Two small RNAs (0.9 and 0.75 kb), named Marek's disease virus (MDV) small RNAs (MSRs) and a 10-kb RNA, all of which map antisense to the MDV ICP4 homolog gene, have been readily detected in MDCC-MSB1 MDV-transformed T-lymphoblastoid cells. These RNAs were not detectable in reticuloendotheliosis virustransformed T cells. When MDV was reactivated by treatment of lymphoblastoid cells with 25 ,ug of iododeoxyuridine per ml, the relative levels of the transcripts decreased. These RNAs were not detected by Northern (RNA) hybridization in productively infected chicken embryo fibroblasts 48 h postinfection; however, they were apparent 140 h postinfection. By using Northern hybridization, RNase protection assays, and primer extension analysis, the MSRs were determined to map antisense to the predicted translational start site of the ICP4 homolog gene. The conclusion most consistent with the data is that the two MSRs are overlapping, spliced RNAs. Both small RNAs contain a latency promoter binding factor consensus recognition sequence located toward their 5' ends as well as two potential ICP4 recognition consensus sequences, one in each orientation. The region contains a number of small open reading frames on each side and within the MSRs. Although the exact endpoints are unknown, the large 10-kb species spans the entire ICP4 homolog region. We believe that this group of RNAs, which map antisense to the ICP4 homolog gene, are latency-associated transcripts of MDV.

Total DNA from Marek's disease virus (MDV)-infected chicken embryo fibroblasts was transfected into freshly plated secondary chicken embryo fibroblasts using calcium phosphate-mediated transfection. Transfection frequencies were dose-dependent and non-linear. The maximum transfection frequencies of nine MDV DNA preparations using 8-25 micrograms total DNA ranged from 45 to 898 plaques per calcium phosphate/DNA precipitate. Approximately 100-200 plaques per 60-mm tissue-culture dish using 1-5 micrograms total DNA from MDV-infected chicken embryo fibroblasts were typically obtained. Transfection was most efficient when the pH of the HEPES buffer was 7.0, no additional carrier DNA was added to the precipitates, and the cultures were exposed for 3 minutes to 15% buffered glycerol 4 hours after the addition of the calcium phosphate/DNA precipitates.

We report the characterization of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome. A deletion mutant (GAA4.8lac) lacks 4.8 kbp of US region DNA, the deleted segment having been replaced by the lacZ gene of Escherichia coli. This deletion results in the loss of the MDV-encoded US1, US10, and US2 homologs of herpes simplex virus type 1, as well as three putative MDV-specific genes, Sorfl, SorfZ, and Sor13. Two mutants containing lacZ insertions in the US1 and US10 genes have been constructed, and we have previously reported a US21ac insertion mutant (J. L.

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