"Out of Africa"

In reference to various pathogens, the subtitle of this presentation could be "Out of Africa." But first, let's make a distinction between two major culprits from Africa. As described in the previous presentation, Ebola is such an acute illness that within approximately three days of infection, people were much aware of the virus's presence. With AIDS, progress was not as quick. It took at least three years to identify the virus that was causing an unusual illness in the U.S. beginning in the late 1970's. This is still much faster than another type of virus that has taken more than 30 years to even begin to identify. I am refering to another class of virus -- termed stealth viruses -- some of which have come out of Africa. In particular, I will review evidence for the African green monkey as being the origin of certain viruses associated with various neurologic illnesses. These viruses may have entered humans through live polio virus vaccines. They are now passing between humans and possibly from humans to animals.

HISTORY OF POLIO

To begin this story, let us first go to Sweden, where in the late 1890s, polio was identified as an epidemic disease. There had been prior reports of occasional individuals who had a paralytic illness consistent with polio, but the disease as an epidemic was first described in 1897 by Dr. Medin, an acute pediatrician from Stockholm. Forty-two cases of an acute paralytic illness were identified. Epidemics of this illness then began to spread throughout Europe. The next major development occurred in Vienna, Austria, where Dr. Carl Landsteiner lived. He is the chemist who described A, B, O blood groups in 1900, and later in the 1940's described the Rhesus (Rh) blood group. Dr. Landsteiner wanted to see if this new polio illness could be transmitted to animals. Similar to other investigators, he used mice, rabbits, and guinea pigs; none came down with an illness. Fortunately at the same time in Vienna, there was a group of psychiatrists including Dr. Sigmund Freud and others, who had extended the work of Charles Darwin in showing brain similarities between primates and humans. These psychiatrists were analyzing the brains of monkeys, and some of these animals were available to Dr. Landsteiner. Specifically, he took the spinal cord from a 9-year-old child who had died from polio and injected ground up spinal tissue into two Old World monkeys. One monkey died very quickly. After 17 days the other monkey, a rhesus monkey, came down with a paralytic illness similar to polio. This important finding was followed by studies indicating that polio was in fact a viral illness. A very long gap followed, up to 1948, before Dr. John Enders in Boston showed that polio virus could be passaged in in vitro cell cultures. This led to immediate interest in making a vaccine. In part, because rhesus monkeys were known to be susceptible to polio, Dr. Jonas Salk chose kidney cell cultures from these monkeys to generate large amounts of the three known strains of polio virus. He used a 1:4000 dilutions for formaldehyde to inactivate the virus for use as a vaccine. In 1954 inactivated polio vaccine was licensed in the United States, leading to a dramatic reduction in the incidence of polio. Dr. Albert Sabin soon followed Dr. Salk. He chose a difference approach -- using an attenuated, or less-virulent, strain of polio as a live polio vaccine. He too initially chose rhesus monkeys. Later, it was realized that the formaldehyde technique used to inactivate polio virus was not successful in inactivating SV-40 virus.

Consequently large numbers, probably millions of people, were inoculated with SV-40 virus. This type of virus is quite prevalent in rhesus monkeys but less so in African monkeys. This lead to the rapid shift from rhesus to African green monkey for both the inactivated and the live vaccines. There was substantial debate about switching from inactivated to live polio vaccine but little discussion about using African green monkeys in place of rhesus monkeys.

The major advantage of live polio vaccine was the rapid onset of immunity; even a single dose could essentially interrupt the progress of an epidemic. In addition, this vaccine strain could spread to individuals who had not actually received the vaccine, and this indirect vaccination provided more comprehensive immunity within the community. In addition, it was much less expensive. On the other hand, as with any live virus vaccine, there is the concern that adventitious or unwanted pathogens might come through the vaccines. There also is the potential that the vaccine virus, such as polio, could mix with other viruses in the cell substrate, giving rise to new viruses as a result of the recombinations. We might think there would be hesitation to use kidney cells straight out of a monkey to start making cell lines than risk using a continuous cell line that might be oncogenic. Even if a cell were only partially on its way to become malignant, oncogenic DNA possibly could be transferred. This concern has long since disappeared, especially with the use of monoclonal antibodies produced in myeloma cells. In other words, when the vaccines were being developed, it was deemed appropriate to use primary kidney cultures from African green monkeys to produce a live polio viral vaccine.

