POSTED BY: Camilla Cracchiolo,
SOURCE: August 28, 1994 lecture
COPYRIGHT: None
Lecture by John Martin, MD PhD at USC
Summary: Dr. John Martin, a pathologist at USC, spoke in Los Angeles
at an event sponsored by the Los Angeles CFIDS Association. The purpose
of the lecture was to raise funds so that he could continue work on
a virus he has isolated and of which he has partially sequenced the
DNA. He has published this information in the American Journal of
Pathology, vol 145, no. 2, August 1994, pp.440-452. The title of the
paper is: "Cytomegalovirus-Related Sequence in an Atypical Cytopathic
Virus Repeatedly Isolated from a Patient with Chronic Fatigue Syndrome".
The authors are listed as Martin, Zeng, Ahmed and Roy. Martin has
stated that he needs $200,000 to continue this work. $100,000 would
go to finishing the DNA sequencing and $100,000 towards working on a
drug to inhibit its growth.
I will give the details of the lecture in the 'Highlights' section
of this report; I want to provide the key information and issuesfirst.
Martin thinks that this virus is very significant: it may bypass
all immune defenses, which would explain why no antibodies show up
in blood samples, and why there appears to be minimal tissue
inflammation when blood tests are done (people with CFS often have
normal red blood cell sedimentation rates and white blood cell
counts, both measures of inflammation in the body.) He suspects
that viruses of this type may be involved in many illnesses, not
only CFS. Various psychiatric conditions, autism, and auto-immune
diseases such as lupus were mentioned as possibly being related to
this virus. Dr. Martin and team have also been able to grow this
virus in the cells of a variety of animals, and suggests
that the ability of this virus to infect non-human species may be
the reason why so many people with CFS have reported CFS-like
illnesses in their pets. He hopes this paper will help defuse the
fights over whether CFS is caused by a virus or not. He feels that,
whether this virus proves to be the cause of CFS or not, he has
shown that it is possible for humans to be infected with a virus in
the central nervous system, and for this virus to not provoke an
inflammatory reaction or to be easily cultured.
There are controversies about this work that will continue even
though this paper has been published. This is the same virus that
Martin previously reported as a spumavirus (a kind of retrovirus
that causes infected cells to form structures that look like air
bubbles and to have a 'foamy' appearance; another name for spuma
viruses is foamy viruses.) He now believes that this virus is a
mutation from cytomegalovirus (CMV), a member of the herpes family,
and *not* a retrovirus. In 1991, the US Centers for Disease Control
ran a double-blind test to evaluate Martin's findings. The CDC
sent Martin blood samples from both people with CFS and healthy
controls, coded by number. Martin was unable to distinguish the samples
from people with CFS from the healthy controls; this virus (or a
similar one) was detected in 47% of the people with CFS and 49% of
the controls.[1] Several other researchers have tried to isolate this
virus, including noted AIDS and CFS researcher Jay Levy in San
Francisco, Gow and Behan in Scotland,[2] Flugal and Komaroff [3]
in the US and Holmes in Australia, but they have been unable to
duplicate Martin's work.
He also talked about 'epione', a substance that he stated inhibits
the growth of this virus in a test tube. It was not clear from his
comments whether epione actually exists, or if they are still
searching for an inhibiting chemical. He refused to state what it
was made of, and was vague about how long it will be to complete
the testing process before it can be tried in humans.
Some other key questions remain to be answered as well:
1. Has he actually isolated a new virus or is he picking up
something from the cells in which this virus was grown?
(this is apparently not uncommon when using the type of PCR
testing he reported). Hopefully this will be addressed as
other researchers attempt to replicate his findings.
2. Is this virus pathogenic (disease causing)? Or is it a harmless
traveller along for the ride? Since we don't know if this virus
was present prior to the patient developing symptoms, we still don't
know how closely it's associated with CFS. Even if Martin's
work is soon replicated, it may take a long time before we know
for certain the answer to this question.
