Pathobiology

Stealth Virus Epidemic in the Mohave Valley:
I. Initial report of virus isolation

W. John Martin and Donovan Anderson

Center for Complex Infectious Diseases, Rosemead CA and
Willow Valley Medical Center, Mohave Valley AZ

Abstract

Increasing number of patients within the Mojave Valley region of the United States are reporting symptoms attributable to atypical neurological illness. Many of these patients had experienced an acute onset gastrointestinal illness during the spring and summer of 1996. Stealth viral cultures performed on the blood of forty of these patients have been uniformly positive, yielding an unequivocal, transmissible cytopathic effect (CPE), in both human and monkey derived cell lines. One patient has died from a stealth CPE positive glioblastoma, while another patient has developed a pleomorphic adenoma of the parotid. Viral culture and epidemiological data support human to human, and probable human to dog transmission of the Mohave stealth virus infection.

Introduction

Viral culture and molecular probe based assays on patients with the chronic fatigue syndrome (CFS) and with more severe neurological and psychiatric illnesses, led to the identification of cytopathic "stealth-adapted" viruses, so named because they do not evoke an effective anti-viral inflammatory response (1-9). The prototype stealth virus, now designated stealth virus-1, is a derivative from an African green monkey simian cytomegalovirus (SCMV). It contains a fragmented, genetically unstable, genome (10, 11). Stealth viruses are molecularly heterogeneous, but do share common in vitro culture characteristics; including the capacity to induce a foamy vacuolating cytopathic effect (CPE), often with syncytia formation, in cells of multiple species (1,2). In vitro development of the CPE is promoted by frequent replacement of the viral culture medium and may also be further enhanced by the addition of boiled supernatants from cultures of known human herpesviruses.

An etiological role for stealth viruses in the pathogenesis of neurological and psychiatric illnesses is strongly suggested by the histologic and electron micrographic findings of vacuolated cells, containing viral-like components, within brain biopsies of several stealth viral culture positive patients (4), as well as in the brains of stealth virus inoculated cats (12). Stealth viral culture positivity has been repeatedly observed in family members of CFS patients, many of whom also display signs and symptoms consistent with a viral encephalopathy (unpublished observations).

Hospital-based and community-based outbreaks of CFS-like illnesses have been reported previously, without the identification of a causative agent (13). A gastrointestinal illness, affecting several hundred individuals in the Mohave Valley region of western Arizona, southeastern California and southern Nevada, occurred during the spring and summer months of 1996. Although the acute gastrointestinal symptoms mostly resolved, many of the patients, as well as additional family relatives and friends, have experienced symptoms commonly encountered in CFS patients. Several patients have had more severe illness, including transient paralysis, major sensory loss, endocrine disorders and persistent gastrointestinal disease. One patient developed a fatal glioblastoma of the frontal lobe. Another patient has developed a pleomorphic adenoma of the parotid. This paper is an initial report of the results of stealth viral cultures on 40 affected patients and on a symptomatic dog belonging to an affected couple.

Methods

Clinical samples: Blood for viral cultures was collected in tubes containing either acid citrate dextrose (ACD) or heparin as an anticoagulant. Serum was obtained from clotted blood. Tumor tissues were placed in sterile media. Cerebrospinal fluid (CSF) was collected in sterile tubes.

Stealth viral cultures: The mononuclear cell fraction was collected from anticoagulated blood by layering 5 ml of blood onto 5 ml of Ficoll-Paque (Pharmacia Biotech, Sweden) and centrifuging for 30 min at 800 g. The cells were washed once and aliquots transferred to culture tubes containing MRC-5 human fibroblasts and RhMK primary rhesus monkey kidney cells (BioWhittaker, Maryland). Within 24 hours, the tubes were rinsed of the majority of non-adherent cells. The cultures were fed at 2 day intervals, or even daily if early toxicity was apparent. For CSF and serum samples, 0.4 ml was diluted into 2 ml of medium added directly to the culture tubes containing MRC-5 or RhMK cells. Routine transfer of the CPE was achieved by either scrapping or using trypsin-EDTA to detach cells and transferring one-fifth to one tenth the volume into fresh tubes of MRC-5 or RhMK cells. Cell free transmission was achieved by using 0.4 ml of centrifuged viral culture supernatant. At least one uninoculated tube was fed in parallel with those containing patient samples. In addition, cells from other patients, outside of the Mojave Valley, were included along with samples from Mojave Valley. The inoculated tubes were viewed microscopically for the appearance of CPE. Cells from several positive cultures were pooled to provide a cell pellet for fixation in glutaraldehyde and examination by electron microscopy.

