The term stealth has been used to define a molecularly heterogeneous grouping of atypically structured cytopathic viruses that can induce multi-system illnesses in both humans and animals, without evoking an anti-viral inflammatory response. A prototype stealth virus has a fragmented, genetically unstable genome, much of which is unequivocally derived from an African green monkey simian cytomegalovirus (SCMV). Genetic sequences coding for the major cytomegaloviral antigens recognized by cytotoxic T cells are lacking in this virus isolate. Since kidney cell cultures from SCMV infected African green monkeys were used in the production of live poliovirus vaccines, it is probable that this particular stealth virus originated in a poliovirus vaccine. Multi-system stealth virus infections with encephalopathy (MSVIE) can present clinically as a spectrum of neurological disorders, including autism, attention deficit and behavioral problems in children; and depression, chronic fatigue, fibromyalgia, and severe motor, sensory and cognitive diseases in adults. Widespread illnesses involving multiple family members and whole communities can be attributed to the spread of stealth viral infections. Histopathological and electron microscopic findings in brain biopsies of severely affected individuals corroborate the vacuolating cytopathic effects (CPE) that develop in stealth virus cultures. Similar changes can also be induced in the brain and in other tissues of stealth virus inoculated animals. Foreign genes, including sequences of cellular and bacterial origins can become incorporated into replicating stealth viruses. This phenomenon appears to be of relatively recent event in microbiology, possibly reflecting the increasing use of live vaccines in humans and in animals. An additional major concern is the isolation of stealth viruses from cancer patients, and the identification of potentially oncogenic cellular sequences adjacent to SCMV sequences in the prototype stealth virus. The apparent capacity of stealth viruses to capture, amplify and mutate cellular oncogenes has expedited efforts to define the modes of stealth virus replication and transmission.

Introduction

Complex arrays of symptoms typify a number of common, chronic, disabling illnesses. To varying extents many patients report: i) Impaired mental capacities, including loss of short term memory, difficulties in verbal expression and/or comprehension, attention deficit and lethargy; ii) altered personality, including a reduced capacity to relate emotionally to others; iii) mood changes, including depression, anxiety and anger; iv) sleep disturbance; v) headaches and; vi) generalized body aches and pains. The medical community is split into those who view these symptoms as an indication of an underlying organic disease process, and those who consider such symptoms to be merely an extension of the normal stresses and strains of everyday living. Clinicians who advocate organic disease have used various diagnostic terms such as chronic fatigue syndrome (CFS), fibromyalgia, depression, Gulf war syndrome, Lyme disease, attention deficit, multiple chemical sensitivity, etc.; without reliable, clear-cut distinguishing clinical criteria. The use of so many ill-defined labels has helped bolster those who hold the opinion that none of these illnesses constitute serious medicine.

Stealth Viruses

I have pursued the concept that an active viral infection of the brain is the underlying cause of CFS and related disorders. The early work was based on weak, but consistently positive, polymerase chain reactions (PCR), and on the development of vacuolating cytopathic effects (CPE) in viral cultures. (1,2) Initial sequence data on a PCR amplified portion of a particular stealth virus isolate showed a significant sequence homology to human cytomegalovirus (HCMV).(3) Other PCR amplified products from the same virus, however, were not related to HCMV or to any other known herpesvirus. Moreover, the cultured virus was clearly distinguishable from HCMV by the appearance of the CPE; wide host range of susceptible cells; lack of reactivity with a specific anti-HCMV monoclonal antibody, and atypical reactivity patterns in PCR assays.(3) With progressive improvements in the stealth viral culture technique, and additional DNA sequencing studies on several viral isolates, it is now well established that atypically structured cytopathic viruses have infected many patients diagnosed with CFS.

Viruses that induced a similar vacuolating cytopathic effect (CPE) in tissue cultures were also detectable in the blood of patients with severe brain diseases.(4) Support for the relevance of the tissue culture method of virus detection was provided by the presence of markedly vacuolated cells in brain biopsies performed in some of these severely ill patients.(2,4) Structures resembling herpes-like viruses were occasionally seen within the vacuolated cells.(4)

A striking feature of the brain biopsies was the absence of a significant inflammatory response.(4) The atypical viruses were clearly not evoking the expected cellular inflammatory reaction from an actively cytopathic virus. To reconcile this finding, I postulated that these cytopathic viruses may have deleted critical genes involved in cellular immune recognition. Accordingly I introduced the term “stealth” to describe such an evasive mechanism.(3,4) While this hypothesis was viewed skeptically several years ago, the results from the most recent DNA sequencing studies have provided considerable support. Indeed, there are now compelling sequence data on a prototypic cytomegalovirus-derived stealth virus to warrant a vigorous public health response to fully characterize the structure and to determine the modes of replication and of transmission of stealth-adapted viruses.

