Determination of the genome composition of influenza virus reassortants using multiplex reverse transcription-polymerase chain reaction followed by fluorescent single-strand conformation polymorphism analysis

Abstract

The influenza virus genome is composed of eight negative-strand RNA segments. In cells coinfected with two or more influenza strains, the genomic RNAs reassort at random, resulting in progeny viruses (reassortants) that contain genes derived from each parent. Genetic reassortment among influenza viruses occurs naturally and plays an important role in viral epidemiology and pathogenicity. The reassortment process is also utilized for the annual production of influenza vaccines. Each year, the two gene segments that encode the major surface antigens of the current virulent, wild-type viruses are reassorted with the remaining six gene segments of a laboratory-derived vaccine or "master donor" strain. As the gene reassortment appears to be random, identifying a progeny virus with the desired gene constellation can be labor-intensive. We developed a streamlined, cost-effective method to genotype influenza viruses that combines multiplex reverse transcription-polymerase chain reaction (RT-PCR) and fluorescent single-strand conformation polymorphism (SSCP) analysis. This method utilizes oligonucleotide primers labeled with one of three fluorescent dyes to generate RT-PCR products for each gene segment in a multiplex configuration. The RT-PCR products of the reassortants, wild-type, and master donor viruses are then electrophoresed under SSCP conditions. The viral origin of each gene segment can be identified by fluorescence and mobility shift patterns of the corresponding RT-PCR products. We demonstrate the utility of this method in differentiating the genes of a master donor strain, several wild-type viruses, and vaccine reassortants.