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article-title
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Complete Genome Sequences of Five Human Coronavirus NL63 Strains Causing Respiratory Illness in Hospitalized Children in China
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alt-title
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Microbiology Resource Announcement
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alt-title
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Zhang et al.
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abstract
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We report the complete genome sequences of five human coronavirus NL63 (HCoV-NL63) strains obtained using next-generation sequencing. The five HCoV-NL63 strains were obtained from hospitalized children with severe acute respiratory infection detected in Guangdong, China. This study provides several complete genomes of HCoV-NL63 and improves our understanding of HCoV-NL63 evolution in China.
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p
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We report the complete genome sequences of five human coronavirus NL63 (HCoV-NL63) strains obtained using next-generation sequencing. The five HCoV-NL63 strains were obtained from hospitalized children with severe acute respiratory infection detected in Guangdong, China. This study provides several complete genomes of HCoV-NL63 and improves our understanding of HCoV-NL63 evolution in China.
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abstract
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ABSTRACT
We report the complete genome sequences of five human coronavirus NL63 (HCoV-NL63) strains obtained using next-generation sequencing. The five HCoV-NL63 strains were obtained from hospitalized children with severe acute respiratory infection detected in Guangdong, China. This study provides several complete genomes of HCoV-NL63 and improves our understanding of HCoV-NL63 evolution in China.
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title
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ABSTRACT
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p
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We report the complete genome sequences of five human coronavirus NL63 (HCoV-NL63) strains obtained using next-generation sequencing. The five HCoV-NL63 strains were obtained from hospitalized children with severe acute respiratory infection detected in Guangdong, China. This study provides several complete genomes of HCoV-NL63 and improves our understanding of HCoV-NL63 evolution in China.
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body
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ANNOUNCEMENT
Human coronavirus NL63 (HCoV-NL63) is a member of the family Coronaviridae, genus Alphacoronavirus, and was first discovered in 2004 (1). HCoV-NL63 is mainly associated with the common cold in children, the elderly, and immunocompromised patients (2, 3). The genome of HCoV-NL63 is about 27 kb with a conserved gene order of 5′-orf1ab-spike (S)-orf3-envelope (E)-membrane (M)-nucleocapsid (N)-poly (A). The species tropism of HCoV-NL63 is determined by spike glycoprotein. HCoV-NL63 and severe acute respiratory syndrome coronavirus (SARS-CoV) share the same cell receptor, angiotensin converting enzyme 2 (ACE-2) (4, 5), for entry into host cells, and HCoV-NL63 is recognized as a common cause of upper respiratory tract infection and has been prevalent worldwide.
Here, nasopharyngeal swab samples were collected from hospitalized children with severe acute respiratory infection in Guangzhou, China, in 2018. This study was performed in strict accordance with human subject protection guidance provided by the Research Ethics Committee of Guangzhou Medical University. The respiratory samples were filtered with 0.22-μm filters, RNA extraction was performed using a Qiagen viral RNA extraction kit, and extracted RNA was used for sequence-independent single-primer amplification (SISPA) (6, 7) as follows: a reverse transcription reaction was performed with SuperScript III reverse transcriptase using a primer containing a fixed sequence, followed by a random hexamer at the 3′ end (FR26RV, GCCGGAGCTCTGCAGATATCNNNNNN). Then, Klenow fragment polymerase (New England Biolabs) was used for DNA synthesis. Finally, PCR amplification was conducted using primers consisting of the fixed portions of the random primers (FR26, GCCGGAGCTCTGCAGATATC). Purified DNA was used for next-generation sequencing (NGS). Libraries were prepared with the Nextera XT kit (Illumina), and paired-end reads (2 × 125 bp) determined using a HiSeq 2500 instrument were used for cleaning and assembling using CLC Genomics Workbench version 11.0. Illumina sequencing yielded about 10 million reads per sample. Reads were assembled into contigs with a de novo assembly model, and the contig sequences were then extracted for subsequent analysis. Partial genome sequences of five HCoV-NL63 strains were obtained by NGS methods. Meanwhile, sets of specific primer pairs were designed and used to amplify the gap region of HCoV-NL63, which was used for genome assemblies using the SeqMan subprogram of the DNAStar software version 7.1.0 with default parameters (Table 1). Finally, five complete genome sequences of HCoV-NL63 were obtained using next-generation sequencing and Sanger sequencing methods together and were designated strains ChinaGD01 (27,531 bp), ChinaGD02 (27,516 bp), ChinaGD03 (27,516 bp), ChinaGD04 (27,532 bp), and ChinaGD05 (27,544 bp). The five HCoV-NL63 strains presented here were aligned using MAFFT version 7.158 (8) and showed 98.5 to ∼99.1% nucleotide homology with the prototype HCoV-NL63 virus (GenBank accession number NC_005831.2) as estimated using MEGA version 5.10 software (9) (Fig. 1).
