ABSTRACT

Because of the sudden onset of "flu-like" symptoms in the vast majority of cases, followed by persistent illness and fatigue over several years, both RNA (retroviruses) and DNA (herpesviruses and enteroviruses) viruses have been suspected to be implicated in the pathogenesis of CFS. In recent years, evidence of the association of some viruses with CFS has progressed, whereas, with some others it has weakened considerably. Thus far, no single virus has been found to be the causative agent of CFS. Reactivation, however, of latent virus or viruses could contribute to the symptomatology of CFS by damaging the immune system either directly or indirectly. In this report we have provided a comprehensive review of the status of research on viral agents which have been investigated for their role in the pathogenesis of CFS.

     The evidence implicating a virus in CFS is specifically based on the sudden onset of "flu-like" symptoms, in the vast majority of cases, followed by persistent illness and fatigue (1-3). Over the years, many different RNA and DNA viruses have been suspected of being associated with CFS, e.g., enterovirus, measles, rubella, influenza, cytomegalovirus (CMV), Epstein-Barr virus (EBV), Human herpesviruses 6 and 7 (HHV-6, HHV-7), hepatitis-C, human T-lymphotropic viruses 1 and 2 (HTLV-I, HTLV-II) -like virus, spuma (foamy) virus and "stealth virus." It has also been suggested that CFS, also known as chronic mononucleosis-like syndrome, NK cell disease, Royal Free Disease, post-viral chronic fatigue syndrome and myalgic encephalitis, may be a neuromuscular disorder, a sequela of an apparent viral illness (2,4). Although reactivation of some viral agents has been observed in CFS patients, thus far, no particular virus has been implicated as the etiological agent in CFS (5). The majority of investigators, and the National Advisory Council of the American Association for CFS, strongly support the hypothesis that CFS may be a result of more than one viral agent, and that the syndrome may be triggered by a variety of organic factors. In the early years of CFS investigation, Professor Peter O. Behan, of the University of Glasgow, Scotland, and Professor Mary Ann Fletcher, of the University of Miami, Florida, proposed that multiple factors are associated with the pathogenesis of CFS. Of particular interest were the immune disturbances which are probably the result of virus infection, and which perhaps account for the clinical features associated with CFS. More recently, Komaroff et al. (1) view CFS as centrally an immunologic disturbance which allows reactivation of latent and ineradicable infectious agents, particularly viruses. The reactivation of such viral agents may only be an epi-phenomenon. Komaroff, however, believes that it is more likely that, once reactivated, these viruses contribute to the morbidity of CFS by directly damaging certain tissues (e.g., pharyngeal mucosa) and indirectly, by eliciting an ongoing immunologic response. On the other hand, Levy (6) believes that, although a viral agent has not been found in CFS, an infectious organism may be the cause of this condition, and may continue to be present in the individual. Levy proposes an alternative concept which he calls "hit and run," based on the lack of recovery of an agent. By this hypothesis, a virus might enter the host, cause immune abnormalities leading to CFS, and then be eliminated. The immune system may, however, not fully recover. According to Levy, it appears that the reduction in NK cell function and/or CD8+ T-cell suppression activity (CD8+ lib+ cells) is involved. Thus the hyperactive immune state resulting from the viral infection, as found in an acute viral illness, continues in the host. Levy further compares CFS with autoimmune disease, where autoimmunity could also be caused by an agent no longer present in the patient, but which has induced a pathogenic course. Thus, according to these hypotheses, CFS appears to be a disorder of the immune system caused initially by an infectious agent. This statement is supported by the fact that frequent immunologic abnormalities are associated with CFS, e.g., depressed NK cell numbers, impaired NK cell function, abnormal CD4+ and CDS+ cell numbers and ratio, T-cell cytotoxicity, abnormalities in the complement cascade, short-term elevation of HLA-DR and CD38+, decreased lymphocyte response to pokeweed mitogen and phytohemagglutinin (PHA), abnormal numbers of B-cells, macro-phages and neutrophil function, IgG subclass deficiencies, skin test allergy and increased expression of ICAM 1 and LFA-1 in the cell (7-10).

