Cloning of RNA-dependent RNA Polymerase (RdRp) Gene from Genotype Dengue Type-2 (New Guinea-C Strain)

Abstract

Dengue virus causes febrile disease in human. Dengue infection causes dengue fever that is not life threatening. However, a severe form of the disease called dengue hemorrhagic fever (DH) or dengue shock syndrome (DSS), proven to be fatal. A positive single stranded RNA virus genome encodes for a single polyprotein precursor and is arranged in the order of NH2-C-prM-E-NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5-COOH. The purpose of this study was to clone NS5 gene that encodes for the RNA-dependent RNA polymerase (RdRp). This enzyme plays an important role in viral RNA replication. The RdRp associated by cofactors produce minus-strand single stranded RNA, which in turn, serves as a template for the production of new plus-strand single stranded genome. The virus RNA was extracted from Aedes albopictus cell line C6/36 that was infected with dengue virus type 2. Then, the extracted virus RNA was used as the template for RT-PCR. A 2.7 kb DNA fragment, representing the RNA-dependent RNA polymerase gene, wassuccessfully amplified using specific primers. The PCR product was then cloned into cloning vector (pGEM-T) and transformed into E. coli JM109.