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Relationships Between Human T-Lymphotropic Virus Type II (HTLV-2)
and Human Lymphotropic Herpesviruses in Chronic Fatigue Syndrome
Project funded by The National CFIDS Foundation, Inc.; Needham, Massachusetts
Background: In 1991 DeFreitas et al. (1) from the Wistar Institute in
Philadelphia reported the detection of HTLV-2 gag gene related DNA sequences
in the lymphocyte DNA of patients with CFS [72% of pediatric patients and 83%
of adult patients] by means of the newly developed polymerase chain reaction (PCR) technique. DNA samples from healthy control individuals were negative
for HTLV-2 gag gene related DNA sequences. Several aspects of these studies
deserve comment.
Strengths of this work include:
• use of a "hot start" PCR technique (2) that maximized the efficiency
of the PCR reaction (Appendix 1)
• the study of well characterized and very ill CFS patients who met the
1988 CDC criteria for CFS (3)
• use of an upstream PCR DNA primer whose sequence would allow equivalent
detection of both the A and B types of HTLV-2.
Weaknesses of these studies include:
• the use of a high GC content (85% GC) DNA probe for detection of the
PCR product
• use of a downstream PCR DNA primer sequence that might specifically fail
to detect the B type of HTLV-2.
Since 1991 there have been five additional published reports of studies of HTLV-2
in CFS. These papers were identified by a search of the National Library of
Medicine for the MESH terms "fatigue syndrome, chronic" and "retroviridae".
Of these reports, one was a non-peer reviewed survey published in the Morbidity
Mortality Weekly Report of the Centers for Disease Control and Prevention (CDC)
(4). Although it is not stated, it is implied that one of the laboratories involved
in this study was that of DeFreitas et al. Using the PCR technique with the
original DeFreitas et al. primers, no differences were found between CFS patients
and controls with respect to the detection of HTLV-2 DNA sequences in lymphocyte
DNA (approximately 60% of both groups were positive for HTLV-2 gag gene related
DNA sequences). The main strength of this study was that the samples were analyzed
in blinded fashion.
Weaknesses of this report include:
• failure to use a "hot start" PCR procedure such as that used by DeFreitas
et al.
• the laboratories involved in the study are not identified
• no technical details concerning the procedures used are provided (especially
with respect to the "modification" of the PCR assay)
• the patients are not clinically described except that they met the 1988
CDC criteria for CFS.
Another of these five reports is that of Gow el al. from laboratories in Scotland
and Germany (5). Again using the original DeFreitas et al. primers, as well
as another HTLV-2 primer pair, no differences were observed between CFS patients
and control subjects (100% of both groups of subjects were positive for HTLV-2
gag gene related DNA sequences).
Weaknesses of this study include:
• the use of a European case definition for CFS (6) which differed substantially
from that used in the United States
• failure to use a "hot start" PCR procedure such as that used by DeFreitas
et al.
• use of the DeFreitas et al. primers with reaction conditions significantly
different from those used by DeFreitas et al
• the DeFreitas et al. detection DNA probe failed to recognize the PCR
product obtained, suggesting nonspecificity of the PCR reaction.
The remaining three publications were from the same group of investigators at
the CDC (7,8,9). These studies which utilized the HTLV-2 specific DNA primers
used by DeFreitas et al. failed to detect HTLV-2 gag gene related DNA sequences
in either CFS patients or control subjects. This finding stands in stark contrast
to the observations of Gow et al. as described above. Strengths of these three
studies included: ‚ they were for the most part performed in a blinded fashion
‚ they attempted to correlate their PCR findings with a variety of epidemiological
factors ‚ they sought a variety of animal retroviruses in addition to HTLV-2.
Weaknesses of these three studies included:
• failure to use a "hot start" PCR procedure such as that used by DeFreitas
et al.
• use of the primers of DeFreitas et al. under experimental conditions
significantly different from those used by DeFreitas et al.
• the results of these studies are somewhat misleading in that the data
reported in all three papers apparently were obtained from a single experiment
involving 21 CFS patients and matched controls. No reference is made to this
fact in any of the three publications.
In conclusion, the results reported by DeFreitas et al. have not been reproduced
by other, independent laboratories. However, these subsequent investigations
differed substantially from the original DeFreitas et al. studies in a number
of ways, including:
• clinical definition of the CFS patients studied
• uniform failure to use a "hot start" procedure with the PCR assay
• use of experimental conditions for the PCR assay that significantly differed
from those used by DeFreitas et al.
Specific Research Proposal
The goals of the proposed studies are:
• to use contemporary PCR techniques in an attempt to confirm the findings
of DeFreitas et al. with respect to the presence of HTLV-2 gag gene related
DNA sequences in the blood leukocytes of patients with CFS
• To correlate the presence of HTLV-2 gag gene related DNA sequences with
the presence of active infections with human herpesvirus six (HHV-6) or Epstein-Barr
virus (EBV)
Patients and Controls
The samples of peripheral blood leukocytes (PBL) to be used in these studies are
currently on hand in our laboratory. They have all been frozen at -70oC for periods
up to 3 years.