     Other than these hypotheses, one must also consider that one viral agent could activate another virus, which, in turn may contribute to disease manifestations. Another possibility could be that there is a direct interaction of two viruses, leading to disease manifestation. Still another possibility exists that a virus may contribute indirectly by inducing certain cytokines production by the activated T-cells or macrophages, e.g., IL-1, IL-2, TNF-alpha, -beta, IL-6 (11-13), These cytokines are known to give rise to a variety of symptoms when administered to individuals (fever, myalgia, nausea, anorexia, fatigue, confusion, hypotension). It is thus conceivable that a virus or combination of several viruses are involved in the pathogenesis of CFS by causing the immune dysregulation, and this, in turn, may lead to reactivation of such viral agents. Therefore, we must investigate the immune system more thoroughly in order to understand the role of virus in CFS.

     In recent years the following RNA and DNA viruses have been investigated in connection with CFS:

     Human herpesviruses, i.e., CMV, EBV, HHV-6 and HHV-7 are ubiquitous agents and their primary infections often result in acute illness (e.g., infectious mononucleosis, roseola infantum) follow by viral latency in various cells of the body. Because of their high prevalence rates of infection in general populations, it is very difficult to prove their causative roles. Because of their high reactivation rates, however, significant and increased antibody titers are highly suggestive that they must play a role in the symptomatology of CFS

     EBV (Human Herpesvirus-4), discovered in 1964, is a member of the gamma herpesvirus sub-family. It is the etiological agent heterophile antibody positive infectious mononucleosis and is transmitted via salivary contact. The oropharynx is the principal site of virus replication and the virus infects B-lymphocytes. The association of EBV with CFS (2,3,9,14-24) was based on:

     In one study, Ablashi et al. (15) found that 25% of the sera &am 300 CFS patients, tested for HHV-6 IgG and EBV-VCA IgG antibody, showed elevated antibodies to both HHV-6 and EBV. In recent studies by Swanink et al. (25) and Ablashi et al. (26) no evidence was found of EBV reactivation in selected CFS patients with high titers of IgG antibody to EBV-VCA and EA. In Swanink's study of 10 CFS patients and 9 healthy controls, immunological regression of in vitro transformed peripheral blood mononuclear cells (PBMC) was equally efficient in patients and controls, whereas, in the study by Ablashi, PBMC from CFS patients failed in culture to show EB virus over a period of three weeks. This suggested that, in these patients, both of these herpesviruses were reactivated. Over the years, evidence of the involvement of EBV in CFS patients is diminishing (22-26). There may, however, be a subset of CFS patients in whom EBV may be a major contributing factor to disease manifestation. Finding antibody to EBV-EA-D suggests that there is reactivation of EBV. It would, therefore, be important to continue to follow these patients looking for an increase or decrease of EA-D antibody, and its corelation with symptoms, particularly after treatment with anti-herpesviral agents, e.g., Acyclovir, Phosphonoformate, Ampligen, and Kutapressin.

     HHV-6 is a member of the herpesviridae and was originally isolated from patients with AIDS and lymphoproliferative disorders in 1986 (27). It is the etiologic agent of Roseola infantum, other febrile illnesses in young children and of non-EBV and non-cytomegalovirus infectious mononucleosis. HHV-6, like human retroviruses, HIV, HTLV-I and II, predominantly infects T-lymphocytes, but can also infect other cell types including fibroblasts, epithelial cells, natural killer cells, rnegacaryocytes, neural cells, and occasionally B-lymphocytes. HHV-6 can be isolated from peripheral blood lymphocytes and with difficulty from saliva. Transmission of HHV-6 is poorly understood (28). There has been considerable interest in investigating its possible role in CFS. As evidenced by the HHV-6 prevalence rate of >85% in the U.S. population, most of us have already been infected with the virus in our first year of life. In most individuals the virus is latent. It may be, thus, that when HHV-6 is reactivated, or during reinfection, it may contribute to CFS. Evidence of the involvement of HHV-6 in CFS (1-3,9,15,19,29-36), compared to that of other human herpesviruses (EBV, CMV, HSV-1 and 2, VZV, HHV-7), is much stronger. The evidence is based on:

