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Thirteen ILTV-specific mabs (IgG1ic) were obtained after immunization of mice with purified virions, and subsequent hybridoma cell cloning.Based on their reactions in immunofluorescence, Western blot, and immunoprecipitation experiments, the mabs could be assigned to 5 different virus proteins or protein groups.Expression of cloned ILTV genes in eucaryotic cells revealed that 2 of the mabs are directed against the 60 kDa glycoprotein gC (UL44 gene product), whereas 6 mabs reacted with another viral glycoprotein (gJ), which is encoded by ORF 5 within the Us genome region.Conflicting with a previous description of the ORF 5 gene product of ILTV as a 60 kDa glycoprotein (gp60), protein bands with apparent molecular masses of 85, 115, 160 and 200 kDa were detected in the present study.The other antibodies recognized ILTV proteins of 15 (2 mabs), 22 (2 mabs), and 38 kDa (1 mab), which were not further characterized up to now. Immune electron microscopy indicated that gC and gJ are localized in the envelope of ILT virions, whereas the 15 kDa protein is a component of the tegument. It was demonstrated hat the epitopes of the glycoprotein-specific mabs are conserved in ILTV isolates from different parts of the world, and that these mabs are suitable for viral antigen detection in tracheas of ILTV infected chickens by immunofluorescence tests or immune histochemistry.Characterization of the ILTV-specific genes ORF A o ORF ENorthern blot analyses revealed properly sized viral mRNAs of five ILTV-specific genes (ORF A to ORF E), which are not conserved in any other known herpesvirus genome.After immunization of rabbits with bacterial fusion proteins monospecific antisera were prepared, which permitted identification of the ORF A to ORF E gene products in ILTV infected cells.In Western blot analyses the viral proteins exhibited apparent masses of 40 kDa (ORF A), 34 kDa (ORF B), 38 and 30 kDa (ORF C), 41 kDa (ORF D), and 44 kDa (ORF E).Comparison of these sizes with that of the respective in vitro translation products, and with calculated molecular masses indicated that the viral ORF B and ORF D gene products initiate at internal start codons, that the ORF C protein is cleaved by cellular proteases, and that the viral ORF D protein is modified after translation. Successful isolation of GFP expressing virus recombinants demonstrated that none of the five ILTV specific genes is essential for replication in cultured cells. When compared to parental wild-type ILTV, almost all deletion mutants exhibited clearly reduced plaque sizes (expect 4ORF B) and virus titers (expect 4ORF C). However, these replication defects might be also caused by a cytotoxic effect of the abundantly expressed GFP, since they could be not complemented in recombinant cell lines which carried the deleted ILTV genes. The relevance of the ORF A to ORF E genes for in vivo replication and virulence of ILTV in chickens remains to be analyzed.