CHALLENGES OF VIRAL DETECTION

The issue of safety from adventitious agents was addressed in various in vitro and in vivo assays for non-polio viruses. Recently, some of those techniques have been enhanced using molecular biological techniques, especially to look for evidence of retroviruses such as simian immunodeficiency virus (SIV) and simian type D viruses. There was also the back-up of looking for any adverse affect in the early clinical trials. As discussed previously by Dr. Shope, there are limitations in these techniques. First, molecular probes will not pick up all viruses, especially if you do not know what sequence you are looking for. Second, the culture tests for viral pathogens may not pick up slow-growing viruses such as cytomegaloviruses. And probably most important of all, an epidemiological approach is very limited when dealing with chronic illness of variable onset. Especially difficult are illnesses that cannot be defined in terms of a clear case definition, do not develop an acute phase following infection, and may begin to pass between humans once the virus is introduced into the community. Even greater difficulty arises with an illness for which there may be multiple causes.

CHRONIC FATIGUE SYNDROME

Probably the most frustrating and the most enigmatic of these diseases is an illness referred to as chronic fatigue syndrome. But this is only part of a bigger picture that could include an entire series of psychiatric disorders, neurogenerative diseases, autoimmune diseases, organ dysfunctions, etc. Although many of these entities have a name -- chronic fatigue syndrome, schizophrenia, depression, autism, etc. -- giving them a name obscures the fact that we do not understand the underlying pathophysiology. We really do not know what is happening. Why does someone change from a normal teenager to a schizophrenic? For many of these illnesses, it is somewhat glib to simply say the cause is genetic or metabolic; even worse, that it is psychological, or all in the mind. We need to come to terms with our ignorance about the cause of many of these common diseases. The also is a notion that many of these diseases are becoming more prevalent in our society and that we are doing little to understand their causes.

I began my work approximately 10 years ago on chronic fatigue syndrome. In my opinion, there really is no satisfactory clinical definition of this disease. Suffice it to say that a person changes from a relatively healthy, energetic individual to someone with an impaired lifestyle. A major symptom is unexplained severe fatigue. Many of these patients also show symptom overlap with common illnesses, with some symptoms explained by a lowered pain threshold. These include diagnoses like fibromyalgia, back pain and pelvic pain syndrome. Other patients show various mood changes from depression, flat effect, and panic disorders. Others have dementia and cognitive dysfunction. This also is a series of illnesses generally called psychosomatic, where there is a dysfunction of the brain's controls over various bodily functions. Again, the names of these diseases are not very helpful, because if anything, they obscure what is really going on.

CHALLENGES OF VIRAL ASSAYS

I decided to identify a viral cause of some of these unexplained illnesses. Politically, the most challenging was chronic fatigue syndrome. The conventional approach of trying to culture something from the blood of affected patients was tired. Interestingly, we observed that in several of these patients, peripheral blood cells included a mild cytopathic effect on a variety of cell lines. The effect was not progressive and was hard to define, but it could be transferred. We could not confirm it using routine serological strains on the cultures, nor could the cause be identified using high-stringency PCR assays. We switched from cultures to direct testing of the blood sample. Again using high-stringency PCR, the assays for herpes viruses and retroviruses were essentially negative. We devised a set of generic herpes viruses primers that could produce weak but consistent positive results shown by all then-known human herpes viruses. The PCR assays were run under low-stringency conditions, resulting in multiple produces from some patients and no responses from several control individuals. In several patients, there were also positive results using a retroviral primer set against the human T lymphocytotropic virus (HTLV) tax gene.

These PCR findings encouraged efforts to go back to the cultures. We improved the culture conditions and began to get a stronger cytopathic effect. The first really strong culture came from a patient with chronic fatigue syndrome. The next was from a patient with acute encephalopathy following a manic depressive illness. The cytopathic effect could be readily transmitted to cells of multiple species including human, monkey, mouse, rat, rabbit, and even insect cells. Viruses could be seen by electron microscopy. Once we had such cultures, we applied the PCR assay to look for the product that might be generated. If the cultures are maintained and nurtured, we could see the beginning of syncytia formation followed by a very strong cytopathic effect. Electron microscopy showed viral particles as well as dense bodies similar to those seen some cytomegalovirus cultures. These structures provide components that go into the formation of viruses. Appearances were suggestive of a herpes type virus, yet staining these cultures with specific antisera against cytomegalovirus, human herpesvirus 6/7 or varicella zoster (Herpes simplex), gave negative results. In the same way, running PCR assays using primers specific for those viruses also gave negative results. On the other hand, the HTLV primer produced two large products that could be cloned and sequenced. Interestingly, both products were generated with a single HTLV primer, which flanked both ends. The internal secquences of one of the products showed a significant match to human cytomegalorvirus. These data were published in The American Journal of Pathology in 1994 ("Cytomegalovirus-related Sequence in an Atypical Cytopathic Virus, Repeatedly Isolated from a Patient with Chronic Fatigue Syndrome"). Having obtained sequence data, we were able to perform a high stringency PCR assay that showed the presence of viral sequences in the patient's blood, confirming the infection. The viral DNA also was isolated from the culture. It was cloned, allowing for more extended sequencing. Each time a sequence was obtained, it was compared to sequences listed within GenBank. For moany animal viruses, only limited sequence data are available. We finally came across sequences that allowed us to identify the true origin of the virus we had cultured. The sequence is related to human cytomegalorvirus, more closely related to rhesus, and very highly related to African green monkey simian cytomegalovirus (SCMV). The same pattern has occured with other origins of the virus. We have preformed PCR assays based on known sequences of SCMV and the cultured virus to confirm similarity but non-identity; the cultured virus differs from SCMV in the type of cytopathic effect, the host range and in vivo pathogenicity studies. But there is sufficient data to unequivocally establish the original derivation of the virus from the African green monkey. This takes us to the fact that the African green monkey was the source of much of the polio vaccination in this country. I don't believe many people have African green monkeys as pets. In our paper describing these studies, we suggest that attention should be given to live polio vaccines. It is worthwhile pursuing this topic because polio vaccines are still being produced in the African green monkey.

