Skip to main content
Journal of Virology logoLink to Journal of Virology
. 1997 Apr;71(4):2980–2987. doi: 10.1128/jvi.71.4.2980-2987.1997

Latency versus persistence or intermittent recurrences: evidence for a latent state of murine cytomegalovirus in the lungs.

S Kurz 1, H P Steffens 1, A Mayer 1, J R Harris 1, M J Reddehase 1
PMCID: PMC191426  PMID: 9060657

Abstract

The state of cytomegalovirus (CMV) after the resolution of acute infection is an unsolved problem in CMV research. While the term "latency" is in general use to indicate the maintenance of the viral genome, a formal exclusion of low-level persistent productive infection depends on the sensitivity of the assay for detecting infectious virus. We have improved the method for detecting infectivity by combining centrifugal infection of permissive indicator cells in culture, expansion to an infectious focus, and sensitive detection of immediate-early RNA in the infected cells by reverse transcriptase PCR. A limiting-dilution approach defined the sensitivity of this assay. Infectivity was thereby found to require as few as 2 to 9 virion DNA molecules of murine CMV, whereas the standard measure of infectivity, the PFU, is the equivalent of ca. 500 viral genomes. Since murine CMV forms multicapsid virions in most infected tissues, the genome-to-infectivity ratio is necessarily >1. This assay thus sets a new standard for investigating CMV latency. In mice in which acute infection was resolved, the viral DNA load in the lungs, a known organ site of CMV latency and recurrence, was found to be 1 genome per 40 lung cells, or a total of ca. 1 million genomes. Despite this high load of CMV DNA, infectious virus was not detected with the improved assay, but recurrence was inducible. These data provide evidence against a low-level persistent productive infection and also imply that intermittent spontaneous recurrence is not a frequent event in latently infected lungs.

