Abstract

The clinical histories and brain biopsy findings of three patients with severe stealth virus encephalopathy are reviewed. The patients initially developed symptoms consistent with a chronic fatigue syndrome (CFS). One patient has remained in a vegetative state for several years, while the other two patients have shown significant, although incomplete, recovery. Histologic and electron microscopic studies revealed vacuolated cells with distorted nuclei and various cytoplasmic inclusions suggestive of incomplete viral expression. There was no significant inflammatory response. Viral cultures provided further evidence of stealth viral infections occurring in these patients.

Introduction

Molecular probe and viral culture based assays on patients diagnosed as having the chronic fatigue syndrome (CFS) led to the identification of a group of cytopathic viruses, provisionally termed stealth viruses because they induce little or no inflammatory reaction and because they have previously gone undetected in conventional viral culture assays [1-3]. While the molecular nature of these viruses remains to be fully elucidated, several of them appear to be derived from human and animal herpesviruses [2,3]. As discussed elsewhere, CFS may be part of a wider spectrum of dysfunctional brain syndromes attributed to stealth virus encephalopathy [4]. Support for this concept has come from the detection of stealth viruses in patients with more severe neurological illnesses. This paper presents the histological and electron microscopic changes seen in brain biopsies performed on three patients with severe stealth viral encephalopathies. In each of these patients, the severe encephalopathy was preceded by a CFS-like illness.

Materials and Methods

Case Reports

Patient 1. The 39 year old patient was an elementary school teacher with an excellent work performance record. In January, 1989 she was surprised by a complaint from a parent that she had misspelled several words on a note sent home with one of her pupils. Growing criticisms concerning her work led her to seek employment at a different school and to attend psychological counseling sessions for a stress-related disorder. During the job transition period, she noted difficulty in verbal and written expression, hesitation in making decisions, and increasing fatigue. Her new appointment was as a part time kindergarten teacher. She found herself unable to cope in the classroom primarily because of fatigue, memory loss and a difficulty in expressing her ideas. She sought medical help and, in September 1989, was referred to a neurologist. He confirmed the expressive dysphasia, but with the exception of dysosmia, he found her sensory and motor functions to be essentially intact. A cerebrospinal fluid (CSF) examination was also normal. An MRI examination, however, showed bilateral confluent periventricular lesions involving both the centrum semiovale and corona radiata; greater on the left than right side. There was no gadolinium enhancement. An EEG showed minimal slowing in the centrotemporal regions, more prominent on the left than on the right. The patient's dysphasia continued to worsen. She experienced marked difficulty in word searching and by December 1989, she frequently failed to correctly name even common items. Her writing had also become disorganized and she could not easily comprehend oral or written communications. Her level of consciousness, however, had not diminished. An MRI performed in January 1990 showed similar changes as those noted earlier, with no evidence of progression. A stereotactic brain biopsy was performed from the left frontal periventricular region of increased MRI signal intensity. The patient subsequently received several courses of both acyclovir and prednisone therapy without apparent benefit. The overall severity of her cognitive impairment remained relatively stable over the next two years. A generalized seizure occurred in early 1992 with delayed recovery of consciousness. This episode was followed by increasing motor difficulties with hyper-reflexia and a diminishing level of consciousness. Eventually her illness progressed to a complete loss of all voluntary limb movements. The patient is awake during the day but shows only minimal emotional recognition of her husband and nursing staff. She is quadriplegic and aphasic and has remained in an essentially vegetative state for over 3 years.

