W. John Martin, M.D., Ph.D.
Presentation made at a meeting entitled:
"Consultation on Detection of Simian Cytomegaloviruses in Human Tissues"
Sponsored by the National Institute of Allergy and Infectious Diseases (NIAID) and held in Room 1A1 Solar Building, 6003 Executive Blvd. Rockville MD 20892, July 1, 1996
The purpose of the meeting is to "evaluate existing data and to discuss approaches for the detection of Simian CMV in human tissues." This issue arose from a presentation made to the Institute of Medicine Vaccine Safety Forum on November 6, 1995 and from reports of stealth viruses infecting patients with neuropsychiatric illnesses. Published data indicate that some stealth viruses have arisen from African green monkey simian cytomegalovirus (SCMV), a potential contaminant of polio vaccines. Requests for immediate FDA/CDC action and for financial support to continue these studies have been denied.
As reflected in the following quotations, vaccine contamination has been a recurring concern with all live vaccines, including polio.
"The use of living organisms for immunization on a large scale is always liable to occasional disasters. It may be possible to be quite certain that the germ we intend to inoculate is harmless, but there is always a possibility of some unwanted and harmful micro-organism finding its way into the material. Cultures or tissue extracts containing living organisms to be used for inoculation cannot be sterilized, so there is no automatic method for which their safety can be assured. In any reputable laboratory every safeguard will be taken to eliminate any possibility of danger from contamination, but all these precautions depend ultimately on human vigilance, which is never infallible."
F. MacFarlane Burnet, 1940
"As monkey kidney tissue is host to innumerable simian viruses, the number found varying in relation to the amount of work expended to find them, the problem presented to the manufacturer is considerable, if not insuperable. As our technical methods improve, we may find fewer and fewer lots of vaccine which can be called free from simian virus."
Hilary Koprowski, communication to Congress, 1961
"If a vaccine program were to be initiated today, one would surely not import wild monkeys from Africa, create short term primary kidney cultures, add a human virus and administer the crude gamish derived from the virally infected cells to virtually every child in the country. Nor would one want to withhold applying the many molecular biological techniques developed over the last 30 years to assess vaccine purity. Yet this is essentially the situation with live polio vaccine and comparable arguments can be made for other human and animal viral vaccines."
W. John Martin, California Dept. of Health sponsored meeting on 20th Century Plagues, March 1, 1996
EVIDENCE OF POLIO VACCINE CONTAMINATION
During the early period of polio vaccine production, greater than half of the rhesus monkey kidney cultures established for polio vaccine production were discarded due to the presence of adventitious viral agents.
In 1959, NIH researcher, Dr. Bernice Eddy went beyond the approved testing protocols and began to test whether rhesus monkey kidney cultures contained a virus able to induce tumors in hamsters. Her positive results were suppressed. Proof of the oncogenicity of a virus frequently present in rhesus monkey kidney cells was subsequently provided in 1960 by Drs. Sweet and Hilleman of Merck. The virus was designated SV-40, since it was the fortieth virus so far found in rhesus monkeys. SV-40 was shown to contaminate many lots of polio and adenoviral vaccines. It was not completely inactivated by the 0.4% formaldehyde used to produce Salk vaccine. There was no recall of the already distributed contaminated lots of polio vaccines. Instead, SV-40 neutralized polio vaccine was simply transferred to African green monkey kidney cultures for the continued production of vaccine.
Between 1961 and 1963, the United States also switched from formaldehyde treated Salk vaccine to Sabin's live attenuated polio vaccine. Again, there is documentation of the willingness of those in-charge of the vaccine program to "turn a blind eye" to the possible presence of contaminating monkey viruses:
"Manufacturing regulations limited the observation of tissue culture control bottles (containing 25% of the monkey tissue used to manufacture polio vaccine) to 14 days - a time period chosen for the specific purpose of passing vaccine lots made in tissue harboring extraneous viruses in 'eclipse'. Longer observation periods (21 or 28 days) were rejected because the expected appearance of contaminants might require rejection of a monopool of the vaccine. The NIH adopted the 14 day time period and manufacturers switched to the untested African green monkey kidney tissue in which SV-40 was not indigenous. Everyone at the meeting agreed that the potential for the presence of a then undetectable virus in African green monkey tissue was great, but since nothing could be detected, the material would pass regulations for production as drawn."