Full Text

The Full Text of this article is available as a PDF (1.5 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Badley J. E., Bishop G. A., St John T., Frelinger J. A. A simple, rapid method for the purification of poly A+ RNA. Biotechniques. 1988 Feb;6(2):114–116. [PubMed] [Google Scholar]
  2. Balthesen M., Dreher L., Lucin P., Reddehase M. J. The establishment of cytomegalovirus latency in organs is not linked to local virus production during primary infection. J Gen Virol. 1994 Sep;75(Pt 9):2329–2336. doi: 10.1099/0022-1317-75-9-2329. [DOI] [PubMed] [Google Scholar]
  3. Balthesen M., Messerle M., Reddehase M. J. Lungs are a major organ site of cytomegalovirus latency and recurrence. J Virol. 1993 Sep;67(9):5360–5366. doi: 10.1128/jvi.67.9.5360-5366.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chong K. T., Mims C. A. Murine cytomegalovirus particle types in relation to sources of virus and pathogenicity. J Gen Virol. 1981 Dec;57(Pt 2):415–419. doi: 10.1099/0022-1317-57-2-415. [DOI] [PubMed] [Google Scholar]
  5. Ebeling A., Keil G. M., Knust E., Koszinowski U. H. Molecular cloning and physical mapping of murine cytomegalovirus DNA. J Virol. 1983 Sep;47(3):421–433. doi: 10.1128/jvi.47.3.421-433.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Fazekas de St Groth The evaluation of limiting dilution assays. J Immunol Methods. 1982 Mar 12;49(2):R11–R23. doi: 10.1016/0022-1759(82)90269-1. [DOI] [PubMed] [Google Scholar]
  7. Grundy J. E., Super M., Lui S., Sweny P., Griffiths P. D. The source of cytomegalovirus infection in seropositive renal allograft recipients is frequently the donor kidney. Transplant Proc. 1987 Feb;19(1 Pt 3):2126–2128. [PubMed] [Google Scholar]
  8. Henry S. C., Hamilton J. D. Detection of murine cytomegalovirus immediate early 1 transcripts in the spleens of latently infected mice. J Infect Dis. 1993 Apr;167(4):950–954. doi: 10.1093/infdis/167.4.950. [DOI] [PubMed] [Google Scholar]
  9. Ho M. Observations from transplantation contributing to the understanding of pathogenesis of CMV infection. Transplant Proc. 1991 Jun;23(3 Suppl 3):104-8, discussion 108-9. [PubMed] [Google Scholar]
  10. Hodgkin P. D., Scalzo A. A., Swaminathan N., Price P., Shellam G. R. Murine cytomegalovirus binds reversibly to mouse embryo fibroblasts: implications for quantitation and explanation of centrifugal enhancement. J Virol Methods. 1988 Dec;22(2-3):215–230. doi: 10.1016/0166-0934(88)90104-8. [DOI] [PubMed] [Google Scholar]
  11. Hudson J. B., Misra V., Mosmann T. R. Cytomegalovirus infectivity: analysis of the phenomenon of centrifugal enhancement of infectivity. Virology. 1976 Jul 1;72(1):235–243. doi: 10.1016/0042-6822(76)90326-3. [DOI] [PubMed] [Google Scholar]
  12. Jonjić S., Mutter W., Weiland F., Reddehase M. J., Koszinowski U. H. Site-restricted persistent cytomegalovirus infection after selective long-term depletion of CD4+ T lymphocytes. J Exp Med. 1989 Apr 1;169(4):1199–1212. doi: 10.1084/jem.169.4.1199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jordan M. C. Latent infection and the elusive cytomegalovirus. Rev Infect Dis. 1983 Mar-Apr;5(2):205–215. doi: 10.1093/clinids/5.2.205. [DOI] [PubMed] [Google Scholar]
  14. Keil G. M., Ebeling-Keil A., Koszinowski U. H. Sequence and structural organization of murine cytomegalovirus immediate-early gene 1. J Virol. 1987 Jun;61(6):1901–1908. doi: 10.1128/jvi.61.6.1901-1908.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Klotman M. E., Henry S. C., Greene R. C., Brazy P. C., Klotman P. E., Hamilton J. D. Detection of mouse cytomegalovirus nucleic acid in latently infected mice by in vitro enzymatic amplification. J Infect Dis. 1990 Feb;161(2):220–225. doi: 10.1093/infdis/161.2.220. [DOI] [PubMed] [Google Scholar]
  16. Kondo K., Kaneshima H., Mocarski E. S. Human cytomegalovirus latent infection of granulocyte-macrophage progenitors. Proc Natl Acad Sci U S A. 1994 Dec 6;91(25):11879–11883. doi: 10.1073/pnas.91.25.11879. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kondo K., Mocarski E. S. Cytomegalovirus latency and latency-specific transcription in hematopoietic progenitors. Scand J Infect Dis Suppl. 1995;99:63–67. [PubMed] [Google Scholar]
  18. Konecki D. S., Brennand J., Fuscoe J. C., Caskey C. T., Chinault A. C. Hypoxanthine-guanine phosphoribosyltransferase genes of mouse and Chinese hamster: construction and sequence analysis of cDNA recombinants. Nucleic Acids Res. 1982 Nov 11;10(21):6763–6775. doi: 10.1093/nar/10.21.6763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Lucin P., Jonjić S., Messerle M., Polić B., Hengel H., Koszinowski U. H. Late phase inhibition of murine cytomegalovirus replication by synergistic action of interferon-gamma and tumour necrosis factor. J Gen Virol. 1994 Jan;75(Pt 1):101–110. doi: 10.1099/0022-1317-75-1-101. [DOI] [PubMed] [Google Scholar]
