Complete genome sequences were determined for 12 human respiratory syncytial virus strains collected from nasopharyngeal samples obtained from children with repeated subgroup B infections. Eight common amino acid polymorphisms in the G, F, and L proteins were identified between the viruses detected in initial and subsequent infections.
ABSTRACT
Complete genome sequences were determined for 12 human respiratory syncytial virus strains collected from nasopharyngeal samples obtained from children with repeated subgroup B infections. Eight common amino acid polymorphisms in the G, F, and L proteins were identified between the viruses detected in initial and subsequent infections.
ANNOUNCEMENT
Human respiratory syncytial virus (RSV; Human orthopneumovirus; family Pneumoviridae) is the leading cause of acute lower respiratory infections in infants and young children (1). RSV is classified into two subgroups, A and B, according to antigen and sequence differences. Although over 80% of children experience at least one RSV infection before 2 years of age, natural infection does not induce lifelong immunity, thus permitting repeated infections (2, 3). Previous reports on repeated infections with homologous RSV subgroups analyzed only partial gene sequences (4–7). Using Sanger sequencing, we previously identified five amino acid substitutions in the G and F genes that may be associated with repeated infections (8). However, mutations in other genes may also be involved in repeated infections. The present study aimed to determine the complete genome sequence of RSV in children with repeated RSV subgroup B (RSV-B) infections.
This prospective cohort study was conducted on children with acute respiratory infections in the Philippines during 2014 to 2017. Previously, repeated RSV-B infections were detected in four children (8). Further analysis identified two additional children with these infections. In the present study, the complete genome sequences of 12 RSV-B strains from initial and subsequent infections were analyzed. This study was approved by the institutional review board of the RITM and the ethics committee of Tohoku University. Viral RNA was extracted from nasopharyngeal samples using the QIAamp viral RNA minikit (Qiagen, Hilden, Germany), and cDNA was transcribed using SuperScript III reverse transcriptase (Thermo Fisher Scientific, Waltham, MA, USA) and RSV-B-specific primers (9). Complete genome sequences were elucidated from six overlapping PCR products (9). Next-generation sequencing of the pooled PCR products was performed using the Illumina HiSeq platform. The samples were processed as paired-end reads (2 × 101 bp), and approximately 30 million reads/sample were obtained. After adaptor reads were removed, sequence assembly and annotation were performed using the genome sequence of strain USA/TH_10590/2014 (GenBank accession no. KU950637) as a reference and the CLC Genomics Workbench v10.1.1 (CLC, Inc., Aarhus, Denmark).
The length of each genome sequence of RSV-B ranged from 15,226 to 15,254 nucleotides, and the average depth of coverage ranged from 159,000× to 311,000× (Table 1). Differences in lengths occurred only in untranslated regions, and we successfully obtained the complete sequences of all coding genes. A comparison of the complete genome sequences between paired initial and subsequent infections from five children revealed eight common nonsynonymous substitutions in the genes encoding the G protein (positions 107, 136, and 252), F protein (positions 173 and 209), and L protein (positions 715, 1712, and 1719). From one child, viral genomes (isolates TB5_CA-14-0525_6 and TB5_CA-16-0946_6) had only one nonsynonymous substitution at position 252 in the G protein and no substitutions in the other seven positions.
TABLE 1.
RSV-B genome sequence information
| Strain | Yr collected |
Length (nt)a | Avg coverage (×) |
GenBank accession no. |
Sequence Read Archive no. |
|---|---|---|---|---|---|
| TB5_CA-14-0368_1 | 2014 | 15,228 | 202,011 | LC384997 | DRX143645 |
| TB5_CA-15-0741_1 | 2015 | 15,230 | 163,217 | LC384998 | DRX143646 |
| TB5_CA-14-0377_2 | 2014 | 15,228 | 194,923 | LC384999 | DRX143647 |
| TB5_CA-15-0711_2 | 2015 | 15,228 | 200,330 | LC385000 | DRX143648 |
| TB8_KW-14-0284_3 | 2014 | 15,228 | 158,738 | LC385001 | DRX143649 |
| TB9_KW-15-0377_3 | 2015 | 15,229 | 207,722 | LC385002 | DRX143650 |
| TB5_CA-15-0849_5 | 2015 | 15,254 | 159,206 | LC385003 | DRX143651 |
| TB5_CA-17-0073_5 | 2017 | 15,226 | 176,616 | LC385004 | DRX143652 |
| TB5_CA-14-0525_6 | 2014 | 15,228 | 172,652 | LC385005 | DRX143653 |
| TB5_CA-16-0946_6 | 2016 | 15,228 | 226,943 | LC385006 | DRX143654 |
| TB6_CA-15-0412_7 | 2015 | 15,228 | 311,497 | LC385007 | DRX143655 |
| TB5_CA-16-0893_7 | 2016 | 15,229 | 191,005 | LC385008 | DRX143656 |
nt, nucleotides.