Patient 2 is a 30 year old dentist described by a friend a jovial, energetic person when he first met her in May 1994. Gradually over the ensuing year, her demeanor changed with increasing expression of fatigue, insomnia, tension headaches and feelings of paranoia. She was dismissed from the dental practice in which she was employed because of aggressive behavior and conflicts with the director of the dental practice. She was hired by a different dental group, but soon began to arrive late for work and occasionally simply stayed at home without notifying her employer. She began to drink heavily and to consume self-prescribed medications. She complained of excessive fatigue and forgetfulness and became irritable and anti-social. In early May, 1995, she saw a neurologist and was referred to a psychiatrist who admitted her to a psychiatric ward. She was noted to have a flat affect and gave evidence of auditory hallucination and delusions. She also had a profound amnesia and some comprehension difficulty. The patient was fully awake and her physical neurological examination was normal. A CSF examination, however, showed 70 lymphocytes per cu mm with normal protein and glucose. The opening pressure was also increased at 260 mm. The patient was transferred to a medical ward where she became increasingly confused and disorientated. A T1 weighted MRI, with and without gadolinium, was performed 5 days later and reported as normal. The patient's level of consciousness continued to deteriorate. Although, she remained afebrile and displayed no localizing neurological signs, it became apparent that she had a severe encephalitis-like illness. An EEG performed 13 days after admission, showed very slow 0.5-1.5 Hz delta background activity with occasional periodic slow sharp-wave discharges prominent in the left frontotemporal area. The patient had no response to intravenous acyclovir therapy. Negative laboratory results included: Routine viral, fungal and mycobacterial cultures; PCR tests for herpes simplex virus; serological assays for encephalitis viruses (Western and Eastern equine, Venezuelan, California and St. Louis); extensive fungal serology; p24 antigen assay for HIV; and testing for rabies. Six weeks after her admission, a brain biopsy from the left frontal lobe was obtained. Over the next month, the patient became more deeply comatose, requiring a tracheotomy for assisted respiration. She became unresponsive even to painful stimulation. Her temperature began to fluctuate widely and on several occasions reached 103o F before whole body cooling was instituted. She also exhibited periodic, repetitive diencephalic release movements consisting of chewing and of leg motion as if riding a bicycle. The patient was tried on the following additional therapies without apparent benefit: Ganciclovir, foscarnet, alpha-interferon and intravenous gamma globulin. She did, however, begin to gradually show signs of consciousness but, for several weeks, remained either in bed or confined to a chair without any voluntary movement or response to non-pain provoking external stimuli. This phase was followed by a period of gradually increasing motor activity in which the patient began to walk short distances and to assist with dressing. Three months after admission, she began to show emotional responses to her family but was cognitively severely impaired. She was unable to speak and showed inconsistent responses to verbal communications. Considerable improvement progressively occurred over the next four months. She regained her ability to speak and to participate in conversations. She still has limited memory of the events leading up to hospital admission. Her memory for current events is also below average. She is receiving speech therapy because of moderately impaired reading comprehension, word finding and word recognition. She is showing increasing interest in intellectual pursuits but still has significant difficulties with problem solving and attention skills. It is considered unlikely that she will return to professional work.

Patient 3. This 30 year old female had for several years been considered by her friends and employers as being "highly strung" and prone to stress. Still she functioned essentially normally until the end of 1994. Her friends then began to notice subtle but progressive personality changes such as undue brooding over minor incidents occurring at work and in her social life. In March of 1995, she resigned from her job without any provocation, yet reacted as if she had been fired. Over the next 3 months, she struggled with increasing insomnia and fatigue. By July 1995, she had become anorexic and irrational in her thought processes. She complained of auditory hallucinations, terrifying nightmares and weakness and numbness of her right arm. Although, she appeared to recognize family members, she could no longer express herself and her speech was garbled. She was taken under restraint to a county hospital. No localizing signs were found on neurological examination. A lumbar puncture obtained shortly after admission showed 20 red blood cells and 33 wbc per cu mm (90% lymphocytes; 9% monocytes and 1% polymorphonuclear cells). A repeat CSF examination 3 days later, showed only 2 wbc per cu mm. CSF protein and glucose levels were normal. A CT scan with contrast and a MRI were both normal. There was no clinical improvement with a 16 day course of intravenous acyclovir. Rather, the patient experienced deepening coma, hyperpyrexia and mild generalized seizure activity, not accompanied by localizing EEG changes. She was treated with Dilantin and phenobarbitone but became even more deeply comatose. As with the previous patient, extensive infectious disease serological studies were non-contributory. She was transferred unconscious and unresponsive to another hospital where a brain biopsy was performed. The patient occasionally awoke from a deep coma but then exhibited facial grimacing and chewing movements, which were only controlled by further sedation. After 5 weeks of coma requiring continual assisted respiration, the patient became conscious. During the next month, she slowly regained the ability to feed herself and to sit up in bed. She was able to speak and to write limited sentences. Communication was difficult, however, because she was emotionally highly labile and regularly expressed anger and frustration at not understanding "what was going on in her head". She was encouraged to walk but her mobility was restricted because of the development of myositis ossificans in the lower right thigh. Dissatisfaction with her daughter's medical care, led her mother to withdrew the patient from the hospital. Over the next 3 months, the patient showed progressive improvement in her physical and mental activities. She was then able to converse effectively and could read for 1-2 hours each day. Her major complaint was forgetfulness such that she had difficulty recalling even events from the previous day. She had little or no memory of the severity of her earlier illness. She consciously limited activities that would require prolonged concentration and also avoided bright sunlight and strong odors since these factors triggered feelings of mental fatigue and depression.