NIH Meeting, circa 1961
"I told Dr. Murray that there was some concern at Lederle about a possible requirement barring the use of African green monkey kidney as the substrate for the growth of attenuated polioviruses...Dr. Murray had stated that the adventitious agents that Dr. Kendall Smith is presumably detecting by his techniques are of little consequence for an oral preparation in that such a large experience exists with the use of oral polio vaccine without any evidence of trouble relating to these agents."
Lederle memo August 23, 1968
"All eleven monkeys studied demonstrated the presence of CMV-like agents. These monkeys all originated from Kenya over a short period of time. Seven of these monkeys would have passed our existing test standards, only one of these monkeys would have passed our test methods using Lederle 130 human diploid cell strain..... We plan to continue to process monkeys at the rate of five per week, probably through October 1972, to provide us with thirty million doses of trivalent poliovirus vaccine...... Unless and until Pfizer's Diplovax is in abundant supply, the BB [Bureau of Biologics] cannot risk Lederle being off the market."
Lederle's "Cytomegalovirus Contingency Plan", 8/4/1972
"I do not believe our problem with the slow release of specific lots of Orimune(R) are a result of a Pfizar influence..... Furthermore, if the Bureau wanted to restrict us they could bring up the subject of CMV (Cytomegalovirus) in our substrate (i.e. African green monkey kidney tissue) which they have not done, even though they have told us the monkeys in the collaborative study performed in 1972 were all positive for this agent."
Memo to the President of American Cyanamid, 1973
An atypical cytomegalovirus, subsequently shown to be an African green monkey simian cytomegalovirus (SCMV), was apparently cultured from a brain biopsy of a 7 year old boy with an atypical encephalopathy. The virus became known as Colburn SCMV.
Dr. Alford and colleagues, 1973
"According to Dr. Petricciani of the BoB, monopool PP3-444 contains 'reverse transcriptase' an enzyme often found to be associated with a certain class of tumor viruses."
American Cyanamid memo, October 1976
"The same agent which is present in 444 giving rA.dT activity can be transferred to FCS-7, MRC-5 and FRhL-1 cells."
Memo from J. Petricciani, 1976
"To our dismay, purified virus RNA was always contaminated by large quantities of unlabeled cellular DNA..... If the presence of cellular DNA in vaccine preparations is a routine occurrence, it would seem possible that there would be a possibility of occasional transfer of genetic information of the viral host species to the vaccine recipient species."
John Dahlberg's review of EM of polio batch PP3-444 January 1977
Not all of the DNA in polio vaccines is from normal monkey cells or from the fetal calf serum used to grow the monkey cells.
Results of G. Aulakh and W. John Martin in 1977
"Stop worrying about it, every time you eat an apple you ingest foreign DNA."
Comments from BoB Director to W. John Martin, 1977
A recipient of an experimental rubella vaccine made in African green monkeys is considered to have been infected with a simian cytomegalovirus.
As remembered by a CDC technician (J.P.)
Significant reverse transcriptase (RT)-like activity is present in 3 of 12 batches of polio vaccine. Six of the 12 batches appear to induced high levels of RT-like activity in cultures of human peripheral blood lymphocytes.
Handwritten notes of a BoB studies dated Aug-Oct, 1985
SV-40 virus is detected in human mesotheliomas, ependymomas, choroid plexus brain tumors and osteogenic sarcomas.
Results from Drs. Carbone, Lednicky and Butel, 1992-1996
DNA sequencing of a virus repeatedly isolated from a patient with chronic fatigue syndrome (CFS) shows that it originated from African green monkey simian cytomegalovirus (SCMV). A molecularly related "stealth" virus is also isolated from a patient with acute encephalopathy and a 4 year history of bi-polar psychosis.