TABLE 1 Primers used for the genome sequencing of HCoV-NL63
Primer Sequence (5′–3′) Target (nucleotide position) or amplification method Size (bp)
1F CCTGGCCTCTTGCTTTTTCACATGT 20504 1,686
1R ACTTCGACGGTTGAGAAACAAATAG 22189 1,686
2F CGCGTTAAGAGTGGTTCACCAGGTG 22059 1,623
2R CAAAGCTGCAAGCCGTCCAGTAATT 23681 1,623
3F TTCAATTCAAGCCGATCAACAAGTT 23624 1,600
3R GTCATCAATTAATCGAAGGAACATC 25223 1,600
4F CGAAGAGCCTGTTGTTGGTATAGTC 25153 1,690
4R AACACGCTTCCAACGAGGTTTCTTC 26849 1,690
5F CCAGGGCTGATAAGCCTTCTCAGTT 26800 754
5R GTGTATCCATATCAAAAACAATATC 27553 754
6F TGAGGATGTTTGTGTTTGTTTTGAC 18864 1,710
6R GTCAGGAACACCTAATTGTAACATA 20573 1,710
7F TGCGTGGTTGGTTGGGTATGGATGT 17234 1,682
7R ACGCTCATACGAACCCTGAATACTA 18915 1,682
8F ATTCAGCAACTGGTTCCTTAGATGT 15479 1,808
8R GTTATCGCCACAAACATGAGCACTT 17286 1,808
9F CTCCCTACTATGACACAGCTGAATC 14005 1,596
9R AGCCGCAAAGAGTCTAAGTGTATCT 15600 1,596
10F GACCGTACAACTATTCAAAGTGTTG 12398 1,659
10R GTTCTTTACCACTAATAGCATACTT 14056 1,659
11F GGGCTATGGCTAATGGTTATACAAG 9801 1,435
11R TTTGCGATATTCATGGCACGCTTCA 11235 1,435
12F ACCCTTCAGAGTGTTGCTTCATCAT 11090 1,378
12R AGTCGAGCTGCACTAGAACCCCTTG 12467 1,378
13F CAACCACTGTAACTAGCTTTCATGG 7758 2,103
13R CTGCCAAAATAGAATAGCACTCAAC 9860 2,103
14F GTCAAAAGGGTGATGCTGAAGAGGC 5424 2,394
14R TCAACTGACCATTCTCAATGTACTT 7817 2,394
15F TAGAGATGAATTGGGTGTTCGTGTT 3424 2,043
15R GGTCCAACATCACCTGTAACAAATT 5466 2,043
16F GCAGATGTTCCAGATGCTTTTCAAT 1637 1,864
16R GCAACTGTACAAGTGTGGTACTAAT 3500 1,864
17F CAGCAATTATGTTCTTCAGGACTTT 565 1,118
17R GTGTAAATGTGCGATAAACTGATTG 1682 1,118
18F CTTAAAGAATTTTTCTATCTATAGA 1 1,056
18R CATGCACCAACACTCCAACTCTCAG 1056 1,056
GSP 1 CGAAGAGCCTGTTGTTGGTATAGTC 3′ RACEa Unknown
AP GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 3′ RACE Unknown
GSP 2 CCAGGGCTGATAAGCCTTCTCAGTT Nested PCR Unknown
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
AAP GGCCACGCGTCGACTAGTACGGGGGGGGGG 5′ RACE Unknown
GSP 1 GTGTAAATGTGCGATAAACTGATTG 5′ RACE Unknown
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
GSP 2 CATGCACCAACACTCCAACTCTCAG Nested PCR Unknown
GSP3 CCATGGCCAAAAACAACATCAAAGT Nested PCR Unknown
a RACE, rapid amplification of cDNA ends.
