Coding-complete genomes of two human coronavirus OC43 strains and one NL63 strain were obtained by metagenomic sequencing of clinical samples collected in 2017 and 2018 in Kilifi, Kenya. Maximum likelihood phylogenies showed that the OC43 strains were genetically dissimilar and that the NL63 strain was closely related to NL63 genotype B viruses. [Abstract copyright: Copyright © 2019 Kamau et al.

Adema, Irene

Agoti, Charles N.

de Laurent, Zaydah R.

Kamau, Everlyn

Luka, Martha M.

Nokes, D. James

Coding-complete genomes of two human coronavirus OC43 strains and one NL63 strain were obtained by metagenomic sequencing of clinical samples collected in 2017 and 2018 in Kilifi, Kenya. Maximum likelihood phylogenies showed that the OC43 strains were genetically dissimilar and that the NL63 strain was closely related to NL63 genotype B viruses

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Genome Sequences of Human Coronavirus OC43 and NL63,Associated with Respiratory Infections in Kilifi, KenyaEverlyn Kamau,a Martha M. Luka,a,b Zaydah R. de Laurent,a Irene Adema,a Charles N. Agoti,a D. James Nokesa,caKEMRI-Wellcome Trust Research Programme, Kilifi, KenyabDepartment of Public Health, Pwani University, Kilifi, KenyacSchool of Life Sciences and Zeeman Institute SBIDER, University of Warwick, Coventry, United KingdomABSTRACT Coding-complete genomes of two human coronavirus OC43 strains andone NL63 strain were obtained by metagenomic sequencing of clinical samples col-lected in 2017 and 2018 in Kilifi, Kenya. Maximum likelihood phylogenies showedthat the OC43 strains were genetically dissimilar and that the NL63 strain wasclosely related to NL63 genotype B viruses.Human coronaviruses (HCoVs) are enveloped viruses within the family Coronaviridaewith positive-sense, single-stranded genomes of up to 32 kb, with a 5= cap structureand 3= polyadenylation tract (1). The 5= two-thirds of the genome encodes the largereplicases 1a and 1b encoding nonstructural proteins, whereas the 3= one-third en-codes the structural proteins (1). Coronaviruses are divided into four genera, namely,Alphacoronavirus (229E and NL63), Betacoronavirus (severe acute respiratory syndrome[SARS], Middle East respiratory syndrome [MERS], OC43, and HKU1), Gammacoronavirus,and Deltacoronavirus (2). The NL63, 229E, HKU1, and OC43 species are endemic inhuman populations, causing relatively mild and severe respiratory disease (2). Up toeight distinct HCoV-OC43 genotypes (3) and three HCoV-NL63 genotypes (4) have beenidentified. However, there is a paucity of HCoV genomes that have been collectedglobally, with a negligible number from Africa.Here, we report coding-complete genomes of two HCoV-OC43 strains and one NL63strain. The isolates NL63_KLF_01_2018 and OC43_KLF_01_2018 were obtained fromacute respiratory infection cases at a local school (from a 7-year-old boy with cough,nasal discharge, and abdominal pain and from a 13-year-old female student withcough, nasal discharge, and fever, respectively). OC43_KLF_02_2017 was isolated from a2-month-old boy presenting at the Kilifi County Hospital with fever and chest in-drawing. Written informed consent for study participation was obtained from parentsor guardians of the patients, and ethical approval was obtained from the KEMRIScientific and Ethics Review Unit.Viral RNA was extracted from nasopharyngeal swabs using TRIzol LS reagent (Invit-rogen) followed by TURBO DNase treatment (Invitrogen), according to the manufac-turer’s instructions. cDNA was synthesized with SuperScript III reverse transcriptase(Invitrogen) with random hexamers, and