Application of the New Generation of Sequencing Technologies for Evaluation of Genetic Consistency of Influenza A Vaccine Viruses

For almost half a century, Sanger sequencing has been the conventional method for sequencing DNA. However, its utility for sequencing heterogeneous viral populations is limited because it can only detect mutations that are present in a significant portion of the DNA molecules. Several molecular methods that quantify mutations present at low levels in viral populations were proposed for evaluation of genetic consistency of viral vaccines; however, these methods are only suitable for single site polymorphisms, and cannot be used to screen for unknown mutations

Evolution of echovirus 11 in a chronically infected immunodeficient patient.

Deep sequencing was used to determine complete nucleotide sequences of echovirus 11 (EV11) strains isolated from a chronically infected patient with CVID as well as from cases of acute enterovirus infection. Phylogenetic analysis showed that EV11 strains that circulated in Israel in 1980-90s could be divided into four clades. EV11 strains isolated from a chronically infected individual belonged to one of the four clades and over a period of 4 years accumulated mutations at a relatively constant rate. Extrapolation of mutations accumulation curve into the past suggested that the individual was infected with circulating EV11 in the first half of 1990s. Genomic regions coding for individual viral proteins did not appear to be under strong selective pressure except for protease 3C that was remarkably conserved. This may suggest its important role in maintaining persistent infection

Deep Sequencing for Evaluation of Genetic Stability of Influenza A/California/07/2009 (H1N1) Vaccine Viruses.

Virus growth during influenza vaccine manufacture can lead to mutations that alter antigenic properties of the virus, and thus may affect protective potency of the vaccine. Different reassortants of pandemic "swine" H1N1 influenza A vaccine (121XP, X-179A and X-181) viruses as well as wild type A/California/07/2009(H1N1) and A/PR/8/34 strains were propagated in embryonated eggs and used for DNA/RNA Illumina HiSeq and MiSeq sequencing. The RNA sequences of these viruses published in NCBI were used as references for alignment of the sequencing reads generated in this study. Consensus sequences of these viruses differed from the NCBI-deposited sequences at several nucleotides. 121XP stock derived by reverse genetics was more heterogeneous than X-179A and X-181 stocks prepared by conventional reassortant technology. Passaged 121XP virus contained four non-synonymous mutations in the HA gene. One of these mutations (Lys226Glu) was located in the Ca antigenic site of HA (present in 18% of the population). Two non-synonymous mutations were present in HA of viruses derived from X-179A: Pro314Gln (18%) and Asn146Asp (78%). The latter mutation located in the Sa antigenic site was also detected at a low level (11%) in the wild-type A/California/07/2009(H1N1) virus, and was present as a complete substitution in X-181 viruses derived from X-179A virus. In the passaged X-181 viruses, two mutations emerged in HA: a silent mutation A1398G (31%) in one batch and G756T (Glu252Asp, 47%) in another batch. The latter mutation was located in the conservative region of the antigenic site Ca. The protocol for RNA sequencing was found to be robust, reproducible, and suitable for monitoring genetic consistency of influenza vaccine seed stocks

Percentage of mutations (≥5%) emerged in A/PR/8/34 viruses 1 and 3 genomes that were subjected to RNA library preparation followed by HiSeq sequencing.

<p>Note: These mutation percentages were calculated per comparison of A/PR/8/34 strains 1 and 3 genomes with the corresponding published sequences. (See accession numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138650#sec002" target="_blank">Materials and Methods</a> paragraph.) nt: Nucleotide, aa: Amino-acid.</p

HiSeq sequencing of RNA Library prepared from A/Ca/07/2009 (H1N1) virus.