     The data from at least three different, independent laboratories outside the U.S. confirmed and extend the findings of Ablashi et al. (15) and Buchwald et al. (29), and others who have reported that the HHV-6 reactivation rate in CFS patients is extremely high, compared to healthy individuals (34,36-37). In a recent study by Yalcin et al. (37), 13 CFS patients and 13 healthy controls from Japan were analyzed for HHV-6 DNA by variant-specific PCR. Seven patients (53.8%) were positive for HHV-6 DNA, while none of the controls was positive. HHV-6 DNA to variant A was detected in the PBMC of 4 patients, another 3 had HHV-6 DNA to variant B. These results suggest activated replication of HHV-6 in CFS patients. Ablashi et al. (26) presented data at the recent Research and Clinical CFS Conference that 65% of CFS patients' PBMC in culture expressed HHV-6 antigens compared to 25% of healthy controls, when tested under code. Using an HHV-6 antigen capture assay to core protein gpl16 (38), 54.7% of culture positive samples were also positive for this protein, compared to only 15% of the healthy controls. Using ELISA to HHV-6 early proteins (P41/38) the mean IgG antibody in CFS patents was 28.3 +/- 3.0, compared to 8.2 +/- 5.8 in healthy donors. These data using three different assays showed a high frequency of HHV-6 reactivation in CFS patients. In more recent findings by Krueger et al. (39) from Germany, 72% of CFS patients exhibiting significantly elevated IgG antibody to HHV-6 was suggestive of ongoing HHV-6 infection. These observations were confirmed by cell culture of patients' PBMC and by antigen capture assay to HHV-6 core protein. By these two assays, 38.6% of patients were positive for virus isolation and core antigen. EBV replication was not detected in these patients. Since HHV-6 is an inducer of monocyte/macrophage cytokines IL-l beta and TNF-alfa (40), it may contribute to CFS in another way. Recently, Lusso et al. (41) showed that HHV-6 can infect and replicate in subsets of NK (natural killer) cells. Because impaired NK cell function and a decrease in NK cell numbers in CFS patients is a consistent finding, it provides evidence that HHV-6 can directly target and kill NK cells, a potential strategy to suppress natural antiviral immunity of the host. There have been no reported studies showing how HHV-6 reactivation contributes to the symptoms of CFS patients, nor has anyone yet reported finding HHV-6 in NK cells of CFS patients.

      HHV-7 was isolated in 1990 from a healthy individual's CD4+ T-lymphocytes after mitogen stimulation (42). HHV-7 was later, independently reported from the peripheral blood mononuclear cells of a CFS patient (43). The prevalence rate of HHV-7 is >85-96% in most of the populations, however, it is approximately 60% in Japan (44). The primary infection of HHV-7 occurs later than HHV-6. Because of its presence in CFS patients, the sera of 30 CFS patients and 17 healthy donors provided by Dr. Komaroff were tested in a blind study for IgG antibody to HHV-7 by IFA. The geometric mean titer of IgG antibody in patients compared to con trols was 87.1 5 3.9 versus 38.9 + 5.5 (P = 0.078 by two-tailed t test). The difference between the titers was not statistically signifi cant, suggesting no elevation of antibody to HHV-7. It did not, however, exclude the association of HHV-7 in some cases of CFS (45). Secchiero et al. (46) detected high titer neutralizing antibody to HHV-7 in sera from patients with CFS, compared to controls, suggesting that HHV-7 in CPS may be related to the immune dys function. More studies are needed to assess whether HHV-7 is reactivated in CFS, as is HHV-6.
 

