Analysis On Characterization Of Immediate Early Gene ORF62of Varicella Vaccine Oka Strains Manufactured In China

Objectives:To investigate the mutations in immediate early (IE) gene ORF62of varicella vaccine Oka (vOka) strains and its possible role(s) in attenuation mechanism by comparing differences of sequences, transcription factor binding motif and promoting activities of ORF62-promoter, as well as sequences of ORF62-coding region and transactivities of ORF62-coded immediate early protein (IE62) between vOka strains manufactured in China and their parent Oka (pOka) strain.Methods:1. ORF62promoter-reporter plasmids and ORF62-expressing plasmids of pOka and three vOka strains (vOka-BK from Changchun BCHT Biotechnology Co., vOka.SH from Shanghai Institute of Biological Product, and vOka-GSK from GlaxoSmithKline Co. as control) were constructed with molecular cloning technique, respectively.2. The ORF62’s promoter region and coding region of three vOka strains and pOka strain were then sequenced and compared with each other.3. The transcription factor binding motif in ORF62promoter of three vOka strains and pOka strain were scanned with TFSEARCH software and compared with each other.4. ORF62-promoter’s activities and ORF62-encoded IE62transactivities upon promoters for immediate early gene ORF4, early gene ORF28and late gene ORF67of three vOka strains and pOka strains were assayed with transient transfection technique and compared with each other.Results:1. Compared with pOka strain, three vOka strains had a consistent T deletion mutation at site110,050in ORF62promoter.2. The consistent T deletion mutation of three vOka strains did not result in any change of transcription factor binding motif.3. Promoter activities of ORF62from three vOka strains were significantly lower than those of pOka strain when activated by either pOka IE62or vOka IE62.4. Compared with pOka strain, three consistent substitution mutation (Tâ†'C at site106262, Tâ†'C at site107136,Tâ†'C at site107252) exsited in ORF62-coding region of three vOka strains, which generated a new cut site for enzyme Sma I, Nae I and BssH II, respectively.5. The transactivities of three vOka strains’ IE62upon ORF4, ORF28and ORF67promoters were significantly higher than those of pOka both in CV-1and Me Wo cells, except that vOka_sH IE62showed significantly lower transactivities upon ORF4promoter than those of pOka strain in CV-1cells.Conclusions:1. The consistent T deletion mutation at site110,050in ORF62promoter of three vOka strains might be responsible for promoter activity reductions and IE62transactivity changes.2. The binding motif of transcription factor Spl was not essential for attenuation of live attenuated varicella vaccine.3. Both pOka and vOka IE62could weakly activate their own ORF62promoters.4. It seemed that type of cells used in transient transfection assays had no big impact for changes of ORF62-promoter activities or IE62transactivities between pOka and vOka strains.