A PRACTICAL APPROACH TO GENETIC SCREENING FOR INFLUENZA-VIRUS VARIANTS

This report describes a quick genetic approach to the screening of inf luenza virus variants, Multiplex reverse transcription (MRT) and multi plex PCR (MPCR) were used to amplify and differentiate the hemagglutin in (HA) genes of different types and subtypes of influenza viruses, He teroduplex mobility assay (HMA) was then used to differentiate strains within the same type and subtype, Three primers complementary to the consensus 3' termini of viral genomic RNA segments of human influenza virus types A, B, and C were used in a single MRT reaction to prime th e synthesis of cDNA of all the viral genome segments, The cDNA was the n amplified in an MPCR containing primers for the HA genes of the H1 a nd H3 subtypes of type A, the HA gene of type B, and the counterpart o f type C virus, Amplicons of the different types and subtypes differ i n size, thus allowing typing and subtyping, The regions amplified cove r most of the HA1 portion of the HA genes and therefore amplicons of v ariants identified by the described BMA can be sequenced directly, In the HMA, the amplicon of an individual strain was mixed with that of a reference strain and heteroduplexes derived from mismatches migrated more slowly than homoduplexes of the same size in electrophoresis, wit h the mobility shift pattern indicating the divergence of amplicons, T he whole process from viral RNA extraction to HMA can be completed wit hin 2 days and thus provides a quick screening before further analysis by hemagglutination inhibition testing and sequencing, In addition, a ll segments of the viral genome can be amplified from a single MRT rea ction, which can yield valuable sources of products for future genetic analyses, This method should facilitate genetic screening and charact erization of influenza virus variants.