Hybridohistochemical analysis of latently infected virus

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In this project, we focused on the latent infection of a gallid herpesvirus, Marek's disease virus (MDV). It was reported that some latently infected viruses may cause tumors and monsters. We observed the gene expression of the latently infected MDV using hybridohistochemistry. Prior to observation of the expression of a virus gene, we estimated the lower limit of the detection using a digoxigenin-labeled cRNA probe. We used mouse renin as a model. The lower limit of the detection was estimated to be the same range of abundance of beta-actin. Using the same method, we observed transcription of sense and antisense strands of Marek's disease virus (MDV) ICP4 homologue gene in MDV-infected chickens (3-4 weeks postinfection). A cRNA probe of MDV ICP4 was hybridized on paraformaldehyde-fixed and paraffin-embedded sections of feather follicle epithelium (FFE) and some lymphocytes in kidney, liver, nerve and spleen. Sense-strand transcripts of the MDV ICP4 gene were abundantly detected in the FFE compared with lymphocytes. Signals for antisense transcripts were seen in some kidney, liver, nerve and spleen lymphoid cells. Northern blot analysis confirmed the existence of transcripts in these tissues. Abundance and the sense/antisense ratio were also confirmed by Northern blot analysis. Immunoblot analysis also confirmed the expression of sense transcripts in FFE,kidney and liver. Thus, the results from lymphocytes suggested that sense and anti-sense transcripts of MDV ICP4 were expressed in some lymphocytes in the kidney, liver and spleen during the latent period. DNA of MDV genome was not detected because of its low copy umber. These data confirmed the reports on MD tumor cells that MDV genome was integrated in host genome and had a nucleosomal structure.