Human noroviruses are responsible for most nonbacterial acute gastroenteritis cases. The GII.2, GII.4, and GII.17 genotypes of human noroviruses have recently arisen as the most frequent genotypes found in humans worldwide. We report here seven nearly complete genomes of these genotypes from patients with acute gastroenteritis in Valencia, Spain.
ABSTRACT
Human noroviruses are responsible for most nonbacterial acute gastroenteritis cases. The GII.2, GII.4, and GII.17 genotypes of human noroviruses have recently arisen as the most frequent genotypes found in humans worldwide. We report here seven nearly complete genomes of these genotypes from patients with acute gastroenteritis in Valencia, Spain.
ANNOUNCEMENT
Human noroviruses are responsible for 18% of all cases of acute gastroenteritis worldwide (1) and are the third most common mortality cause in children under 5 years old (2). The Norovirus genus belongs to the Caliciviridae family and is classified into six distinct genogroups (GI to GVI), which are further subdivided into different genotypes (3). Norovirus strains are subjected to continuous processes of punctual mutations and/or recombination. Therefore, new strains emerge periodically (4, 5). In the last few years, a new recombinant strain has emerged (GII.P4.NewOrleans2009_GII.4.Sydney2012). Together with this variant, GII.17 and GII.2 are responsible for the majority of outbreaks worldwide (6, 7).
We report here seven nearly complete genome sequences of human GII noroviruses (GII.2, GII.4, and GII.17) initially detected by conventional reverse transcriptase PCR (RT-PCR) in feces collected from both adults and children. Samples were from sporadic cases and outbreaks of acute gastroenteritis from November 2015 to September 2017. All patients were treated in different hospitals in Valencia, Spain.
Viral RNA was extracted from 250 μl of 10% fecal suspension in phosphate-buffered saline using TRIzol reagent (Invitrogen). Total RNA was sent to Macrogen, Inc., for library construction using a TruSeq mRNA library prep kit (Illumina) and sequencing on a HiSeq platform (Illumina). Raw read quality control was assessed with FastQC version 0.11.5 (8). Reads were quality trimmed using seqtk trimfq version 1.2-r101-dirty (9), and genomes were assembled using SPAdes version 3.11.1 (10) and MEGAHIT version 1.2.4 (11). The obtained contigs were annotated using BLASTn against the full viral genome database from NCBI. Contigs assigned as norovirus were further genotyped using the norovirus genotyping tool (12). All genome sequences contained three open reading frames (ORFs).
Table 1 summarizes the most relevant information of each of the sequences. The isolate 3453 sequence had a 99.22% (7,468/7,527 [number of nucleotides identical to the reference sequence compared to the total number of nucleotides of the obtained sequence]) nucleotide similarity with the isolate NORO_187_01_10_2015 GII.2 strain (MH218655). The isolate 3351 sequence had a 99.63% (7,505/7,533) nucleotide similarity with the Hu/GII.P17_GII.17/KR/2014/CAU-265 strain (KU561253). The isolate 3587 sequence shared 99.5% (7,520/7,558) nucleotide similarity with the Hu/GII.17/142700/Shanghai/2014 strain (KT380915). The sequence of sample 3618 had a 99.11% (7,475/7,542) nucleotide similarity with the Hu/GII.P17_GII.17/KR/2014/CAU-265 strain (KU561253). The isolate 3546 sequence had a 98.55% (7,430/7,539) nucleotide similarity with the GII.P4 New Orleans 2009_GII.4 Sydney 2012 GII isolate NORO_141_02_11_2014 (MH218612). The sequence of sample 3549 shared a 99.55% (7,512/7,546) nucleotide similarity with the GII.Pe_GII.4 Sydney 2012 NORO_42_01_09_2014 strain (MH218701). The isolate 3552 sequence had a 98.45% (7,419/7,536) nucleotide similarity with the GII.P4 New Orleans 2009_GII.4 Sydney 2012 NORO_141_02_11_2014 strain (MH218612).
