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Stealth Viruses: A Bridge Between Molecular Virology Abstract
Stealth viral infections of the brain may explain the increasing prevalence of dysfunctional brain syndromes in modern society. This paper formulates this concept by providing coherent overviews of both molecular virology and clinical neuropsychiatry. Cytopathic viruses which have undergone a stealth-adaptation as a means of bypassing the cellular immune defense mechanisms, cause persistent systemic infections, which frequently involve the brain. The terms multisystem stealth viral infection with- and multisystem stealth viral infection without- encephalopathy (MSVIE and MSVIE, respectively), have been suggested to help emphasize the encephalopathic illness seen in a subset of stealth viral infected patients. Patients with MSVIE can manifest a wide range of symptoms, including those that meet criteria for a primary psychiatric diagnoses. Infected patients have been labeled as having schizophrenia, manic depression, dementia, autism, attention deficit and major personality disorders. The clinical and therapeutic significance of a stealth viral cause of psychiatric illness is briefly discussed.
Introduction
Dysfunctional brain syndromes are erroneously viewed as comprising two distinct groups of illnesses: neurological and psychiatric. Neurologists mainly address diseases that can be attributed to discrete anatomic lesions, with readily elicited physical signs pertaining to the affected region of the brain. Although the therapeutic options are usually limited, the causes of these illnesses have a rational basis in terms of well defined neuroanatomical lesions. As opposed to neurologists, psychiatrists and other mental health personnel, mostly address diseases lacking precise anatomic localization or biologic explanation. These diseases are expressed in terms of varying degrees of altered emotions, behaviors and cognitive processes; functions that are viewed as expressions of the mind rather than of the organic brain. The availability of mind-altering drugs has helped shift the therapeutic emphasis for these diseases from simply trying to coerce the patient to change his or her ways (psychotherapy) to the somewhat more successful (if still empirical) psychopharmacological approach. Major additional strides could be expected if there were a conceptual framework to better understand the biological basis and the diverse clinical manifestations of psychiatric illnesses. This review addresses this need.
Brain Function
The brain is unique among the body=s organs in the spatial distribution of its many functions. Unlike other organs, damage to one area of the brain can not readily be compensated for by heightened activities of other brain areas. Moreover, individual components of the brain participate in complex neural networks which can subserve a variety of integrated functions. Even minimal damage to neurological tissue has the potential for profound effect compared to similar damage in extra-neural tissues. Not only is the brain tissue spatially complex, it is hampered by the inability of mature neuronal cells to replicate and to replace neurons damaged as a result of either illness or normal senescence.
Assessment of Brain Function
The brain is responsible for motor, sensory, autonomic and cognitive functions. It also determines personality, mood, self-perception and social interactions. Assessment of gross deficits of sensory and motor functions is readily achieved in routine neurological examinations. Office testing for more subtle sensory and motor changes, and for possible derangements of the autonomic nervous system are rarely, if ever, employed by psychiatrists ( ). Such tests have also been disregarded by many neurologists as providing inconsequential Asoft signs.@ Complex assays, such as tilt-table testing for orthostatic hypotension ( ), can provide a quantitative measure of a specific autonomic function, but are unsuitable for everyday clinical practice. Neuroimaging techniques, such as computerized EEG, PET scans and functional MRI, can also provide measures of brain function, but they, too, are unsuitable for routine psychiatric practice. Furthermore, the etiological foundations for the minor changes that may be seen in psychiatric patients are not yet established (4). Neuropsychiatric testing for minor personality disorders and for mild cognitive impairments requires an in depth knowledge of the individual=s pre-illness performance; information which is rarely available. One-time testing will usually not reveal the early changes in personality or cognitive abilities that patients themselves or their friends may perceive.
Diagnostic Labels
In spite of the shortcomings in assessments of many brain functions, psychiatrists have managed to categorize psychiatric illnesses into distinct clinical entities by grouping symptoms into a variety of syndromes. (5). These groupings obscure the fact that many symptoms are common to various disease categories. Moreover, the naming of an illness tends to overlook the considerable variability that can exist in the actual symptoms, and especially their relative severity between patients and even in a single patient over time. The lack of true diagnostic precision in reflected in such terms as Aco-morbidity@ and Aborderline condition@. The assumption that different syndromes have different underlying etiologies has also hampered efforts to find causes of mental illnesses.
