ABSTRACT
Here, we report the coding-complete genome sequence of a clinical sample of influenza virus obtained from a pig at a livestock farm in Karaganda, Central Kazakhstan, during a pig study in 2020. Isolate A/Swine/Karaganda/04/2020 (H1N1) belongs to clade 1A.3.2.2 lineage 1A, which includes the 2009 H1N1 pandemic strains.
ANNOUNCEMENT
Influenza A viruses (IAV) belong to the genus Alphainfluenzavirus in the family Orthomyxoviridae and cause one of the most important respiratory diseases in pigs as well as humans (1–3). An important role in the emergence of pandemic strains can be played by genetic reassortment between human and avian influenza viruses in pigs, since swine are equally susceptible to both human and avian influenza viruses (4).
In 2020 at a livestock farm in Karaganda, Kazakhstan, nasopharyngeal swabs were collected from pigs 2 to 5 months of age. All manipulations with animals were approved by the Institutional Animal Care and Use Committee from the Research and Production Center for Microbiology and Virology, Almaty, Kazakhstan. Viral RNA was extracted from a single sample using the PureLink viral RNA/DNA minikit (Invitrogen). The IAV segments were amplified by reverse transcriptase PCR (RT-PCR) using the protocol proposed by Zhou et al. (5). Library preparation was performed using the Nextera DNA Flex library prep kit, and sequencing was performed using the MiSeq reagent kit v2 (Illumina) according to the manufacturer’s protocols. For A/Swine/Karaganda/04/2020 (H1N1), a total of 623,688 reads were generated with an average length of 255 nucleotides. Default parameters were used for all the following analytical software tools. After read quality trimming using UGENE v39.0 (6), the reads were assembled de novo using SPAdes v3.15.2. The resulting contigs were used to conduct a BLASTn search for reference sequences in the GenBank database. The search was restricted to only Influenza A virus sequences (taxid, 11320). Mapping against the 8 segments of the reference strains was performed using BWA software (Table 1).
TABLE 1.
Reference sequences for all gene segments of isolate A/Swine/Karaganda/04/2020 (H1N1)
| Segment | Reference strain | Identity at nucleotide level (%) | GenBank accession no. |
|---|---|---|---|
| 1 | A/Baltimore/R0252/2018 (H1N1) | 99.19 | MH637811.1 |
| 2 | A/Missouri/51/2017 (H1N1) | 99.35 | MH083791.1 |
| 3 | A/Arizona/75/2017 (H1N1) | 99.41 | MH083356.1 |
| 4 | A/USA/SC7097/2018 (H1N1) | 99.10 | MK168543.1 |
| 5 | A/Baltimore/R0264/2018 (H1N1) | 99.42 | MH637700.1 |
| 6 | A/Baltimore/R0252/2018 (H1N1) | 98.29 | MH637715.1 |
| 7 | A/Swine/France/43-180020/2018 (H1N1) | 99.22 | MT379017.1 |
| 8 | A/Baltimore/R0258/2018 (H1N1) | 99.21 | MH637476.1 |
The genome assembly of A/Swine/Karaganda/04/2020 (H1N1) was 13,611 bp long (average coverage, 40×). An influenza virus sequence annotation tool (7) was used to automatically generate annotations for the 8 segments of the strain.
Using the Swine H1 Clade Classification Tool from the Influenza Research Database (IRD) (8), the Kazakh isolate was determined to belong to clade 1A.3.2.2 lineage 1A. This clade has a wide global distribution, which includes the 2009 H1N1 pandemic strains. The phylogenetic tree for the hemagglutinin gene was constructed using the likelihood criterion for optimization, model HKY85, and with 500 replicas for bootstrapping using the IRD Web server. The tree was downloaded in a Newick format file, and MEGA7 (9) was used for visualization, collapsing, and coloring of some of the branches (Fig. 1).
FIG 1.
(A) Total phylogenetic tree with global swine H1 clade classification. (B) Subtree of clade 1A influenza hemagglutinin (HA) genomes. Strain A/Swine/Karaganda/04/2020 (H1N1) is marked with a rhombus.
The obtained genome sequences will be used to study virus circulation and genetic variation for inducing the reversal of drug sensitivity (10). Swine strains can be reintroduced into the human population after a certain period and cause a pandemic, as illustrated by the influenza A(H1N1)pdm09 virus.
Data availability.
The data for the 8 segments of strain A/Swine/Karaganda/04/2020 (H1N1) are available at NCBI under GenBank accession numbers MZ363969.1 to MZ363976.1. The raw sequence reads can be accessed through the Sequence Read Archive (SRA) database under accession number SRR15011445 and BioProject accession number PRJNA742842.
