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

Application of semi-nested pcr rflp for detection and genotyping GBV-c among HBV-infected patients

GBV-C is a virus transmissible via blood transfusion and sexual routes. Because of the shared transmission routes in GBV-C and hepatitis B virus [HBV] we have attempted to detect GBV-C in HBsAg-positive patients using the more sensitive semi-nested PCR. We used restriction fragment length polymorphism [RFLP] to determine genotype. A total of 100 serum samples were collected from HBsAg-positive patients from 1389-1390. After designing specific primers and optimizing the semi-nested PCR, sequences of PCR products were analyzed by Neb Cutter software. Restriction enzyme sites were determined and suitable enzymes selected for RFLP. Semi-nested PCR shows acceptable sensitivity for the detection of GBV-C and its genotype determination. This technique is more affordable than other techniques. There appears to be a high rate of GBV-C infection among Iranian HBV patients. In this study, the majority of patients co-infected with GBV-C were of HBV genotype 2, which is similar to the pattern seen in the US and Europe

novel sensitive multiplex RT-PCR ELISA method for detection of HIV-1/HCV co-infection

Despite sensitive antibody based blood donor screening, infection can be transmitted during window period. Therefore sensitive methods based on nucleic acid test [NATs] have been considered. The aim of this project was to design a more sensitive method for detection of PCR products and diagnosis of HIV-1/HCV co- infection accurately.After designing specific primers and probes, the Multiplex RT-PCR method was optimized and the PCR products were labeled with Digoxigenin. The PCR product was denatured under alkaline condition and was hybridized with the specific probe that had a biotin at 5' end, and then was added to streptavidin coated wells. After washing an antibody against DIG, conjugated with alkaline phosphates enzyme was used, following second washing, the substrate [ABTS] solution was added to each reaction well. Development of green color shows the positive where as no color shows negative results. 35 samples were tested with the developed method including 27 positive samples, 8 confirmed negative and 4 standard panels. False negative or positive reactions were not observed. This method had acceptable sensitivity and specificity for detecting HCV and HIV-1 infections during window period, also the method can be quantified which can be used for the flow-up and treatment of patients. In addition to the very high sensitivity of the test, it is cost effective and takes less time to perform