NOVEMBER 6th, 1995

Dr. W. John Martin. Thank you very much for this opportunity to discuss with you work on atypical herpes viruses in chronic neurological diseases.

I will begin with clinical descriptions of two patients, and then move to in vitro culture work, and conclude with data showing that some atypical herpesviruses have sequences of African green monkey simian cytomegalovirus origin.

This first slide is the attempts at drawing a clock and a house by a 39-year-old school teacher from Palm Springs. This was in 1990. Her illness began one year earlier, when she merely noticed the inability to spell some words. She was treated for a stress-related disorder, but her condition deteriorated within the year. An MRI showed bilateral periventricular lesions. She was referred for a stereotactic brain biopsy. The next slide illustrates the brain biopsy. Noteworthy, is the absence of any inflammatory response. Electron microscopy, however, showed vacuolated glial cells, as seen on the next slide, with some atypical structures consistent with herpesviruses.

I chose to mention this patient because the biopsy was performed way back in 1990. I have encountered a number of patients since then with atypical neurological illnesses attributable to a viral encephalopathy. One of the last, but unfortunately not the last brain biopsy that I have examined, is this biopsy from a 30 year old dentist who was asymptomatic midway through last year. Her condition slowly deteriorated, especially affecting her work performance and social demeanor. Again, her initial illness was simply ascribed to stress. Essentially she had what is called chronic fatigue syndrome. Her illness became quite severe in May of this year. Following a request to her mother for help, she was admitted to a psychiatric hospital with a psychosis. Her condition further deteriorated and she became deeply comatose. She had a brain biopsy which showed the characteristic vacuolated cellular appearance seen in this slide. For two months, she remained deeply comatose. She has since, however, shown significant improvement but is still quite incapacitated.

I will now describe the in vitro culture findings of viral induced cytopathic effects. This next slide demonstrates the typical effects seen when blood and other samples are cultured with human fibroblasts. The fibroblasts develop vacuolated changes similar to those in the brain biopsies.

I want to emphasise that in the various patients from whom we have had positive cultures, routine testing for human viruses have yielded negative results. It is only by careful culturing that we pick up the cytopathic effect. By regular refeeding, the cytopathic effect progresses to yield large syncytial cells, that can look like foamy cells. Such cultures clearly show a very marked cytopathic effect. Strongly positive cultures have now been seen using blood, brain and CSF samples from a large number of neurologically impaired patients.

The important question is, what is causing the in vitro cytopathic effect, and can we relate it to what is going on in the brains of patients, with milder degrees of brain dysfunction than those in whom brain biopsies were performed.

We now have answers to some of these questions. This is the in vitro cytopathic effect on human fibroblasts induced with cerebrospinal fluid of a 23 year old woman admitted in early 1991 to the Los Angeles County Hospital. Her illness had began when she was 19 years old, with an initial diagnosis of schizophrenia, subsequently changed to bipolar manic depression. She deteriorated in early 1991, to the extent that she became comatose, developed seizures and had a brief cardiac arrest. She was treated initially as if she had a herpes simplex encephalitis. But as with several other patients, a confounding finding was the lack of any inflammatory reaction in her cerebrospinal fluid. So the conclusion was reached that she probably had overdosed on some medication. This particular patient has never recovered from her comatose state. Similar to the school teacher, this patient has remained in a vegetative state. She is awake, but has had no cerebral functions since 1991 when she was admitted to the hospital. I'll come back to this patient in a while.

Cultures showing these types of vacuolating cells have been obtained from patients who have had less severe, but still persisting, neuropsychiatric illness. Many of these patients have been simply characterized clinically as having the chronic fatigue syndrome.

This is one such patient's in vitro culture showing the vacuolated cytopathic effect. In her case, electron microscopy revealed numerous viral particles. They looked like herpesviruses, particularly cytomegalovirus even showing typical dense bodies. Yet when we performed specific serological and polymerase chain reaction based tests to see if they were human cytomegalovirus, human herpesvirus 6, Epstein-Barr virus, varicella zoster or human herpes simplex virus, we obtained negative results.

Another striking feature of the cytopathic effect seen with this virus, was that it was not restricted to human cells, but instead had an extremely wide host range. The cytopathic effect could be transmitted to cells from a variety of species, even down to the level of insect cells, inducing the same transmissible vacuolating cytopathic effect.