The samples will be of two types:
• The first set of PBL samples were obtained as part of a cross-sectional
study of CFS patients performed by our laboratory in collaboration with Drs. Anthony
Komaroff (Boston, Massachusetts) and Daniel Peterson (Incline Village, Nevada).
The samples consist of PBL from 20 patients with CFS and from 20 matched healthy
control individuals. Samples will be analyzed in a blinded fashion.
• The second set of PBL samples will be PBL from CFS patients submitted to
our laboratory for virological analysis. These samples were submitted from three
physicians who specialize in the treatment of CFS patients: i.e. 10 samples from
Dr. Joseph John (Robert Wood Johnson Medical School; New Brunswick, New Jersey),
10 samples from Dr. Paul Cheney (Bald Head Island, North Carolina) and 10 samples
from Dr. Patricia Salvato (Houston, Texas).
• Thus the samples to be analyzed will be derived from 50 CFS patients and
20 healthy controls.
Detection of HTLV-2 gag Gene Related DNA Sequences
The PCR systems to be used in these studies can be summarized as follows:
• The original DNA primer system of DeFreitas et al. will be used with one
modification. In place of the radioactive probe procedure used in their original
studies, we will use a nested PCR system (Appendix 2)to provide adequate sensitivity
and specificity. Also, a pool of two downstream primers differing in HTLV-2 subtype
specific nucleotide sequences will be used in order to allow equivalent detection
of both types of the virus.
• A newly designed nested PCR system targeting a different region of the
HTLV-2 gag gene will be used in addition to the primer system of DeFreitas et
al. This PCR system was designed with contemporary nucleotide sequence analysis
software, which allows more precise prediction of binding affinity and specificity,
using the gag gene sequences from six strains of HTLV-2A and five strains of HTLV-2B.
This strategy allowed us to design primers that will certainly detect both types
of HTLV-2 with equivalent efficiency.
• DNA will be extracted and purified from patient and control samples of
blood leukocytes by means of a commercially obtained, widely used kit (QIAmp DNA
Mini Blood Kit; QIAGEN Inc; Valencia, California) (Appendix 3).
• Detection of PCR products in both PCR systems will be accomplished by agarose
gel electrophoresis with ethidium bromide staining.
Relationship Between the Presence of HTLV-2 gag Gene Related Sequences and
Active Lymphotropic Herpesvirus Infections
Numerous studies have established that herpesviruses and retroviruses can intimately
interact at the subcellular level (10,11,12). These interactions range from
cross-transactivation
of each other's genes to actual integration of the retrovirus genome into the
much larger herpesvirus genome. There are, in addition, data suggesting that active
infections by HHV-6 and/or EBV may be involved in the pathogenesis of CFS (K Knox
and D Carrigan; data not shown). Therefore, it will be of interest to correlate
our findings concerning HTLV and these two viruses in the studies proposed here.
Specifically, we will:
• correlate the detection of HTLV-2 gag gene DNA sequences with the results
obtained with the same sample in a rapid HHV-6 culture assay. All specimens to
be studied have already been analyzed by this rapid culture procedure with numerous
positive samples being identified.
• correlate the detection of HTLV-2 gag gene DNA sequences with the results
obtained with the same sample with a serum PCR procedure specific for EBV DNA.
Serum (or plasma) samples matched to the PBL specimens in these studies are available
in frozen form in our laboratory. The serum PCR procedure has been successfully
applied to samples from CFS patients (K Knox and D Carrigan; data available upon
request.
REFERENCES
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Z, Palladino M, Woodward JP AND Koprowski H. Retroviral sequences related to human
T lymphotropic virus type II in patients with chronic fatigue immune dysfunction
syndrome. Proc Natl Acad Sci 1991; 88:2922?2926.
2. Defreitas E and Hilliard B. Method and compositions for diagnosing and treating
chronic fatigue immunodysfunction syndrome. International Patent Application Number
PCT/US91/06238; April, 1992.
3. Holmes GP, Kaplan JE, Gantz NM et al. Chronic fatigue syndrome: a working case
definition. Ann Intern Med 1988; 108:387-389.
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persons with chronic fatigue syndrome, 1992. Morb Mortal Wkly Rep. 1993;42:189?190.
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6. Behan PO and Bakheit AMO. Clinical spectrum of postviral fatigue syndrome.
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LB. AssessmSent of a retrovirus sequence and other possible risk factors for the
chronic fatigue syndrome in adults. Ann Intern Med. 1993;118:241?245.
8. Folks TM, Heneine W, Khan A, Woods T, Chapman L and Schonberger L. Investigation
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9. Heneine W, Woods TC, Sinha SD, Khan AS, Chapman LE, Schonberger LB, Folks TM.
Lack of evidence for infection with known human and animal retroviruses in patients
with chronic fatigue syndrome. Clin Infect Dis. 1994; 18 Suppl 1:S121?S125.
10. Isfort R, Jones, D, Kost R, Witter R and Kung HJ. Retrovirus insertion into
herpesvirus in vitro and in vivo. Proc Nat Acad Sci 1992; 89:991-995.
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Trends Microbiol 1994; 2:174-177.
12. Robinson D, Liu JL, Jones D et al. Avian leukemias and lymphomas: Interplay
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