Description of CPE. As reported previously (2), stealth virus CPE in MRC-5 cells begins with a rounding of the normally elongated, spindle shaped fibroblasts. The somewhat enlarged cells tend to adhere to form tight clusters often with readily apparent syncytia formation. The cytoplasm acquires a foamy, somewhat vacuolated and slightly granular appearance. Over several days, the cell clusters may increasingly show a brownish pigmentation suggestive of lipofuscin accumulation. Considerable cell destruction can also occur with the surviving clusters forming distinct islands. A striking feature of many stealth viral cultures, is that the intensity of the CPE tends to reduce over time, possibly reflecting the accumulation of viral inhibitory components in the culture supernatant. The CPE inhibitory effect can be seen by the outgrowth of viable cells from the periphery of cellular clusters, especially in infrequently fed cultures. Supernatant factors may also explain a more generalized slowing of overall metabolism in virus positive cultures. Whereas the cells in control cultures will deteriorate and die within 3-4 weeks from excessive metabolic activity, the residual normal appearing cells in a positive culture appear to be relatively quiescent, allowing these cultures to out-survive the control cultures. This longevity feature can be seen even in cultures in which focal viral CPE can no longer be demonstrated. Essentially, similar cellular changes can be seen in other cell lines, including RhMK cultures.

Clinical Illness

The patients studied have shown a wide range of illnesses of varying severity. Many could recall a gastrointestinal "flu-like" illness occurring between February and July of 1996. This illness was characterized by acute onset of nausea, diarrhea, fever and generalized achiness. Similar illness successively occurred among different family members. Symptoms gradually lessened over several weeks, except in the few patients with a more persistent gastrointestinal disturbance. Even in the patients who did improve, few actually regained a sense of normal well being. Either from about the time of their initial illness, or developing over the ensuring several months, the patients began to experience episodes of neurological dysfunction. The manifestations would be of varying duration and would change over time. Prominent among the symptoms were numbness, paresthesia, sensory loss, gait disturbance, clumsiness, memory loss, sleep disturbance, attention deficit, headaches and personality changes. Several patients also developed signs and symptoms of one or more non-neurological illness, including anti-nuclear antibody, hyper- or hypothyroidism, pericarditis, liver disease, hypertension or arthritis. One patient developed a frontal lobe glioblastoma which was surgically resected but the patient subsequently died. Another patient developed a pleomorphic adenoma of the parotid which was also surgically resected. Several dogs belonging to a couple became sick with neurological signs.

Results

Forty human blood samples received yielded strikingly positive stealth viral cultures. Using mononuclear cells, the beginnings of the CPE were usually apparent at 24 hours and within several days extended throughout most of the culture. Although, generally similar to the CPE described for other stealth viruses, the positive cultures from these patients showed several features which, when taken together, allowed a distinction from previously isolated stealth viruses. Prominent among these differences are; the rapid development of CPE ; the greater response seen in MRC-5 cultures compared to RhMK cultures; the relatively early accumulation of lipofuscin-like pigmentation within large foci of affected cells; and the striking outgrowth of relatively normal cells from these foci, especially in unfed positive cultures.

The same type of CPE was also seen in cultures inoculated with the three CSF samples that were available from blood culture positive patients. Similarly, a serum sample from another positive patient yielded a positive, although delayed, CPE. Serum that was received from one of the three sick dogs belonging to a culture positive couple, also yielded a CPE essentially indistinguishable from that seen in the cultures from the dog owners. Positive, although delayed, transmission of CPE was also achieved using the cell free supernatant from positive cultures.

One of the patient developed an aggressive glioblastoma of the left frontal lobe. A cell lysate from the tumor yielded the same characteristic CPE as did a blood sample obtained from the patient prior to his death. Similarly, a pleomorphic adenoma of the parotid gland which developed in another patient was culture positive, as was a blood sample from the same patient.

Selected cultures were used to further define the nature of the cytopathic agent. Several cultures were maintained by serial passage using detached cells. CPE could also be transferred using cell free supernatant. Electron microscopy showed markedly vacuolated cells with structures suggestive of partial viral genome expression, including occasional viral like particles and defined patches of enhanced staining seen on both internal and plasma membranes. Fig 1,2.

Discussion

These preliminary findings are presented since they highlight several previously unreported observations relating to stealth viral infections, and because they may help hasten the development of anti-stealth viral therapies. While more formal epidemiological studies need to be undertaken, it is obvious that a communicable illness exist within the Mojave Valley community. It had an acute onset with primary gastrointestinal symptoms This was followed by a protracted course, generally presenting with a diverse range of relatively mild neurological symptoms similar to those encountered with patients diagnosed as having CFS. They also correspond to symptoms seen in veterans labeled as having the Gulf War Syndrome (GWS). Indeed, several of the individuals involved in the Mojave Valley outbreak had served in the Persian Gulf. As discussed below, however, a number of individuals involved in this disease outbreak have had severe symptoms, extending well beyond those typically associated with CFS or GWS. This is especially so for the patient who has since died from a glioblastoma and for the patient with a pleomorphic adenoma of the salivary gland.

Proof of a viral cause for CFS and GWS has been hampered by inadequate viral culture methods and by the subjective nature of many of the clinical symptoms experienced by CFS/GWS patients. The first problem has been largely addressed by using the culture methods described for stealth viruses. Blood from all of the affected patients readily induced a striking CPE within 1-2 days of culture. The CPE was sufficiently distinctive to strongly suggest a common source. The electron microscopic appearance of the cells from infected cultures confirmed the foamy vacuolated changes that are typically seen with stealth viral cultures. While fully formed intact viral particles have yet to be identified, the changes seen are consistent those seen with other stealth viral isolates. Products have been generated using the polymerase chain reaction (PCR), but have yet to be sequenced. These studies should help establish the original derivation of the virus and should help provide the molecular tools to assess viral load, sources of viral transmission and presence of virus in tissue sections.