I emphasize that the virus is pathogenic for animals. It will induce encephalopathy in cats, drastically affecting their behavior. Yet looking at the brain histologically, there is no inflammatory reaction. We can see vacuolated cells and viral particles by electron microscopy. The effects are quite subtle, however, and ordinarily would receive only minor mention by a neuropathologist looking primarily for either malignancy or for an inflammatory change. The acute illness is followed by a chronic illness. Although the virus is widespread in the animal, the major clinical manifestations are neurologic. In preliminary observations, if animals are preimmunized with viral infected cells, they do not come down with an illness. This might be because they have established an antibody barrier to the virus getting to the brain.

STEALTH VIRUSES

The animal studies have produced an impetus for human studies. Historically, we see the same changes in human brain biopsies as in the cats. We also have cultured virus from biopsies. Histologically, the most interesting aspect is the failure of vacuolated brain cells to evoke a significant inflammatory reaction, leading to the designation of "stealth viruses." While these viruses need to be more fully sequenced, it is known that relatively few components of viruses act as potent inducers of a cellular inflammatory reaction. Those components appear to be deleted, or at least, mutated, in stealth viruses. Therefore, they can bypass the cellular immune system, which does not respond. Stealth viruses take much more effort to grow than a fully intact virus. They probably don't do much damage in many organs of the body unless there's a secondary complication such as autoimmunity. But they can disrupt the very complex integrated function of the brain, such that a small amount of viral damage could cause profound clinical effects. Inappropriate responses could occur as a result of viral damage.

CLINICAL MANIFESTATIONS

In this presentations, we focused on the origin of stealth viruses and patients who have been identified. Let me assure you there are many patients now identified by culture as having stealth virus infections. Some are extremely sick and some have been in vegetative states for the past several years. A school teacher in Palm Springs, California, has been in a nursing home for the past several years. She had a brain in 1991 showing typical vacuolated cell change. There is a girl who is in a vegetative state. She was first diagnosed at Los Angeles County Hospital as schizophrenic. Her diagnosis was changed to a manic-depressive illness. In 1991, she came into the County Hospital with what clinically thought to be Herpes simplex encephalitis. But the cerebrospinal fluid showed no inflammatory reaction. Clinically, the diagnosis shifted to "must be a drug overdose." There are several medical professionals, some of whom voluntarily gave up their licences because they could not practice medicine. One has essentially lived the life of a demented individual for the past several years. There is a dentist who was perfectly normal one year ago, a jovial fun person. She had an absolute change of character throughout last year, lost her job and became paranoid. She was admitted to a psychiatric hospital. Her clinicians finally realized there was something more organic going on. She became comatose and a brain biopsy was performed. It revealed the same characteristic pattern of minimal inflammation and widespread minor cellular changes. Her culture was positive for stealth-adapted viruses. Another case involves an immigrant who died within a year of being admitted with an unexplained encephalopathy. A series of these cases is now being complied, for which there's no evidence on brain biopsy and culture for atypical cytopathic viruses. Some, but certainly not all, are suggestive of an African green monkey origin. In other words, this is only one of a wide variety of possible sources of stealth-adapted viruses. Of interest, rubella vaccines were first grown in dog kidney cells and subsequently in duck cells. Other live vaccines are produced in foreign species. Once such viruses are in humans, they can pass from human to human.

SUMMARY

Stealth viruses can probably produce clinical manifestations beyond chronic fatigue syndrome. The concept presented here is that chronic fatigue syndrome is part of a spectrum of neurologic disorders associated with, at least in some cases, a viral infection potentially derived as an inadvertent consequence of live viral vaccines. The challenge now is for the U.S. Food and Drug Administration, CDC, and state and county health departments to look at the prevalence of this kind of an infection.