This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dezzutti, C. S.
Right arrow Articles by Cowan, E. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dezzutti, C. S.
Right arrow Articles by Cowan, E. P.

 Previous Article  |  Next Article 

Clinical and Diagnostic Laboratory Immunology, July 2003, p. 715-717, Vol. 10, No. 4
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.4.715-717.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection of Human T-Lymphotropic Virus (HTLV) tax Sequences in New York City Blood Donors Seronegative for HTLV Types 1 and 2

Charlene S. Dezzutti,1* Patricia C. Guenthner,1 Sylvester Daniel,2 Ursula Utz,2,{dagger} Thania Cabrera,1 James H. Marshall,1 Celso Bianco,3 Renu B. Lal,1 and Elliot P. Cowan2

Division of AIDS, STD, and TB Laboratory Research, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,1 Center for Biologics Evaluation and Research, Food and Drug Administration, Rockville, Maryland 20852,2 America's Blood Centers, Washington, D.C. 200053

Received 13 January 2003/ Returned for modification 11 March 2003/ Accepted 13 March 2003


arrow
ABSTRACT
 
A potential public health concern is the reported detection of the human T-lymphotropic virus (HTLV) tax gene in the lymphocytes of up to 11% of a low-risk group of New York City blood donors (NYBD). This study aimed to independently confirm the prevalence of HTLV tax sequences in 293 NYBD. All NYBD tested negative for antibodies to HTLV types 1 and 2 and HTLV Tax. HTLV tax sequences were not detected in the NYBD lymphocytes. These data demonstrate the lack of HTLV-1 tax in this group of NYBD at low risk for HTLV infection.


arrow
TEXT
 
Human T-lymphotropic virus type 1 (HTLV-1) is etiologically associated with two major diseases, adult T-cell leukemia and HTLV-1-associated myelopathy-tropical spastic paraparesis (HAM-TSP) (3). HTLV-2 is a closely related retrovirus but has not been etiologically associated with any disease. Worldwide, HTLV infects 15 to 20 million people (2, 8) via sexual (primarily male-to-female), vertical (mother-to-child), and parenteral (drug use and blood transfusion) routes. Because of the high risk of transfusion-related transmission, the United States began to screen donated blood for HTLV-1 infection in 1988 and for HTLV-2 infection in 1997 by means of enzyme immunoassays (EIAs). These assays have high specificity and sensitivity for HTLV-1 and -2 and have effectively prevented HTLV transmission through blood and its components. In the United States, the incidence rate of HTLV in blood donors is estimated to be 1.59 per 100,000 person-years (4), and the residual risk of transmitting HTLV infection by transfusing screened blood is estimated to be 1 in 641,000 units (15).

Two papers by Zucker-Franklin et al. (19, 20) have reported HTLV-1 and -2 tax gene sequences in the lymphocytes of healthy blood donors, i.e., persons who had no known risk factors for HTLV-1 or -2 and who were serologically HTLV negative according to licensed screening assays. In the first study, 11 of 100 randomly selected blood donors at the New York University Medical Center Blood Bank had the HTLV tax sequence. Further, 8 of these 11 had antibodies to the Tax protein as determined by the Western blot assay (20). A subsequent study of 250 healthy persons (210 blood donors and 40 volunteers at New York University Medical Center) reported that 22 persons tested positive for the HTLV tax sequence. Plasma samples from these 22 blood donors were Western blot reactive to the HTLV Tax protein (19). These studies led to the speculation that some healthy blood donors might harbor a defective virus containing only HTLV tax sequences and that such blood was escaping detection by standard serological assays for HTLV-1 and -2.

To address this issue, a comprehensive, multicenter study was designed to determine the prevalence of HTLV tax sequences in 100 randomly selected HTLV-seronegative blood donors from the Baltimore, Md.-Washington, D.C., area (1). The samples were processed and prepared by the American Red Cross and sent without identification of donor to four testing centers. The PCR procedure used by the original investigators was followed, and HTLV tax sequences were not detected in any of the donors.

Despite these findings, a question remained about whether the demographics of the blood donors from New York City might differ from those of the donors in the Baltimore-Washington area. Therefore, for this study, we obtained during the fall of 1999 heparinized blood from randomly selected New York City blood donors (NYBD) who were seronegative for HTLV-1 and HTLV-2 and tested the samples for the presence of HTLV tax sequences by PCR and for anti-Tax antibodies. To ensure that we could detect with 95% assurance at least one person with HTLV tax sequences, in accordance with reported prevalence rates, 300 NYBD specimens were collected. Plasma samples and lymphocyte cell pellets were isolated from whole blood as previously described (11) and stored at -80°C until they were tested. Seven of the 300 NYBD samples were not usable because of sample clotting and were not included in this analysis. Additionally, heparinized blood was obtained from nine HTLV-1-infected persons and processed in a similar manner to serve as PCR-positive controls.

