ABSTRACT
We report 19 nearly complete genome sequences of influenza C virus isolated from clinical samples recovered from children in the Philippines between 2014 and 2019.
ANNOUNCEMENT
Influenza C virus (ICV), a member of the Orthomyxoviridae family, consists of an antisense single-stranded RNA genome in seven segments: polymerase basic 2 (PB2), polymerase basic 1 (PB1), polymerase 3 (P3), hemagglutinin-esterase (HE), nucleoprotein (NP), matrix (M), and nonstructural (NS). HE protein determines the antigenic properties of ICV, and currently, there are six different lineages (1). ICV mainly causes mild upper respiratory tract illnesses in children. However, sometimes it also causes lower respiratory tract illnesses, such as bronchitis, bronchiolitis, and pneumonia (2–5). ICV has been recognized as a common respiratory pathogen in humans (6–10). In our previous study, we analyzed ICV strains isolated between 2009 and 2013 in the Philippines. We determined the complete sequences of coding regions (CDS) of the HE, M, and NS gene segments and partial sequences of the four internal gene segments; we observed that the composition of the internal genes of the Philippines strains differed from that of Japanese strains (11). In this study, we aimed to determine the complete genome sequences of all seven gene segments of ICV in children in the Philippines between 2014 and 2019. The study was approved by the Ethics Committee of Tohoku University Graduate School of Medicine, Japan, and the Institutional Review Board of the RITM, Philippines.
We conducted a cohort study on Biliran Island from 2014 to 2019 and collected nasal or nasopharyngeal swabs from children with acute respiratory illness (12). We also collected samples from hospitalized children with severe pneumonia in a hospital-based study on Palawan Island from 2014 to 2016 (13). ICV was isolated and propagated in Madin–Darby canine kidney cell culture using a microplate method (11, 14), and total RNA in the cell culture supernatant was extracted using a QIAamp viral RNA minikit (Qiagen, Hilden, Germany). DNA libraries were generated using the TruSeq stranded mRNA LT sample prep kit and sequenced (100-bp paired-end reads) on a NovaSeq 6000 S4 flow cell on the Illumina NovaSeq 6000 platform at Macrogen Japan Corporation (Tokyo, Japan). CLC Genomics Workbench version 20.0.4 (Qiagen, Hilden, Germany) was used for data analysis. Quality control of the raw reads, including removal of adapter sequence and low-quality reads, was performed using the default settings of CLC Workbench. Consensus sequences were extracted from the mapped reads after simultaneous mapping of the reads to the following reference sequences: GenBank accession number NC_006307 for PB2, NC_006308 for PB1, NC_006309 for P3, NC_006310 for HE, NC_006311 for NP, NC_006312 for M, and NC_006306 for NS.
The average coverage per sample ranged from 5,390 to 152,413 reads (Table 1). The minimum and maximum depths of coverage in the CDS of all samples were 143 and 551,997 reads, respectively. The full length of the CDS was covered at more than 100× depth. Based on a phylogenetic analysis of the HE gene, the samples were divided into two lineages: C/Kanagawa and C/Sao Paulo (Fig. 1).
TABLE 1.
Information on complete genome sequences of influenza C virusa
| Strain | Yr | No. of reads | % GC | Avg coverage (×) | % of coding regions covered at ≥100× | Sequence Read Archive accession no. | GenBank accession no. | Gene length (bp) |
||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| PB2 | PB1 | P3 | HE | NP | M | NS | ||||||||
| C/Palawan/068/2014 | 2014 | 44,582,786 | 52.35 | 62,205 | 94.47 | SRR14537525 | MZ331007–MZ331013 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB5-195/2014 | 2014 | 43,211,150 | 54.41 | 13,916 | 94.39 | SRR14537524 | MZ330944–MZ330950 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB5-241/2014 | 2014 | 53,471,664 | 53.69 | 34,393 | 94.47 | SRR14537514 | MZ330951–MZ330957 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB2-162/2014 | 2014 | 57,572,618 | 55.40 | 9,978 | 94.41 | SRR14537513 | MZ330881–MZ330887 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB2-180/2014 | 2014 | 48,560,152 | 54.76 | 5,390 | 94.40 | SRR14537512 | MZ330888–MZ330894 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB8-250/2014 | 2014 | 49,854,394 | 54.55 | 20,153 | 94.37 | SRR14537511 | MZ330993–MZ330999 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB2-204/2014 | 2014 | 52,851,942 | 53.35 | 