  20. Maciejewski J. P., Bruening E. E., Donahue R. E., Mocarski E. S., Young N. S., St Jeor S. C. Infection of hematopoietic progenitor cells by human cytomegalovirus. Blood. 1992 Jul 1;80(1):170–178. [PubMed] [Google Scholar]
  21. Mercer J. A., Marks J. R., Spector D. H. Molecular cloning and restriction endonuclease mapping of the murine cytomegalovirus genome (Smith Strain). Virology. 1983 Aug;129(1):94–106. doi: 10.1016/0042-6822(83)90398-7. [DOI] [PubMed] [Google Scholar]
  22. Mercer J. A., Wiley C. A., Spector D. H. Pathogenesis of murine cytomegalovirus infection: identification of infected cells in the spleen during acute and latent infections. J Virol. 1988 Mar;62(3):987–997. doi: 10.1128/jvi.62.3.987-997.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Messerle M., Bühler B., Keil G. M., Koszinowski U. H. Structural organization, expression, and functional characterization of the murine cytomegalovirus immediate-early gene 3. J Virol. 1992 Jan;66(1):27–36. doi: 10.1128/jvi.66.1.27-36.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Minton E. J., Tysoe C., Sinclair J. H., Sissons J. G. Human cytomegalovirus infection of the monocyte/macrophage lineage in bone marrow. J Virol. 1994 Jun;68(6):4017–4021. doi: 10.1128/jvi.68.6.4017-4021.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mocarski E. S., Jr, Abenes G. B., Manning W. C., Sambucetti L. C., Cherrington J. M. Molecular genetic analysis of cytomegalovirus gene regulation in growth, persistence and latency. Curr Top Microbiol Immunol. 1990;154:47–74. doi: 10.1007/978-3-642-74980-3_3. [DOI] [PubMed] [Google Scholar]
  26. Mutter W., Reddehase M. J., Busch F. W., Bühring H. J., Koszinowski U. H. Failure in generating hemopoietic stem cells is the primary cause of death from cytomegalovirus disease in the immunocompromised host. J Exp Med. 1988 May 1;167(5):1645–1658. doi: 10.1084/jem.167.5.1645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Pollock J. L., Virgin H. W., 4th Latency, without persistence, of murine cytomegalovirus in the spleen and kidney. J Virol. 1995 Mar;69(3):1762–1768. doi: 10.1128/jvi.69.3.1762-1768.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Pomeroy C., Hilleren P. J., Jordan M. C. Latent murine cytomegalovirus DNA in splenic stromal cells of mice. J Virol. 1991 Jun;65(6):3330–3334. doi: 10.1128/jvi.65.6.3330-3334.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reddehase M. J., Balthesen M., Rapp M., Jonjić S., Pavić I., Koszinowski U. H. The conditions of primary infection define the load of latent viral genome in organs and the risk of recurrent cytomegalovirus disease. J Exp Med. 1994 Jan 1;179(1):185–193. doi: 10.1084/jem.179.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Reddehase M. J., Weiland F., Münch K., Jonjic S., Lüske A., Koszinowski U. H. Interstitial murine cytomegalovirus pneumonia after irradiation: characterization of cells that limit viral replication during established infection of the lungs. J Virol. 1985 Aug;55(2):264–273. doi: 10.1128/jvi.55.2.264-273.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Roizman B., Sears A. E. An inquiry into the mechanisms of herpes simplex virus latency. Annu Rev Microbiol. 1987;41:543–571. doi: 10.1146/annurev.mi.41.100187.002551. [DOI] [PubMed] [Google Scholar]
  32. Shakhov A. N., Nedospasov S. A. Molekuliarnoe klonirovanie genov, kodiruiushchikh faktory nekroza opukholei. Polnaia posledovatel'nost' nukleotidov genomnoi kopii FNO-alpha myshi. Bioorg Khim. 1987 May;13(5):701–705. [PubMed] [Google Scholar]
  33. Vilcek J., Lee T. H. Tumor necrosis factor. New insights into the molecular mechanisms of its multiple actions. J Biol Chem. 1991 Apr 25;266(12):7313–7316. [PubMed] [Google Scholar]
  34. Weiland F., Keil G. M., Reddehase M. J., Koszinowski U. H. Studies on the morphogenesis of murine cytomegalovirus. Intervirology. 1986;26(4):192–201. doi: 10.1159/000149701. [DOI] [PubMed] [Google Scholar]
  35. Yu Y., Henry S. C., Xu F., Hamilton J. D. Expression of a murine cytomegalovirus early-late protein in "latently" infected mice. J Infect Dis. 1995 Aug;172(2):371–379. doi: 10.1093/infdis/172.2.371. [DOI] [PubMed] [Google Scholar]
  36. Yuhasz S. A., Dissette V. B., Cook M. L., Stevens J. G. Murine cytomegalovirus is present in both chronic active and latent states in persistently infected mice. Virology. 1994 Jul;202(1):272–280. doi: 10.1006/viro.1994.1343. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Virology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES