1887

Abstract

Peripheral blood leukocytes (PBL), namely polymorphonuclear leukocytes (PMNL), are the major carrier of human cytomegalovirus (HCMV) in the blood of immunocompromised patients with HCMV viraemia. By using monoclonal antibodies (MAbs) directed against different early and late viral proteins, we showed that the protein accumulating in PBL, originally reported to be an immediate early (IE) gene product, is the 65K lower matrix early structural protein (ep65). This protein is detectable by immunofluorescence before IE proteins during early stages of the replication cycle of HCMV in permissive human embryonic lung fibroblast cells. However, the appearance of ep65 in the nucleus within 1 h post-infection in the presence of cycloheximide indicates that it represents uptake from the virus inoculum rather than newly synthetized protein. The ep65 MAbs staining PBL did not react with Vero cells infected with a recombinant vaccinia virus encoding the major IE gene (IE1) product, whereas MAbs reactive with the 72K major IE protein stained only faintly a small number of infected PBL. A group of four ep65 MAbs was tested in competitive binding assays to show that ep65 possesses at least three distinct epitopes. These were recognized by all four MAbs in AD169-infected Vero cell cultures when fixed with formaldehyde, whereas only one MAb recognizing a distinct epitope was reactive with methanol-acetone (MA)-fixed AD169-infected Vero cells. In formalin-fixed PBL the number of infected cells stained by the four ep65 MAbs was about twofold that found using MA-fixed cells. Using fluorescence-activated cells orter-purified leukocyte subpopulations from viraemic patients with different levels of viraemia, the ratio of ep65-positive to ep65-negative cells was found to be 1:100 to 1:100000 for PMNL, and only 1:10000 to 1:100000 for mononuclear cells.

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1992-02-01
2024-03-29
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References

