BACKGROUND:Influenza virus reverse genetics has reached a level of sophistication where one can confidently generate virus entirely form cloned cDNA. This system makes it possible to generate attenuated live virus vaccine candidate. We tried to generate human influenza A viruses encoding altered viral NS1 proteins in various available cell lines. MATERIALS AND METHODS:Eight (HA, NA, NP, M, NS, PBI, PB2, PA) viral and four (PB1, PB2, PA, NP) expression plasmids were generated from A/T exas/ 36/91 influenza virus by RT-PCR and cloning with POL-I and pGEM-T vector. Two NS1 mutant cDNA (NS1-126delta, NS1-99delta) were also generated. We transfected these plasmids into the 293T/MDCK, 293, CEF and Vero cells and incubated with culture media for 2-3 days. And then, we inoculated cell soups into the embryonated eggs. After 3-4 days of incubation, we harvested allantoic fluid and checked viral titer by HA assay. Finally we did RT-PCR and sequencing to confirm the virus. RESULTS:Finally we got the NS1 mutant A/Texas influenza viruses from 293T/MDCK cells, but not from FDA approved cells. However, whereas 293 cells are capable of being transfected and of growing the NS1 mutant viruses with low titer, CEF cells are only capable of growing this mutant viruses. CONCLUSION:293 and CEF cells could not be used alone for acquiring NS1 mutant A/Texas influenza viruses. However, 293/CEF co-culture seems to be a resonable next step for NS1 mutant virus rescue for human using.
Cell Line ; Clone Cells ; Cloning, Organism ; Coculture Techniques ; Culture Media ; DNA, Complementary ; Eggs ; Humans* ; Influenza, Human* ; Orthomyxoviridae ; Ovum ; Plasmids ; Reverse Genetics ; Vero Cells

BACKGROUND:Influenza virus reverse genetics has reached a level of sophistication where one can confidently generate virus entirely form cloned cDNA. This system makes it possible to generate attenuated live virus vaccine candidate. We tried to generate human influenza A viruses encoding altered viral NS1 proteins in various available cell lines. MATERIALS AND METHODS:Eight (HA, NA, NP, M, NS, PBI, PB2, PA) viral and four (PB1, PB2, PA, NP) expression plasmids were generated from A/T exas/ 36/91 influenza virus by RT-PCR and cloning with POL-I and pGEM-T vector. Two NS1 mutant cDNA (NS1-126delta, NS1-99delta) were also generated. We transfected these plasmids into the 293T/MDCK, 293, CEF and Vero cells and incubated with culture media for 2-3 days. And then, we inoculated cell soups into the embryonated eggs. After 3-4 days of incubation, we harvested allantoic fluid and checked viral titer by HA assay. Finally we did RT-PCR and sequencing to confirm the virus. RESULTS:Finally we got the NS1 mutant A/Texas influenza viruses from 293T/MDCK cells, but not from FDA approved cells. However, whereas 293 cells are capable of being transfected and of growing the NS1 mutant viruses with low titer, CEF cells are only capable of growing this mutant viruses. CONCLUSION:293 and CEF cells could not be used alone for acquiring NS1 mutant A/Texas influenza viruses. However, 293/CEF co-culture seems to be a resonable next step for NS1 mutant virus rescue for human using.
Cell Line ; Clone Cells ; Cloning, Organism ; Coculture Techniques ; Culture Media ; DNA, Complementary ; Eggs ; Humans* ; Influenza, Human* ; Orthomyxoviridae ; Ovum ; Plasmids ; Reverse Genetics ; Vero Cells