     Another DNA virus with related sequences to CMV, designated the "stealth virus," was reportedly cultured from a 43-year-old patient by Martin et al. (47). Although viral particles resemble CMV, specific antisera for CMV, HSV and HHV-6 failed to react with cells infected with stealth virus. The PBLs from CFS patients after separation of the mononuclear cells were initially cultured on human foreskin fibro blasts. Cytopathic effects (CPE) were observed, and CPE was transfer able to subsequent cultures. The authors claim that they also cultured the virus from the patient during a 1991 hospital admission. The plasmid 1.5-5-4 from the virus showed partial homology with only CMV, by PCR. Morphology of the virus revealed enveloped viral particles, consistent with a herpesvirus. CPE, however, resembled foamy cell syncytia. The authors also suggested that the virus is present in CFS patients and is non-inflammatory and neurotropic. Since this is only one report from one case, the precise association of this virus in CFS needs further investigation and confirmation by others, as well as isolation from other CFS patients.

     Enteroviruses belong to the family Picoma viridae, and include poliovirus (3 serotypes), Coxsackie A virus (23 serotypes), Coxsackie B virus (6 serotypes), echoviruses (30 sereotypes) and the newer enteroviruses (68-71 serotypes). Human hepatitis virus has also been classified as enterovirus 72. Enteroviruses are small, non-enveloped, icosahedral, single-stranded RNA viruses with 7.5 kilbase genome. Enteroviruses are responsible for a variety of illnesses, with approximately 80% of the infections being asymptomatic. Coxsakie A viruses produce aseptic meningitis, herpangina and hand, foot and mouth disease. Coxsackie B virus, which has been linked to CFS, produces myocarditis and pericarditis, generalized disease in new barns, pneumonia, rashes and common colds. The region of highest homology between different enteroviruses is a 5'LTR, and most investigators choose this DNA probe for PCR work.

     The majority of work on the role of enteroviruses in CFS patients has been done in the UK (48-49). The evidence of circulating anti gen and IgM complexes was found in the majority of CFS patients, with the virus being isolated in 22% of patients. Archard et al. (48) studied 96 CFS patients using molecular hybridization with entero virus group-specific probes to RNA isolated from quadriceps muscle biopsies. Only 20 patients were found positive for enterovi rus RNA, suggesting viral replication. Gow et al. (49) used PCR to detect enteroviral RNA sequences in muscle biopsies from 53% of CFS patients and from 15% of controls. Landay et al. (9) found a higher seroprevalence rate of coxsackie B virus in CFS patients, however, Miller et al. (50) detected no differences between the prevalence rates for CFS patients and controls for Coxsackie B by IgM and neutralizing antibodies. The data presented by Gow et al. (51) at the International CFS Conference held in Albany, NY, in 1992, on the detection of enterovirus sequences and virus from patients with post-viral fatigue syndrome did not find any specific enterovirus type involved in CFS. Contrary to their earlier studies, however, the present data does not show any significant differences between patients and controls for the presence of enterovirus (51).

      Swanink et al. (52) reported at the CFS Research and Clinical Conference that no differences were detected between CFS patients and controls for enteroviral antibodies by complement fixation assay and antigen capture ELISA, since they found similar preva lence rates of VP-1 antigen in sera from patients and controls. Furthermore, enterovirus was not isolated either by direct culture or culture acid elution in any of the stool specimens in Swanink's data, therefore supporting the recent findings of Gow et al. (52), which hypothesize that enteroviruses do not play any role in the etiology of CFS.

