Retrovirus – like structures in cfs

SUMMARY

Structures consistent in size, shape and character with all identifiable stages of a Lentvirus replicative cycle were observed by electron microscopy in 12-day peripheral-blood lymphocyte cultures from 10 of 17 Chronic Fatigue Syndrome patients and not in controls. Attempts to identity a lymphoid phenotype containing these structures by immunogold labeling failed and the results of reverse-transcriptase assay of culture supernatants were equivocal. The study was blind and case-controlled, patients being paired with age, sex and ethnically matched healthy volunteers. Pre-screening of subjects included the common metabolic and immunological disorders, functional conditions and a virus screen against hepatitis B and C, Epstein-Barr Virus, Cytomegalovirus and Human Immunodeficiency Virus.

INTRODUCTION.

The outbreak of CFS amongst back-country farmers in Western Otago, Southern New Zealand, which occurred in the mid- 1980's, displayed features suggesting an infectious aetiology in this particular group. Before the condition was identified with CFS, sufferers referred to the condition amongst themselves as 'Poor Mans' AIDS' and the absence of common colds was widely noted. Indeed, the advent of a cold was held to be a sign of recovery (Snow, pers corn.). This was ascribed to high levels of circulating interferon (IFN-0:), a finding which was later noted elsewhere (Lever et al, 1988; Gin et al, 1989) and to which fi~rther weight is now added by a recent study using a rat model ( Saphier, 1994). Since IFN-0: is induced only by intracellular, foreign, nucleic acid, it inferred the presence of a chronic, persistent, virus infection. In addition, the symptoms were, as the patients suggested, markedly similar to the AIDS-Related Complex (ARC). Accordingly, with the emergence of findings suggesting immune disturbances in CFS patients (Behan et al, 1985; Tosato et al, 1985; Lloyd et al, 1989), evidence was sought for the presence of a chronic, lymphotropic virus in CFS patients' peripheral blood lymphocytes (PBL). The observations reported here are from the fourth of a series of case-controlled clinical trials. Unlike the earlier trials, the patients taking part in the present study were longer-term cases who had all been suffering from CFS for more than 4 years.

METHODS

Volunteers

Subjects comprised 20 CFS patients who meet the current CDC CFS case definition (Fukuda et al, 1994) with a duration of symptoms m excess of 4 years (mean duration 6.1 years) paired, age, sex and ethnically matched with 20 healthy controls. Ages ranged from 17 to 51 years (mean 35.3, SD 11.4). PBL specimens were taken by antecubital venepuncture between 11 am and noon to minimise circadian variation in circulation kinetics (Ritchie et al, 1983). Subjects were screened for common metabolic and immunological disorders, fimctional conditions and for the viruses HIV, Epstein-Barr virus, Cytomegalovirus, Hepatitis B and Hepatitis C. They were also ranked clinically on a crudely subjective 10-point scale based on the degree of curtailment of their physical activity. All experimental procedures were carried out blind. Three pairs were disqualified from the final group which comprised 4 male and 13 female pairs. Of those disqualified, one patient had an equivocal reaction to Hepatitis C screening, later shown to be negative, one control partner showed cytological evidence of chronic infection and another control partner proved to be a sibling of the patient. Informed consent was obtained from all participants after the nature of the procedures was fully explained.

Specimens

Twelve-day PBL cultures were established in RPMI 1640 medium containing 10% Foetal calf serum (Gibco) in 25 cm2 tissue culture flasks (Falcon). They were treated with 1 ug mL-1 of the mitogen, Concanavilin A and 4 days later with 4.5 ng nL-1 interleukin-2. Culture sup ernatants were assayed for reverse-transcriptase (RT) activity using a digoxygenin-biotin labeled kitset assay (Boehringer-Mannhieim). The cells were examined for the presence of virus-like structures by electron microscopy (EM) and labeled with immunogold-conjugated antiphenotypic monoclonal antibody markers for CD2+, CD4+, CD8+, B and Macrophage (Canterbury Health Labs) in an attempt to identity the phenotype(s) of the leucocyte(s) in which the structures were observed. Attempts to label PBLs with Natural Killer cell (NK) markers failed. Specimens for EM were embedded in Agar 100 resin, sectioned at 80 nm, stained with uranyl acetate, counterstained with lead citrate and viewed on 3 nm nickel grids.