FIG 1 Phylogenetic analysis of HCoV-NL63 based on complete genomes. The complete genomes of 69 coronavirus references were obtained from the GenBank database, and multiple alignments were performed using MAFFT version 7.158 with default parameters. The phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstraps in MEGA version 5.10 with default parameters. The numbers at the nodes represent bootstrap support. Bootstrap values greater than 70% were considered statistically significant for grouping. The HCoV-NL63 strains presented in this study are marked with red circles. Only two complete genome sequences of HCoV-NL63 associated with acute respiratory illness have been obtained and reported in China. The complete genome sequence data from our study will provide insight into the evolution and genetic diversity of HCoV-NL63 in China.
Data availability.
The complete genome sequences of the five newly identified HCoV-NL63 strains have been deposited in GenBank under the accession numbers MK334043, MK334044, MK334045, MK334046, and MK334047. The sequencing reads are available in the SRA database under BioProject accession number PRJNA601331.
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sec
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ANNOUNCEMENT
Human coronavirus NL63 (HCoV-NL63) is a member of the family Coronaviridae, genus Alphacoronavirus, and was first discovered in 2004 (1). HCoV-NL63 is mainly associated with the common cold in children, the elderly, and immunocompromised patients (2, 3). The genome of HCoV-NL63 is about 27 kb with a conserved gene order of 5′-orf1ab-spike (S)-orf3-envelope (E)-membrane (M)-nucleocapsid (N)-poly (A). The species tropism of HCoV-NL63 is determined by spike glycoprotein. HCoV-NL63 and severe acute respiratory syndrome coronavirus (SARS-CoV) share the same cell receptor, angiotensin converting enzyme 2 (ACE-2) (4, 5), for entry into host cells, and HCoV-NL63 is recognized as a common cause of upper respiratory tract infection and has been prevalent worldwide.
Here, nasopharyngeal swab samples were collected from hospitalized children with severe acute respiratory infection in Guangzhou, China, in 2018. This study was performed in strict accordance with human subject protection guidance provided by the Research Ethics Committee of Guangzhou Medical University. The respiratory samples were filtered with 0.22-μm filters, RNA extraction was performed using a Qiagen viral RNA extraction kit, and extracted RNA was used for sequence-independent single-primer amplification (SISPA) (6, 7) as follows: a reverse transcription reaction was performed with SuperScript III reverse transcriptase using a primer containing a fixed sequence, followed by a random hexamer at the 3′ end (FR26RV, GCCGGAGCTCTGCAGATATCNNNNNN). Then, Klenow fragment polymerase (New England Biolabs) was used for DNA synthesis. Finally, PCR amplification was conducted using primers consisting of the fixed portions of the random primers (FR26, GCCGGAGCTCTGCAGATATC). Purified DNA was used for next-generation sequencing (NGS). Libraries were prepared with the Nextera XT kit (Illumina), and paired-end reads (2 × 125 bp) determined using a HiSeq 2500 instrument were used for cleaning and assembling using CLC Genomics Workbench version 11.0. Illumina sequencing yielded about 10 million reads per sample. Reads were assembled into contigs with a de novo assembly model, and the contig sequences were then extracted for subsequent analysis. Partial genome sequences of five HCoV-NL63 strains were obtained by NGS methods. Meanwhile, sets of specific primer pairs were designed and used to amplify the gap region of HCoV-NL63, which was used for genome assemblies using the SeqMan subprogram of the DNAStar software version 7.1.0 with default parameters (Table 1). Finally, five complete genome sequences of HCoV-NL63 were obtained using next-generation sequencing and Sanger sequencing methods together and were designated strains ChinaGD01 (27,531 bp), ChinaGD02 (27,516 bp), ChinaGD03 (27,516 bp), ChinaGD04 (27,532 bp), and ChinaGD05 (27,544 bp). The five HCoV-NL63 strains presented here were aligned using MAFFT version 7.158 (8) and showed 98.5 to ∼99.1% nucleotide homology with the prototype HCoV-NL63 virus (GenBank accession number NC_005831.2) as estimated using MEGA version 5.10 software (9) (Fig. 1).