3. Is this virus a new 'stealth virus' or is it just another member
of the herpes family (perhaps HHV-8)? And if so, is it any more
significant than any other herpes virus in CFS, possibly
representing
a reactivation of an old herpes infection rather than actually
causing CFS?However, Martin's research is very interesting, and if
it
can be
replicated has tremendous implications for people with diseases of
the central nervous system. The paper published in Journal of
Pathology is very detailed, so it should be easy for other
researchers to duplicate his techniques.
Before I get into the highlights section, I want to share some of
the things I found out while trying to interpret Martin's paper.
For those people who don't understand what PCR is (polymerase chain
reaction, the method Martin used to find the viral DNA), here is a
passage from _Microbiology: Principles and Applications_ byJacquelyn
Black:
"PCR allows us to produce rapidly (amplify) a billion copies of
DNA without needing a living cell. These large quantities are
then easily analyzed..... What happens in PCR amplification of
DNA? A large piece, or mixture of pieces, of DNA is cut up into
smaller pieces by ...enzymes. It is necessary to know the base
composition of the ends of the exact piece of DNA you wish to
replicate. In less than 24 hours, automated synthesizing equipment
can make...primer molecules that will pair complementarily with the
ends of the desired section of DNA. A primer is a molecule that
will serve as a starting point for DNA synthesis....When the
thermal cycling has produced billions of copies of the desired
piece of DNA, the DNA is easily detected (as in a clinical
diagnostic test) or analyzed for total base sequence. Cutting
a large pice of DNA up into smaller pieces, sequencing the PCR
amplified quantities of these, and then looking for overlaps at
the ends will allow us finally to determine the swquence of the
entire original DNA piece"
I discussed the Martin paper with several pathologists and viral
researchers. Here is what one of pathologist said about the kind
of testing Martin used:
"They used a low stringency method of amplification to get the
products to examine. In PCR reactions stringency determines the
fit of the primer to the DNA template. You could imagine this as
being the same as buying shoes: High stringency means you will
only accept a size 9 width E shoe, low stringency means that
although
you need a 9E, you will accept a size 11 because it doesn't pinch.
Low stringency means you cannot be entirely certain what you are
amplifying. You just have to assume that they were amplifying
something in the virus they were culturing, but it is possible that
they got a sequence from the culture cells (they say that tests were
done using Rhesus monkey kidney cells, MRC-5 (human lung
fibroblasts),
and MRHF (human foreskin fibroblasts), but all of these cells are
from
closely related animals so there may be a homologous sequence in
these
cells that isn't present in hamsters, ducks etc, which the virus
wouldn't grow in."
Highlights: The event began by an introduction from Heather Hunter,
the group leader of the Los Angeles CFIDS Association, a patient support
group. The Los Angeles CFIDS Association is not a fundraising
organization, but would like to see a CFIDS fundraising organization
start in Southern California, similar to the CFIDS Foundation in
San Francisco. They are very enthusiastic about Dr. Martin's
research. Ms. Hunter went over Dr. Martin's credentials: he
originally obtained his MD and PhD in Australia. He travelled
to the US and England. In the mid-seventies he became Director of
the viral oncology lab of the Bureau of Biologics at the National
Institutes of Health. Between 1980 and 1985, he became a
pathologist and obtained certifications in immunopathology and
medical microbiology. He became professor of pathology at the USC
School of Medicine in 1985 and has been working on CFS researchsince
1987.
Dr. Martin began his lecture by introducing the other members of the
research team: Khalid Ahmed, who did all the actual cultures (over
3000); Dr. Zeng, described as an expert in polymerase chain
reaction (PCR), the technique used to identify the virus; Sally
Spelling (sp?), a pre-med student and volunteer and advocate for
autistic children; Nicholas Chelnikov (sp?) a biologist from the
University of Moscow.
He stated that the USC Medical Center may have been the site of the
first breakout of CFS back in 1934. Over 1000 individuals were at
that time diagnosed as having 'atypical polio-like syndrome', which
went on to all the hallmarks of what we now know as Chronic
FatigueSyndrome.