double-stranded cDNA was synthesized withKlenow polymerase (5 U; New England BioLabs). Libraries were prepared using theNextera XT kit (Illumina) according to the manufacturer’s instructions and sequencedusing the MiSeq reagent kit v2 (500 cycle) (Illumina) on an Illumina MiSeq sequencer.Sequencing reads (paired, 2  250 bp) were filtered, kmer normalized, and de novoassembled as previously described (5) by using the settings specified in Table 1.Reference OC43 and NL63 genomes (GenBank accession numbers AY391777 andNC_005831, respectively) were used to transfer annotations to assembled contigs usingGeneious (R9). MAFFT v.7.221 (6) was used for sequence alignment using the param-Citation Kamau E, Luka MM, de Laurent ZR,Adema I, Agoti CN, Nokes DJ. 2019. Genomesequences of human coronavirus OC43 andNL63, associated with respiratory infections inKilifi, Kenya. Microbiol Resour Announc8:e00730-19. https://doi.org/10.1128/MRA.00730-19.Editor Jelle Matthijnssens, KU LeuvenCopyright © 2019 Kamau et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Everlyn Kamau,ekamau@kemri-wellcome.org.Received 8 July 2019Accepted 22 October 2019Published 14 November 2019GENOME SEQUENCEScrossmVolume 8 Issue 46 e00730-19 mra.asm.org 1Downloaded from https://journals.asm.org/journal/mra on 07 June 2024 by 2001:630:40:70e0::9a89.eters “–localpair –maxiterate 1000.” Maximum likelihood phylogenies were estimated inRAxML v.8 (7) using the general time-reversible (GTR) nucleotide substitution modeland gamma distribution of among site rate variation.Characteristics of the three coronaviruses are listed in Table 1, and their genomeorganization was typical of NL63 and OC43 (8). Other viral contigs belonging toFIG 1 Maximum likelihood phylogenetic tree of 175 OC43 (A) and 56 NL63 (B) genome sequences. Reference and contemporarysequences were retrieved from GenBank. Sequences from Kilifi, Kenya, are colored red. Only nonoverlapping bootstrap values of 70 areshown in the phylogeny. Scale bars indicate expected nucleotide substitutions per site.TABLE 1 Characteristics of the HCoV OC43 and NL63 genomes and samples from Kilifi, KenyaaIsolateGenomesize (nt)GCcontent (%)No. ofreadsb N50 (bp)cAvgdepth ()dNo. ofcontigsrRT-PCReCT valueGenBankaccession no.NL63_KLF_01_2018 27,125 34.5 754,456 19,834 348.596 127 20.88 MN026166OC43_KLF_01_2018 30,777 36.6 622,954 24,566 502.009 240 20.18 MN026164OC43_KLF_02_2017 30,318 36.6 1,072,826 27,724 983.988 2,219 21.07 MN026165a QUASR parameters, -d – q –l 125 –m 30; SPAdes parameters, — careful -k 77, 99, 127; QUAST parameters, minimum contig length, 500, ambiguity, one, and thresholdfor extensive misassembly size, 1,000, bowtie2 parameters, -q –S –local. nt, nucleotide; rRT-PCR, real-time reverse transcription-PCR; CT, threshold cycle.b Short read length of the raw sequence data ranged from 35 to 250 bases.c N50 length is calculated by summing the lengths of contig assembly of a particular sample from the longest to the shortest and determining the point at which 50%of the assembly size is reached.d Calculated by dividing the per-position coverage output (described in the text) by respective genome length.e The rRT-PCR assay, including primers and probe sequences used for HCoV detection, has been described previously (9).Kamau et al.Volume 8 Issue 46 e00730-19 mra.asm.org 2Downloaded from https://journals.asm.org/journal/mra on 07 June 2024 by 2001:630:40:70e0::9a89.respiratory syncytial virus (RSV), adenovirus, influenza, and rhinovirus were identifiedusing the BLASTn program, albeit in low frequencies (0.1% of total contigs persample). The two OC43 genomes had 