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

Abstract

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.

Establishment of BALB/C mouse models of influenza A H1N1 aerosol inhalation

“…To establish animal models of H1N1 aerosol inhalation by simulating the natural infection, we chose to expose BALB/C mice to influenza A/California/07/2009, which has caused global pandemic outbreaks since 2009 and is responsible for the increased H1N1‐associated morbidity and mortality in China to date. Influenza A/California/7/2009 is still the prototype H1N1 strain recommended by the WHO for the current seasonal vaccine, and recent studies have revealed that other clades of H1N1 viruses are antigenically similar to the A/California/7/2009 strain …”

Coding-Complete Genome Sequences of Six Influenza Type A Strains Circulating in Lithuania in the 2009–2010 Epidemic Season

“…All sequences were aligned, using MAFFT v7.388, against the A/California/07/2009 reference strain circulating among people at that time ( 9 – 12 ). We observed a high degree of similarity (>99%) among all six strains as well as the reference strain (similarity levels are presented in Table 1 ).…”

Nuclease pre-treatment increases efficiency of whole genome sequencing of Influenza B virus in respiratory specimens

“…Trimmed sequence reads of each sample were analyzed by using Kraken (Wood & Salzberg, 2014) and CLARK (31-mers) (Ounit et al, 2015) in order to identify and estimate the proportion of mapped reads of known bacterial, viral, and human genome sequences. Trimmed sequence reads of each sample were taxonomically classified by using high accuracy Kraken tool (Majid et al, 2015;Wood & Salzberg, 2014). However, since Kraken does not provide a human database, CLARK (Ounit et al, 2015) was used to identify and estimate the proportion of mapped reads of known bacterial, viral, and human genome sequences.…”