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Keywords:

  • CV-B4 E2;
  • murine foetal thymus organ cultures;
  • T-cell differentiation

Abstract

The infection of foetal thymus with coxsackievirus B4 (CV-B4) E2 has been studied ex vivo by using CD-1 mice on foetal day 14, as a ready source of organs for experimentation to investigate the hypothesis of the role of thymic viral infections in the pathogenesis of type 1 diabetes. The replication of CV-B4 E2 in murine foetal thymus organ cultures has been demonstrated by evaluating the levels of positive- and negative-stranded viral RNA in cells by using a real-time quantitative RT-PCR method and by determining titres of infectious viral particles in culture supernatants for 7 days post-infection (p.i.). Staining of tissue sections with an anti-cytokeratin antibody and haematoxylin–eosin showed that CV-B4 infection had no visible effect on cell survival and organ integrity. Cell counts in mock- and virus-infected foetal thymus organ cultures increased from day 1 through day 7, and live cell numbers were comparable in both conditions as shown by Trypan blue exclusion test and 7-amino-actinomycin D staining of thymocytes. Compared with controls on day 7 p.i., cytofluorometric analyses on cells from CV-B4 E2-infected foetal thymus organ cultures displayed a marked increase in the percentage of the most immature CD3CD4CD8 thymocytes, and a decrease in the percentage of immature CD3CD4+CD8+ cells, together with an increase in the percentage of mature CD3+CD4+ and CD3+CD8+ cells. These data show that CV-B4 E2 disturbs T-cell maturation and differentiation processes in infected murine foetal thymus organ cultures and provide evidence of a suitable system to investigate the effect of viruses in T-cell differentiation. J. Med. Virol. 80:659–666, 2008. © 2008 Wiley-Liss, Inc.