Martin stated that his own interest in CFS began as an effort to
bridge basic research and clinical medicine; trying to bring PCR
technology into the patient setting. He recognized early on that
CFS has devastating consequences to the people who have it; he also
is interested in other neurologic and psychiatric illnesses, and
began looking at the possibility that viruses could be involved. He
discussed a number of patients that he has cultured viruses out of,
who had very severe illnesses but where there was no clear
understanding of the cause.
He first described a 39 year old school teacher who was eventually
diagnosed as having CFS. (This is not the woman from whom the virus
Martin wrote up for Journal of Pathology was grown.) This woman was
a very conscientious worker. She began making many spelling and
grammatical errors early on in the course of her illness, and lost
her teaching job because of this. She had seen many doctors and
psychologists for stress management and tried taking a part time
job as a kindegarten teacher. She was unable to handle even this
lightened work load. All of her examinations were normal, but she
managed to find a neurologist who took her situation seriously and
did not stop at a physical exam. This doctor ordered an MRI scan,
and the MRI had very noticeable 'unidentified bright objects'. Dr.
Martin showed a slide of this woman's MRI. The UBOs were quite
apparent to even an untrained eye such as my own; he described these
as being around the ventricles. Dr. Martin stated that in the last
few years, neurologists have begun to recognize 'sub-cortical
brain pathology', where the physical examination is normal, yet
clearly something abnormal is happening in the brain. She was
referred to USC for evaluation; a brain biopsy was taken. He then
showed the slide of the biopsy specimen to the audience; no signs of
inflammation, although he stated that 'a little gliosis' was noted.
This specimen was further examined with an electron microscope, and
clear
abnormalities were seen here. The vacuoles were enlarged (vacuoles
are spaces inside the protoplasm of cells, which store various
enzymes and other materials) and dark particles that looked like
viruses were seen lining the inside of them. (If you want to know
what viruses look like, I recommend the Scientific American book
_Viruses_ by Arthur Levine. The slide that Dr. Martin showed was
very similar to pictures in that book.) He stated that a virus has
been repeatedly cultured from this woman's blood and spinal fluid.
At the same time that Martin was dealing with this patient, another
came along with a similar situation. This one was a 23 year old
woman, with an initial diagnosis of schizophrenia. She was confined
in the County psychiatric hospital and was given lithium to which
she somewhat responded. This went on for 4 years. In 1991, she
came to the County hospital with seizures and an acute onset of
viral-like symptoms. However, on examination, her spinal fluid was
perfectly normal: no elevations of white blood cells or other
indications of infection. This lack of inflammation heavily
influenced her diagnosis, and she was diagnosed as having suffered
from a 'drug overdose'. This woman went into a cardiac arrest, had
massive brain damage and went into a completely vegetative state.
She has been this way for the last 4 years, she was sent to a nursing
home. She also has had repeated positive viral cultures in Martin's
lab and again, she had the big foamy cells and the appearance of virus
lining the vacuoles.
The next case report was from a 33 year old man who was previously very
healthy and hard working. This person had an acute onset of headaches
and loss of appetite. He progressed over the next two weeks to
confusion,
became reclusive, and non-cooperative with his family. He came to the
County ER and initially was given a psychiatric diagnosis and
admitted to the psych hospital at the medical center. He was
transferred to the medical ward, deteriorated and had an abnormal
MRI. This time there were bright spots in the basal ganglia (part
of the limbic system that Jay Goldstein and others think is injured
in CFS.) His neurologic symptoms were diffuse, rather than
localized to one side or area of the body. He was put on heavy
antibiotic medication for bacteria, fungi and viruses. Again, the
spinal fluid showed no signs of inflammation. He was sent to a
convalescent hospital and then home. CFS like symptoms of memory
loss, getting lost in familiar areas and severe fatigue persisted.