The 293 NYBD specimens were HTLV-1 and -2 serologically negative according to the Vironostika HTLV-I/II Microelisa System (bioMérieux, Durham, N.C.) (Fig. 1). To determine if antibodies to HTLV-1 Tax could be detected, plasma specimens were assayed with a peptide-based HTLV-1 Tax EIA (6). Twenty of the 293 plasma samples were initially reactive; however, when the plasma samples were preincubated with excess Tax peptides and the EIA was rerun, all samples remained reactive, while HTLV-positive plasma reactivity was removed by the preincubation. These data indicate that the reactivity of the 20 plasma samples was not specific to HTLV-1 Tax (Fig. 1); therefore, these specimens were considered negative for HTLV-1 Tax by EIA.



View larger version (13K):
[in this window]
[in a new window]
  		FIG. 1. HTLV serology and PCR analysis of NYBD specimens. Plasma samples from NYBD were screened by the Vironostika HTLV-I/II Microelisa System and subsequently by an HTLV anti-Tax EIA. When an excess of Tax peptide was used to clear HTLV Tax-specific reactivity, 20 of the initially reactive HTLV tax-containing plasma samples were determined upon retesting to have nonspecific reactivity. These samples were subsequently considered serologically to be HTLV tax negative. Cell pellets were lysed and used in two HTLV tax PCR methods, both resulting in no HTLV tax-positive NYBD specimens. Eight of nine HTLV-positive specimens were positive for tax gene sequences by PCR.

For PCR analysis, lymphocyte cell pellets were processed in a blind fashion according to the protocol of Pancake et al. (11), with nine HTLV-1-positive cell pellets randomly placed among the 293 NYBD cell pellets. Cell lysates were subjected to two 30-cycle PCR assays for HTLV tax. The PCR products were separated through a 2% agarose gel and subjected to Southern blotting. HTLV tax-specific bands were detected by hybridization to a digoxigenin-labeled SK45 probe (Genius; Roche Applied Science, Indianapolis, Ind.) and developed. Eight of the nine HTLV-1-positive samples were positive for tax by this PCR method. None of the 293 NYBD specimens was positive for HTLV tax sequences (Fig. 1), which we confirmed by using a nested-PCR tax assay (18). The nested-PCR tax assay also confirmed the presence of the tax gene in the same eight of nine HTLV-1-positive samples previously tested. Using this nested-PCR method, we were able to detect one HTLV tax sequence per 100,000 cells, which was similar to the results of the two 30-cycle PCRs.

The published prevalence rate of 11% for HTLV tax sequences in healthy blood donors from the New York City area (19, 20) suggested that our screening of 293 NYBD would detect approximately 32 individuals harboring the HTLV tax sequence. However, no individuals tested positive for HTLV tax sequences or for antibodies to the HTLV Tax protein. Our work has therefore confirmed the findings of the earlier multicenter study (1). Furthermore, the results indicate that low-risk blood donors, regardless of their geographical locations, do not have HTLV tax sequences. The reasons for the discrepancy between our study and the previous studies examining NYBD are not clear. Methods for processing the blood specimens, handling the cell pellets, and performing the PCR were identical in the present and all previous studies (11, 19, 20). Therefore, the inability to detect HTLV tax sequences in the lymphocytes in the present study cannot be ascribed to procedural differences. Moreover, we used a highly sensitive, nested-PCR method for tax to confirm our original PCR results.

Defective HTLV proviruses that code for the tax gene in the absence of other viral genes are readily found in confirmed HTLV-infected adult T-cell leukemia and HAM-TSP patients and asymptomatic carriers (10). It remains unclear how these defective tax-only viruses may contribute to viral pathology. Shuh et al. (16) described such a defective virus and showed that expression of tax in vitro depends on trans-activating factors produced from intact proviruses and that the tax-only virus is not expressed alone. Interestingly, two other studies described persons who are seronegative or seroindeterminate for HTLV-1 and HTLV-2 and who have an illness resembling HAM-TSP; in both studies, the HTLV tax gene sequences were detected while other gene sequences were not (13, 17). While defective tax-only proviruses exist in some high-risk groups, a more recent study shows that lymphocytes of serologically indeterminate blood donors do not harbor defective HTLV proviruses (9). Further, several other studies show that low-risk HTLV-seronegative or -seroindeterminate persons, such as blood donors, with no clinical HTLV symptoms had no evidence of HTLV infection (5, 7, 14). Collectively, these data suggest that seronegative or seroindeterminate persons with HTLV-like illnesses and/or risk factors for HTLV infection may have defective HTLV proviruses, may harbor low copy numbers of HTLV, or may have a novel retrovirus with partial homology to HTLV and therefore warrant further study. Conversely, low-risk, symptom-free, seronegative or seroindeterminate persons are not HTLV infected.