98.4% (28,654 sites) pairwise identity but clustered indistinct branches of the genome-based phylogeny (Fig. 1A), while the NL63 sequencegrouped with global NL63 genotype B sequences (Fig. 1B). Extensive variability, includ-ing insertions, deletions, and nonsynonymous substitutions, was observed in the Sgene between Kilifi and other global strains for both NL63 and OC43 viruses. Further-more, the number of codons and the pattern of putative N-glycosylation (N-X-S/T)varied in the spike protein between the two Kilifi OC43 strains. These new complete-coding genomes increase the available data from Africa and will be useful for futuremolecular epidemiology studies.Data availability. The raw sequence data were deposited in the Sequence ReadArchive (SRA) under BioProject accession number PRJNA547576 and BioSample accessionnumbers SAMN11969662, SAMN11969663, and SAMN11969664. The genome se-quences generated here were deposited in GenBank under accession numbersMN026164, MN026165, and MN026166.ACKNOWLEDGMENTSWe acknowledge VEC laboratory staff at the KEMRI-Wellcome Trust Research pro-gram and study personnel involved in recruitment and evaluation.This work was funded by the Wellcome Trust grant number 102975.The manuscript was submitted for publication with permission from the director ofthe Kenya Medical Research Institute.REFERENCES1. Wevers BA, van der Hoek L. 2009. Recently discovered human coronavi-ruses. Clin Lab Med 29:715–724. https://doi.org/10.1016/j.cll.2009.07.007.2. Su S, Wong G, Shi W, Liu J, Lai ACK, Zhou J, Liu W, Bi Y, Gao GF. 2016.Epidemiology, genetic recombination, and pathogenesis of coronavi-ruses. Trends Microbiol 24:490 –502. https://doi.org/10.1016/j.tim.2016.03.003.3. Zhu Y, Li C, Chen L, Xu B, Zhou Y, Cao L, Shang Y, Fu Z, Chen A, Deng L,Bao Y, Sun Y, Ning L, Liu C, Yin J, Xie Z, Shen K. 2018. A novel humancoronavirus OC43 genotype detected in mainland China. Emerg MicrobesInfect 7:173. https://doi.org/10.1038/s41426-018-0171-5.4. Dominguez SR, Sims GE, Wentworth DE, Halpin RA, Robinson CC, TownCD, Holmes KV. 2012. Genomic analysis of 16 Colorado human NL63coronaviruses identifies a new genotype, high sequence diversity in theN-terminal domain of the spike gene and evidence of recombination. JGen Virol 93:2387–2398. https://doi.org/10.1099/vir.0.044628-0.5. Kamau E, Agoti CN, Ngoi JM, de Laurent ZR, Gitonga J, Cotten M, Phan MVT,Nokes DJ, Delwart E, Sanders E, Warimwe GM. 2019. Complete genomesequences of dengue virus type 2 strains from Kilifi, Kenya. Microbiol ResourAnnounc 8:e01566-18. https://doi.org/10.1128/MRA.01566-18.6. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment soft-ware version 7: improvements in performance and usability. Mol Biol Evol30:772–780. https://doi.org/10.1093/molbev/mst010.7. Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis andpost-analysis of large phylogenies. Bioinformatics 30:1312–1313. https://doi.org/10.1093/bioinformatics/btu033.8. Weiss SR, Navas-Martin S. 2005. Coronavirus pathogenesis and the emergingpathogen severe acute respiratory syndrome coronavirus. Microbiol Mol BiolRev 69:635–664. https://doi.org/10.1128/MMBR.69.4.635-664.2005.9. Hammitt LL, Kazungu S, Welch S, Bett A, Onyango CO, Gunson RN, ScottJA, Nokes DJ. 2011. Added value of an oropharyngeal swab in detectionof viruses in children hospitalized with lower respiratory tract infection. JClin Microbiol 49:2318 –2320. https://doi.org/10.1128/JCM.02605-10.Microbiology Resource AnnouncementVolume 8 Issue 46 e00730-19 mra.asm.org 3Downloaded from https://journals.asm.org/journal/mra on 07 June 2024 by 2001:630:40:70e0::9a89.