He eventually developed acute illness and was admitted to a
community hospital with seizures and died. Martin doesn't mention
if this man had viral cultures or pathology slides.
Martin is culturing viruses out of many people who have been given
serious neurologic diagnoses: MS, Parkinson's disease, childhood
schizophrenia and autism. Often one family member will be diagnosed
as having CFS and another will have one of the above illnesses.
He then went on to discuss the woman from whom the virus he reported
was cultured. She was a health care worker, and was working in a
physician's office. She reported an acute onset of symptoms to her
boss and he took it seriously and hospitalized her. Again, the
normal spinal fluid. The doctors concluded that she may have had a
viral encephalitis or menigitis, but they didn't know for certain.
She has been ill for several years, unable to work, has had her
house repossessed. Martin did molecular probing on her blood and
found evidence that a virus might be there. He then made a strong
effort to culture this, and succeeded in growing out the virus.
Again, a foamy cell effect on slides. This virus was grown out of
her blood and spinal fluid 15 out of 18 times. They also ran other
tests: 6 out of 6 specimens were positive for virus when looked at
with an electron microscope, and 8 out of 8 specimens were positive
on PCR. Cultures grown in the lab were then compared to
her blood and the same DNA sequences were found in the blood, making
it extremely unlikely that these viral cultures were growing out
contaminants from the lab. When looked at with an electron microscope,
the virus isolated looks physically similar to herpes viruses (it has a
similar envelope). But her spinal fluid remains completely normal
looking,
and Dr. Martin urges neurologists to be aware that a person may have
a viral infection in the brain and to not stop investigations with a
normal spinal fluid. This virus does not react to any of the
antibodies normally used to test for herpes family viruses. The virus
persists in this patient. They took this virus, regrew it in animal
cells, and then cloned them. They have 620 clones with 'important'
DNA sequences.They then subjected the virus to PCR to sequence the DNA.
They isolated two products, each about the same size. The first
showed no similarity to any other viruses that have been identified.
However, the second showed some similarity to CMV, about 58%
homology. It's important to note that this fragement contained
1500 base pairs; the whole CMV genome is over 200,000 base pairs
long. Thus, this is only a very small fragment; however, Martin
says that it's sufficient to establish some relation to CMV while
confirming that it is not CMV. Interestingly, the parts of CMV
that were missing in Martin's virus contained parts that help
the immune system to recognize and act against CMV. Also, unlike
CMV which is restricted to human hosts, this virus did grow in all
the animals he tested. He wants to test pets of people with CFS who
have reported that their pets developed lethargy and other symptoms
of CFS about the same time the owner did.THE QUESTION AND ANSWER PERIOD:
Before Dr. Martin took questions, he discussed the possibility of
developing a drug to counteract this virus. He calls this compound
Epione, although it wasn't clear from his comments whether he had
actually found an agent that inhibits this virus in the test tube,
or if he was still looking for it. The process would involve first
finding a substance that inhibits this virus, then testing it on
animals then getting FDA approval for tests on humans. However, he
suggested that he might get permission to use it in humans who were
close to death sooner than normal. For example, the person
mentioned earlier in this document who died was someone he would
liked to have tried some compound on. Since he has isolated this
virus from people with many different neurologic problems, what
line of research he pursues will depend a lot on what he gets
funding for. If the CFS community raises the money, then he will
pursue CFS connection. If it comes from people who want psychiatric
illnesses or autism studied, then that's what he will look at. He
wants to raise $200,000. $100,000 will go toward sequencing
the virus and the other $100,000 will to developing an antiviralagent.
The other researchers who worked on the paper were also introduced.
Dr. Ahmed discussed how he cultured the virus, which he mentioned
was an unusual technique. Unfortunately, he didn't go into detail
on this. He did say that he has done more than 3000 of thesecultures.
Since many people have the same questions that were asked at the
lecture, I've tried to stick pretty closely to Martin's answers.
This makes the report a lot longer, but hopefully it will be more
informative as well.1. How likely is it that people will be cured?
