Development of Pyrosequencing-Based Assay for Analyzing the Origin of Genes in Preparing Reassortant LAIV Candidates

Abstract

Live attenuated influenza vaccine (LAIV) is an effective measure to prevent influenza infection. The vaccine strain for LAIV is derived by reassortment of epidemic influenza strain with cold-adapted master donor virus (MDV) in developing chicken embryos. Molecular analysis of genomic composition of generated reassortants to select appropriate candidate for LAIV is a critical stage of vaccine strain development. Method of analysis permitting quick and precise detection of the genome composition of reassortants is required. The purpose of this study was to develop pyrosequencing-based assay using PyroMark Q24 for analysis of the origin of genes in preparing reassortant LAIV candidates for influenza A vaccine strains based on A/Leningrad/134/17/57 master donor virus and influenza B vaccine strains based on B/USSR/60/69 or B/Leningrad/14/77/55 master donor viruses, respectively. Objectives included development of primers set for genotyping of influenza A and B reassortants by pyrosequencing, establishing optimal dispensation order and assessment of effectiveness of protocol application for detecting the source of genes in virus samples. We have shown that a unified genome analysis protocol using PyroMark Q24 system is able to detect the source of internal genes in reassortant influenza strain by pyrosequencing. It allows analyzing the genome composition by a sensitive and specific innovative method in the first stages of vaccine strain development.