Genome sequences of human coronavirus OC43 and NL63, associated with respiratory infections in Kilifi, Kenya

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Genome Sequences of Human Coronavirus OC43 and NL63,Associated with Respiratory Infections in Kiliﬁ, KenyaEverlyn Kamau,a Martha M. Luka,a,b Zaydah R. de Laurent,a Irene Adema,a Charles N. Agoti,a D. James Nokesa,caKEMRI-Wellcome Trust Research Programme, Kiliﬁ, KenyabDepartment of Public Health, Pwani University, Kiliﬁ, KenyacSchool of Life Sciences and Zeeman Institute SBIDER, University of Warwick, Coventry, United KingdomABSTRACT Coding-complete genomes of two human coronavirus OC43 strains andone NL63 strain were obtained by metagenomic sequencing of clinical samples col-lected in 2017 and 2018 in Kiliﬁ, Kenya. Maximum likelihood phylogenies showedthat the OC43 strains were genetically dissimilar and that the NL63 strain wasclosely related to NL63 genotype B viruses.Human coronaviruses (HCoVs) are enveloped viruses within the family Coronaviridaewith positive-sense, single-stranded genomes of up to 32 kb, with a 5= cap structureand 3= polyadenylation tract (1). The 5= two-thirds of the genome encodes the largereplicases 1a and 1b encoding nonstructural proteins, whereas the 3= one-third en-codes the structural proteins (1). Coronaviruses are divided into four genera, namely,Alphacoronavirus (229E and NL63), Betacoronavirus (severe acute respiratory syndrome[SARS], Middle East respiratory syndrome [MERS], OC43, and HKU1), Gammacoronavirus,and Deltacoronavirus (2). The NL63, 229E, HKU1, and OC43 species are endemic inhuman populations, causing relatively mild and severe respiratory disease (2). Up toeight distinct HCoV-OC43 genotypes (3) and three HCoV-NL63 genotypes (4) have beenidentiﬁed. However, there is a paucity of HCoV genomes that have been collectedglobally, with a negligible number from Africa.Here, we report coding-complete genomes of two HCoV-OC43 strains and one NL63strain. The isolates NL63_KLF_01_2018 and OC43_KLF_01_2018 were obtained fromacute respiratory infection cases at a local school (from a 7-year-old boy with cough,nasal discharge, and abdominal pain and from a 13-year-old female student withcough, nasal discharge, and fever, respectively). OC43_KLF_02_2017 was isolated from a2-month-old boy presenting at the Kiliﬁ County Hospital with fever and chest in-drawing. Written informed consent for study participation was obtained from parentsor guardians of the patients, and ethical approval was obtained from the KEMRIScientiﬁc and Ethics Review Unit.Viral RNA was extracted from nasopharyngeal swabs using TRIzol LS reagent (Invit-rogen) followed by TURBO DNase treatment (Invitrogen), according to the manufac-turer’s instructions. cDNA was synthesized with SuperScript III reverse transcriptase(Invitrogen) with random hexamers, and double-stranded cDNA was synthesized withKlenow polymerase (5 U; New England BioLabs). Libraries were prepared using theNextera XT kit (Illumina) according to the manufacturer’s instructions and sequencedusing the MiSeq reagent kit v2 (500 cycle) (Illumina) on an Illumina MiSeq sequencer.Sequencing reads (paired, 2  250 bp) were ﬁltered, kmer normalized, and de novoassembled as previously described (5) by using the settings speciﬁed in Table 1.Reference OC43 and NL63 genomes (GenBank accession numbers AY391777 andNC_005831, respectively) were used to transfer annotations to