The results from this study and the earlier multicenter study (1) suggest that the prevalence of HTLV tax sequences in low-risk blood donors is not as high as previously reported (19, 20) and provide additional support that the risk of transmission of HTLV by screened blood products is remote (12, 15).


arrow
ACKNOWLEDGMENTS
 
We thank the CDC Blood Safety Working Group for its support of this work.

The use of trade names is for identification only and does not constitute endorsement by the U.S. Department of Health and Human Services, the Public Health Service, or the Centers for Disease Control and Prevention.


arrow
FOOTNOTES
 
* Corresponding author. Mailing address: Centers for Disease Control and Prevention, 1600 Clifton Rd., NE, Mailstop G19, Atlanta, GA 30333. Phone: (404) 639-1034. Fax: (404) 639-1174. E-mail: cdezzutti{at}cdc.gov. Back

{dagger} Present address: Division of Extramural Research, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Rockville, MD 20892. Back


arrow
REFERENCES
 
    1
  1. Cowan, E. P., G. J. Nemo, A. E. Williams, R. K. Alexander, A. Vallejo, I. K. Hewlett, R. B. Lal, C. S. Dezzutti, D. Gallahan, K. George, B. A. Pancake, D. Zucker-Franklin, P. R. McCurdy, and E. Tabor. 1999. Absence of human T-lymphotropic virus type I tax sequences in a population of normal blood donors in the Baltimore, MD/Washington, DC, area: results from a multicenter study. Transfusion 39:904-909.[CrossRef][Medline]
    2. 2
  2. de The, G., and R. Bomford. 1993. An HTLV-I vaccine: why, how, for whom? AIDS Res. Hum. Retrovir. 9:381-386.[Medline]
    3. 3
  3. Ferreira, O. C., Jr., V. Planelles, and J. D. Rosenblatt. 1997. Human T-cell leukemia viruses: epidemiology, biology, and pathogenesis. Blood Rev. 11:91-104.[CrossRef][Medline]
    4. 4
  4. Glynn, S. A., S. H. Kleinman, G. B. Schreiber, M. P. Busch, D. J. Wright, J. W. Smith, C. C. Nass, A. E. Williams, et al. 2000. Trends in incidence and prevalence of major transfusion-transmissible viral infections in US blood donors, 1991 to 1996. JAMA 284:229-235.[Abstract/Free Full Text]
    5. 5
  5. Khabbaz, R. F., W. Heneine, A. Grindon, T. M. Hartley, G. Shulman, and J. Kaplan. 1992. Indeterminate HTLV serologic results in U.S. blood donors: are they due to HTLV-I or HTLV-II? J. Acquir. Immune Defic. Syndr. 5:400-404.
    6. 6
  6. Lal, R. B., C. Z. Giam, J. E. Coligan, and D. L. Rudolph. 1994. Differential immune responsiveness to the immunodominant epitopes of regulatory proteins (tax and rex) in human T cell lymphotropic virus type I-associated myelopathy. J. Infect. Dis. 169:496-503.[Medline]
    7. 7
  7. Lal, R. B., D. L. Rudolph, J. E. Coligan, S. K. Brodine, and C. R. Roberts. 1992. Failure to detect evidence of human T-lymphotropic virus (HTLV) type I and type II in blood donors with isolated gag antibodies to HTLV-I/II. Blood 80:544-550.[Abstract/Free Full Text]
    8. 8
  8. Madeleine, M. M., S. Z. Wiktor, J. J. Goedert, A. Manns, P. H. Levine, R. J. Biggar, and W. A. Blattner. 1993. HTLV-I and HTLV-II world-wide distribution: reanalysis of 4,832 immunoblot results. Int. J. Cancer 54:255-260.[Medline]
    9. 9
  9. Morozov, V. A., H. Ellerbrok, C. Fleischer, H. H. Brackmann, and G. Pauli. 2002. Defective human T-cell leukaemia virus type 1 (HTLV-1) genomes: no evidence in serologically indeterminate German blood donors but new type detected in established cell lines. J. Med. Virol. 66:102-106.[CrossRef][Medline]
    10. 10
  10. Morozov, V. A., S. Lagaye, G. P. Taylor, E. Matutes, and R. A. Weiss. 2000. Chimeric matrix proteins encoded by defective proviruses with large internal deletions in human T-cell leukemia virus type 1-infected humans. J. Virol. 74:3933-3940.[Abstract/Free Full Text]