Etiologies
In a similar way that diagnostic labels have tended to artificially sub-divide a spectrum of neuropsychiatric illnesses, the proponents of various etiologic theories have also tended to be exclusive rather than inclusive. The notion that organic brain illness has to be either genetic, infectious, auto-immune or toxic, precludes the known interactions between all of these components. The aging process itself can slowly erode the limited functional reserves that may have survived an earlier insult, leading to a delay in the clinical expression of an illness years after the initiating event has occurred. Of the four etiologic categories listed above, an infectious cause has the promise of being the most readily targeted for therapy, as well as the added concern of being potentially transmissible between individuals. Viral infections of the brain could present in many different ways depending simply on its localization to different regions of the brain and on the varying levels of the various capacities for various brain functions, that the patient had prior to becoming infected. It could also render an individual susceptible to normally tolerated environmental factors and other stressors of brain function.
Viruses and Psychiatric Illnesses
The digression of psychiatry from basic molecular biology is seen in the minimal attention currently given to the potential role of viral infections in psychiatric illnesses. Historically, such conditions as encephalitis lethargica (5), subacute sclerosing panencephalitis ( ) and general paresis of the insane (6) were belatedly accepted as infectious. The reality of AIDS dementia is also now unquestioned (7). On the other hand early attempts to detect viruses in patients with schizophrenia (schizoviruses) and other major psychiatric illnesses, failed to provide convincing and readily reproducible findings (8). In spite of the availability of more sensitive technologies (9), such as the polymerase chain reaction (PCR), few psychiatrists are intellectually poised to consider viral infections as a likely cause of their patients= illnesses.
The prevailing model of a viral brain infection is that of Herpes simplex virus (HSV) encephalitis (10). Typically the patient will present with an acute onset (<2 weeks from the initial symptoms to severe illness); have a progressively diminishing level of consciousness; show localizing signs, often to the temporal lobes, on clinical, radiologic and EEG examinations; and have a marked CSF pleocytosis with increased protein levels. Relatively mild meningitis/encephalitis-like illnesses are also commonly encountered in General Practice. If pursued vigorously, serological assays, changes in CSF and stool cultures will sometimes indicate enteroviral infection. The illnesses are considered to be short lasting without sequella. The notion of a persisting, sub-acute, non-inflammatory viral encephalopathy is rarely considered clinically or tested for using either viral cultures or molecular probe based assays.
Virus Classifications
One reason for the lack of consideration of viruses in psychiatric illnesses is the plethora of different viruses that in many ways seem unconnected to each other. Emphasizing differences rather than similarities has impeded a clear overview of molecular virology in much the same way that the over categorization of dysfunctional brain syndromes has confounded, rather than simplified, psychiatry. A working model to help bypass the complex viral classifications schemes that are currently in place can be arrived at through the simple principle that viruses must replicate. While larger, more complex DNA viruses code their own DNA dependent DNA polymerase, smaller DNA viruses need to make use of either the cells own DNA polymerases, or alternatively the polymerase of a larger DNA virus. Many of the very small DNA viruses, such as parvoviruses, which lack their own polymerase will only replicate within either already dividing cells, or cells co-infected with a virus that has a DNA polymerase. Some small DNA viruses (e.g. papovaviruses) replicate through the activation of cellular proliferation. With the exception of hepatitis D virus (an RNA virus that the cell misreads as DNA), conventional RNA viruses generally need to provide their own polymerase. Negative single-stranded RNA viruses encode their own RNA dependent RNA polymerase, which is packaged along with the viral genome. Positive single-stranded (ss) RNA viruses and most double stranded (ds)RNA viruses, synthesized their polymerase soon after infection occurs. Retroviruses are negative strand RNA viruses which encode a reverse transcriptase RNA dependent DNA polymerase which can replicate its RNA into ds DNA. Integration of the ds DNA into the cellular genome, allows for reformation of ss RNA through the action of cellular DNA dependent RNA polymerase. DNA copies of ss RNA viral sequences could be similarly replicated by cells in which endogenous retroviral genes, containing a functional RNA dependent DNA polymerase, were activated.
Viral survival also depends on the ability to spread to other cells. Simple viruses can rely merely on passive uptake or hitching a ride within other viruses. The survival of viruses between cells is generally achieved by enclosing the viral geome within a insoluble protein structure formed by the self-assembly (aggregation) of proteins, giving rise to the viral capsid or coat. More complex viruses can incorporate sequences coding for molecules which become embedded into cellular membranes. This provides a viral envelope which enables the viral particle to more effectively interact with the surface of cells destined to become infected. The actual genes encoding the capsid, polymerase and envelope proteins can exist on a single nucleic acid molecule or, in the case of viruses with segmented genomes, can exist as separate cooperative genetic elements. Complex viruses may possess additional protein components which serve to facilitate their metabolic interactions with the host cell. These interactions are more readily understood in terms of the specific relatedness of viral genes to their cellular counterparts. The viral-host gene interactions explain many of the distinctive cytopathic effects (CPE) manifested by viruses, beyond those simply attributed to metabolic competition.