18,793 | 94.51 | SRR14537510 | MZ330895–MZ330901 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB2-293/2014 | 2014 | 45,432,860 | 53.49 | 20,277 | 94.39 | SRR14537509 | MZ330902–MZ330908 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Palawan/061/2015 | 2015 | 44,671,646 | 53.57 | 31,770 | 94.46 | SRR14537508 | MZ331000–MZ331006 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB5-498/2016 | 2016 | 61,000,696 | 54.27 | 63,616 | 94.45 | SRR14537507 | MZ330965–MZ330971 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB5-536/2016 | 2016 | 62,519,482 | 52.71 | 81,834 | 94.42 | SRR14537523 | MZ330972–MZ330978 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB5-625/2016 | 2016 | 57,831,760 | 49.51 | 100,708 | 94.52 | SRR14537522 | MZ330979–MZ330985 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB5-736/2016 | 2016 | 48,786,384 | 53.59 | 38,654 | 94.51 | SRR14537521 | MZ330986–MZ330992 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB4-024/2017 | 2017 | 62,691,494 | 52.84 | 88,608 | 94.55 | SRR14537520 | MZ330909–MZ330915 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB5-312/2017 | 2017 | 62,459,368 | 54.84 | 60,857 | 94.47 | SRR14537519 | MZ330958–MZ330964 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB4-067/2017 | 2017 | 62,583,132 | 55.01 | 47,907 | 94.48 | SRR14537518 | MZ330916–MZ330922 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB4-130/2018 | 2018 | 42,157,534 | 51.59 | 79,847 | 94.48 | SRR14537517 | MZ330923–MZ330929 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB4-169/2018 | 2018 | 49,578,338 | 49.16 | 152,413 | 94.58 | SRR14537516 | MZ330930–MZ330936 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
| C/Biliran/TB4-271/2019 | 2019 | 47,001,394 | 56.25 | 10,687 | 94.50 | SRR14537515 | MZ330937–MZ330943 | 2,325 | 2,265 | 2,130 | 1,968 | 1,698 | 1,125 | 862 |
The average coverage and percentage of coding regions covered at ≥100× were calculated for the complete sequence of the coding regions as follows: nucleotides 22 to 2346 for polymerase basic 2 (PB2), nucleotides 18 to 2282 for polymerase basic 1 (PB1), nucleotides 22 to 2151 for polymerase 3 (P3), nucleotides 22 to 1989 for hemagglutinin-esterase (HE), nucleotides 30 to 1727 for nucleoprotein (NP), nucleotides 26 to 1150 for matrix (M), and nucleotides 27 to 888 for the nonstructural (NS) gene.
FIG 1.
Phylogenetic tree of the influenza C virus hemagglutinin-esterase (HE) gene. For this analysis, the length of the coding region of the HE gene was 1,926 nucleotides (positions 64 to 1989). The phylogenetic tree was constructed using the neighbor-joining method in MEGA7 software, using T92 as the substitution model with gamma-distributed site heterogeneity. The bootstrap values were calculated from 1,000 replicates, and values greater than 75% are shown at the branches of the tree. Strains analyzed in this study are marked with circles: green circles for lineage C/Kanagawa and red circles for lineage C/Sao Paulo.
These nearly complete genome sequences reveal the ICV lineages that circulated between 2014 and 2019 in the Philippines.
Data availability.
The nearly complete genome sequences of 19 ICV isolates have been deposited in GenBank (accession numbers MZ330881 to MZ331013). The raw sequencing reads were deposited in the NCBI Sequence Read Archive under BioProject accession number PRJNA719209.
ACKNOWLEDGMENTS
We thank the staff at the Research Institute for Tropical Medicine (RITM), Ospital ng Palawan, Biliran Provincial Hospital, Kawayan and Caibiran Rural Health Units, and the Virology Department of Tohoku University for their support.
This study was financially supported by the Japan Initiative for Global Research Network on Infectious Diseases (J-GRID) from the Japan Agency for Medical Research and Development (AMED) (grant number JP19fm0108013), Science and Technology Research Partnership for Sustainable Development (SATREPS) at AMED, Japan International Cooperation Agency (JICA) (grant number JP16jm0110001), and the Japan Society for the Promotion of Science (JSPS) KAKENHI (grant number JP19KK0204).
Contributor Information
Clyde Dapat, Email: clyde@med.tohoku.ac.jp.
Simon Roux, DOE Joint Genome Institute.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The nearly complete genome sequences of 19 ICV isolates have been deposited in GenBank (accession numbers MZ330881 to MZ331013). The raw sequencing reads were deposited in the NCBI Sequence Read Archive under BioProject accession number PRJNA719209.