Brief Overview of DNA Sequencing Studies on a Prototype Stealth Virus

The prototype stealth virus was cultured from both blood and cerebrospinal fluid (CSF) of a CFS patient.(3) The virus induced a foamy vacuolating CPE in cells of many species, including humans, monkeys, cats, mice and even insects. Electron microscopy of infected cells revealed the presence of numerous atypical herpesvirus-like particles.(3) DNA was extracted from the material pelleted from culture supernatants by ultracentrifugation (3B series), and from agarose gel banded viral DNA extracted from the ultracentrifuged pellet of material released from lysed virus infected cells (C16 series).(5) The DNA was cut with restriction enzymes (EcoRI for the 3B series, and SacI for the C16 series). It was ligated into pBluescript plasmids and sequenced. The sequence data were analyzed for matching similarity with known viral, bacterial and cellular sequences, using the BlastN and BlastX Programs of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine.(6) As noted above, the sequence of a PCR amplified product statistically matched to HCMV. Similar to other herpesviruses, HCMV is a long, linear, double stranded DNA molecule. Each strand of the HCMV has approximately 235,000 nucleotides. The DNA comprises two major segments, designated unique long (UL) and unique short (US). The two segments are flanked by series of repeat sequences, referred to as terminal and as internal repeats of the UL and US segments. The unique regions of HCMV potentially code for 187 proteins, designated as UL1 to UL151, and US1 to US36.(7) Approximately two-thirds of the DNA clones obtained from the stealth virus contained at least a partial sequence that matched statistically to some portion of the HCMV genome.(5,8) There was an uneven distribution of clones matching to different regions of the HCMV genome. Several regions of the HCMV genome were over represented, including the UL36, UL52, UL84 and US28 genes, while other regions were not identifiable within any of the clones.(8) Minor sequence variations were noted between clones that matched to the same region of the HCMV genome. This microheterogeneity suggested that the stealth viral genome was genetically unstable and prone to replication errors.(5)

Clones corresponding to the gene that code for the UL83 (pp65, lower matrix) protein of HCMV were noticeably absent.{8) This protein provides the major target for anti-HCMV cytotoxic T lymphocytes (CTL).(9) Also missing were clones corresponding to the UL55 gene encoding glycoprotein B, the second major target for anti-HCMV CTL.(10} Other changes were noted in the organization of the immediate early gene product that constitutes another target for anti-HCMV CTL. The apparent absence and/or mutations of these genes is consistent with the concept that stealth-adaptation involved the loss of genes coding critical targets for CTL recognition.(8) Additionally, it was noted that the stealth virus genome existed as multiple fragments approximately 20 kilobases in length rather than as a single genetic molecule.(3)

The statistical quality of the matching of the stealth virus sequences to HCMV was less than would be expected if the virus had actually been derived from HCMV.(3) Limited portions of the genomes of rhesus and of African green monkey cytomegaloviruses have been sequenced. For those stealth virus genes that could be aligned with rhesus monkey cytomegalovirus (RhCMV), there was far better homology to RhCMV than to HCMV.(8,11) Several of the stealth viral sequences corresponded to four of the five known regions of the African green monkey simian cytomegalovirus (SCMV).(8,11) For these sequences, nearly identical matching occurred, leaving little doubt that the virus had, in fact, come from SCMV.(11) The significance of an origin from African green monkeys relates to the long established practice of using kidney cells from these animals for live poliovirus vaccine production.