Results of W. John Martin 1991-1995
SUMMARY OF EXPERIMENTS LEADING TO DETECTION OF STEALTH VIRUSES
The project was initiated in 1988 to see if patients diagnosed as having CFS were virally infected. Using the polymerase chain reaction (PCR), a primer set reactive with all human herpesviruses, gave weak positive signals with blood from some CFS patients, but not with blood of laboratory control personnel. CFS patients also not infrequently, gave a low level response using primers designed to amplify the tax gene of human T lymphocytotropic viruses.
The potential significance of the marginal responses seen in CFS patients became clearer in 1990 when strikingly positive PCR responses occurred with cerebrospinal fluid (CSF) samples from two patients and on a brain biopsy of a third patient. All three patients had unexplained severe encephalitis-like illnesses. The brain biopsy came from a 39 year old school teacher who had gradually lost her capacity for written and oral communication, but was otherwise alert with no localizing neurological signs. She did, however, have an abnormal MRI with periventricular lesions. The brain biopsy showed minimal cellular changes with no inflammation. Complete and incomplete forms of herpesvirus-like particles were present in several of the glial cells examined by electron microscopy. Some of the virus positive cells showed marked vacuolated changes and lipid accumulation. This patient has since further deteriorated and is in a vegetative state. An MRI performed in March 1996, shows severe atrophy with marked ventricular dilatation. The PCR positive CSF samples tested in 1990, were both acellular, indicating an absence of inflammation. One came from a premature infant with delayed neural development. This child gradually improved but still performs at a below average level. His mother has had an MRI for headaches and other symptoms which have developed over the last several years. The other CSF sample came from an adolescent with residual severe brain dysfunction three years after what was labeled an atypical herpes simplex encephalitis. His younger brother also had an unexplained encephalitis-like illness with coma from which he had recovered.
I had made earlier attempts to grow human herpes virus-6 (HHV-6) from CFS patients using cord blood lymphocytes. Routine human fibroblast cultures from CFS patients were also established. The fibroblasts frequently showed a delayed, transient cytopathic effect (CPE) somewhat suggestive of human CMV. Buoyed by the PCR findings in the severe encephalopathy cases, attempts were again made to culture a virus from a PCR positive patient. I became aware of a 43 year old healthcare worker who had not fully recovered from an unexplained subacute encephalitis-like illness in July of 1990. Her blood tested positive using both the HTLV and the herpesvirus primer sets. An additional blood sample was collected for culture. After 6 weeks, enlarged, rounded, vacuolated cell syncytia appeared in both human and monkey derived cultures. The CPE could be passed to many other cell types and to cells of multiple species. Electron microscopy showed numerous complete and incomplete herpesvirus-like viral particles. The foamy, vacuolated cells did not, however, stain with antisera specific for the immediate-early gene of human CMV. Nor did the cells stain with antisera specific for HSV, HHV-6/7, VZV, adenoviruses or enteroviruses. PCR assay using the HTLV tax gene primer gave two well defined products. Sequencing of one of the PCR products (GenBank accession # U09212) identified a region of partial sequence homology to the UL34 gene of human CMV. The sequence of the other PCR product (GenBank accession # U09213) was suggestive of a possible herpesvirus origin but could not be aligned with any of the known herpesviruses. Viral DNA was isolated and cloned. A region of sequence homology to African green monkey simian cytomegalovirus (SCMV) was noted in early 1994 and reported to CDC. Dr. Gary Hayward of Johns Hopkins University submitted additional sequence data on SCMV to GenBank in December 1994. From his sequence data, it was unequivocal that the CFS patient's virus had originated from SCMV. This conclusion was reported to FDA in March of 1995.
In 1991, I coined the term "stealth" to describe the viruses that were being identified in blood and CSF cultures, and which had been seen on electron microscopy of the brain biopsy. This term was used primarily because it denoted a cytopathic infection occurring in the absence of inflammation. The SCMV-derived virus isolated from patient D.W., was designated stealth virus-1. She has remained quite disabled with fatigue and cognitive impairment. Positive cultures are still obtainable from this patient but complete viruses are no longer seen by electron microscopy and no longer react in PCR assays using HTLV primers.