Viral Pathogenesis
All viruses can mediate cellular changes by altering the normal metabolic balance within the cell through over utilization of the cells energy resources. While this can eventually lead to cell death, an earlier cost can be the failure of the cell to perform all of its normal functions. Continued metabolic drain on the cell can lead to a loss of essential components such as ATP required to maintain basic mitochondrial energy generation. In addition, viruses can have other specific effects including stimulating the proliferation of already resting cells and/or interfering with the normal maturation process , both of which can help promote tumorigenesis (11). As might be expected, many viruses can subvert several of the cells metabolic processes to help favor those required for viral replication and assembly. Many of these more specialized interactions between viruses and host cells depend on the genetic makeup and preexisting gene expression of the cell. These factors, together with the availability of cell surface receptors can help explain the restricted permissiveness of many cell types to only certain viral infections.
The cell can also respond in a manner that is detrimental to viral survival. Nucleic acids are subject to degradation by nucleases. Through the interferon pathway, ds RNA can lead to activation of RNase. (12). The cell can also provide some protection of the viral genome, for example, the negatively charged nucleic acid can be partially stabilized by attracting positively charged polyamine molecules such as spermine and spermidine. Polyamines are derived from S-adenosylmethionine (SAMe). Diversion of SAMe towards polyamine synthesis can deplete the cells capacity for methylation reactions, lipid metabolism, detoxification and growth.
Viral Immunity
The immune system can also both reduce and enhance the extent of viral damage. Antibodies can provide an effective blockade preventing viruses from gaining access to normally permissive cells. In particular, antiviral antibodies can help prevent viruses passing from the blood to the brain. Cellular immunity can reduce viral load by destroying infected cells prior to the release of infectious viral particles (13). On the other hand, cellular immunity against viral antigens or against modified or inappropriately expressed cellular antigens can lead to immune damage of a cell beyond that achieved by the virus itself (14).
Viruses have evolved various mechanisms to help evade the immune system. One such mechanism is the deletion of the genes coding for the major antigens recognized by the cellular immune system (15). This mechanism of bypassing the cellular immune defenses has been referred to as stealth adaptation.
Stealth Viruses
A corollary of the clonal selection theory of immunology is that to be effectively recognized, a viral infected cell must restrict the number of different viral antigens presented to the cellular immune system. Even with large complex viruses, relatively few viral components actually serve as effective targets for cellular immune defenses (16). By deleting or mutating the genes, viruses can avoid effective immune recognition. As might be expected, such downsized or stealth viruses may lose some of their efficiency in replication and cause only limited damage to infected tissues. As discussed above, however, even limited viral damage can have devastating consequences if it involves critical regions of the brain. As also discussed above, there are limited basic requirements for viral survival (essentially a capsid coding gene and, in the case of RNA viruses, a mechanism for RNA replication). It is not difficult to envision how large viruses, and/or combinations between viruses, could yield a multitude of varying sized derivative viruses, which would lack major immunogenic targets for effective cellular immunity. Stealth viral sequences can comprise both DNA and RNA. An interesting observation is the apparent genetic instability and fragmentation of a stealth viral DNA genome. A potential mechanism of stealth viral DNA replication is through the bridging of viral fragments with long RNA molecules. This scaffolding effect could be inhibited in the presence of short RNA molecules competing with the longer RNA molecule for binding to one of the fragments.
Stealth Viral Infection
Because they bypass effective cellular immunity, stealth viral infections can persist within the body. Not only have steal th-adapted viruses evaded the cellular immune system, but they have also gone unnoticed by investigators relying on an inflammatory response as a sign of infection (17). Stealth brain infections have been described in humans (17-20). Illness has also been seen in naturally infected cats, as well as in cats inoculated with stealth viruses obtained from infected humans. The predominant histological characteristic in both humans and in the animal model, is the presence of occasional cells with distinctly vacuolated cytoplasm and distorted abnormal nuclei (17-18). The affected cells may show varying granules positive with periodic acid Schiff stain. The animal studies confirmed that the cellular changes were not confined to the brain but that signs of infection could be found in various organs (21). The predominant clinical manifestations in the animals were, as expected, neurobehavioral, consistent with the unique susceptibility of the brain to limited degrees of viral damage.
