Rapid confirmation by RFLP of transfer to vaccine candidate reassortment viruses of the principal 'high yield' gene of influenza A viruses.
Influenza vaccines must be revised constantly on almost a yearly basis because of the sequential mutations (antigenic drift) that occur as the virus responds to immunologic pressure. New, high yield (hy) reassortant viruses have proved essential to meet production needs for the supply of new vaccines. We have devised a method for simple, rapid and precise identification of the principal influenza A virus RNA segment (RNA 7) associated with hy and transferred from the hy donor virus, A/PR/8/34 (H1N1). The method entails the use of a single restriction enzyme, Bsgl, in analysis by restriction fragment length polymorphism (RFLP) of reverse transcriptase-polymerase chain reaction (RT-PCR)-generated DNA amplicons. The method clearly distinguishes the RNA coding for the M proteins of the donor virus from that of representative and epidemiologically significant human wild type viruses of the past 60 years. In the course of this methodological study further evidence has been found of the variability of the so-called 'invariant' and stable M1 and M2 proteins of the virus. Another finding of potentially basic significance that merits further study is the occurrence of a consistent change at the same amino acid (aa) site of the donated RNA 7 upon its transfer to reassortant viruses.
Brett
Ian
I
Department of Microbiology and Immunology, Room 315, Basic Sciences Building, New York Medical College, Valhalla, NY 10595, USA.
Werber
John
J
Kilbourne
Edwin D
ED
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002642:Chick Embryo; D004279:DNA, Viral; D015252:Deoxyribonucleases, Type II Site-Specific; D005814:Genes, Viral; D006801:Humans; D009980:Influenza A virus / Q000235:genetics*; D007252:Influenza Vaccines / Q000235:genetics*; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D012367:RNA, Viral / Q000032:analysis*; D011995:Recombination, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013997:Time Factors
D004279:DNA, Viral; D007252:Influenza Vaccines; D012367:RNA, Viral; C100361:endodeoxyribonuclease BsgI; D015252:Deoxyribonucleases, Type II Site-Specific