Gene analysis of reassortant influenza virus by RT-PCR followed by restriction enzyme digestion.
An amplification system for nearly full length cDNA coding the eight influenza virus segments of A type (H1N1, H2N2, H3N2) and B type influenza viruses is described. Each of the segments of PB1, PB2, PA, NP, M, and NS can be amplified using one 5' primer and one 3' primer for A-type influenza viruses. The RT-PCR amplification system was applied to define the gene composition of three subtype cold-recombinant, live attenuated influenza viruses. Each segment of the attenuated influenza virus could be identified as deriving from segments of the Ca donor or wild virus by comparing the representative restriction enzyme digestion patterns of the three PCR products obtained from the Ca donor, the cold-live attenuated influenza viruses and the wild virus. This RT-PCR method, using RT-PCR followed by digestion of PCR products with restriction enzymes, was very beneficial for analyzing the genome of reassortant influenza viruses.
Sakamoto
S
S
Chemo-Sero-Therapeutic Research Institute, Shimizu Laboratory 668, Kumamoto, Japan.
Kino
Y
Y
Oka
T
T
Herlocher
M L
ML
Maassab
F
F
D001483:Base Sequence; D018076:DNA, Complementary; D004279:DNA, Viral / Q000032:analysis* / Q000378:metabolism; D015245:Deoxyribonuclease BamHI / Q000378:metabolism; D019003:Deoxyribonuclease HpaII / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D006801:Humans; D009980:Influenza A virus / Q000235:genetics*; D009981:Influenza B virus / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000032:analysis*; D016865:Reassortant Viruses / Q000235:genetics*
D018076:DNA, Complementary; D004279:DNA, Viral; D012367:RNA, Viral; D015245:Deoxyribonuclease BamHI; D019003:Deoxyribonuclease HpaII; D015252:Deoxyribonucleases, Type II Site-Specific; C024400:GTYRAC-specific type II deoxyribonucleases