Rapid Strategy for Screening by Pyrosequencing of Influenza Virus Reassortants - Candidates for Live Attenuated Vaccines

Svetlana V Shcherbik,Tatiana L Bousse,Nicholas C Pearce,Marnie L Levine,Julie M Villanueva,Alexander I Klimov,Andrew Pekosz

doi:10.1371/journal.pone.0092580

Abstract

BackgroundLive attenuated influenza vaccine viruses (LAIVs) can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV) and a seasonal wild-type (wt) virus. The vaccine candidates contain hemagglutinin (HA) and neuraminidase (NA) genes derived from the circulating wt viruses and the remaining six genes derived from the MDV strains. Rapid, efficient selection of the viruses with 6∶2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses.Methodology/Principal FindingsThis manuscript describes a new approach for the genotypic analysis of LAIV reassortant virus clones based on pyrosequencing. LAIV candidate viruses were created by classical reassortment of seasonal influenza A (H3N2) (A/Victoria/361/2011, A/Ohio/02/2012, A/Texas/50/2012) or influenza A (H7N9) (A/Anhui/1/2013) wt viruses with the MDV A/Leningrad/134/17/57(H2N2). Using strain-specific pyrosequencing assays, mixed gene variations were detected in the allantoic progenies during the cloning procedure. The pyrosequencing analysis also allowed for estimation of the relative abundance of segment variants in mixed populations. This semi-quantitative approach was used for selecting specific clones for the subsequent cloning procedures.Conclusions/SignificanceThe present study demonstrates that pyrosequencing analysis is a useful technique for rapid and reliable genotyping of reassortants and intermediate clones during the preparation of LAIV candidates, and can expedite the selection of vaccine virus candidates.

Highlights

The influenza virus is a globally important respiratory pathogen which causes significant morbidity and mortality in humans and animals
Development and Evaluation of Pyrosequencing Assays The RT-PCR fragments, amplified with pyrosequencing primers designed for each gene of the Master Donor Virus (MDV)-H3N2 and the MDV-H7N9 pairs (Tables 1 and 2), contained the targets with strain-specific signature nucleotides
The target sequences for MDV-H3N2 and MDV-H7N9 genotyping are shown in Table 5 and Table 6, respectively

Summary

Introduction

The influenza virus is a globally important respiratory pathogen which causes significant morbidity and mortality in humans and animals. Influenza vaccination is the most effective method for preventing influenza virus infection and its potentially severe complications [1,2]. Accumulation of mutations in genes encoding for the viral surface proteins leading to antigenic drift require that the WHO recommendations for influenza vaccine viruses be updated on a biannual basis to provide protection against contemporary seasonal influenza virus strains. LAIVs appeared to be more effective in preventing influenza infection than trivalent IIV [1,5,6,7,8]. Live attenuated influenza vaccine viruses (LAIVs) can be generated by classical reassortment of gene segments between a cold adapted, temperature sensitive and attenuated Master Donor Virus (MDV) and a seasonal wild-type (wt) virus. Efficient selection of the viruses with 6:2 genome compositions from the large number of genetically different viruses generated during reassortment is essential for the biannual production schedule of vaccine viruses

Methods

Results

Conclusion