RESULTS:

RT Assay:

Evidence of RT activity was equivocal. Activity was detected in 5 patients and 1 control at levels between 0.001 and 0.027 ng mL-1 but the differences failed to reach significant levels by paired or unpaired analysis (data not shown).

EM Studies:

Structures m aggregate consistent in size and appearance with every stage of a lentivirus Ilfe cycle were identified from 10 of the 34 subjects. When the codes were broken all 10 proved to be from CFS patients. There was no correlation between the patients' percieved severity of symptoms and the presence of particle-like structures (table 1). Of the retroviruses used for comparisons, the majority of these structures most closely resembled Visnavirus morphologically. Although immunogold-labeled cells were identified, none contained structures resembling virus particles.

Structures resembling laminar bodies were observed in grids from 5 patients but were clearly defined and separate from other intracytoplasmic material in only one. The structures were 120 nm in diameter, discreet, 40 nm wider than, and anatomically remote from, elements of Golgi apparatus (fig 1). Others resembling immature intracellular forms were observed in grids from 2 patients (fig 2) and structures consistent with budding particles at various stages of development were observed in 4 more (fig 3). Structures resembling immature enveloped particles were observed in grids from 2 patients (Fig 4) and others resembling mature, free enveloped particles from a further 8 (Fig 5). They ranged from 90 to 110 nm in diameter.

DISCUSSION.

Studies of CFS aimed at investigating possible infectious aetiologies have been plagued by the associated presence of a series of relatively low-grade or opportunistic pathogens. Following their isolation from CFS patients, causal roles have been assigned to a wide range of microbiota including Candida albicans, Borrelia burgdorfrri , Brucellae, EBV, Enteroviruses and Herpesvirus Horninis Type 6 (Bell, 1994; Swaninck et al,1994; Coyle et al,1994; Calabrese etal, 1992; Kionoff 1992; Renfro etal, 1989). None of them have survived close scrutiny as causal agents and now it seems likely that they are all epiphenomena. For this reason the data offered above, while provocative, must be viewed with considerable caution.

The presence of low-grade or opportunistic pathogens, by their very nature, suggests an underlying failure of immunosurveillance mechanisms in CFS and this has been borne out by reports of NK cell dysfimction (Caligiuri et at, 1987; Morrison et at, 1991; Ho-Yen et at, 1991; Gupta and Veyuvegula, 1991; Qjo-Amaize et at, 1994). Nevertheless, while the condition is consistent with an aetiological scenario based on a lymphotropic retrovirus, there is no hard evidence that any known human retrovirus is associated with CFS (Guim et at, 1993). Early studies in this laboratory indicated the presence of low levels of RT activity in CFS patients (Holmes, 1992) but they were not evident in the present trials.

More importantly, if these structures truly represent a retrovirus, immunogold phenotypic markers failed to identity a target cell. In this context it is unfortunate that the NK marker either failed to attach to NK cells or did not accept the antispecies conjugate, since the available evidence would favour NK cells as the target phenotype. With any new virus isolate the major priority is to grow it in the laboratory which, of course, requires in vitro culture of an appropriate host cell. The structures could still be artefactual but that likehhood is reduced by the lact that they were found consistently in patients and not in controls. However, even if they are virus, morphological criteria alone are insufficient to positively identity them as retroviruses. Furthermore, if they are retroviruses, identifying and growing them is insufficient in itself to ascribe to them a causal role in CFS. While they merit reconsideration as such, they could still be epiphenoma or reactivated endogenous forms. Notwithstanding such extensive caveats, the findings suggest that a new human lymphotropic retrovirus should be considered as a possible candidate for an aetiological agent in at least some cases of CFS.

This final reservation is made because it is beginning to seem likely that there are aetiologically distinct forms of CFS with different time courses and recovery rates. The prognosis for 'epidemic' forms, for example, appears to be much better than for 'sporadic' cases. Indeed, the assignment of separate aetiological categories for the condition is currently under consideration by task forces for the British Medical Research council and the American CDC.

Michael J Holmes Damian S Diack, Richard A Easingwood, John P Cross and Bronwyn Carlisle

Acknowledgments

The authors wish to thank the Association of New Zealand ME Societies (Inc) for funding this study, Dr P 0 Snow and Dr R Sinclair for collecting CFS cases and the patient and control volunteers for taking pan in it.

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