    11. 11
  11. Pancake, B. A., D. Zucker-Franklin, and E. E. Coutavas. 1995. The cutaneous T cell lymphoma, mycosis fungoides, is a human T cell lymphotropic virus-associated disease. A study of 50 patients. J. Clin. Investig. 95:547-554.
    12. 12
  12. Prati, D., C. Capelli, P. Rebulla, F. Mozzi, P. Bosoni, C. De Mattei, G. Sirchia, et al. 1998. The current risk of retroviral infections transmitted by transfusion in patients who have undergone multiple transfusions. Arch. Intern. Med. 158:1566-1569.[Abstract/Free Full Text]
    13. 13
  13. Ramirez, E., L. Cartier, M. Rios, and J. Fernandez. 1998. Defective human T-cell lymphotropic virus type I (HTLV-I) provirus in 10 Chilean seronegative patients with tropical spastic paraparesis or HTLV-I-associated myelopathy. J. Clin. Microbiol. 36:1811-1813.[Abstract/Free Full Text]
    14. 14
  14. Rouet, F., L. Meertens, G. Courouble, C. Herrmann-Storck, R. Pabingui, B. Chancerel, A. Abid, M. Strobel, P. Mauclere, and A. Gessain. 2001. Serological, epidemiological, and molecular differences between human T-cell lymphotropic virus type 1 (HTLV-1)-seropositive healthy carriers and persons with HTLV-I Gag indeterminate Western blot patterns from the Caribbean. J. Clin. Microbiol. 39:1247-1253.[Abstract/Free Full Text]
    15. 15
  15. Schreiber, G. B., M. P. Busch, S. H. Kleinman, J. J. Korelitz, et al. 1996. The risk of transfusion-transmitted viral infections. N. Engl. J. Med. 334:1685-1690.[Abstract/Free Full Text]
    16. 16
  16. Shuh, M., S. A. Hill, and D. Derse. 1999. Defective and wild-type human T-cell leukemia virus type I proviruses: characterization of gene products and trans-interactions between proviruses. Virology 262:442-451.[CrossRef][Medline]
    17. 17
  17. Soldan, S. S., M. D. Graf, A. Waziri, A. N. Flerlage, S. M. Robinson, T. Kawanishi, T. P. Leist, T. J. Lehky, M. C. Levin, and S. Jacobson. 1999. HTLV-I/II seroindeterminate Western blot reactivity in a cohort of patients with neurological disease. J. Infect. Dis. 180:685-694.[CrossRef][Medline]
    18. 18
  18. Tuke, P. W., P. Luton, and J. A. Garson. 1992. Differential diagnosis of HTLV-I and HTLV-II infections by restriction enzyme analysis of 'nested' PCR products. J. Virol. Methods 40:163-173.[CrossRef][Medline]
    19. 19
  19. Zucker-Franklin, D., and B. A. Pancake. 1998. Human T-cell lymphotropic virus type 1 tax among American blood donors. Clin. Diagn. Lab. Immunol. 5:831-835.[Abstract/Free Full Text]
    20. 20
  20. Zucker-Franklin, D., B. A. Pancake, M. Marmor, and P. M. Legler. 1997. Reexamination of human T cell lymphotropic virus (HTLV-I/II) prevalence. Proc. Natl. Acad. Sci. USA 94:6403-6407.[Abstract/Free Full Text]

Clinical and Diagnostic Laboratory Immunology, July 2003, p. 715-717, Vol. 10, No. 4
1071-412X/03/$08.00+0     DOI: 10.1128/CDLI.10.4.715-717.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Bassani, S., Toro, C., Jimenez, V., Rodes, B., Soriano, V. (2006). Can the Level of Immunosuppression in Human Immunodeficiency Virus-Infected Patients Affect the Reliability of Human T-Cell Lymphotropic Virus Type 2 Serological Diagnosis?. CVI 13: 160-161 [Abstract] [Full Text]  

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Dezzutti, C. S.
Right arrow Articles by Cowan, E. P.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Dezzutti, C. S.
Right arrow Articles by Cowan, E. P.

Copyright © 2009 by the American Society for Microbiology.   For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»