<p>*: Count of nucleotide location started 20 nucleotides (UTR) before the starting codon AUG.</p><p>Note: These mutation percentages were calculated per comparison of A/California/07/2009 (H1N1) genome with its published sequence. (See accession numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138650#sec002" target="_blank">Materials and Methods</a> paragraph.) aa: Amino acid, nt: Nucleotide.</p

Identification and quantification of defective virus genomes in high throughput sequencing data using DVG-profiler, a novel post-sequence alignment processing algorithm

© This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication. Most viruses are known to spontaneously generate defective viral genomes (DVG) due to errors during replication. These DVGs are subgenomic and contain deletions that render them unable to complete a full replication cycle in the absence of a co-infecting, non-defective helper virus. DVGs, especially of the copyback type, frequently observed with paramyxoviruses, have been recognized to be important triggers of the antiviral innate immune response. DVGs have therefore gained interest for their potential to alter the attenuation and immunogenicity of vaccines. To investigate this potential, accurate identification and quantification of DVGs is essential. Conventional methods, such as RT-PCR, are labor intensive and will only detect primer sequence-specific species. High throughput sequencing (HTS) is much better suited for this undertaking. Here, we present an HTS-based algorithm called DVG-profiler to identify and quantify all DVG sequences in an HTS data set generated from a virus preparation. DVG-profiler identifies DVG breakpoints relative to a reference genome and reports the directionality of each segment from within the same read. The specificity and sensitivity of the algorithm was assessed using both in silico data sets as well as HTS data obtained from parainfluenza virus 5, Sendai virus and mumps virus preparations. HTS data from the latter were also compared with conventional RT-PCR data and with data obtained using an alternative algorithm. The data presented here demonstrate the high specificity, sensitivity, and robustness of DVG-profiler. This algorithm was implemented within an open source cloud-based computing environment for analyzing HTS data. DVG-profiler might prove valuable not only in basic virus research but also in monitoring live attenuated vaccines for DVG content and to assure vaccine lot to lot consistency

Mutation percentages (≥5%) emerged in HA and NA of X-181 viruses passaged in eggs.

<p>Notes: The percentages obtained from RNA library followed by HiSeq sequencing are presented without parentheses or brackets. The percentages obtained from RNA library followed by MiSeq sequencing are presented in parentheses. The percentages obtained from DNA library followed by HiSeq sequencing are presented in brackets. These mutation percentages were calculated per comparison with the corresponding expanded progenitor. (See accession numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138650#sec002" target="_blank">Materials and Methods</a>.) aa: Amino-acid, nt: Nucleotide.</p

Percentage of mutations (≥5%) emerged in the genome of A/PR/8/34 strain (CBER stock) obtained by sequencing five times its RNA library by HiSeq and MiSeq.

<p>Notes: MiSeq values that differ from HiSeq's values are presented in parentheses. These mutation percentages were calculated per comparison to their published sequences. (See accession numbers in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138650#sec002" target="_blank">Materials and Methods</a> paragraph.) nt: Nucleotide, aa: Amino-acid.</p

A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H₂O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H₂O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H₂O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.

<p>A: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Phusion DNA polymerase, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: 1 kb DNA Ladder, 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H₂O). B: Simultaneous RT-PCR amplification of the eight segments of A/California/07/2009 (H1N1) vaccine viruses using Thermo Scientist Extensor Mix, 1: 1 kb DNA Ladder, 2: X-181-M1, 3: X-179A-M1, 4: X-181-M2, 5: X-179A-M2, 6: X-181-M3, 7: X-179A-M4, 8: Negative control (H₂O), 9: 1 kb DNA Ladder, 10: 121XP-M4, 11: X-181-M4, 12: X-179A-M3, 13: X-179A-M5, 14: X-179A, 15: X-181, 16: Negative control (H₂O). C: The distribution amount of sequencing reads between the eight segments of influenza virus using different approaches of library preparation followed by Illumina HiSeq sequencing.</p

Viruses of A/California/07/2009 vaccines, and A/PR/8/34 and wild type A/California/07/2009 viruses used for deep sequencing analysis.

<p>*: A/PR/8/34 virus stock was passaged 13 times in eggs, and the last passaged virus was passaged one time (passage 14) separately in 2 different eggs, the 2 harvested viruses are designated as A/PR/8/34-1 and A/PR/8/34-3.</p><p>Note: CBER: Center for Biologics Evaluation and Research at US Food and Drug Administration.</p