TABLE 1.
Assembly characteristics for the norovirus contigs obtaineda
| Isolate | GenBank accession no. | Polymerase genotypeb | Capsid genotypeb | ORF1 ntc | ORF2 ntc | ORF3 ntc | No. of norovirus reads/raw reads (%) | Coverage (×) | Genome coverage (%) (genome length [bp])d | GC content (%) | GenBank accession no. of most similar strain (nt lacking in the 5′ end) |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 3453 | MK789430 | GII.P2 | GII.2 | 1–5094 | 5075–6703 | 6703–7482 | 4,529,781/31,181,056 (14.5) | 2,675.12 | 99.9 (7,536/7,546) | 49.8 | MH218655 (GTGAATGAAG) |
| 3351 | MK789431 | GII.P17 | GII.17 | 1–5097 | 5078–6700 | 6700–7499 | 1,360,087/94,666,148 (1.4) | 2,587.67 | 99.9 (7,533/7,549) | 48.2 | KU561253 (GTGAATGAAGATGGCG) |
| 3587 | MK789432 | GII.P17 | GII.17 | 5–5113 | 5094–6716 | 6716–7495 | 67,237,010/70,923,098 (94.8) | 7,674.57 | 100 (7,558/7,558) | 48.1 | KT380915 |
| 3618 | MK789433 | GII.P17 | GII.17 | 1–5099 | 5080–6702 | 6702–7481 | 15,904,593/46,226,582 (34.4) | 6,316.85 | 99.8 (7,542/7,556) | 48.2 | KU561253 (GTGAATGAAGATGG) |
| 3546 | MK789434 | GII.P4 New Orleans 2009 | GII.4 Sydney 2012 | 1–5093 | 5074–6696 | 6696–7502 | 39,211,847/90,843,600 (46.1) | 7,716.42 | 99.9 (7,540/7,551) | 49.9 | MH218612 (GTGAATGAAGA) |
| 3549 | MK789435 | GII.Pe | GII.4 Sydney 2012 | 5–5104 | 5085–6707 | 6707–7513 | 90,198,955/90,548,480 (99.6) | 7,792.28 | 100 (7,569/7,569) | 49.6 | MH218701 |
| 3552 | MK789436 | GII.P4 New Orleans 2009 | GII.4 Sydney 2012 | 1–5090 | 5071–6693 | 6693–7499 | 68,287,930/88,189,676 (77) | 7,755.68 | 99.8 (7,536/7,550) | 49.8 | MH218612 (GTGAATGAAGATGG) |
nt, nucleotides.
Polymerase and capside genotypes were assigned using the norovirus genotyping tool (12).
The ORFs were automatically assigned by GenBank after sequence submission.
The total expected genome length is determined relative to the sequence annotated as “full genome” with the higher similarity after BLAST search.
In summary, our study describes seven nearly complete genome sequences of different genotypes of norovirus isolates, providing additional important information to use as reference for phylogenetic and evolutionary studies.
Data availability.
The GenBank accession numbers for the norovirus genomes are MK789430, MK789431, MK789432, MK789433, MK789434, MK789435, and MK789436. The sequence data are available in the Sequence Read Archive (SRA) under BioProject number PRJNA497363.
ACKNOWLEDGMENTS
This work was supported by Spanish government (Ministerio de Economia y Competitividad) grants AGL2014-52996-C2-2-R and RYC-2013-12442 to J.R.-D. R.G.-R. is the recipient of a postdoctoral grant from the Valencian government (APOST/2017/037), and S.V.-V. is the recipient of a predoctoral fellowship from the Valencian government (ACIF/2016/437). A.F.-T. is the beneficiary of a predoctoral fellowship from the Valencian government (ACIF/2018/303). J.D.S.R.D.A. received financial support from the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior-Brasil (CAPES), finance code 001.
