An infectious illness, attributed to atypically structured cytopathic "stealth" viruses, occurred in 1996 in the Mohave Valley region of the United States. A stealth virus infected child from this region has developed a severe non-inflammatory, vacuolating (spongiform) encephalopathy. The illness initially presented as a behavioral problem without overt neurological signs. Extensive investigations, including repeated magnetic resonance imaging (MRI), two brain biopsies and stealth virus cultures, have helped define the disease process occurring in this child. Modest clinical benefit with possible retardation of disease progression occurred during a 6 weeks course of ganciclovir therapy. The potential contributing role of stealth virus infections in children presenting with behavioral problems needs to be addressed.

The potential emergence of new pathogens is a constant threat to human and to animal welfare. While epidemics of acute infectious illnesses are readily recognized, chronic infectious diseases have not infrequently been initially ascribed to other causes (1,2). This situation applies especially to diseases for which strict clinical diagnostic criteria are lacking, and in which social stress appears to provide an alternative explanation (3,4). For example, suggestions of an infectious cause of the chronic fatigue syndrome have been viewed skeptically by much of the medical profession (5) and, unfortunately, also by many Public Health officials (6). This response attended the report of a viral-like illness appearing in the Mohave Valley in early 1996 (7). The type of virus implicated in this outbreak has been previously designated "stealth," to help underscore the ability of certain atypically structured viruses to induce a cytopathic effect (CPE), yet fail to evoke an effective inflammatory response (8-15).

Several hundred stealth virus infected patients from the Mohave Valley and surrounding regions, have since been identified, many with complex neurological and neuropsychairtic illnesses. In the absence of more extensive investigations, it has been difficult to confidently attribute the symptoms in each of these patients to a direct stealth virus infection of the brain. This paper presents the findings, obtained on MRI and brain biopsy, of a virus culture positive child, with a severe vacuolating (spongiform) encephalopathy.

Clinical History: The early clinical manifestations of the patient’s illness were retrospectively identified by his second grade schoolteacher. At the beginning of the school year in September 1997, his writing skills were less than expected from his first grade performance. A slight hearing problem, that had been present since birth, was seemingly causing growing problems in communicating classroom instructions. More noticeably, the child was beginning to change emotionally. With increasing frequency, he displayed episodic signs of social ineptness, sadness, anger and diminished motivation. By early 1998, his behavior was such that he was twice temporarily suspended from school. Between bouts of impulsive, defiant, and inappropriate aggressive behavior, he would express remorse and frustration at losing control. He soiled his clothes from uncontrolled diarrhea and urinated once on the bathroom floor. On one occasion, he complained to his teacher of having a stiff neck. On another day, he said one of his legs felt weak. He occasionally complained of muscle and joint pains and also mentioned he had "blood in his pee." He became easily fatigued and would often sleep during afternoon classes. He had a brief flu-like illness with headaches and vomiting. He failed to complete homework assignments, and had difficulties following class readings. Yet at other times he appeared to function normally. The inconsistent nature of his complaints and his inattention to school work suggested he was "acting out," possibly in response to his parents separation and filing for divorce. He was labeled as having an attention deficit disorder and the decision was made to more strictly enforce school discipline. The child was noted to be somewhat clumsy during a party held for his 8^th birthday on March 24, 1998. In April 1998, he began to complain of seeing double when tired or stressed. Headaches and morning vomiting recurred. He became more aggressive and, in a rage reaction, even threatened to knife his brother. A medical examination included a CT scan that was reported as abnormal. He was then referred to a regional neurologist specializing in children.

The detailed neurological examination conducted on April 17, 1998, confirmed slight weakness of the right lateral rectus muscle (6^th nerve). Otherwise muscle tone and strength were reported as normal, as was the sensory examination. The fundi revealed possible blurring of the disc margins, providing suggestive evidence of papillodema. An MRI performed the same day showed "fairly extensive white matter disease present with a frontal lobe predominance." The findings were suggestive of a leukodystrophy. A repeat MRI performed on May 22, 1998 was "not considerably altered" but thought to "more likely reflect a butterfly glioma than a leukodystrophy." Cerebrospinal fluid (CSF) examinations were unremarkable.