TABLE 1 Primers used for the genome sequencing of HCoV-NL63
Primer Sequence (5′–3′) Target (nucleotide position) or amplification method Size (bp)
1F CCTGGCCTCTTGCTTTTTCACATGT 20504 1,686
1R ACTTCGACGGTTGAGAAACAAATAG 22189 1,686
2F CGCGTTAAGAGTGGTTCACCAGGTG 22059 1,623
2R CAAAGCTGCAAGCCGTCCAGTAATT 23681 1,623
3F TTCAATTCAAGCCGATCAACAAGTT 23624 1,600
3R GTCATCAATTAATCGAAGGAACATC 25223 1,600
4F CGAAGAGCCTGTTGTTGGTATAGTC 25153 1,690
4R AACACGCTTCCAACGAGGTTTCTTC 26849 1,690
5F CCAGGGCTGATAAGCCTTCTCAGTT 26800 754
5R GTGTATCCATATCAAAAACAATATC 27553 754
6F TGAGGATGTTTGTGTTTGTTTTGAC 18864 1,710
6R GTCAGGAACACCTAATTGTAACATA 20573 1,710
7F TGCGTGGTTGGTTGGGTATGGATGT 17234 1,682
7R ACGCTCATACGAACCCTGAATACTA 18915 1,682
8F ATTCAGCAACTGGTTCCTTAGATGT 15479 1,808
8R GTTATCGCCACAAACATGAGCACTT 17286 1,808
9F CTCCCTACTATGACACAGCTGAATC 14005 1,596
9R AGCCGCAAAGAGTCTAAGTGTATCT 15600 1,596
10F GACCGTACAACTATTCAAAGTGTTG 12398 1,659
10R GTTCTTTACCACTAATAGCATACTT 14056 1,659
11F GGGCTATGGCTAATGGTTATACAAG 9801 1,435
11R TTTGCGATATTCATGGCACGCTTCA 11235 1,435
12F ACCCTTCAGAGTGTTGCTTCATCAT 11090 1,378
12R AGTCGAGCTGCACTAGAACCCCTTG 12467 1,378
13F CAACCACTGTAACTAGCTTTCATGG 7758 2,103
13R CTGCCAAAATAGAATAGCACTCAAC 9860 2,103
14F GTCAAAAGGGTGATGCTGAAGAGGC 5424 2,394
14R TCAACTGACCATTCTCAATGTACTT 7817 2,394
15F TAGAGATGAATTGGGTGTTCGTGTT 3424 2,043
15R GGTCCAACATCACCTGTAACAAATT 5466 2,043
16F GCAGATGTTCCAGATGCTTTTCAAT 1637 1,864
16R GCAACTGTACAAGTGTGGTACTAAT 3500 1,864
17F CAGCAATTATGTTCTTCAGGACTTT 565 1,118
17R GTGTAAATGTGCGATAAACTGATTG 1682 1,118
18F CTTAAAGAATTTTTCTATCTATAGA 1 1,056
18R CATGCACCAACACTCCAACTCTCAG 1056 1,056
GSP 1 CGAAGAGCCTGTTGTTGGTATAGTC 3′ RACEa Unknown
AP GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 3′ RACE Unknown
GSP 2 CCAGGGCTGATAAGCCTTCTCAGTT Nested PCR Unknown
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
AAP GGCCACGCGTCGACTAGTACGGGGGGGGGG 5′ RACE Unknown
GSP 1 GTGTAAATGTGCGATAAACTGATTG 5′ RACE Unknown
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
GSP 2 CATGCACCAACACTCCAACTCTCAG Nested PCR Unknown
GSP3 CCATGGCCAAAAACAACATCAAAGT Nested PCR Unknown
a RACE, rapid amplification of cDNA ends.
FIG 1 Phylogenetic analysis of HCoV-NL63 based on complete genomes. The complete genomes of 69 coronavirus references were obtained from the GenBank database, and multiple alignments were performed using MAFFT version 7.158 with default parameters. The phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstraps in MEGA version 5.10 with default parameters. The numbers at the nodes represent bootstrap support. Bootstrap values greater than 70% were considered statistically significant for grouping. The HCoV-NL63 strains presented in this study are marked with red circles. Only two complete genome sequences of HCoV-NL63 associated with acute respiratory illness have been obtained and reported in China. The complete genome sequence data from our study will provide insight into the evolution and genetic diversity of HCoV-NL63 in China.
Data availability.
