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

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.

Generation and Characterization of Live Attenuated Influenza A(H7N9) Candidate Vaccine Virus Based on Russian Donor of Attenuation

“…AF which exhibited antigenic specificity of wt virus were combined and a cloning by limiting dilution procedure was carried out in the presence of antiserum at 25°C. 6:2 reassortants were selected as described previously [ 34 ].…”

“…Pyrosequencing analysis was performed using the PyroMark Q96 ID Platform (Qiagen, Carlsbad, CA, USA) following the manufacturer’s instructions. RT-PCR with biotinylated primers, gel analysis of the RT-PCR product, sample preparation, pyrosequencing reaction, and data analysis were done as described previously [ 34 ]. The primers were designed based on sequences of the A/Anhui/01/2013 and MDV using PSQ Assay Design software (Qiagen).…”

Biotic concerns in generating molecular diagnosis matrixes for 4 avian viruses with emphasis on Marek’s disease virus

“…In this study degenerated primers were designed to amplify a portion of the fusion gene responsible for virulence, then a 30-bp region encompassing the cleavage site was pyrosequenced. Pyrosequencing analysis was a useful technique also for genotyping AIV reassortants in the search of vaccine virus candidates (Shcherbik et al, 2014).…”

Разработка Холодоадаптированного Реассортантного Вируса Гриппа А/H6n1 На Основе Донора Аттенуации a/Ленинград/134/17/57 (H2N2) И Его Генотипирование Методом Анализа Кривых Плавления Высокого Разрешения (HRM-анализ)

“…Для быстрого анализа состава генома потенциальных вакцинных кандидатов применяют также ПЦР в реальном време-ни с использованием гидролизируемых олигонукле-отидных зондов [12] и пиросеквенирования [13]. Для упрощения процедуры скрининга вакцинных кан-дидатов мы предлагаем использовать ПЦР в режиме реального времени с анализом кривых плавления вы-сокого разрешения (HRM-анализ).…”