Studies on genetically engineered chicken DF-1 cell for efficient production of influenza virus

Abstract

Influenza virus has been known to cause pandemics in poultry species leading to mortality and massive economic losses in the poultry industry. Therefore, methods have been devised for quick isolation, amplification, and identification of influenza subgroups. Several strategies have been used for quick and abundant amplification of the virus, including an egg-based system and cell-based system. The egg-based system is a well-established system; however, it offers several notable disadvantages especially in the process of vaccine production. Therefore, a cell-based system has been developed as a viable alternative strategy for the amplification of the virus. The establishment of a cell-based system is critical because the virus replicates efficiently in species-specific host cells. This system provides several advantages as follows: there is no need for virus adaptation to the cell for proliferation, and virus-host factors can be studied in a species-specific manner. Also, the engineering of cells could provide an improved system that could mimic the conditions for the replication of the virus, similar to in vivo conditions. Moreover, an added advantage of a cell-based system is that it could reduce the cost during the process of viral amplification for vaccine production. In this study, plasmid-based reverse genetics was established for the rescue of influenza virus in cell culture. After that, it was demonstrated that the influenza virus codon usage bias between influenza and the host species affects the viral polymerase activity. Furthermore, genetically engineered cell lines were established that could be used for the amplification of the influenza virus in the absence of trypsin. In the first study, chicken polymerase I promoter was identified, amplified, and inserted into a plasmid to produce the virus in the avian cells. The viral genomes were first codon optimized for the chicken codon usage bias and inserted into the plasmid harboring chicken polymerase I promoter. Then, viral polymerase activity was evaluated by establishing a luciferase assay based system. It was confirmed that the influenza virus could be rescued and that the polymerase activity was correlated to the translation of the proteins of the polymerase complex based on the codon usage of the chicken. Interestingly, compared to the wild-type virus, the codon optimized virus polymerase complex showed significantly higher activity at 24h post-transfection. In the next study, the expression of host factors, such as TMPRSS2 and TMPRSS4 involved in the cleavage of the hemagglutinin protein of the virus and ST3GAL1 involved in the cell receptor recognition by the hemagglutinin protein, in various chicken tissues that are routinely infected with the influenza virus were evaluated compared to wild-type DF-1 cells. From the results, it was revealed that the expression levels of TMPRSS2 and TMPRSS4 were very low in wild-type DF-1 cells. Therefore, a piggyBac transposon vector was constructed for integration and overexpression of each host factor in the wild-type DF-1 cells. After that, the expression levels of TMPRSS2, TMPRSS4, and ST3GAL1 were evaluated by qRT-PCR of the transfected cells. Interestingly, TMPRSS2 and ST3GAL1 showed increased expression in the transfected cells, while TMPRSS4 had a relatively low expression. The proliferation of engineered cells was further analyzed, and it was found that the overexpression of host factors TMPRSS2, TMPRSS4, and ST3GAL1 had no negative effect on the cells. Each engineered cell line was challenged with the influenza virus (PR8-H5N8 PB2-627E), and the results showed an increased viral titer in the engineered cells, even in the absence of trypsin compared to wild-type DF-1 cells. Furthermore, a cell line expressing both TMPRSS2 and ST3GAL1 was established, and this cell line was challenged with the influenza virus (PR8-H5N8 PB2-627E). The results showed an increased viral titer in the cells co-expressing TMPRSS2 and ST3GAL1 compared with the WT DF-1 cell line treated with trypsin. Collectively, these results indicate that this system could be used for the rescue and amplification of the influenza virus even in the absence of trypsin, which is critical in cell-based systems used for the propagation of the influenza virus. This system and the engineered cells could be applied to other fields of research, such as the study of species-specific host factors that interact with the influenza virus during infection and in vaccine production without the addition of trypsin, thus reducing the cost of production.