne WeIsch, Elizaoeth G. Furr, Ramamurthy Charubala, and Wolfgang Pfleiderer
Authors: D.V. Ablashi*+, S. Marsh*, M. Handy*, J. Whitman*, D. Viza**, O. Krueger***, and P.H. Levine****
*Advanced Biotechnologies Inc, Columbia, MD
+Georgetown University School of Medicine, Washington, DC
**Laboratoire d'Immunobiologie, Faculte' de Medecine, Paris, France
***Laboratory of Immunopathology, Pathology Institute, University of Köln, Köln Germany
****George Washington University School of Medicine, Washington, DC
Objective: Human herpesvirus-6 (HHV-6), the etiologic agent of exanthem subitum, some febrile illnesses, and EBV and CMV negative mononucleosis, has been implicated in the pathogenesis of CFS. We evaluated sera/plasma from CFS patients and normal healthy donors for antibody to HHV-6, HHV-7, and HHV-8, as well as peripheral blood mononuclear cells for viral antigens.
Methods: We tested serial samples of peripheral blood mononuclear cells (PBMC) from some of the CFS patients and normal donors for the presence of HHV-6 using an HHV-6 antigen capture assay and virus isolation. Two patients exhibiting active HHV-6 infection in their PBMC were treated with HHV-6 specific transfer factor (TF). Two CFS patients and two healthy blood donors were followed over a period of 30 months for HHV-6 infection.
Results: Using IFA, sera/plasma from 35 CFS patients and 25 normal donors showed that, compared to healthy controls, titers to HHV-6 IgG antibody were only elevated in CFS patients, and no differences either in the antibody distribution or titers were evident between controls and CFS patients for HHV-7 and HHV-8. Furthermore, by the ELISA, IgM antibody to HHV-6 early protein (p4l/38), a marker for virus persistence or reactivation, was detected in 57% of the CFS patients compared to only 8% in the controls. HHV-6 core protein (gp116/64/54) was detected by antigen capture ELISA in 45% of the CFS patients' sera compared to 18% of the controls, further supporting a significantly higher HHV-6 reactivation in CFS patients. Two CFS patients' peripheral blood mononuclear cells (PBMC) collected over a 30-month period showed the presence of HHV-6 core antigen in the plasma at various time intervals, and HHV-6 could be isolated from the PBMC. However, the HHV-6 profile of these patients varied, one having persistent-latent HHV-6 infection, and the other indicating viral reactivation. HHV-6 Variant A was predominant in CFS patients' PBMC (66%) compared to controls where 2/3 isolates were HHV-6 Variant B (33%). No HHV-6 antigens or virus could be detected in the PBMC of two CFS patients after the treatment with HHV-6-specific-TF, further suggesting that HHV-6-TF could be used in conjunction with other therapy in treating CFS patients. Moreover, CFS patients analyzed independently at the University of Köln, Laboratory of Immunopathology, showed that > 75% had active
HHV-6 infection compared to only two of the patients with active HHV-7, and none had HHV-8 infection.
Conclusion: The above data support the concept that HHV-6 reactivation or persistent-latent infection is significantly higher in CFS patients compared to healthy donors, suggesting its role in the pathogenesis of CFS. Since IgG antibody prevalence and titers to HHV-7 and HHV-8 were similar to that of the controls, these two herpesviruses did not appear to be associated with CFS. Additional longitudinal studies are necessary to correlate HHV-6 infection and disease manifestation to assess the role HHV-6 plays as a co-factor in CFS.
Authors: Konstance K Knox Ph.D.*; Joseph H. Brewer, M.D.**, and Donald R. Carrigan, Ph.D*.
*Herpesvirus Diagnostics, Inc. and Institute for Viral Pathogenesis; 12346 W. Layton Ave.; Greenfield, Wisconsin 532281 and
**Infectious Diseases; St. Luke's Hospital; Kansas City, Missouri.
Objective: Data from a number of laboratories have suggested a role for HHV-6 in the pathogenesis of chronic fatigue syndrome (CFS). In the studies described here we sought to explicitly test the hypothesis that a portion of patients with CFS have persistent, active HHV-6 infections.
Methods: Blood samples from patients with CFS were evaluated by a rapid HHV-6 culture procedure. This technique diagnoses active HHV-6 infections by detecting transfer of the virus from the patient's blood leukocytes to a target human cell line. CFS patients from two CFS-oriented clinics, a large infectious disease practice, and blood samples from CFS patients submitted to our laboratory from physicians and clinics around the United States were studied. Clinical characteristics of and multiple blood samples from a group of CFS patients from the infectious disease practice were evaluated in detail.