Results

Brain Histology and Electron Microscopy

Case 1. The abnormal MRI obtained from this patient in January 1990 is shown in Fig 1. Routine histological examination of the stereotactic biopsy was reported as showing mild gliosis with focal gemistocytic change and a minor increase in microglia. A slightly reduced myelin content was also demonstrated on myelin staining. A more detailed examination of additional sections revealed numerous cells with distorted nuclei and discernible vesicular and granular cytoplasmic changes (Figs. 2,3). There was no evidence of a lymphocytic inflammatory response. The biopsy specimen was processed for electron microscopy. Occasional cytoplasmic herpesviral-like particles were seen within vacuolated (lipid containing) glial cells (Fig. 4). More typically, multiple cells could be seen in which there were collections of abnormal, membrane bound, structures which may reflect aberrant expression of a viral genome (Figs. 5,6). Empty, degenerating myelin sheaths provided evidence of axonal loss and demyelination (Fig. 5). Mitochondrial damage was also apparent in many of the vacuolated cells (Figs. 5,6).

Case 2. The brain biopsy showed very minimal inflammation within the meningeal lining. No inflammation was detected within the parenchyma of the brain. Several of the neural cells showed degenerate changes while others showed more subtle evidence of nuclear and cytoplasmic vacuolization (Fig. 7). Finely granular, periodic acid Schiff (PAS) positive inclusions were seen in some of the cells. Electron microscopy confirmed the presence of vacuolated, degenerate cells. Although relatively few viral-like particles could be seen (Fig. 10), most of the affected cells showed extensive accumulations of unusual appearing granular material along with prominent mitochondrial disruption.

Case 3. Again, the brain tissue was striking in the lack of any evidence of inflammation. Finely granular PAS positive material was present, however, within cells with distorted nuclei and subtle, but discernible, cytoplasmic and nuclear vacuolated changes (Fig. 11). Occasional cell syncytia were seen (Fig. 12). Electron microscopy confirmed the presence of vacuolated cells containing abnormal collections of granular material and of small ring shaped structures with translucent cores. Patches of thickened membranes and occasional discrete viral-like particles were also seen (Fig. 13).

Stealth Viral Cultures

The brain biopsies from patient 1 was not cultured for stealth viruses. Positive stealth viral cultures were, however, obtained from the brain biopsies of patients 2 and 3. Blood samples from all of the patients were repeatedly positive for stealth viruses by culture, as were CSF samples collected from patients 2 and 3. A urine culture from patient 2 was also positive. In the culture studies, material from the patients was added to human (MRC-5 and MRHF) fibroblasts and to rhesus monkey (RMK) derived kidney cells. A positive culture was identified by a cytopathic effect (CPE) characterized by rounding and enlargement of clusters of fibroblasts which begin to show a vacuolated foamy cell appearance with evidence of syncytia formation. Figure 14 shows the phase contrast appearance of an early positive culture in MRHF cells from patient 3, while figure 15 shows a stained cell pellet from a positive MRHF culture of patient 2. In each of the three cases described in this paper, the CPE either from brain biopsy, CSF or blood was unequivocal and readily transmissible to secondary and tertiary cultures. Detailed molecular and immunological studies of each of the viral isolates have yet to be performed.

Discussion

The cases presented in this paper were chosen to highlight the severity of illness seen in a sub-set of patients in whom stealth viruses have been detected and to provide a description of the major histopathological and electron microscopic findings seen on brain biopsy. They were also chosen to help emphasize the clinical continuum, even within individual patients, between vague cognitive dysfunction, often accompanied by unexplained fatigue, and much more devastating neurological illness. Each of the patients could have legitimately be labeled as having a CFS-like illness earlier during the course of the disease. This diagnosis was clearly inappropriate as the disease evolved, but these cases do serve to illustrate the difficulty of precisely defining CFS as a discrete clinical entity [4].

