Permissible Variation in the 3′ Non-Coding Region of the Haemagglutinin Genome Segment of the H5N1 Candidate Influenza Vaccine Cirus NIBRG-14

Rachel E Johnson,Michelle Hamill,Ruth Harvey,Othmar G Engelhardt,James S Robertson,Carolyn Nicolson,Andrew Pekosz

doi:10.1371/journal.pone.0036241

Rachel E Johnson, Michelle Hamill + Show 5 more

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https://doi.org/10.1371/journal.pone.0036241

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Abstract

The candidate H5N1 vaccine virus NIBRG-14 was created in response to a call from the World Health Organisation in 2004 to prepare candidate vaccine viruses (CVVs) to combat the threat of an H5N1 pandemic. NIBRG-14 was created by reverse genetics and is composed of the neuraminidase (NA) and modified haemagglutinin (HA) genes from A/Vietnam/1194/2004 and the internal genes of PR8, a high growing laboratory adapted influenza A(H1N1) strain. Due to time constraints, the non-coding regions (NCRs) of A/Vietnam/1194/2004 HA were not determined prior to creating NIBRG-14. Consequently, the sequence of the primers used to clone the modified A/Vietnam/1194/2004 HA was based upon previous experience of cloning H5N1 viruses. We report here that the HA 3′ NCR sequence of NIBRG-14 is different to that of the parental wild type virus A/Vietnam/1194/2004; however this does not appear to impact on its growth or antigen yield. We introduced additional small changes into the 3′NCR of NIBRG-14; these had only minor effects on viral growth and antigen content. These findings may serve to assure the influenza vaccine community that generation of CVVs using best-guess NCR sequences, based on sequence alignments, are likely to produce robust viruses.

Highlights

Influenza A viruses have a single-stranded negative sense RNA genome composed of eight individual RNA segments
When sequence information is available in the public domain for the non-coding regions (NCRs), information regarding their derivation is often unavailable
For the rapid development of an H5N1 candidate vaccine virus in 2004, the modified HA and the NA genome segments of A/ Vietnam/1194/2004 had to be cloned into a reverse genetics plasmid with no prior knowledge of their true NCR sequences

Summary

Introduction

Influenza A viruses have a single-stranded negative sense RNA genome composed of eight individual RNA segments. The 39 and 59 NCRs of influenza A viruses are important for virus replication [1,2,3] with the terminal 12 or 13 nucleotides of the 39 and 59 NCRs, respectively, being highly conserved among the eight RNA segments and different influenza A virus strains [1,4,5,6]. Beyond these conserved nucleotides the sequences of the NCRs are segment specific with variable levels of conservation amongst different viruses. It is frequently not possible to regard the NCR sequences as genuine as it is not known whether the sequences have been determined de novo or represent the sequence of primers used during PCR and/or cloning

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