A molecularly related virus (stealth virus-2) was isolated in February 1991 from the CSF of a patient with an acute severe encephalopathy. The 23 year old female patient (B.H.) had a 4 year history of a manic depressive illness. Her encephalopathy was attributed to an attempted suicide from drug overdose, for which no evidence was subsequently found. A diagnosis of viral infection was excluded mainly because of the paucity of cells in her CSF and the lack of a response to acyclovir therapy. The patient has remained in a vegetative state since 1991. DNA sequencing of a PCR amplified product from her infected cultures showed that it was similar, but not identical, to the SCMV-derived stealth virus-1. Numerous other stealth viral isolates have been obtained from patients with various types of illnesses and from some apparently normal controls. I am awaiting resources to perform a true prevalence study as well as sequencing analyses on several of the isolates.
Restriction enzyme digests of stealth virus-1 have been cloned and partially sequenced. The genome appears to comprise multiple fragments of approximately 20 kilobase pairs. The virus displays an unusually high degree of sequence microheterogeneity suggesting infidelity of DNA replication or an alternative replication strategy. The concept of a fragmented, unstable viral genome is consistent with electron microscopic observations on several additional stealth viral isolates. It has an important implication for the limited utility of molecular techniques to screen for these viruses. While replication errors may help explain recovery from infection in the absence of an inflammatory response, it does raise some concern about the potential oncogenicity of stealth viruses.
Stealth virus-1 induces an acute neurological illness when inoculated into cats. The early manifestations developed within a week and included gingivitis, bloody ocular and nasal discharge, lymphadenopathy, pupil dilatation with photophobia (squinting in response to light) and nuchal hair loss from rubbing against the cage. There was a reduction in body temperature beginning in the second week which averaged 0.6oF at 2 weeks and 0.8oF at weeks 3 and 4. Most striking was the marked behavioral changes in all of the virus-inoculated cats. They lost the playfulness that was present prior to injection and became reclusive and irritable. They resisted being handled and the animal caretakers resorted to wearing leather gloves. By palpation, the enlarged lymph nodes and various muscle groups were identified as being painful for the animals. The severity of the illness peaked at around 4 weeks with definite improvement noted in the cat necropsied at 6 weeks. A cat that was maintained to 15 weeks had resumed normal activities by week 10 and appeared to be symptom-free. Histological examination of animals' brain tissue showed foci of cells with cytoplasmic vacuolization and an absence of any inflammatory reaction. Electron microscopy confirmed the presence of occasional herpes-like viral particles along with accumulations of viral-like granular and membranous structures suggestive of subgenomic viral expression. A cat injected with heated stealth virus-1 did not become sick upon receiving unheated stealth virus-1, but did succumb to neurological illness when subsequently injected with the stealth virus isolated from another patient.
Unpublished surveys suggests that the pets of patients with CFS have a higher than expected incidence of neurological illness. This observation is consistent with a human to animal disease transmission. Furthermore, blood from a symptomatic cat transmitted disease to a healthy recipient cat. The brains of both animals showed vacuolating cellular changes characteristic of stealth viral infection.
STEALTH VIRUSES ISOLATED FROM PATIENTS WITH SEVERE ENCEPHALOPATHY
Since beginning these studies, many patients have been identified with complex neuropsychiatric and rheumatic illnesses that exceeded the normal bounds of existing medical specialities. The finding of a positive stealth viral culture in such patients may help unravel the true cause of their illness and provide a fresh approach to therapy and disease prevention. Children with autism and other pervasive developmental disorders have also shown consistent evidence for stealth viral infection. Clinical observations and interviews with many of the neurologically affected patients, lend support to the concept of a spectrum of potentially infectious neuropsychiatric illnesses. The distinction between patients labeled as having different illnesses may simply reflect infection of different regions of the brain and varying psychological reactions to the resulting dysfunctions. Support for a viral encephalopathy etiology in some of these patients has been provided by detailed histologic examinations of additional brain biopsies which reveal similar cytoplasmic and nuclear changes as those seen in the brains of infected cats. In one of the biopsies, there was an additional vasculitis component, while in another, there was an overall low level inflammatory reaction. Electron microscopy readily confirms the cellular vacuolization, often accompanied by mitochondrial disruption. Although intact viruses may be rare, prominent accumulations of viral-like materials can usually be seen.
