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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.


1. DeFreitas E, Hilliard B, Cheney PR, Bell DS, Kiggundu E, Sankey D, Wroblewska 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.

4. Gunn WJ, Komaroff AL, Bell DS et al. Inability of retroviral tests to identify persons with chronic fatigue syndrome, 1992. Morb Mortal Wkly Rep. 1993;42:189?190.

5. Gow JW, Simpson K, Schliephake A, Behan WM, Morrison LJ, Cavanagh H, Rethwilm A and Behan PO. Search for retrovirus in the chronic fatigue syndrome. J Clin Pathol. 1992; 45:1058?1061.

6. Behan PO and Bakheit AMO. Clinical spectrum of postviral fatigue syndrome. Brit Med Bull 1991; 47:793-808.

7. Khan AS, Heneine WM, Chapman LE, Gary HE Jr, Woods TC, Folks TM and Schonberger 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 of retroviral involvement in chronic fatigue syndrome. Ciba Found Symp. 1993; 173:160?166.

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.

11. Isfort RJ, Witter R and Kung HJ. retrovirus insertion into herpesviruses. Trends Microbiol 1994; 2:174-177.

12. Robinson D, Liu JL, Jones D et al. Avian leukemias and lymphomas: Interplay between retroviruses and herpesviruses. Leukemia 1997; 11 (suppl 3):176-178.


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