ACKNOWLEDGMENT
The sequencing, genome assembly, annotation, and bioinformatics analysis were funded by grant 325/1 of the program “Development and Application of New Technologies of Genomic and Bioinformatic Analysis, Contributing to the Conservation of Biodiversity, Increasing the Productivity of Animals and Plants, Protecting Organisms from the Damaging Effects of Mutagens and Improving the Quality of Life of the Population,” provided by the Ministry of Education and Science of the Republic of Kazakhstan.
Contributor Information
Tatiana I. Glebova, Email: taty1962@mail.ru.
John J. Dennehy, Queens College CUNY
REFERENCES
- 1.World Health Organization. Influenza (avian and other zoonotic). https://www.who.int/health-topics/influenza-avian-and-other-zoonotic#tab=tab_1.
- 2.Klivleyeva NG, Ongarbayeva NS, Baimukhametova AM, Saktaganov NT, Lukmanova GV, Glebova TI, Sayatov MK, Berezin VE, Nusupbaeva GE, Aikimbayev AM. 2021. Detection of influenza virus and pathogens of acute respiratory viral infections in population of Kazakhstan during 2018–2019 epidemic season. Infektsiia Immun 11:137–147. (In Russian.) doi: 10.15789/2220-7619-DOI-1348. [DOI] [Google Scholar]
- 3.Saktaganov NT, Klivleyeva NG, Ongarbayeva NS, Glebova TI, Lukmanova GV, Baimukhametova AM. 2020. Study on antigenic relationships and biological properties of swine influenza А/H1N1 virus strains isolated in Northern Kazakhstan in 2018. Sel'skokhozyaistvennaya Biologiya 55:355–363. doi: 10.15389/agrobiology.2020.2.355eng. [DOI] [Google Scholar]
- 4.Van Reeth K, Vincent AL. 2019. Influenza viruses, p 576–593. In Zimmerman JJ, Karriker LA, Ramirez A, Schwartz KJ, Stevenson GW, Zhang J (ed), Diseases of swine. John Wiley and Sons, Inc, Ames, IA. [Google Scholar]
- 5.Zhou B, Donnelly ME, Scholes DT, St George K, Hatta M, Kawaoka Y, Wentworth DE. 2009. Single-reaction genomic amplification accelerates sequencing and vaccine production for classical and swine origin human influenza A viruses. J Virol 83:10309–10313. doi: 10.1128/JVI.01109-09. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Okonechnikov K, Golosova O, Fursov M, UGENE team . 2012. Unipro UGENE: a unified bioinformatics toolkit. Bioinformatics 28:1166–1167. doi: 10.1093/bioinformatics/bts091. [DOI] [PubMed] [Google Scholar]
- 7.Bao Y, Bolotov P, Dernovoy D, Kiryutin B, Tatusova T. 2007. FLAN: a Web server for influenza virus genome annotation. Nucleic Acids Res 35:W280–W284. doi: 10.1093/nar/gkm354. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Zhang Y, Aevermann BD, Anderson TK, Burke DF, Dauphin G, Gu Z, He S, Kumar S, Larsen CN, Lee AJ, Li X, Macken C, Mahaffey C, Pickett BE, Reardon B, Smith T, Stewart L, Suloway C, Sun G, Tong L, Vincent AL, Walters B, Zaremba S, Zhao H, Zhou L, Zmasek C, Klem EB, Scheuermann RH. 2017. Influenza Research Database: an integrated bioinformatics resource for influenza virus research. Nucleic Acids Res 45:D466–D474. doi: 10.1093/nar/gkw857. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870–1874. doi: 10.1093/molbev/msw054. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Joubert M, Reva ON, Korotetskiy IS, Shvidko SV, Shilov SV, Jumagaziyeva AB, Kenesheva ST, Suldina NA, Ilin AI. 2019. Assembly of complete genome sequences of negative-control and experimental strain variants of Staphylococcus aureus ATCC BAA-39 selected under the effect of the drug FS-1, which induces antibiotic resistance reversion. Microbiol Resour Announc 8:e00579-19. doi: 10.1128/MRA.00579-19. [DOI] [PMC free article] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The data for the 8 segments of strain A/Swine/Karaganda/04/2020 (H1N1) are available at NCBI under GenBank accession numbers MZ363969.1 to MZ363976.1. The raw sequence reads can be accessed through the Sequence Read Archive (SRA) database under accession number SRR15011445 and BioProject accession number PRJNA742842.