A frontal lobe brain biopsy was performed in late May, 1998. Histologically, the white matter was described as having a "Swiss cheese" appearance. Prominent fat staining was seen on the frozen section. The slides were reviewed at several medical centers with an overall impression of a spongy (vacuolar) myelinopathy. There was "no evidence of an inflammatory response." The gray matter was considered unremarkable. The possibility of a glioma was not excluded, but was not supported by a 4-locus search for "loss of heterozygosity" when comparing DNA extracted from the "normal gray matter" with that of the "abnormal white matter." Biochemical studies also excluded X-linked adrenoleukodystrophy.

The child was referred to several major medical centers for further evaluations. An MRI performed on June 17, 1998, again showed no evidence of disease progression. It did confirm the "marked diffuse deep white matter and some T2 signal hyperintensity involving the entire frontal lobe bilaterally crossing and expanding the corpus callosum through its anterior and body. No evidence of post gadolinium contrast enhancement (was) noted." A stereotactic brain biopsy was performed from the left frontal lobe. Again it was reported as showing a vacuolating myelinopathy with additional opinion of possibly "an inflammatory gliomatosis without the inflammation." Electron microscopy on the biopsy sample confirmed the presence of "atypical astrocytes with interlacing bundles of intermediate filaments within the cytoplasmic ‘bellies.’ Myelin blebbing (was) also apparent." Diagnostic "considerations included a demyelinating/dysmyelinating process and a tumor such as a gliomatosis."

The child’s clinical condition slowly deteriorated. By early September, 1998, he was unable to walk unaided because of muscle weakness, especially on the right side. He was also experiencing recurrent severe headaches. He was dysphasic, especially with regards to naming objects, and had an impaired memory of recent events. He had photopbobia, paraesthesia and was constantly tired. His schoolteacher remarked that she was unable to make "emotional contact" during a recent home visit. Therapy had included steroids, prescribed on the suggestion of papillodema, opiate patches for headaches, intravenous gamma globulin and vitamins. In late September 1998, he was started on intravenous ganciclovir (250 mg bid for 7 days, followed by 250mg daily for 5 additional weeks). Significant overall clinical improvement was noted by his mother during the second week of therapy, and confirmed by the treating neurologist. Muscle strength in the right upper arm increased by 20-30% and diplopia was far less frequent. Although the illness has appeared to stabilized, the child has experienced several episodes of dehydration from anorexia, requiring brief periods of hospitalization. The child is prone to emotional outbursts, depressed and still experiences headaches, relieved only with narcotics. He is confined to a wheelchair for any outings and efforts at schooling have been discontinued.

The tissue block from the first brain biopsy, and unstained slides and electron microscopy grids from the second brain biopsy, were provided for review. The sections were stained with periodic acid Schiff (PAS) reagent. The histological section showed spongiform change with readily apparent large vacuoles and numerous smaller vacuoles. (Fig. 1A and B) While relatively intact (Fig. 1C), the gray matter showed blood vessels with unevenly deposited PAS-positive materials (Fig. 1D). Atypical cells, including apparent syncytia, and cells displaying nuclear and cytoplasmic vacuolization with PAS-positive inclusions, could also be identified (Fig. 1E and F).

Electron microscopy confirmed the presence of markedly vacuolated cells (Fig. 2), cell syncytia (Fig. 3), myelin sheath blebbing (Fig. 4), and various atypical inclusion-like bodies (Fig.4). Occasional particles, consistent with a herpes-like virus, were noted. (Fig.4).

An MRI showing bilateral involvement of the frontal lobes with extension across the corpus callosum is shown in Fig. 5. Another view of the brain showed hypointense areas involving basal ganglia (not shown).

Mononuclear cells were isolated using Ficoll-Paque (Pharmacia, Sweden) separation of whole blood that had been collected two days previously in an ACD solution A vacutainer (Becton Dickinson, New Jersey). An aliquot of the cells was fixed and examined by electron microscopy. Early degenerative changes, accompanied by vacuolization, was noted (Fig. 6). A further aliquot of mononuclear cells was co-cultured with human MRC-5 fibroblasts. ⁷ A marked vacuolating CPE with considerable cell destruction and syncytia formation, was detectable within 24 hours. A previously collected CSF sample, also gave a positive, although less intense, CPE. The CPE from both the blood and CSF were transferable to secondary and to tertiary cultures. Representative photomicrographs contrasting the appearance of normal fibroblasts with the cellular changes seen in the secondary culture of the patient’s sample, are provided in Figs. 7 and 8. Electron microscopy of the positive culture showed vacuolated degenerating cells with extensive deposits of membranous materials in lamella-like structures (Fig. 9). Virus particles were not seen.

