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

ANNOUNCEMENT

Belonging to the family Orthomyxoviridae, genus Alphainfluenzavirus, the influenza type A virus causes annual human and animal influenza. The manifestation of the disease may take the form of seasonal waves of cases, local epidemics, or global pandemics caused by the emergence of antigenically different strains (1).

Lack of immunity to such strains in the human population is caused by continuous genetic variation of the virus, which is based on phenomena such as reassortment (exchange of genetic segments between two different viruses), antigenic shift (changes of genetic segments encoding the major surface antigens), and antigenic drift (slow accumulation of small variations in sequences encoding the major surface antigens) (2, 3). Therefore, it is important to not only keep tracking the current genetic diversity of these viruses but also take a look at changes that occurred in the past to have a full set of data for accurate estimation of the antigenic drift and what course it may take (4, 5).

Viral RNA was isolated, using the RNeasy purification kit (Qiagen), from oropharyngeal swabs collected by the National Public Health Surveillance Laboratory of Lithuania during the 2009–2010 epidemic season from patients showing influenza symptoms such as fever, coughing, headache, and chills. The Vilnius Regional Bioethics Committee approved the study protocol; this was deemed minimal risk, and the requirement for written consent was waived. The samples were positively diagnosed as A(H1N1) with a reverse transcription-quantitative PCR (RT-qPCR) assay using primers and probes recommended by the World Health Organization (6, 7). For all A(H1N1) genomic segments, reverse transcription and PCR amplification were performed using PathAmp FluA reagents (8). Postreaction cleanup was done on Agencourt AMPure XP magnetic beads (Beckman Coulter). We performed full genome sequencing on the MiSeq platform (Illumina) with v3 sequencing chemistry. For genomic library preparation, a Nextera XT DNA sample preparation kit (Illumina) was used. Paired-end reads of the target size 2 × 300 bp were generated (isolate 1167, 2,514,242 reads; isolate 1172, 1,185,476 reads; isolate 1241, 1,758,300 reads; isolate 1305, 1,675,062 reads; isolate 1319, 2,687,104 reads; isolate 1324, 1,299,004 reads). Data were gathered as fastq files and further analyzed with Geneious Prime v20.2 software (Biomatters) using integrated tools (trimming, i.e., deleting 3′ and 5′ ends with an error probability limit of 0.05) and the BBNorm package v38.84 (https://sourceforge.net/projects/bbmap) (error correction and normalization, i.e., correcting substitution errors based on quality, discarding reads that have a probable coverage of 2× or less, and reducing the number of reads that have a probable coverage of 40× or more). De novo assembly of contigs was performed using a medium sensitivity script. Final contigs were trimmed to the size of the reference genome (GenBank accession numbers are provided in the footnote of Table 1).

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). These analyses revealed several single-nucleotide polymorphisms that alter the amino acid sequences in two major surface glycoproteins, namely, hemagglutinin (HA) and neuraminidase (NA), i.e., V24I (isolate 1305), R45K (isolates 1319 and 1324), V47G (isolates 1319 and 1324), S74N (isolate 1167), P83S (all isolates), S203T (all isolates), A215V (isolate 1305), I321V (isolates 1167, 1172, 1241, and 1305), I321F (isolates 1319 and 1324), and E374K (all isolates except isolate 1167) in HA and G41V (isolate 1172), I46T (isolate 1167), V106I (all isolates), and N248D (all isolates) in NA. The S74N, P83S, S203T, and A215V substitutions are localized in epitope regions of HA or the immediate vicinity (13). The V47G and S74N substitutions were characterized earlier in our findings, in A(H1N1) isolates collected in Taiwan in 2010 to 2011 (14). None of the isolates harbored the NA drug resistance mutations in the NA gene (15).