assembled contigs usingGeneious (R9). MAFFT v.7.221 (6) was used for sequence alignment using the param-Citation Kamau E, Luka MM, de Laurent ZR,Adema I, Agoti CN, Nokes DJ. 2019. Genomesequences of human coronavirus OC43 andNL63, associated with respiratory infections inKiliﬁ, Kenya. Microbiol Resour Announc8:e00730-19. https://doi.org/10.1128/MRA.00730-19.Editor Jelle Matthijnssens, KU LeuvenCopyright © 2019 Kamau et al. This is anopen-access article distributed under the termsof the Creative Commons Attribution 4.0International license.Address correspondence to Everlyn Kamau,ekamau@kemri-wellcome.org.Received 8 July 2019Accepted 22 October 2019Published 14 November 2019GENOME SEQUENCEScrossmVolume 8 Issue 46 e00730-19 mra.asm.org 1 on December 16, 2019 by guesthttp://mra.asm.org/Downloaded from eters “–localpair –maxiterate 1000.” Maximum likelihood phylogenies were estimated inRAxML v.8 (7) using the general time-reversible (GTR) nucleotide substitution modeland gamma distribution of among site rate variation.Characteristics of the three coronaviruses are listed in Table 1, and their genomeorganization was typical of NL63 and OC43 (8). Other viral contigs belonging toFIG 1 Maximum likelihood phylogenetic tree of 175 OC43 (A) and 56 NL63 (B) genome sequences. Reference and contemporarysequences were retrieved from GenBank. Sequences from Kiliﬁ, Kenya, are colored red. Only nonoverlapping bootstrap values of 70 areshown in the phylogeny. Scale bars indicate expected nucleotide substitutions per site.TABLE 1 Characteristics of the HCoV OC43 and NL63 genomes and samples from Kiliﬁ, KenyaaIsolateGenomesize (nt)GCcontent (%)No. ofreadsb N50 (bp)cAvgdepth ()dNo. ofcontigsrRT-PCReCT valueGenBankaccession no.NL63_KLF_01_2018 27,125 34.5 754,456 19,834 348.596 127 20.88 MN026166OC43_KLF_01_2018 30,777 36.6 622,954 24,566 502.009 240 20.18 MN026164OC43_KLF_02_2017 30,318 36.6 1,072,826 27,724 983.988 2,219 21.07 MN026165a QUASR parameters, -d –q –l 125 –m 30; SPAdes parameters, —careful -k 77, 99, 127; QUAST parameters, minimum contig length, 500, ambiguity, one, and thresholdfor extensive misassembly size, 1,000, bowtie2 parameters, -q –S –local. nt, nucleotide; rRT-PCR, real-time reverse transcription-PCR; CT, threshold cycle.b Short read length of the raw sequence data ranged from 35 to 250 bases.c N50 length is calculated by summing the lengths of contig assembly of a particular sample from the longest to the shortest and determining the point at which 50%of the assembly size is reached.d Calculated by dividing the per-position coverage output (described in the text) by respective genome length.e The rRT-PCR assay, including primers and probe sequences used for HCoV detection, has been described previously (9).Kamau et al.Volume 8 Issue 46 e00730-19 mra.asm.org 2 on December 16, 2019 by guesthttp://mra.asm.org/Downloaded from respiratory syncytial virus (RSV), adenovirus, inﬂuenza, and rhinovirus were identiﬁedusing the BLASTn program, albeit in low frequencies (0.1% of total contigs persample). The two OC43 genomes had 98.4% (28,654 sites) pairwise identity but clustered indistinct branches of the genome-based phylogeny (Fig. 1A), while the NL63 sequencegrouped with global NL63 genotype B sequences (Fig. 1B). Extensive variability, includ-ing insertions, deletions, and nonsynonymous substitutions, was observed in the Sgene between Kiliﬁ and other global strains for both NL63 and OC43 