An epidemic illness involving at least 10% of the approximately sixteen thousand residents in the Mohave Valley and neighboring towns in California and Arizona, was identified during the spring and summer of 1996 (7). It manifested as an outbreak of gastrointestinal dysfunction with nausea, vomiting, and diarrhea. While the symptoms cleared in the majority of those affected, significant numbers of patients began to experience a complex array of neurocognitive impairments, mood and personality changes, recurrent headaches, altered sensory-motor functions, and an atypical post-exertion fatigue. The outbreak was reported to Public Health authorities and, in spite of a visit by a State Public Health Officer, the illnesses were deemed either non-existent or attributed to other processes, such as diabetes.

Blood samples from these patients have consistently induced a vacuolating CPE in tissue cultures of human and/or animal fibroblasts. In a double blind study, only 1 of 8 control blood samples tested positive, compared to 18 of 19 blood samples from symptomatic patients. The one negative sample came from a patient with relatively mild symptoms. Similar CPE has been regularly observed in blood samples from patients throughout the United States, variously diagnosed as having chronic fatigue syndrome, fibromyalgia, Gulf war illness, chronic Lyme disease, schizophrenia, depression, autism, attention deficit hyperactivity disorder and various malignancies (8-10, 13-15 and unpublished observations). Several culture positive patients with severe, and even a fatal neurological illness, in whom brain biopsies have been obtained, have been reported (10-13).

The stealth virus infecting the child described in this paper has not been molecularly characterized. Studies on a prototype stealth virus have indicated a major contribution from a herpesvirus, specifically an African Green Monkey simian cytomeglovirus (8,11,12). Stealth viruses are able to assimilate various cellular and other genes (17). and unpublished data. They appear to be genetically unstable and as a group are molecularly heterogeneous (12,17). They are best detected on the bases of characteristic vacuolating CPE they induce in culture.

The clinical, histological, electron microscopic and culture results with the patient are similar to those previously described and are consistent with a non-inflammatory viral encephalopathy (8,10,16). The severity of the illness is clearly exceptional, compared with the usual virus culture positive patient. Moreover, the overt neurological nature of the disease process is unquestionable. There is, however, considerable overlap between the earlier manifestations of the disease in this child and conditions loosely included in terms such as attention deficit, oppositional defiant behavior, aggression and childhood depression (18). For over seven months, the child was regarded as simply responding to a disrupted family situation. Even with the knowledge of an abnormal CT scan, the neurological findings were essentially limited to a slight degree of ocular paresis and suggestive blurring of the optic discs.

The mother of the patient contracted an acute illness with pulmonary and gastrointestinal features in 1996. She subsequently developed a severe depression and repeated migraine headaches. Although she has been able to resume part-time work, she has had difficulties coping physically and emotionally with her child’s illness and her divorce.

Illness within several family members has been noted for a number of the Mohave Valley residents. A not infrequently encountered situation is for accelerated dementia to occur in an elderly member, chronic fatigue and/or depression in an adult, and learning and behavioral problems in one or more of the children. Several middle aged adults with complex multi-system illnesses have died. Enrollment of children in "Special Educational Programs" has now exceeded 15% in the various school districts (personnel communication), compared to a National average of somewhere between 5 - 9%. The prospect that many children in the Mohave Valley and throughout the United States may have a contagious stealth virus infection has to be addressed.

The diverse and varying clinical manifestations of patients identified on having a stealth virus infection have posed difficulties in establishing a clinical case definition for formal epidemiological surveys. Tissue culture does, however, provide a reliable assay for infection, even if it is shown that not all culture positive individuals are presently symptomatic. Cultures can also be used to help identify routes of transmission, including the potential involvement of animals (16). The Mohave Valley stealth virus has induced illness in mice (unpublished) and positive cultures have been obtained from household pets of Mohave Valley residents (7). The similarity of the histological changes seen in the brain biopsy of the child to those seen in various spongiform degenerative diseases in both humans and animals (19) should help reopen the issue of whether a virus has been overlooked in the formulation of the prion hypothesis (20).

Stealth viruses have been found in association with various cancers, including glioblastoma (7), salivary gland tumors (7, 21) lymphoma, and multiple myeloma (unpublished). This may reflect the incorporation, and possibly mutation, of growth regulatory genes (oncogenes), involved in the stealth virus replicative process (15, 21). Further discussion on these topics can be found on the internet at www.ccid.org.