Molecular Signature of High Yield (Growth) Influenza A Virus Reassortants Prepared as Candidate Vaccine Seeds

<div><p>Background</p><p>Human influenza virus isolates generally grow poorly in embryonated chicken eggs. Hence, gene reassortment of influenza A wild type (wt) viruses is performed with a highly egg adapted donor virus, A/Puerto Rico/8/1934 (PR8), to provide the high yield reassortant (HYR) viral ‘seeds’ for vaccine production. HYR must contain the hemagglutinin (HA) and neuraminidase (NA) genes of wt virus and one to six ‘internal’ genes from PR8. Most studies of influenza wt and HYRs have focused on the HA gene. The main objective of this study is the identification of the molecular signature in all eight gene segments of influenza A HYR candidate vaccine seeds associated with high growth <i>in ovo.</i></p><p>Methodology</p><p>The genomes of 14 wt parental viruses, 23 HYRs (5 H1N1; 2, 1976 H1N1-SOIV; 2, 2009 H1N1pdm; 2 H2N2 and 12 H3N2) and PR8 were sequenced using the high-throughput sequencing pipeline with big dye terminator chemistry.</p><p>Results</p><p>Silent and coding mutations were found in all internal genes derived from PR8 with the exception of the M gene. The M gene derived from PR8 was invariant in all 23 HYRs underlining the critical role of PR8 M in high yield phenotype. None of the wt virus derived internal genes had any silent change(s) except the PB1 gene in X-157. The highest number of recurrent silent and coding mutations was found in NS. With respect to the surface antigens, the majority of HYRs had coding mutations in HA; only 2 HYRs had coding mutations in NA.</p><p>Significance</p><p>In the era of application of reverse genetics to alter influenza A virus genomes, the mutations identified in the HYR gene segments associated with high growth <i>in ovo</i> may be of great practical benefit to modify PR8 and/or wt virus gene sequences for improved growth of vaccine ‘seed’ viruses.</p></div

Recurrent Coding Changes in HYRs.

<p>Single letter amino acid code is used.</p><p>The recurrent coding changes were found in the ‘internal’ genes of PR8 origin and in the HA genes from the respective wt viruses.</p

Total number of recurrent silent and recurrent coding changes in HYRs.

<p>The majority of PR8 derived recurrent silent changes are found in NS1 (22) followed by PB2, NP, PB1-F2, and PA. The majority of the PR8 derived recurrent coding changes are found in NS2 followed by NS1, NP, PB1-F2 and PB1. HA had a total of 17 recurrent coding changes involving more than one amino acid position (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone-0065955-t009" target="_blank">Table 9</a>). NA and M genes had no recurrent changes.</p

Structural model of H3N2 HA (monomer) with coding changes found in the H3N2 HYRs in this study.

<p>The structure was generated by PyMOL Molecular Graphics System (<a href="http://www.pymol.org" target="_blank">www.pymol.org</a>). Single letter amino acid code is used. The numbers in the parentheses indicate recurrent changes (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone-0065955-t009" target="_blank">Table 9</a>); underlined changes were reported earlier <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Robertson1" target="_blank">[25]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Kumari1" target="_blank">[27]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Stevens1" target="_blank">[29]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Kilbourne3" target="_blank">[43]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Widjaja1" target="_blank">[45]</a> and also found in our study. ¹indicates unique changes found in the HA1 in this study and ² indicates unique changes found in the HA2 region.</p

HYR internal genes with recurrent coding changes.

<p>Single letter amino acid code is used.</p><p>The recurrent coding changes were found in the ‘internal’ genes of PR8 origin and are given in bold.</p

Total number of nucleotide changes in HYRs.

<p>Total Changes: The majority of the nucleotide changes (Silent and Coding, black bars) in the internal genes was found in the NS gene followed by PB1, PB2, NP, and PA. The HA gene had a total of 33 nucleotide changes; in contrast NA had only 2 nucleotide changes, both coding changes. M gene had no nucleotide change in any of the 23 HYRs. Silent Changes: Silent nucleotide changes were found in the internal genes of the HYR derived from hy donor virus, PR8 (blue bars). No silent changes were found in the internal genes derived from wt viruses except in NYMC X-157 PB1 derived from wt virus NY/04 which had 13 silent changes. HA had 3 silent nucleotide changes (green bar). NA and M genes had no silent changes in any of the 23 HYRs. Coding Changes: Among the internal proteins, the majority of the coding changes (orange bars) were found in PR8 derived NS (14 total; 7 in NS1 and 7 in NS2), followed by PB1 (10 total; 7 in PB1 and 3 in PB1-F2), NP (9) and PA (8). PB2 had only 1 coding change in contrast with 20 silent changes. M had no coding changes in any of the 23 HYRs. HA had 30 coding changes; in contrast NA had only 2 coding changes (red bars).</p

Total nucleotide changes in H3N2 HYRs.

<p>Silent changes are in regular font; Coding changes are in bold font with corresponding aa.</p><p>changes in the parentheses.</p><p>X-157 was a mixture of two populations of HYRs, one with ^a PR8 PB1 and a second with ^b NY/04 PB1.</p

HYR hemagglutinins with recurrent coding changes.

a<p>Single letter amino acid code used.</p>b<p>WT parent virus CA/2009 had either G or D; the HYRs had D at position 222.</p><p>Amino acid numbering was according to HA subtypes (refer <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone-0065955-t001" target="_blank">Table 1</a> for HYR subtype).</p

Gene composition and number of silent and coding changes per gene in HYRs.

a<p>Genes derived from respective wt viruses are given in italics.</p>b<p>Black bold font are genes derived from the hy donor virus PR8.</p>c<p>Includes the coding change Cys42Phe in the PB1-F2 of the X-163 series of HYRs.</p>d<p>RT-PCR/RFLP analysis showed X-53 PB1 derived from NJ/76 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065955#pone.0065955-Fulvini1" target="_blank">[36]</a>, however sequence data showed X-53 PB1 is a mixture of both PR8 and NJ/76 PB1 genes and also had more numbers of ambiguities, hence not included in the analysis.</p>e<p>NYMC X-157 PB1was a mixture of both PR8 and NY/04 PB1 genes.</p><p><b>A</b>/<i>A</i>- PB1 is a mixture of PR8 and NY/04; <b>A</b> (PR8 PB1) had only one silent change, <i>A</i> (NY/04 PB1) had 13 silent and 2 coding changes.</p

Influenza A virus HYRs analyzed in this study.

a<p>HYRs prepared in E.D. Kilbourne laboratory, others at Bucher laboratory at NYMC.</p>b<p>Used in seasonal influenza vaccine production.</p>c<p>SOIV- swine origin influenza virus.</p>d<p>X-53 and X-53a were used in the 1976 swine influenza vaccine production and X-179A was used in the 2009 H1N1pdm vaccine production.</p>e<p>X-135 was developed as a pre-pandemic vaccine seed candidate in the event of a reappearance of an H2N2 subtype in the population.</p