Background:In the recent decade, the reverse genetics method has been broadly used for rescue of negative-stranded RNA viruses from cDNA or viral minigenomes. This technique has been applied to study different steps in virus replication and virus-host interactions. Reverse genetics could also be implemented for design of new vaccines. The T7 RNA polymerase activity as well as virus (nucleocapsid protein) N, (phosphoprotein) P and (Large) L proteins are necessary to rescue the virus or viral minigenome. Measles virus is a negative-stranded non-segmented RNA virus. There are useful vaccine strains to prevent measles disease. Objectives:Here, we describe the construction of a new helper cell line for rescue of mea... More
Background:In the recent decade, the reverse genetics method has been broadly used for rescue of negative-stranded RNA viruses from cDNA or viral minigenomes. This technique has been applied to study different steps in virus replication and virus-host interactions. Reverse genetics could also be implemented for design of new vaccines. The T7 RNA polymerase activity as well as virus (nucleocapsid protein) N, (phosphoprotein) P and (Large) L proteins are necessary to rescue the virus or viral minigenome. Measles virus is a negative-stranded non-segmented RNA virus. There are useful vaccine strains to prevent measles disease. Objectives:Here, we describe the construction of a new helper cell line for rescue of measles virus minigenome. The helper cell line stably expresses T7 RNA polymerase as well as measles virus N and P proteins by tricistronic mRNA. Materials and Methods:For rescue of measles virus minigenome a stable helper cell line by using tricistronic expression vector was developed which expressed T7 RNA polymerase as well as measles virus N and P proteins. To construct the tricistronic expression vector, T7 RNA polymerase gene was cloned after cytomegalovirus (CMV) promoter and measles virus N and P proteins were under control of IRES (internal ribosome entry site) sequences. Results:Our results indicated that measles virus minigenome could be rescued in this constructed helper cell line. Conclusions:Through this system, the measles virus minigenome was rescued. Further studies are necessary to improve the rescue efficiency. This may be possible by replacing the CMV promoter with the T7 promoter.