Even when fully developed, the clinical picture was not typical of what is generally expected from viral infections within the brain. Thus, the traditional concept of viral encephalitis is that of an acute illness developing rapidly over several days in a patient with a diminishing level of consciousness; localizing signs on neurologic examination and on MRI and marked CSF pleocytosis [5]. Especially in cases of severe encephalitis, for example due to Herpes simplex virus, brain biopsy shows an intense lymphocytic inflammatory response with extensive cellular destruction [5]. The three cases described in this paper clearly did not conform to the expected clinical or histological characteristics of Herpes simplex encephalitis. The illnesses were much more protracted. Even with the onset of severely disabling symptoms, the level of consciousness was not noticeably impaired and there were few if any localizing neurological signs. Transient modest cellular responses were seen in the CSF of two of the three patients, while only one patient showed MRI changes.

Histologically, the predominant cellular changes seen on brain biopsies were nuclear distortion, cytoplasmic vacuolization and accumulations of PAS positive granular material. Electron microscopy confirmed the vacuolated cellular changes. At least part of the cytoplasmic changes appeared to reflect lipid accumulation, possibly a result of impaired mitochondrial function. In contrast to a more typical herpesviral infection, there was a scarcity of fully formed (intact) viral particles. Rather, there was an abundance of granular and other particulate materials within the vacuolated cells. Unpublished studies have suggested that these materials may reflect the dysregulated production of viral proteins from fragments of the stealth viral genome. Some of these components may be responsible for the positive PAS staining seen in many of the affected cells.

A striking histological feature was the lack of widespread inflammation within the brain. The brain biopsies did not even show the limited vasculitis that has been seen in some other cases of stealth viral encephalopathy [6]. The subtle cellular changes and absence of inflammation correlate with similar findings observed in stealth virus inoculated cats [7].

The subtle histological changes seen in the brain biopsies also correlate well with the vacuolated CPE induced in human fibroblasts and in monkey kidney cell cultures [2]. Both the in vitro CPE and the in vivo cellular findings may simply reflect a generalized disruption of cellular metabolism as a consequence of diversion of cellular resources to the synthesis of viral coded components. As discussed elsewhere, varying degrees of severity of viral induced dysfunction of neural cells could result in a wide spectrum of neurological diseases, including the severe diseases experienced by the patients described in this paper [4,8].

Two of the patients showed quite remarkable, even if incomplete, restoration of brain function. In view of the paucity of inflammation, it is unlikely that the recovery is immunologically mediated. Rather, the recovery may be associated with the production of viral inhibitory components, similar to those detected in infrequently fed stealth viral cultures (unpublished observations). Further characterization of these components may offer a novel therapeutic approach to hasten the recovery from a stealth viral encephalopathy.

References

1. Martin WJ: Viral infection in CFS patients; in Hyde B (ed): The Clinical and Scientific Basis of Myalgic Encephalomyelitis/Chronic Fatigue Syndrome. Ottawa, Nightingale Res Found, 1992, pp 325-327.

2. Martin WJ, Zeng LC, Ahmed K, Roy M: Cytomegalovirus-related sequences in an atypical cytopathic virus repeatedly isolated from a patient with the chronic fatigue syndrome. Am J Path 1994; 145:440-451.

3. Martin WJ, Ahmed KN, Zeng LC, et al: African green monkey origin of the atypical cytopathic stealth virus isolated from a patient with chronic fatigue syndrome. Clin Diag Virol 1995;4:93-103.

4. Martin WJ: Stealth viruses as neuropathogens. CAP Today 1994;9:67-70.

5. Whitley RJ, Schlitt M: Encephalitis caused by herpesviruses, including B virus; in Scheld WM, Whitley RJ, Duroek DT (eds): Infections of the Central Nervous System. New York, NY, Raven Press, 1991, pp 41-86.

6. Martin WJ: Stealth virus encephalopathy: Report of a fatal case complicated by cerebral vasculitis. Pathobiology 1996 (in press).

7. Martin WJ, Glass RT: Acute encephalopathy induced in cats with a stealth virus isolated from a patient with chronic fatigue syndrome. Pathobiology 1995;63:115-118.

8. Martin WJ: Stealth virus isolated from an autistic child. J Aut Dev Dis 1995; 25:223-224.

9. Martin WJ: Simian cytomegalovirus-related stealth virus isolated from the CSF of a patient with bi-polar psychosis and acute encepathology. Pathobiology 1996 (in press).