Psychiatric Illnesses
One of the earliest isolates of a stealth virus came from a patient with a 4 year history of a psychotic bi-polar manic depression. Her condition deterioated acutely in January, 1991 with what appeared clinically to be a viral encephalitis. The absence of significant cellular changes in the cerebrospinal fluid led to a revised diagnosis of a attempted suicide from drugs complicated by cerebral anoxia from a transient cardiac arrest. This patient has remained in a vegetative state since this episode. Positive stealth viral cultures have been seen in patients presenting with an array of other severe psychiatric disorders including acute toxic psychosis, aggressive violent behavior, schizophrenia, dementia, autism, attention deficit, anxiety attacks, violent social behaviors and drug addiction (22 and unpublished observations). Because of the possibility of the developing brain repairing viral induced damage, the presence of stealth viruses in children labeled as autistic has been of special concern. The incidence of autism has increased significantly since it was first described in 1942. All but 1 of 20 autistic children referred for stealth viral testing have shown positive stealth viral cultures. Stealth viral infections have also been linked to abrupt onset of severe learning disorders with attention deficit, poor memory and oppositional defiant behavior.
Adult patients have unwittingly contributed to their diagnosis of a psychotic disorder by desperately trying to explain symptoms unfamiliar to their physicians. For example a diagnosis of schizophrenia was suggested when one patient described her headache as a large screw boring into the back of my head. Another patient confronted his physician with the idea that his illness was like a number of men inside my head not knowing what the others are doing. Unsuspecting patients present themselves as being outside the limits of accepted medical diagnoses, and are offended when this leads to talk of a serious psychiatric disease.
One culture positive patients labeled as schizophrenic come from family in which both parents were diagnosed as having CFS and a grand parent as an atypical Parkinsons disease. Several examples have been encountered where family members of an autistic child or of a schizophrenic adolescent, have progressively succumbed to dysfunctional brain illness. Often their symptoms have been originally ascribed to the stress of having to cope with an psychiatrically affected family member. The diagnosis was changed when objective neurological signs of an encephalopathy were elicited and their blood samples yielded positive stealth viral cultures. Additional family studies have confirmed the existence of a pervasive infection with varying severity and clinical manifestations.
Culture positive patients, labeled CFS and/or FMS, not uncommonly exhibit mild psychiatric symptoms. These individuals have been forced to choose between the social stigma and limited medical and disability insurance coverage of a psychiatric diagnosis versus the uphill battle of trying to convince physicians and friends of the reality of a nebulous medical illness.
Additional Manifestations
Stealth viral culture positive patients, whether presenting with a psychiatric or neurological illness, will commonly show signs and symptoms consistent with a systemic viral infections involving various organs, including the liver, salivary glands, thyroid and genital organs. Autoimmune damage, probably evoked in response to viral induced cellular changes, may account for the prevalence of anti-nuclear and anti-thyroid antibodies seen in many stealth viral infected patients. In some patients, including a patient who died from his illness, a vasculitis component was present, possibly resulting from direct viral infection of blood vessels (20). Since brain damage is essentially additive, patients with a viral encephalopathy, leaky bowel and impaired liver detoxification, are particularly susceptible to environmental neurotoxins. These include many of the toxic chemicals used for medical, veterinary and agricultural use. Indeed multiple chemical sensitivity can be a presenting manifestation of a stealth viral encephalopathy.
Possible Vaccine Origins of Stealth Viruses
While stealth adapted viruses have presumably existed for a long time, the increasing incidence of many of the diseases referred to above, suggests an additional recent source of these viruses. DNA sequence data on the stealth virus isolated from a patient with acute encephalopathy following a 4 year history of a manic-depressive illness, indicate an origin from African green monkey simian cytomegalovirus (SCMV) (23-24). Short term kidney cell cultures from African green monkeys have been used since the early 1960's to produce live polio virus vaccines. The probable presence of SCMV in polio vaccines was largely ignored even though in 1972, all 11 monkey kidney culture tested using sensitive indicator cell lines, showed the presence of SCMV. Only 4 of these isolates would have been detected using the standard detection procedures which remained in place despite of the above finding.