viruses. Further-more, the number of codons and the pattern of putative N-glycosylation (N-X-S/T)varied in the spike protein between the two Kiliﬁ OC43 strains. These new complete-coding genomes increase the available data from Africa and will be useful for futuremolecular epidemiology studies.Data availability. The raw sequence data were deposited in the Sequence ReadArchive (SRA) under BioProject accession number PRJNA547576 and BioSample accessionnumbers SAMN11969662, SAMN11969663, and SAMN11969664. The genome se-quences generated here were deposited in GenBank under accession numbersMN026164, MN026165, and MN026166.ACKNOWLEDGMENTSWe acknowledge VEC laboratory staff at the KEMRI-Wellcome Trust Research pro-gram and study personnel involved in recruitment and evaluation.This work was funded by the Wellcome Trust grant number 102975.The manuscript was submitted for publication with permission from the director ofthe Kenya Medical Research Institute.REFERENCES1. Wevers BA, van der Hoek L. 2009. Recently discovered human coronavi-ruses. Clin Lab Med 29:715–724. https://doi.org/10.1016/j.cll.2009.07.007.2. Su S, Wong G, Shi W, Liu J, Lai ACK, Zhou J, Liu W, Bi Y, Gao GF. 2016.Epidemiology, genetic recombination, and pathogenesis of coronavi-ruses. Trends Microbiol 24:490–502. https://doi.org/10.1016/j.tim.2016.03.003.3. Zhu Y, Li C, Chen L, Xu B, Zhou Y, Cao L, Shang Y, Fu Z, Chen A, Deng L,Bao Y, Sun Y, Ning L, Liu C, Yin J, Xie Z, Shen K. 2018. A novel humancoronavirus OC43 genotype detected in mainland China. Emerg MicrobesInfect 7:173. https://doi.org/10.1038/s41426-018-0171-5.4. Dominguez SR, Sims GE, Wentworth DE, Halpin RA, Robinson CC, TownCD, Holmes KV. 2012. Genomic analysis of 16 Colorado human NL63coronaviruses identiﬁes a new genotype, high sequence diversity in theN-terminal domain of the spike gene and evidence of recombination. JGen Virol 93:2387–2398. https://doi.org/10.1099/vir.0.044628-0.5. Kamau E, Agoti CN, Ngoi JM, de Laurent ZR, Gitonga J, Cotten M, Phan MVT,Nokes DJ, Delwart E, Sanders E, Warimwe GM. 2019. Complete genomesequences of dengue virus type 2 strains from Kiliﬁ, Kenya. Microbiol ResourAnnounc 8:e01566-18. https://doi.org/10.1128/MRA.01566-18.6. Katoh K, Standley DM. 2013. MAFFT multiple sequence alignment soft-ware version 7: improvements in performance and usability. Mol Biol Evol30:772–780. https://doi.org/10.1093/molbev/mst010.7. Stamatakis A. 2014. RAxML version 8: a tool for phylogenetic analysis andpost-analysis of large phylogenies. Bioinformatics 30:1312–1313. https://doi.org/10.1093/bioinformatics/btu033.8. Weiss SR, Navas-Martin S. 2005. Coronavirus pathogenesis and the emergingpathogen severe acute respiratory syndrome coronavirus. Microbiol Mol BiolRev 69:635–664. https://doi.org/10.1128/MMBR.69.4.635-664.2005.9. Hammitt LL, Kazungu S, Welch S, Bett A, Onyango CO, Gunson RN, ScottJA, Nokes DJ. 2011. Added value of an oropharyngeal swab in detectionof viruses in children hospitalized with lower respiratory tract infection. JClin Microbiol 49:2318–2320. https://doi.org/10.1128/JCM.02605-10.Microbiology Resource AnnouncementVolume 8 Issue 46 e00730-19 mra.asm.org 3 on December 16, 2019 by guesthttp://mra.asm.org/Downloaded from 

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Genome sequences of human coronavirus OC43 and NL63, associated with respiratory infections in Kilifi, Kenya

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