Construction of a minigenome rescue system for measles virus, AIK-c strain

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. 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. Our results indicated that measles virus minigenome could be rescued in this constructed helper cell line. 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

Construction of an eGFP expression plasmid under control of T7 promoter and IRES sequence for assay of T7 RNA polymerase activity in mammalian cell lines

Recently, the use of T7 RNA polymerase instead of other viral and cellular promoters is increasing due to high efficacy of transcription in the cell cytoplasm by this polymerase. In order to translate the transcripts produced by T7 RNA polymerase in mammalian cell lines, it is necessary to include Internal Ribosome Entry Site [IRES] sequences. In addition, if sequence of poly A signal would be included after interested gene, the rate of expression could be increased in the cells. For expression of eGFP in HEK-293 and T7-BHK cells by T7 RNA polymerase, the sequence of eGFP as well as IRES sequences upstream of eGFP gene and poly A signal were inserted into a pUC57 plasmid. On the other hand, gene of T7 RNA polymerase was cloned into modified pIRES2-EGFP plasmid. Then, the constructed plasmids were transfected into HEK-293 cells. T7-BHK cell was used for control of T7 RNA polymerase activity. Our results showed that using T7 RNA polymerase for expression of foreign genes in mammalian cell lines is highly efficient. Highly efficient eGFP expression in HEK-293 cells showed that T7 RNA polymerase could be used for cytoplasmic RNA transcription such as production of anti-cancer proteins and oncolytic viral genomic RNA by reverse genetics

Determining influenza virus shedding at different time points in madin-darby canine kidney cell line

Monitoring of influenza virus shedding and optimization of multiplicities of infection [MOI] is important in the investigation of a virus one step growth cycle and for obtaining a high yield of virus in vaccine development and conventional basic diagnostic methods. However, eluted infectious viruses may still be present immediately after virus inoculation and when cells are washed following virus cultivation which may lead to a false positive virus infectivity assay. In this experimental study, we investigated influenza virus progeny production in Madin-Darby canine kidney [MDCK] cells with five different MOI at determined time points. The results were analyzed by end point titration tests and immunofluorescence assay. Higher titers of eluted virus were observed following a high MOI inoculation of virus in cell culture. Most probably, this was the result of sialic acid residues from viral hemagglutin in proteins that were cleaved by neuraminidase glycoproteins on the surface of the influenza virus, which promoted viral spread from the host cell to the culture supernatant or during endocytosis, where viruses recycle to the cell surface by recycling endosomes which culminated in virus shedding without replication. We demonstrated that the pattern of influenza virus progeny production was dose-dependent and not uniform. This production was influenced by several factors, particularly MOI. Understanding the exact features of viral particle propagation has a major impact in producing high virus yields in the development of vaccines. Use of lower MOI [0.01] could result in accurate, precise quantitative assays in virus diagnosis and titration methods