Dr. Martin: This is an important question, because it's possible
that there may be permanent tissue damage even if an antiviral
agent is found. One hopeful sign is that this virus does not provoke
a lot of inflammation, which means that the immune system does not cause
damage on top of what the virus may do. He gave hepatitis as an
example of a viral illness where most of the liver damage was caused
by the immune response rather than the hepatitis virus itself.
Electron microscopy on cells taken from infected animals reveals
that the cells remain basically intact, even though the largevacuoles
are
seen.
2. Since the virus is neurotropic, how do you feel this relates to
the high cytokine and low natural killer cell levels immunologists
are finding in CFS?
Dr. Martin: There clearly are conflicting hypotheses regarding the
cause of chronic fatigue syndrome. The immune changes are
relatively modest however, when compared to illnesses like TB. It's
not there aren't immune defects, but I feel these are secondary to
having a chronic viral infection. The proof, of course, will be if
you can eradicate the problem. There is more hope at this stage of
eradicating a virus than there is of correcting an immune imbalance.
And it's important to remember that there are many chronic fatigue
patients who have no immune changes, yet have CFS.
3. How do we relate this research to the paper Dr. Ablashi
published showing that there was no difference between blood samples
from people with CFS and healthy controls when tested for yourvirus?
Dr. Martin: I've known Dr. Ablashi for over 15 years now, in fact I
just saw him in Vancouver a few weeks ago. Again, I return to the
fact that we have kept our focus on the idea that there are viruses
that can infect the central nervous system without provoking the
usual immune response, and yet can cause significant dysfunction.
We've tested a number of families, and we have found that everyone
in the family may test positive for the virus, yet have different
problems. One may have a CFS like condition, another may be
autistic and another not affected at all. It's very important to
finish sequencing the virus, so that we can better understand why
some people with virus are quite ill and others are not. I do not
see stealth viruses as only infecting the brain. The idea is that
the virus is sufficiently handicapped that if it hits another organ,
such as the liver, it doesn't significantly impair the function of
that organ because of the reserve capacity the organ has. It's when
you put that virus into the brain, particularly a part like the
limbic system which is very delicately balanced, a little bit of
viral damage can cause a lot of deranged sensation like fatigue,
pain, cognitive dysfunctions and so forth. The arguement is that
this virus may affect a large number of people but not cause
symptoms. But this doesn't mean that therefore no one has problems.
I think it's very important to look into this question. It's
important to realize that a person may go to a doctor and the doctor
says 'you're depressed' and we treat that as though it is a final
answer. But it still doesn't tell us *why* that person is
depressed. Is there something in the brain that we should be
looking at? I went to a conference on autism recently. Everyone
there was focusing on managing the autistic child and there was no
focus asking why that child had autism in the first place. It's
inappropriate to say that 5 years ago, or 10 years ago, people
couldn't find a virus, so there's no need to look for one. There's
been such enormous advances in molecular techniques and viral
culturing in the last few years. I think the first priority should
be to apply these techniques to depression and other problems
instead of just telling the person 'you're depressed'. I want to
emphasize that stealth viruses do exist.
4. How many patients have you seen? The paper only refers to one.
Dr. Martin: We've seen an enormous number. We hope that we can develop
a generic treatment for stealth viruses that will address manyillnesses.
5. Since this research seems so important, why can't you get USC to
fund you? $200,000 sounds like a lot to us patients, but is a very
small amount for a research grant from a place like USC.
Dr. Martin: I'm very grateful to USC. The university environment
is what makes this kind of scientific exchange possible. We have to
get the work sufficiently established for publication purposes
first. The university is not in the business of giving money;
however it has been focusing its efforts on AIDS and cancer
research, and I have heard from the fundraising office about our
research that this may be another major catagory as well. Our
task now is to convince people who may not be aware of CFS to give a
large amount of money. There's always discussion that some people
who are quite well known may have this illness themselves. Right
now we have to sequence the virus.