To the best of our knowledge, this is the first report on the complete genome sequences of RSV-B strains detected in repeated infections, revealing three additional substitutions in the L protein.
Data availability.
The 12 RSV-B genome sequences have been deposited at GenBank and the Sequence Read Archive (SRA) under the accession numbers listed in Table 1.
ACKNOWLEDGMENTS
We thank the staff of the Tohoku-RITM Collaborating Research Center on Emerging and Re-emerging Infectious Diseases in Biliran and the RITM and the Virology Department of Tohoku University Graduate School of Medicine for sample collection and laboratory work.
This work was supported by the Japan Initiative for Global Research Network on Infectious Diseases from the Japan Agency for Medical and Research and Development (AMED; grant no. JP18fm0108013), the Science and Technology Research Partnership for Sustainable Development from the AMED and Japan International Cooperation Agency (grant no. JP16jm0110001), and the Japan Society for the Promotion of Science Grant-in-Aid for Scientific Research (grant no. JP16H02642).
REFERENCES
- 1.Shi T, McAllister DA, O'Brien KL, Simoes EAF, Madhi SA, Gessner BD, Polack FP, Balsells E, Acacio S, Aguayo C, Alassani I, Ali A, Antonio M, Awasthi S, Awori JO, Azziz-Baumgartner E, Baggett HC, Baillie VL, Balmaseda A, Barahona A, Basnet S, Bassat Q, Basualdo W, Bigogo G, Bont L, Breiman RF, Brooks WA, Broor S, Bruce N, Bruden D, Buchy P, Campbell S, Carosone-Link P, Chadha M, Chipeta J, Chou M, Clara W, Cohen C, de Cuellar E, Dang D-A, Dash-Yandag B, Deloria-Knoll M, Dherani M, Eap T, Ebruke BE, Echavarria M, de Freitas Lázaro Emediato CC, Fasce RA, Feikin DR, Feng L, et al. 2017. Global, regional, and national disease burden estimates of acute lower respiratory infections due to respiratory syncytial virus in young children in 2015: a systematic review and modelling study. Lancet 390:946–958. doi: 10.1016/S0140-6736(17)30938-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Glezen WP, Taber LH, Frank AL, Kasel JA. 1986. Risk of primary infection and reinfection with respiratory syncytial virus. Am J Dis Child 140:543–546. [DOI] [PubMed] [Google Scholar]
- 3.Henderson FW, Collier AM, Clyde WA Jr, Denny FW. 1979. Respiratory-syncytial-virus infections, reinfections and immunity. A prospective, longitudinal study in young children. N Engl J Med 300:530–534. doi: 10.1056/NEJM197903083001004. [DOI] [PubMed] [Google Scholar]
- 4.Parveen S, Broor S, Kapoor SK, Fowler K, Sullender WM. 2006. Genetic diversity among respiratory syncytial viruses that have caused repeated infections in children from rural India. J Med Virol 78:659–665. doi: 10.1002/jmv.20590. [DOI] [PubMed] [Google Scholar]
- 5.Scott PD, Ochola R, Ngama M, Okiro EA, James Nokes D, Medley GF, Cane PA. 2006. Molecular analysis of respiratory syncytial virus reinfections in infants from coastal Kenya. J Infect Dis 193:59–67. doi: 10.1086/498246. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Zlateva KT, Vijgen L, Dekeersmaeker N, Naranjo C, Van Ranst M. 2007. Subgroup prevalence and genotype circulation patterns of human respiratory syncytial virus in Belgium during ten successive epidemic seasons. J Clin Microbiol 45:3022–3030. doi: 10.1128/JCM.00339-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Yamaguchi M, Sano Y, Dapat IC, Saito R, Suzuki Y, Kumaki A, Shobugawa Y, Dapat C, Uchiyama M, Suzuki H. 2011. High frequency of repeated infections due to emerging genotypes of human respiratory syncytial viruses among children during eight successive epidemic seasons in Japan. J Clin Microbiol 49:1034–1040. doi: 10.1128/JCM.02132-10. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Okamoto M, Dapat CP, Sandagon AMD, Batangan-Nacion LP, Lirio IC, Tamaki R, Saito M, Saito-Obata M, Lupisan SP, Oshitani H. 2018. Molecular characterization of respiratory syncytial virus in children with repeated infections with subgroup b in the Philippines. J Infect Dis 218:1045–1053. doi: 10.1093/infdis/jiy256. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Agoti CN, Otieno JR, Munywoki PK, Mwihuri AG, Cane PA, Nokes DJ, Kellam P, Cotten M. 2015. Local evolutionary patterns of human respiratory syncytial virus derived from whole-genome sequencing. J Virol 89:3444–3454. doi: 10.1128/JVI.03391-14. [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 12 RSV-B genome sequences have been deposited at GenBank and the Sequence Read Archive (SRA) under the accession numbers listed in Table 1.