Results: The cross-sectional (one blood sample per patient) incidence of active HHV-6 infection was 37% (128/349) in the CFS patients, with the incidence being similar at all four sources of samples (range 25% to 47%). This incidence of active HHV-6 infection was significantly higher than the 0% seen in 26 normal controls (p < 0.05). To assess the possibility that HHV-6 infections may be episodic or variable with respect to viral load in patients with CFS, seven patients whose first blood samples were negative for active HHV-6 infection were retested at intervals ranging from 4 to 12 weeks after the initial sample was obtained. Three of the seven patients (43%) were found to be positive for active HHV-6 infection with the second sample. This finding suggested that active HHV-6 infections may be intermittently detectable in patients with CFS. This possibility was examined by testing at least four blood samples from each of four patients with CFS over periods of time ranging from 1 to 5 months. The consecutive blood samples from the four patients were found to be HHV-6 positive 58% (22/38) of the time with the positivity rates for the individual patients ranging from 40% (4/10) to 69% (9/13). These observations suggest that the active HHV-6 infections in patients with CFS are either intermittent or variable with respect to their viral load. Thus, an individual patient's HHV-6 infection status must be assessed using multiple blood samples obtained over a period of weeks or months. Also, the 37% estimate of the incidence of active HHV-6 infections in patients with CFS should be held as a minimal value since the true incidence may be higher (60% to 70%). In the course of these studies it was observed that many HHV-6 positive CFS patients had central nervous system (CNS) involvement in their disease. To formally address this, 25 patients with CNS disease (abnormal SPECT or MRI scans, sensory abnormalities, cognitive defects, etc.) seen in the infectious disease practice were evaluated for active HHV-6 infections. Fourteen of the 25 patients (56%) were HHV-6 positive. This incidence of HHV-6 infection was higher
(p < 0.08) than that seen in total population of unselected CFS patients (37%), suggesting that the selection for CFS with CNS involvement co-selected for active HHV-6 infections. Confirmation of CNS infection with HHV-6 in some patients with CFS was obtained by the detection of HHV-6 DNA in the cerebrospinal fluid (CSF) of 20% (7/35) of the CFS patients studied.
Conclusion: These studies demonstrate that a sizable proportion (30% to 70%) of patients with CFS suffer from an active persistent infection with HHV-6, which may account for all, or many, of the clinical manifestations of their disease. Active HHV-6 infections may be especially prevalent in CFS patients with CNS involvement, consistent with the highly neuroinvasive nature of HHV-6.
Authors: Robert J. Suhadolnik*, Daniel L Peterson**, Paul R Cheney+, Susan E. Horvath*, Nancy L Reichenbach*, Karen O'Brien**, Vincent Lombardi**, Suzanne WeIsch++, Elizaoeth G. Furr+, Ramamurthy Charubala***, and Wolfgang Pfleiderer***
*Temple University School of Medicine, Philadelphia, PA
**Sierra Internal Medicine Associates, Incline Village, NV
+The Cheney Clinic, Charlotte, NC
++Sierra Nevada College, Incline Village, NV;
***Universität Konstanz, Konstanz, Germany.
Objective: We have previously demonstrated a statistically significant dysregulation in key components of the 2',5'-oligoadenylate [2-5A] synthetase / RNase L pathway in individuals with chronic fatigue syndrome (CFS) [1,2]. In CFS, 2-5A synthetase is present predominantly in its activated form, bioactive 2-5A levels are elevated, and RNase L activity is upregulated compared with healthy controls. A novel low molecular weight (LMW) RNase L (37 kDa) has been discovered in CFS . These studies were designed to determine the extent of these phenomena in less severely disabled individuals with CFS and to identify clinical correlates to these biochemical findings.
Methods: Extracts of peripheral blood mononuclear cells [PBMC] were prepared from 56 well-defined individuals who met the CDC case definition for CFS and from 27 age and gender-matched healthy controls from two clinical sites [1,2]. RNase L activity, bioactive 2-5A concentration and LMW RNase L were assayed in PBMC extracts as described [1,2,3].
Results: Consistent with our earlier reports, immune activation was observed in extracts of PBMC from CFS patients compared to matched healthy controls. Statistically significant increases were observed in RNase L activity (p < 0.001), bioactive 2-5A concentration (p < 0.0001) and LMW RNase L (p < 0.007) in CFS patients compared to healthy controls. Karnofsky performance scores [KPS] (patient mean = 57.3) correlated to RNase L activity, 2-5A concentration and LMW RNase L (p < 0.002, p < 0.025, p < 0.05, respectively). In subsets (n = 100) selected for the highest RNase L activity or highest level of LMW RNase L, there was statistically significant positive correlation to alpha interferon levels (p < 0.01 and p < 0.01).
Summary: Our working hypothesis has been that the characteristic clinical signs and symptoms of CFS are associated with the dysregulation of the 2-5A synthetase / RNase L pathway. The results presented here confirm and extend our published reports on the biochemical dysregulation of the 2-5A synthetase / RNase L pathway in individuals who are severely disabled with CFS. Evidence is provided indicating that the RNase L enzyme dysfunction in CFS is more profound and complex than previously reported.
1. R.J. Suhadolnik et al., Clinical Infectious Diseases 18: S96-S 104 (1994).
2. R.J. Suhadolnik et al., In Vivo 8: 599-604 (1994).
3. R.J. Suhadolnik et al., Journal of lnterfrron & Cytokine Research 17: 377-385 (1997).
Acknowledgments: Supported in part by research grants from the CFIDS Association of America.