     Retroviruses belong to the family Retro viridae and, as such, they are enveloped, negative-stranded RNA viruses with a genome size of 9 kilobases. Human retroviruses are relatively unstable and are quickly inactivated by alcohol, detergents and 0.5% sodium hypo-chlorite solutions. HTLV-I (Figure 2) has been implicated in T-cell leukemia and has been linked to chronic neurologic diseases (tropical spastic parapareses). HTLV-I is poorly contagious and is not easily transmitted by cell-free body fluids. Transmission can take place, however, through blood cell transfusion, contaminated needles, sexual contact and from mother to child through breast milk. Another illness called HTLV-I-associated myelopathy (HAM) has been observed in all parts of the world.

     HTLV-II was first isolated in 1982 from patients with a T-cell variant of hairy cell leukemia. Very little is known about the virus. Recent reports suggest that the virus is prevalent in drug users in New York, New Orleans, England, Milan, Italy, and in other population centers worldwide. HTLV-I and HTLV-II are closely related and show antibody cross reactivity. HTLV-II has not yet been linked to any disease. HIV, although a human retrovirus, is a lentivirus, and a considerable amount of lentiviruses have been reported from animals (horses, cattle, sheep, etc.).

     Among the retroviruses, two major subgroups have been studied in CFS, i.e., HTLV-II-like and spuma viruses (29,32,53-60). First, antibody to HTLV-I and -II were studied by investigators in the laboratory of Dr. Robert Gallo at the National Cancer Institute, NIH, and they found no evidence of HTLV-I or -II in sera obtained from CFS patients from Lake Tahoe. DeFreitas et al. also tested sera from CFS patients from Lake Tahoe and found no antibody to HTLV-I (55). Ablashi and associates found two sera positive, one for HTLV-I and the other for HTLV-II when testing 70 sera provided by Dr. Kornaroff from his CFS patients (unpublished data). The seroprevalence rate of HTLV-I in the U.S. population is <0.01%; however, the rate in Native Americans is >20%. The seroprevalence of HTLV-II is even less, but in IV drug users the rate is between 20-30%. Further studies on antibodies to HTLV-I were performed by Buchwald et al. (29) and Levine et al. (32) in CFS patients and controls. None of the patients or controls exhibited antibody to HTLV-I, nor did Buchwald find evidence of any human retrovirus using PHA-stimulated peripheral blood mononuclear cells(29).
     DeFreiata et al. (55) reported in April 1991 that HTLV_IIlike viral sequences were detected in the peripheral blood lymphocytes obtained from adults and children with CFS. Such vial sequences were not detected in the lymphocytes of healthy individuals. These investigators did detect the antibody to viral gag proteins, by West ern blot, in the sera of CFS patients which contained viral sequences. The finding of an HTLV-II-like virus raised many questions, such as whether the virus is transmitted sexually and how infectious is this virus. Independent reports from CDC (58), from Scotland by Gow et al. (59), and Levy (personal communication) found no evidence of a retrovirus in CFS patients. In fact, no evidence of animal retroviruses has been found in lymphocytes of CFS patients. At the International Conference on CFS/ME in 1992, He neine and co-workers (58) and Gunn and co-workers (60) reported the lack of evidence of a retrovirus in CFS patients. In Gunn's study, which was later published, samples were collected from four different centers, with the study performed on coded samples by Oncore Analytics, Houston, Texas. In the original CARA assay, the CFS patients and healthy controls were 40% positive for both, and in the new CARA, 3% of patients were positive compared with 1.5% of controls. Dr. Gunn's study (60) concluded that the tests performed by Oncore Analytics did not distinguish between CFS cases and matched controls in a blind study. Dr. Gunn further stated that the present results, and those reported by others, did not support the theory of a retroviral etiology in CFS, but such a possibility still exists. Since the test detected HTLV-II-like sequences in patients and controls, one may ask whether these sequences may be nonspecific and cellular rather than viral.