  1. Borysiewicz L. K., Hickling J. K., Graham S., Sinclair J., Cranage M. P., Smith G. L., Sissons J. G. P. 1988; Human cytomegalovirus-specific cytotoxic T cells. Journal of Experimental Medicine 168:919–931
    [Google Scholar]
  2. Britt W. J., Auger D. 1986; Identification of a 65, 000 dalton virion envelope protein of human cytomegalovirus. Virus Research 4:31–36
    [Google Scholar]
  3. Britt W. J., Vugler L. 1987; Structural and immunological characterization of the intracellular forms of an abundant 68000 M r human cytomegalovirus protein. Journal of General Virology 68:1897–1907
    [Google Scholar]
  4. Campbell M. E., Palfreyman J. W., Preston C. M. 1984; Identification of herpes simplex virus DNA sequences which encode a transacting polypeptide responsible for stimulation of immediate early transcription. Journal of Molecular Biology 180:1–19
    [Google Scholar]
  5. Clark B. R., Zaia J. A., Balce-Directo L., Ting Y. P. 1984; Isolation and partial chemical characterization of a 64, 000-dalton glycoprotein of human cytomegalovirus. Journal of Virology 49:279–282
    [Google Scholar]
  6. Dankner W. M., McCutchan J. A., Richman D. D., Hirata K., Spector S. A. 1990; Localization of human cytomegalovirus in peripheral blood leukocytes by in situ hybridization. Journal of Infectious Diseases 161:31–36
    [Google Scholar]
  7. Davis M. G., Huang E. -S. 1985; Nucleotide sequence of a human cytomegalovirus DNA fragment encoding a 67-kilodalton phosphorylated viral protein. Journal of Virology 56:7–11
    [Google Scholar]
  8. Davis M. G., Mar E. -C., Wu Y. -M., Huang E. -S. 1984; Mapping and expression of a human cytomegalovirus major viral protein. Journal of Virology 52:129–135
    [Google Scholar]
  9. Dummer J. S., White L. T., Ho M., Griffith B. P., Hardesty R. L., Bahnson H. T. 1985; Morbidity of cytomegalovirus infection in recipients of heart or heart-lung transplants who received cyclosporine. Journal of Infectious Diseases 152:1182–1191
    [Google Scholar]
  10. Fiala M., Cone L. A., Chang C. M., Mocarski E. S. 1986; Cytomegalovirus viremia increases with progressive immune deficiency in patients infected with HTLV-III. AIDS Research 2:175–181
    [Google Scholar]
  11. Geballe A. P., Leach F. S., Mocarski E. S. 1986; Regulation of cytomegalovirus late gene expression: γ genes are controlled by post-transcriptional events. Journal of Virology 57:864–874
    [Google Scholar]
  12. Gerna G., Revello M. G., Dovis M., Petruzzelli E., Achilli G., Percivalle E., Torsellini M. 1987; Synergistic neutralization of rubella virus by monoclonal antibodies to viral haemagglutinin. Journal of General Virology 68:2007–2012
    [Google Scholar]
  13. Gerna G., Revello M. G., Percivalle E., Zavattoni M., Parea M., Battaglia M. 1990; Quantification of human cytomegalovirus viremia by using monoclonal antibodies to different viral proteins. Journal of Clinical Microbiology 28:2681–2688
    [Google Scholar]
  14. Gleaves C. A., Smith T. F., Shuster E. A., Pearson G. R. 1984; Rapid detection of cytomegalovirus in MRC-5 cells inoculated with urine specimens by using low-speed centrifugation and monoclonal antibody to an early antigen. Journal of Clinical Microbiology 19:917–919
    [Google Scholar]
  15. Goins W. F., Stinski M. F. 1986; Expression of human cytomegalovirus late gene is posttranscriptionally regulated by a 3′-end processing event occurring exclusively late after infection. Molecular and Cellular Biology 6:4202–4213
    [Google Scholar]
  16. Griffiths P. D., Panjwani D. D., Stirk P. R., Ball M. G., Ganczakowski M., Blacklock H. A., Prentice H. G. 1984; Rapid diagnosis of cytomegalovirus infection in immunocompromised patients by detection of early antigen fluorescent foci. Lancet ii:1242–1245
    [Google Scholar]
  17. Irmiere A., Gibson W. 1983; Isolation and characterization of a noninfectious virion-like particle released from cells infected with human strains of cytomegalovirus. Virology 130:118–133
    [Google Scholar]
  18. Nowak B., Gmeiner A., Sarnow P., Levine A. J., Fleckenstein B. 1984; Physical mapping of human cytomegalovirus genes: identification of DNA sequences coding for a virion phosphoprotein of 71kDa and a viral 65-kDa polypeptide. Virology 134:91–102
    [Google Scholar]
  19. Pande H., Lee T. D., Churchill M. A., Zaia J. A. 1990; Structural analysis of a 64-kDa major structural protein of human cytomegalovirus (Towne): identification of a phosphorylation site and comparison to pp65 of HCMV (AD169). Virology 178:6–14
    [Google Scholar]
  20. Randall R. E., Dinwoodie N. 1986; Intranuclear localization of herpes simplex virus immediate-early and delayed-early proteins: evidence that ICP4 is associated with progeny virus DNA. Journal of General Virology 67:2163–2177
    [Google Scholar]
  21. Reichert C. M., O’Leary T. S., Levens D. L., Simrell C. R., Macher A. M. 1983; Autopsy pathology in the acquired immune deficiency syndrome. American Journal of Pathology 112:357–382
    [Google Scholar]
  22. Revello M. G., Percivalle E., Zavattoni M., Parea M., Grossi P., Gerna G. 1989a; Detection of human cytomegalovirus immediate-early antigen in leukocytes as a marker of viremia in immunocompromised patients. Journal of Medical Virology 29:88–93
    [Google Scholar]
  23. Revello M. G., Zavattoni M., Percivalle E., Grossi P., Gerna G. 1989b; Correlation between immunofluorescent detection of human cytomegalovirus immediate-early antigens in polymorphonuclear leukocytes and viremia. Journal of Infectious Diseases 160:159–160
    [Google Scholar]
  24. Revello M. G., Percivalle E., Zannino M., Rossi V., Gerna G. 1991; Development and evaluation of a capture ELISA method for determination of IgM antibody to the human cytomegalovirus major DNA binding protein. Journal of Virological Methods (in press)
    [Google Scholar]
  25. Roby C., Gibson W. 1986; Characterization of phosphoproteins and protein kinase activity of virions, noninfectious enveloped particles, and dense bodies of human cytomegalovirus. Journal of Virology 59:714–727
    [Google Scholar]
  26. Saltzman R. L., Quirk M. R., Jordan M. C. 1988; Disseminated cytomegalovirus infection. Molecular analysis of virus and leukocytes interactions in viremia. Journal of Clinical Investigation 81:75–81
    [Google Scholar]
  27. Somogyi T., Michelson S., Masse M. -J. O. 1990; Genomic localization of a human cytomegalovirus protein with protein kinase activity (PK68). Virology 174:276–285
    [Google Scholar]
  28. Stinski M. F., Thomsen D. R., Stenberg R. M., Goldstein L. C. 1983; Organization and expression of the immediate early genes of human cytomegalovirus. Journal of Virology 46:1–14
    [Google Scholar]
  29. Van der Bij W., Schirm J., Torensma R., van Son J., Tegzess A. M., The T. H. 1988a; Comparison between viremia and antigenemia for detection of cytomegalovirus in blood. Journal of Clinical Microbiology 26:2531–2535
    [Google Scholar]
  30. Van der Bij W., Torensma R., van Son W. J., Anema J., Schirm J., Tegzess A. M., The T. H. 1988b; Rapid immunodiagnosis of active cytomegalovirus infection by monoclonal antibody staining of blood leukocytes. Journal of Medical Virology 25:179–188
    [Google Scholar]
  31. Wunderli W., Kagi M. K., Gruter E., Auracher J. D. 1989; Detection of cytomegalovirus in peripheral leukocytes by different methods. Journal of Clinical Microbiology 27:1916–1917
    [Google Scholar]
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