Whatever the origin and nature of the putative viral agent causing the CPE, the patients from whom this agent has been cultured do not present with a uniform pattern of disease. Only with detailed questioning and astute clinical observations, could one determine that most patients have a degree of mild brain dysfunction, commonly associated with CFS/GWS and related illnesses. Several patients, however, showed unmistakable signs of additional neurological lesions. Other patients have also developed non-neurological illnesses, such as diabetes and arthritis. These observations are in keeping with the overall concept that CFS/GWS are simply parts of a spectrum of dysfunctional brain syndromes and that many patients with these illnesses can also show signs consistent with a systemic viral infection. The issue of probable human to human transmission is also supported by prior studies in which positive stealth viral cultures have been obtained from several members of a family, even though clinically they presented with non-identical illnesses.

The question of potential human to animal transmission of stealth viral infection has also been addressed previously. Stealth viruses have been isolated from symptomatic animals belonging to CFS patients. Furthermore, stealth viruses isolated from humans, induced an acute disease when inoculated into cats. Although, only one symptomatic dog from the Mohave Valley has been studied to date, its serum gave the same characteristic CPE as did blood from its symptomatic owners.

Controlled therapeutic studies on stealth viral positive individuals may help determine how best to treat patients with persistent stealth viral infections. The finding that many patients have a relatively mild disease may be related to the in vitro regression of CPE that can be seen, especially in infrequently fed cultures. Understanding the nature of the virus inhibitory component(s) that appear to accumulate in such cultures may lead to new therapeutic insights. The potential role of antibodies in restricting the spread of infection also needs to be defined. The apparent animal transmission of disease should provide an important model to further studies on the prevention and therapy of the Mohave stealth virus.

Acknowledgment

Supported in part by funds from the Theodore and Valda Stanley Foundation,; CAN, Cure Autism Now; and private donations.

References

1. Martin W.J. Viral infection in CFS patients. in "The Clinical and Scientific Basis of Myalgic Encephalomyelitis Chronic Fatigue Syndrome." Byron M. Hyde Editor. Nightingdale Research Foundation Press. Ottawa Canada pp 325-327, 1992.

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. 145: 441-452, 1994.

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

4. Martin WJ. Severe stealth virus encephalopathy following chronic fatigue syndrome-like illness: Clinical and histopathological features. Pathobiology 64:1-8, 1996.

5. Martin WJ. Stealth viral encephalopathy: Report of a fatal case complicated by cerebral vasculitis. Pathobiology 64:59-63, 1996.

6. Martin WJ. Simian cytomegalovirus-related stealth virus isolated from the cerebrospinal fluid of a patient with bipolar psychosis and acute encephalopathy. Pathobiology 64:64-66, 1996.

7. Martin W.J. Stealth viruses as neuropathogens. College of American Pathologist's publication "CAP Today" 8 67-70, 1994

8. Martin W.J. Detection of viral related sequences in CFS patients using the polymerase chain reaction. in "The Clinical and Scientific Basis of Myalgic Encephalomyelitis Chronic Fatigue Syndrome." Byron M. Hyde Editor. Nightingdale Research Foundation Press. Ottawa Canada pp 278-283, 1992.

9. Gollard RP, Mayr A, Rice DA, Martin WJ. Herpesvirus-related sequences in salivary gland tumors. J. Exp. Clin. Can. Res. 15: 1-4, 1996.

10 Martin WJ. Genetic instability and fragmentation of a stealth viral genome. Pathobiology 64:9-17, 1996.

11. Martin WJ, Ahmed KN, Zeng LC, Olsen J-C, Seward JG, Seehrai JS. African green monkey origin of the atypical cytopathic 'stealth virus' isolated from a patient with chronic fatigue syndrome. Clin. Diag. Virol. 4: 93-103, 1995.

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

13. A bibliography of M.E./CFS Epidemics in "The Clinical and Scientific Basis of Myalgic Encephalomyelitis Chronic Fatigue Syndrome." Byron M. Hyde Editor. Nightingdale Research Foundation Press. Ottawa Canada pp 176-186, 1992.

Legend to Figures:

Figure 1. Low magnification electron micrograph of an extensively vacuolated MRC-5 cell. Variously sized spherical and irregularly shaped structures can be seen within some of the vacuoles and also external to the vacuoles. Some of these structures are consistent with viral coded materials.

Figure 2. Higher magnification of a vacuolated cell from a positive culture showing vacuoles containing various formed structures. Disrupted mitochondria can also be seen. On close examination, spherical structures consistent with incompletely formed viral particles can be seen especially in the region of the plasma membrane. Patches of enhanced staining along the plasma membrane and along some of the inner membranes, are also present..