Other stealth viruses also appear by electron microscopy to have a relatedness to herpesviruses, but so far their sequence data have yet to be linked to that of known human or animal viruses. Fresh animal tissues have been used for a variety of human live viral vaccines, including dog and duck kidney cells for rubella vaccines. SV-40 virus was present in many of the polio vaccines lots produced in kidney cultures from Rhesus monkeys. A diverse array of animal cell lines have also been used for the many animal vaccines that have been developed. It is not unreasonable to suggest that the vaccine viruses may have contributed genetic elements to contaminating herpes and other viruses to facilitate the emergence of replicating, non-immunogenic (stealth-adapted) viruses. Once within the human population, stealth viruses can be passed via direct human to human and human to animal to human transmission routes.
Detection of Stealth Viruses
The most reliable method for detecting the diversity of stealth viruses is to co-culture the patient blood with a variety of indicator cell types and observe the cultures for the induction of a transmissible CPE (18). Typically, rhesus monkey kidney cells and a human fibroblast cell line such as MRC-5 cells are inoculated with the patients mononuclear cells and observed for 2-4 weeks. Frequent refeeding of the cultures can help promote the development of the CPE. It is quite unusual (<10%) to observe a rapidly developing CPE in blood samples from randomly selected hospital outpatients. Conversely, it is unusual not to observe a strong positive CPE in cultures from patients with otherwise unexplained neurological or behavioral disorders.
The stealth virus CPE is best characterized by the formation of foci of enlarged, rounded cells, often with the suggestion of syncytia. Proliferation foci of affected cells can occasionally be seen. The actual appearance of the CPE differs between cultures and is best followed by repeated examination of individual cultures by the same observer. The CPE can be transferred to fresh cultures. Positive cultures can be further examined by staining cell smears or sectioned cell pellets using the patients and other sera. Electron microscopic studies can also be performed. Cell derived DNA and RNA can also be used for molecular characterization. A series of PCR primer sets based on previously characterized stealth viruses can be used to screen for SCMV derived DNA and RNA sequences. The primers can also be used to test for DNA and RNA dependent polymerases. Finally, the viral cultures can be used to test the effects of various anti-viral therapies.
As noted above, stealth viral infections are not necessarily confined to the brain and indeed blood samples are routinely used for stealth viral cultures. Other serological signs of viral infections can include unusually high levels of anti-herpesvirus antibodies. This may reflect the presence of the stealth virus or the two-way cross stimulation that can be seen between stealth and conventional herpesviruses. Broadly reactive herpesviral primers can also be used in low stringency PCR based assays on DNA and RNA directly isolated from the patients blood. Cloning and sequencing of the PCR products can be used to design of more specific primer sets. The possible role of stealth adapted herpesviruses in secondary activation of parvo- and papovaviruses, including monkey-derived SV40, can also be assessed using serological and molecular probe based assays for these agents.
Therapy for Stealth Viruses
The molecular heterogeneity between stealth viruses will undoubtedly pose a problem with their therapy. An important in vitro observation, however, is that viral growth is promoted by regular refeeding of the cultures. This has been equated with the accumulation of inhibitory component(s) of possible therapeutic use. The term has been applied to these putative inhibitors. Preliminary data are consistent with RNA molecules acting as inhibitors.
Some stealth viral isolates have responded to alpha interferon and a partial clinical response was seen in a patient being treated for a stealth virus disease. Occasional patients, including some children, have been given oral Acyclovir with suggestive improvement. More recently, oral and intravenous ganciclovir have been tried in individual patients with apparent, if modest, beneficial effect. Neither agent is likely to be effective in arresting replicating RNA viral genomes. Inhibitors of RNA dependent polymerases warrant in vitro, and if effective, in vivo testing.
Knowledge of the stealth virus induced metabolic derangements should lead to appropriate nutritional therapies. In attempting metabolic resuscitation, it is important to consider the potential adverse effects that could result if one were to merely feed the virus rather than restoring normal cellular function. The in vitro cultures provide a valuable system to evaluate this possible dilemma.
Summary
Stealth viral encephalopathy could account for many psychiatric illnesses currently attributed to functional derangements of the mind. Psychiatric patients are generally not adequately tested for clinical evidence of organic brain disease. Detailed clinical examination focusing on more subtle changes in brain functions can help establish a diagnosis of encephalopathy. Patients with objective neurological signs should have stealth viral cultures and should also be tested for other manifestations of systemic viral infection. Viral isolates should also be characterized in terms of their composition, replication strategy and origin. Culture positive patients should be assigned to anti-viral treatment groups and should also receive other supportive therapies.
Acknowledgment: The work was supported in part by the Stanley Foundation.
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