The question arose as to how, in the absence of several major structural genes, the SCMV-derived virus was able to retain and/or regain its cytopathic activity. As noted above, about one-third of the cloned sequences of the stealth virus did not match to known cytomegalovirus–related genes. As additional sequence data became available, it was apparent that some of these additional genes were of cellular origins.(12) It appeared that the stealth virus had "captured, amplified and mutated" genes from infected cells. Since the original publication of these findings, several additional cellular sequences have been identified. Many of the incorporated cellular genes have repetitive and reiterated sequences.(12)

Of paramount importance is the finding of an assimilated cellular gene with potential oncogenic activity. The gene known as "melanoma growth stimulatory activity” or GRO-alpha, is present in three copies within a region of the SCMV-derived stealth virus, that codes for genes corresponding to HCMV genes UL141, UL144 and UL145.(13) It is known that the cellular GRO-alpha gene contains several introns, which are normally excised in the formation of mRNA. Since the GRO-alpha genes within the stealth virus lack introns, the genes must have been incorporated as RNA. This important finding implicates reverse transcription in the formation of stealth viral DNA. Supportive data for the role of an RNA intermediate in the replication of certain stealth viruses has also been obtained in assays comparing RNA and DNA based PCR assays on stealth virus infected cultures.(14)

In addition to incorporated cellular sequences, the prototype stealth virus has assimilated a diverse array of genes derived directly from bacteria and probably also from fungi.15 Again, it appears that the virus has reconstructed itself in several ways, including utilizing certain portions of bacterial genomes. The term viteria has been coined to refer to viruses of animal origin that have incorporated genes from bacteria, and vifungus when the genes are predominately acquired from a fungus. The alignments of the captured bacterial genes are quite unlike that in known bacteria. This finding, along with other data, implies a dynamic progressive process yielding novel life forms. Furthermore, analyses of the actual bacterial genes indicate that many are involved in rather unique metabolic functions, while others provide mechanisms for gene insertions and replication.(15) Preliminary indications are that the stealth-adapted viruses can contribute unique metabolic functions to infected bacteria, and that the viruses can pass freely between animal cells and bacteria.

Clinical and Public Health Implications

The current understanding of stealth viruses is that of replicating, cytopathic agents that can reconstruct themselves in a variety of ways using combinations of cellular, viral, bacterial and even fungal genes. They can essentially go unnoticed by the cellular immune defense mechanisms because they lack critical antigens targeted by CTL. They are, therefore, appropriately termed stealth. They constitute a molecularly heterogeneous grouping of atypically structured cytopathic viruses, with a reduced capacity to evoke an anti-viral inflammatory reaction due to the deletion and/or mutation of critical genes encoding the major targets for cellular immunity. Stealth viruses have been found in association with a number of brain diseases, including CFS, fibromyalgia, Gulf war syndrome, depression, schizophrenia, amyotrophic lateral sclerosis, Parkinsonism, dementia, autism, behavioral problems and attention deficit hyperactivity syndrome. (1-4, 16-18) Stealth viruses have also be recovered from patients with allergies, multiple chemical sensitivity, multiple myeloma, melanoma, lymphoma, breast cancer, brain tumors and tumors of the salivary gland. (17,18)

The overall prevalence of stealth virus infection in various communities has not been formally established. On the basis of small series, from 10-20% of the adult population may be infected. Most of the observed reactions given by control samples are relatively mild compared to the strikingly strong positive CPE seen with blood samples of neurologically impaired patients. The application of traditional epidemiological tools to clinically monitor stealth viral infections is rendered difficult because of the wide variability of the clinical manifestations.(17) Thus, one cannot easily establish a clinical case definition. Furthermore, as described in a recent publication, even when an illness is patently present, as evidenced by grossly abnormal MRI and CT scans, a routine clinical neurological examination can be essentially normal.(19) Many types of illnesses, in addition to those alluded to above, could be included under the proposition of being potentially caused by a stealth virus infection. While bypassing cellular immunity, stealth-adapted viruses and viteria may still be able to elicit various types of antibodies. This is because antibodies can be formed against viral components that are not necessarily expressed on the surface of an infected cell. IgE, IgA and IgG4 antibodies to certain bacterial antigens encoded within viteria might account for the high frequency of food and other allergic reactions noted among stealth virus infected patients. Antigenic cross-reactions may also have a confounding effect in the interpretation of various serological assays for conventional bacterial pathogens such as Borrelia burgdorferi, mycoplasma, chlamydia and rickettsiae. Stealth virus induced damage to various organs could, in susceptible individuals, evoke auto-immune reactions. The potential presence of growth regulatory oncogenes may also lead to cancer development. The majority of multiple myeloma patients tested, for example, are stealth virus infected.