     Spuma viruses (Latin term for foamy viruses) (Figure 3) were first identified in the 1950s as contaminating viral agents which caused cytopathic effects in cultures of rhesus monkey kidney cells. The cytopathology of infected cells is characterized by extensive forma tions of intracellular vacuoles in multinucleated syncytial cells. The first human spuma virus was identified in 1971. Dr. Martin, at the University of California, reports finding spuma virus in a significant number of CFS cases (56). Dr. Levy, in whose laboratory foamy viruses were studied, previously found no evidence of spuma virus in CPS patients. Flugal et aL (57) also found no evidence of spuma virus in CFS or in controls. Test on cell cultures from CFS patients and controls for the cytopathic effects of human spuma virus conducted by Dr. Martin on coded samples did not differentiate between patients and controls for the presence of human spuma virus. This data showed that human spuma virus is not associated with CFS (60).

     At the CFS Research and Clinical Conference 1994, Diack et al. (61) reported the identification of retrovirus particles from the pe ripheral blood lymphocytes of 10/34 CPS patients and none of the controls. The majority of viral particles were similar to the ultra structure of visna virus (lentivirus) and murine leukemia virus. According to the authors, virus-like structures were compatible with various maturation stages of lentivirus. No reverse transcrip tion activity or possible target cell phenotype was found. Considerable work is needed to prove that these ultra-structures resembling a retrovirus are not artifacts.

     More recently, Dale et al. (62) found no hepatitis C virus infection in CFS patients.

      The above review of RNA and DNA viruses in CFS does not identify any specific virus in the etiology of CFS. It is most likely that CFS has more than one causative factor. Some of these may trigger the pathologic changes of CFS, either directly or indirectly, and may not be required to maintain the syndrome. It can be con cluded that the end result of viral infection in CFS is the dysregula tion of the immune system or vice versa. The data reviewed here also show that it is difficult to prove the role of a herpesvirus in CFS, since most of us carry them throughout our lives. Some envi ronmental or organic factors, however, are responsible for the reac tivation of a virus from a latent state. If the virus is reactivated, as suggested by Komaroff, this may contribute directly or indirectly to the pathogenesis of CFS, the end result of which are the abnormali ties of the immune system. The reason for the immune dysfunction in individuals with CFS is unknown, but the immune system must be brought back into balance to enable the reversal of the symptoms associated with this disorder (63). However, individuals with CFS have two basic changes in immune status, e.g., chronic immune activation and poor immune cell function with decreased natural killer cell cytotoxic activity (13). The study of lymphokines in CFS patients holds many promising leads into the pathophysiology of CFS. It is also evident that longitudinal follow-up studies are needed to define the role of any virus in the symptomatology of CFS by correlating the severity of symptoms to viral reactivation. These studies are also needed to help us understand the underlying mechanism leading to the pathogenesis. It is also possible that we may not yet have found the agent which is etiologic, as suggested by Levy.

     If we believe that a reactivated virus or viruses contribute to the symptomatology of CFS, antiviral agents should be recommended to treat the viral infection and to see if such a treatment would offer relief in the disease manifestations.

The authors thank Ms. Louise Lane of Advanced Biotechnolo gies Inc., Columbia, MD, for excellent secretarial assistance. We would also like to thank Dr. A. Komaroff of the Harvard School of Medicine, Boston, MA, Dr. J. Levy of the University of California, San Francisco, CA, and Ms. Orvalene Prewitt of the National Chronic Fatigue Syndrome and Fibromyalgia Association, Kansas City, MO for their helpful comments in the preparation of this article.
 

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Dharam V. Ablashi is affiliated with the Advanced Biotechnologies, Inc., Columbia, MD 21046, and is Adjunct Professor of Microbiology, The Georgetown University School of Medicine, Washington, DC 20007.

Kristine L. Ablashi is affiliated with the Corresponding Office of the American Association for Chronic Fatigue Syndrome and the International Institute of Immunopathology, Olney, MD 20832.

Bernhard Kramarsky, John Bernbaum and James E. Whitman, Jr. are affiliated with the Advanced Biotechnologies, Inc., Columbia, MD 21046.

Gary R. Pearson is affiliated with the Department of Microbiology and Immunology, The Georgetown University School of Medicine, Washington, DC 20007.