A promising clinical aspect of these infections is the marked improvement that has occurred in some of the severely ill patients. A better understanding of the mechanisms involved in their recovery process offers some hope for the many patients in whom their tragic disease simply persists for years. Detailed clinical summaries of twelve such patients, including several healthcare providers who may have been occupationally infected, was provided to the FDA and to the CDC in June 1995.
GOVERNMENTAL RESPONSE TO REPORTS OF ATYPICAL VIRAL INFECTIONS
No specific enquiries were received about any of the patients reported to the FDA and CDC. Nor was the requested financial support provided to study the prevalence of SCMV-derived stealth virus infection in humans or to perform sequencing on additional viral isolates. FDA has sided with American Cyanamid to legally resist making the polio vaccine lots administered to a child who has an unexplained neurological disease, (W.W.), available for independent analysis for contaminating viruses. FDA and CDC appear reluctant to openly address whether vaccines could have contributed to the increasing incidence of dysfunctional brain syndromes, including CFS. The possibility that monkey viruses may have entered into the nation's blood supply certainly warrants the attention of FDA.
The Gulf War syndrome has stricken many American servicemen, but has largely been played down as a possible infectious disease. Considering all of the counterproliferation efforts to combat biological terrorism, the Military should be concerned with the quality of the vaccines and of blood products, including gamma globulin preparations, given to its troops.
Most individuals are unaware of the enormity of the world's trading in vaccines and blood products. As in other large industries, the public needs reassurance that the agencies designed to act as watch-dogs do not simply become shields for questionable business practices. Legislation allowing the bypassing of FDA regulations of blood products exported from this Country, including testing for HIV, undoubtedly contributed to the spread of this infection throughout the world. It would be a sad commentary, if the grand contributions of Dr. Salk and Dr. Sabin to the eventual elimination of polio were to be marred by inattention to problems not fully appreciated at the time of their discoveries.
Immediate scientific disclosure of non-published studies relating to the safety of polio vaccines conducted by FDA and/or by American Cyanamid (Lederle).
Independent (non-Governmental and non-industry) scientific review of the production, source material and testing protocols utilized in the currently licensed live and inactivated polio vaccines.
A statement to the public that until these data have been reviewed and recommendations implemented, there should be no compulsory polio vaccination.
Testing of blood products, including gamma globulin preparations for stealth viruses and for SV-40.
Prevalence study on stealth and SV-40 viral infections in the general population and in various disease groups.
Notification through the MMWR of the need to report cases of atypical encephalopathic illnesses.
A waiver given along with any product submitted for IND or NDA approval giving the FDA the authority, and indeed the obligation, to inform the medical and scientific communities of any information it gathers related to the product's safety.
1. Martin W.J. Stealth viruses as neuropathogens. CAP Today 8 67-70, 1994.
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, 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.
5. Cristaudo A, et al. Molecular biology studies on mesothelioma tumor samples: preliminary data on H-ras, p21 and SV40. J. Environ Path 14: 29-34, 1995.
6. Lednicky JA et al. Natural simian virus strains are present in human choroid plexus and ependymoma tumors. Virology 212: 710-717, 1995.
7. Carbone M, Rizzo P. Procopia A., et al. SV40-like sequences in human bone tumors. Oncogene (in press).
8. Martin WJ. Severe stealth virus encephalopathy following chronic fatigue syndrome-like illness: Clinical and histopathological features Pathobiology July 1996
9. 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.
10. Gollard RP, Mayr A, Rice DA, Martin WJ. Herpesvirus-related sequences in salivary gland tumors. J Exp Clin Can Res.15: 1-4, 1996.
11. Martin WJ. Genetic instability and fragmentation of a stealth viral genome. Pathobiology July 1996.
12. Martin WJ. Simian cytomegalovirus-related stealth virus isolated from the CSF of a patient with bi-polar psychosis and acute encephalopathy. Pathobiology (in press).
13. Martin WJ. Stealth viral encephalopathy: Report of a fatal case complicated by cerebral vasculitis. Pathobiology (in press).
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