Being stealth virus infected may also render a person unusually susceptible to neurotoxic chemicals and to routine vaccinations, that are well tolerated by non-infected individuals. For example, stealth virus testing proved positive for several individuals sustaining an apparent adverse neurological reaction to Lindane treatment prescribed for head lice. In a small sample of children, an adverse effect of vaccination with combined diphtheria/petussis/tetanus (DPT) and polio vaccines occurred in children born of mothers with a preexisting CFS-like illness. A major implication of an infectious etiology of CFS relates to the potential spread of infection. Infection can pass between humans and between humans and animals.(20) Clinical diversity in disease manifestation can often be seen in family members and other contacts of an infected patient. Household pets are commonly reported to be symptomatic.(17) As if the potential inter-species spread of infection was not bad enough, the potential transmission through infected bacteria raises an alarming prospect of an essentially unstoppable spread of illness.(15) Further information relating to stealth viruses and viteria is available from the World Wide Web at www.ccid.org

References

1. Martin WJ. Detection of viral related sequences in CFS patients using the polymerase chain reaction; in Hyde BM (ed): The Clinical and Scientific Basis of Myalgic Encephalomyelitis Chronic Fatigue Syndrome. Ottawa, Nightingdale Research Foundation Press, 1992, pp 278-284.

2. Martin WJ. Viral infections in CFS patients; in Hyde BM (ed): The Clinical and Scientific Basis of Myalgic Encephalomyelitis Chronic Fatigue Syndrome. Ottawa, Nightingdale Research Foundation Press, 1992, pp 325-327.

3. 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;145:441-452.

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

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

6. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25:3389-3402.

7. Chee,M.S., Bankier,A.T., Beck,S., Bohni,R., Brown,C.M., Cerny,R., Horsnell,T., Hutchison III,C.A., Kouzarides,T., Martignetti,J.A., Preddie,E., Satchwell,S.C., Tomlinson,P., Weston,K.M. Barrell,B.G. Analysis of the protein-coding content of the sequence of human cytomegalovirus strain AD169. Curr. Top. Microbiol. Immunol. 1990;154: 125-169

8. Martin WJ. Stealth adaptation of an African green monkey simian cytomegalovirus. Exp Mol Path. 1999;66:3-7.

9. Wills MR, Carmichael AJ, Mynard K, Jin X, Weekes MP, Plachter B, Sissons JG. The human cytotoxic T-lymphocyte (CTL) response to cytomegalovirus is dominated by structural protein pp65: frequency, specificity, and T-cell receptor usage of pp65-specific CTL. J Virol. 1996;70:7569-79

10. Hopkins JI, Fiander AN, Evans AS, Delchambre M, Gheysen D, Borysiewicz LK. Cytotoxic T cell immunity to human cytomegalovirus glycoprotein B. J Med Virol. 1996;49:124-31.

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. 1995;4:93-101.

12. Martin WJ. Cellular sequences in stealth viruses. Pathobiology 1998;66:53-58

13. Martin WJ. Melanoma growth stimulatory activity (MGSA/GRO-alpha) chemokine genes incorporated into an African green monkey simian cytomegalovirus (SCMV)-derived stealth virus. Exp Mol Path. 1999;66: 15-18.

14. Martin WJ. Detection of RNA sequences in cultures of a stealth virus isolated from the cerebrospinal fluid of a health care worker with chronic fatigue syndrome. Pathobiology 1997;65:57-60.

15. Martin WJ. Bacteria related sequences in a simian cytomegalovirus-derived stealth virus culture. Exp Mol Path. 1999;66:8-14.

16. Martin WJ. Stealth virus isolated from an autistic child. J Autism Dev Disord. 1995;25:223-224.

17. Martin WJ, Anderson D. Stealth virus epidemic in the Mohave Valley. I. Initial report of virus isolation. Pathobiology 1997;65:51-56.

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

19. Martin WJ, Anderson D. Stealth virus epidemic in the Mohave Valley. Severe vacuolating encephalopathy in a child presenting with a behavioral disorder. Exp Mol Path. 1999;66:19-30.

20. 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.