Simple and rapid strategy for genetic characterization of influenza B virus reassortants.
Genetic reassortment of influenza viruses is widely used for creating viruses with specific phenotypes. Reassortment of two influenza viruses, each with eight RNA segments potentially yields as many as 256 gene segment combinations. Therefore, confirmation that progeny viruses possess genomes corresponding to the specified phenotypes can be laborious and time-consuming. To establish a convenient method for genotyping influenza virus reassortants, we adapted single-strand conformation polymorphism analysis (SSCP) using standard laboratory equipment. By varying the concentration of polyacrylamide between 4-6% and the concentration of glycerol between 5-8% in the gel, together with adding PCR primers to the DNA sample during the denaturing step, optimal conditions can be found for SSCP with little effort. The described method has high accuracy and reliability, and provides a tool for rapid, cost-effective genetic screening and assessment of the purity and genetic stability of the reassortant viruses. This method should be useful in basic research applications and in preparing reassortant viruses for vaccine use.
Lugovtsev
Vladimir Y
VY
Division of Viral Products, Center for Biologics Evaluation and Research, Food and Drug Administration, 8800 Rockville Pike, Bethesda, MD 20892, USA. Lugovtsev@cber.fda.gov
Vodeiko
Galina M
GM
Strupczewski
Caryn M
CM
Levandowski
Roland A
RA
D001482:Base Composition; D009981:Influenza B virus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D018807:Polymorphism, Single-Stranded Conformational; D016865:Reassortant Viruses / Q000235:genetics*; D015203:Reproducibility of Results