6. How long would it take to get Epione through the whole FDAprocess?
Dr. Martin: I think a lot of the criticism of the FDA is unjustified.
If there is proof that something works, it's possible to get through
the FDA process pretty fast. Contacts I've had so far have indicated a
strong willingness to set up a pre-IND meeting with the FDA to
review it. The responsibility on my part is to sufficiently define
and standardize what we hope to do so they can scientificallyevaluate
it.
7. How long will all this take? Weeks? Months? Years? (note:
this question didn't come out well on the tape, so it's not exact)
Dr. Martin: We're working as a hard and as fast as we can, and see us
developing a testable hypothesis in a short period of time. We're not
going to spend 5 years looking at the virus and another 5 looking for an
inhibitor. We've already spent that 5 years looking at the virus.
It will go faster if we have funding. The more funding we have, the
greater the percentage of time our researchers can spend on this
project instead of others.
8. What do you see as your worst scenario and your best scenario?
Dr. Martin: The patient who died in June: I only found out he died in
preparation for this meeting. I call around every few months to see
what's happening with these people. Had I known that this person
had been admitted to a community hospital, had I had two or three
weeks notice, I would have pushed hard that some treatment be tried.
In that situation, where the person is close to death, and where
there is no treatment, it is possible to contact the FDA and get
approval to try something in that one individual. A lot depends on
what happens. With maximum resources it should move very, veryfast.
9. Yes, but what is very, very fast?
Dr. Martin: I think if Epione works, it will go fast. If it doesn't
work, then it's back to the drawing board.10. (This question not
audible.)
Dr. Martin: The conventional wisdom would argue that if you have a
virus that causes disease, then all patients with the illness would
have the virus, all people with the virus would have the disease.
This only holds, however, if you have a well-defined entity called
'the disease' and a well-defined entity called 'the virus'. What we
have done in the study that was done before (presumably he is
referring to the CDC study here----Camilla) we can have samples sent to
us from clinicians who define the patient as having chronic fatigue
syndrome vs controls. In the case of the study done before, the
controls in one group consisted of children who were in the same
apparent epidemic in Lyndonville NY, friends of patients who had CFS
(done in NY) who were individuals who found themselves in middle age
pumping iron in a gym trying to get fit. We couldn't go back to the
clinicians and ask them about the patients because it was all double
blinded and the clinicians didn't know: only the person who held the
code could break it. We have seen people who can be healthy and
still have a virus that produces a foamy cell. What we don't know
is the nature of the virus in that individual. Is it the absence of
a neurologic problems such as CFS merely indicative that the virus
is in their body but not in the brain? Is it the same as what we're
having or is it slightly different? The appropriate way to
study this would be to look at the whole family. If we say that
someone who doesn't have CFS has a virus, but that virus is in their
brain or central nervous system, I have a very hard time dismissing
that as normal. We couldn't do the follow up that I wanted to do.
We could have given up on this project a long time ago: we could
have just said 'gee, the virus is present in a normal person;
therefore it is not relevant to a disease'. But it's clear that
it's not normal to have virus in your brain and people were dying
from what looked like viral illnesses.
11. Could you explain Epione? Is it synthetic or natural? What's
in it? A lot of us have chemical sensitivities and need to knowthis.
Dr. Martin: No, it would take too long and people are starting to leave.
There's going to be steps and what we want in the end is a safe and
effective therapy.12. So what's in it then?
Dr. Martin: What it is is something inhibitory to the virus in a test
tube. (at this point, some people in the audience got angry and kept
trying to get him to tell them what was in Epione. The tape wasn't
clear enough to type).
The issue is how to procede and to demonstrate that this virus is
implicated in the cause of disease. One way to do this is to
develop an inhibitory substance, give it to people with the illness
and see if they improve. To be honest, we don't know what the
nature of the inhibitor is. Once we can identify it, then the next
step is to find a way to synthesize it.
--Q & A continued in Part 2
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