The wide host range allowed me to undertake animal studies with Dr. Tom Glass of the University of Oklahoma. We wanted to see what would happen if we inoculated the virus into cats. What will be published soon, is the description of the severe acute encephalopathy which occurred in the cats inoculated with this virus. We did the study in cats becaus of epidemiological data and anecdotal reports of unexplained illness in pets of chronic fatigue syndrome patients.

The virus was pathogenic and able to induce an acute encephalopathy in animals. The inoculated cats changed dramatically from friendly, frisky animals to very irritable, disturbed animals. Yet routine histopathological analysis of the brains of the animals showed rather subtle changes, for which most neuropathologists would exclude infection, especially since the changes occurred in the absence of inflammation.

If one looks for cellular details, one can see the same characteristic vacuolated cells with distorted nuclei as seen in the human brainn biopsies mentioned before. Moreover, by electron microscopy, one can see viral particles often accompanied by a lot of extraneous viral-like products. It is the lack of inflammation that led to the term "stealth virus, referring to viruses that were disregarded by the immune system yet retain the ability to disrupt cellular function, particularly within the brain.

The next major issue was to come to grips with what kind of viruses were we dealing with and where had they come from.

I, therefore, proceeded with molecular studies. I was able to isolate DNA from the viral cultures, clone it and begin to do sequence analysis. Last year, we reported that the initial sequence data clearly indicated a relationship to cytomegalovirus.

Only limited sequence data are available on animal cytomegaloviruses, unlike human cytomegalovirus which has been completely sequenced. What we did find was only a 50 to 60 percent homology to human cytomegalovirus. But by serology and PCR it clearly was not identical to human cytomegalovirus.

This situation persisted until we could obtain additional sequence data covering regions of the virus where there were corresponding sequence data available for various animal cytomegaloviruses.

This analysis led to a definitive indication that the virus we were characterizing had a very high level of sequence homology to the simian cytomegalovirus of African green monkeys. The African green monkey was, therefore, the origin of the atypical cytopathic stealth virus repeatedly cultured from this patient.

Having had the nucleotide data, one could translate it to amino acid data, and again do a direct sequence comparison with the known herpesviruses. These various letters indicate the amino acids in the stealth virus. One can see with one base change or amino acid change, the sequence is identical over this region to simian African green monkey cytomegalovirus. In contrast, there are additional changes from human cytomegalovirus, murine cytomegalovirus, etc. We can quantitate the matches of the stealth virus with various cytomegaloviruses using computer-assisted analysis that are described in quantitative terms using FastA and BlastN programs. A FastA value over 100 is significant. The homology to human cytomegalovirus is reflected in the score of 348. It has a greater homology to rhesus monkey cytomegalovirus, but an extraordinary homology to African green monkey simian cytomegalovirus. This is also expressed in the BlastN scores. Ten to minus 8 for human CMV: Ten to minus 63 for rhesus CMV; and ten to minus 245 for African green monkey CMV. It is irrefutable that the sequence is from African green monkey cytomegalovirus.

The point is, now that we have sequence data - we can begin to do polymerase chain reactions on the blood and cerebrospinal fluids of patients and see if they are carrying monkey virus.

The patient with the chronic fatigue syndrome who has been sick since 1990 is still sick. We have taken her blood and run PCR assays for simian CMV. One can see products formed using primers reactive with simian CMV.

We did the same thing with cerebrospinal fluid from the woman now in a vegetative state, who had the manic depressive illness at age 19 and became comatose at age 23. Her CSF still gives a positive PCR assay.

I am quite confident that the virus we are describing is present in at least these patients. It is not exactly the same as simian cytomegalovirus, in the sense that there are DNA sequence changes that have occurred, possibly as part of a stealth adaptation. We can discern differences in the pattern of the PCR reactivity with these viruses, compared to intact monkey virus. The cytopathic effect is also more subtle, and differs from that with the Colburn isolate of simian CMV. Most importantly, is the ability to grow in a wide inter-species manner. They are potentially a zoonosis threat that could infect man and animals.

The African green monkey origin is a major concern with regards to polio vaccines. Therefore, requests have gone repeatedly to CDC, FDA, to begin to look at the issue of live polio vaccines as a potential source of the inadvertent introduction of monkey cytomegalovirus stealth adapted viruses to the human population. Thank you.

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