REFERENCES
- 1.Ahmed SM, Hall AJ, Robinson AE, Verhoef L, Premkumar P, Parashar UD, Koopmans M, Lopman BA. 2014. Global prevalence of norovirus in cases of gastroenteritis: a systematic review and meta-analysis. Lancet Infect Dis 14:725–730. doi: 10.1016/S1473-3099(14)70767-4. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Lanata CF, Fischer-Walker CL, Olascoaga AC, Torres CX, Aryee MJ, Black RE. 2013. Global causes of diarrheal disease mortality in children <5 years of age: a systematic review. PLoS One 8:e72788. doi: 10.1371/journal.pone.0072788. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3.Kroneman A, Vega E, Vennema H, Vinjé J, White PA, Hansman G, Green K, Martella V, Katayama K, Koopmans M. 2013. Proposal for a unified norovirus nomenclature and genotyping. Arch Virol 158:2059–2068. doi: 10.1007/s00705-013-1708-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Bull RA, Eden JS, Rawlinson WD, White PA. 2010. Rapid evolution of pandemic noroviruses of the GII.4 lineage. PLoS Pathog 6:1–10. doi: 10.1371/journal.ppat.1000831. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Siebenga JJ, Vennema H, Renckens B, de Bruin E, van der Veer B, Siezen RJ, Koopmans M. 2007. Epochal evolution of GGII.4 norovirus capsid proteins from 1995 to 2006. J Virol 81:9932–9941. doi: 10.1128/JVI.00674-07. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Chan MCW, Hu Y, Chen H, Podkolzin AT, Zaytseva EV, Komano J, Sakon N, Poovorawan Y, Vongpunsawad S, Thanusuwannasak T, Hewitt J, Croucher D, Collins N, Vinjé J, Pang XL, Lee BE, De Graaf M, Van Beek J, Vennema H, Koopmans MPG, Niendorf S, Poljsak-Prijatelj M, Steyer A, White PA, Lun JH, Mans J, Hung TN, Kwok K, Cheung K, Lee N, Chan PKS. 2017. Global spread of norovirus gII.17 Kawasaki 308, 2014–2016. Emerg Infect Dis 23:1354–1359. doi: 10.3201/eid2308.161138. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 7.Ao Y, Cong X, Jin M, Sun X, Wei X, Wang J, Zhang Q, Song J, Yu J, Cui J, Qi J, Tan M, Duan Z. 2018. Genetic analysis of re-emerging GII.P16–GII.2 noroviruses in 2016–2017 in China. J Infect Dis 218:133–143. doi: 10.1093/infdis/jiy182. [DOI] [PubMed] [Google Scholar]
- 8.Schmieder R, Edwards R. 2011. Quality control and preprocessing of metagenomic datasets. Bioinformatics 27:863–864. doi: 10.1093/bioinformatics/btr026. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9.Li H. 2015. Seqtk. https://github.com/lh3/seqtk.
- 10.Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, Pyshkin AV, Sirotkin AV, Vyahhi N, Tesler G, Alekseyev MA, Pevzner PA. 2012. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 19:455–477. doi: 10.1089/cmb.2012.0021. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Li D, Liu CM, Luo R, Sadakane K, Lam TW. 2015. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics 31:1674–1676. doi: 10.1093/bioinformatics/btv033. [DOI] [PubMed] [Google Scholar]
- 12.Kroneman A, Vennema H, Deforche K, Avoort HVD, Peñaranda S, Oberste MS, Vinjé J, Koopmans M. 2011. An automated genotyping tool for enteroviruses and noroviruses. J Clin Virol 51:121–125. doi: 10.1016/j.jcv.2011.03.006. [DOI] [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 GenBank accession numbers for the norovirus genomes are MK789430, MK789431, MK789432, MK789433, MK789434, MK789435, and MK789436. The sequence data are available in the Sequence Read Archive (SRA) under BioProject number PRJNA497363.