The complete genome sequences of the five newly identified HCoV-NL63 strains have been deposited in GenBank under the accession numbers MK334043, MK334044, MK334045, MK334046, and MK334047. The sequencing reads are available in the SRA database under BioProject accession number PRJNA601331.
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title
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ANNOUNCEMENT
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p
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Human coronavirus NL63 (HCoV-NL63) is a member of the family Coronaviridae, genus Alphacoronavirus, and was first discovered in 2004 (1). HCoV-NL63 is mainly associated with the common cold in children, the elderly, and immunocompromised patients (2, 3). The genome of HCoV-NL63 is about 27 kb with a conserved gene order of 5′-orf1ab-spike (S)-orf3-envelope (E)-membrane (M)-nucleocapsid (N)-poly (A). The species tropism of HCoV-NL63 is determined by spike glycoprotein. HCoV-NL63 and severe acute respiratory syndrome coronavirus (SARS-CoV) share the same cell receptor, angiotensin converting enzyme 2 (ACE-2) (4, 5), for entry into host cells, and HCoV-NL63 is recognized as a common cause of upper respiratory tract infection and has been prevalent worldwide.
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p
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Here, nasopharyngeal swab samples were collected from hospitalized children with severe acute respiratory infection in Guangzhou, China, in 2018. This study was performed in strict accordance with human subject protection guidance provided by the Research Ethics Committee of Guangzhou Medical University. The respiratory samples were filtered with 0.22-μm filters, RNA extraction was performed using a Qiagen viral RNA extraction kit, and extracted RNA was used for sequence-independent single-primer amplification (SISPA) (6, 7) as follows: a reverse transcription reaction was performed with SuperScript III reverse transcriptase using a primer containing a fixed sequence, followed by a random hexamer at the 3′ end (FR26RV, GCCGGAGCTCTGCAGATATCNNNNNN). Then, Klenow fragment polymerase (New England Biolabs) was used for DNA synthesis. Finally, PCR amplification was conducted using primers consisting of the fixed portions of the random primers (FR26, GCCGGAGCTCTGCAGATATC). Purified DNA was used for next-generation sequencing (NGS). Libraries were prepared with the Nextera XT kit (Illumina), and paired-end reads (2 × 125 bp) determined using a HiSeq 2500 instrument were used for cleaning and assembling using CLC Genomics Workbench version 11.0. Illumina sequencing yielded about 10 million reads per sample. Reads were assembled into contigs with a de novo assembly model, and the contig sequences were then extracted for subsequent analysis. Partial genome sequences of five HCoV-NL63 strains were obtained by NGS methods. Meanwhile, sets of specific primer pairs were designed and used to amplify the gap region of HCoV-NL63, which was used for genome assemblies using the SeqMan subprogram of the DNAStar software version 7.1.0 with default parameters (Table 1). Finally, five complete genome sequences of HCoV-NL63 were obtained using next-generation sequencing and Sanger sequencing methods together and were designated strains ChinaGD01 (27,531 bp), ChinaGD02 (27,516 bp), ChinaGD03 (27,516 bp), ChinaGD04 (27,532 bp), and ChinaGD05 (27,544 bp). The five HCoV-NL63 strains presented here were aligned using MAFFT version 7.158 (8) and showed 98.5 to ∼99.1% nucleotide homology with the prototype HCoV-NL63 virus (GenBank accession number NC_005831.2) as estimated using MEGA version 5.10 software (9) (Fig. 1).
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table-wrap
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TABLE 1 Primers used for the genome sequencing of HCoV-NL63
Primer Sequence (5′–3′) Target (nucleotide position) or amplification method Size (bp)
1F CCTGGCCTCTTGCTTTTTCACATGT 20504 1,686
1R ACTTCGACGGTTGAGAAACAAATAG 22189 1,686
2F CGCGTTAAGAGTGGTTCACCAGGTG 22059 1,623
2R CAAAGCTGCAAGCCGTCCAGTAATT 23681 1,623
3F TTCAATTCAAGCCGATCAACAAGTT 23624 1,600
3R GTCATCAATTAATCGAAGGAACATC 25223 1,600
4F CGAAGAGCCTGTTGTTGGTATAGTC 25153 1,690
4R AACACGCTTCCAACGAGGTTTCTTC 26849 1,690
5F CCAGGGCTGATAAGCCTTCTCAGTT 26800 754
5R GTGTATCCATATCAAAAACAATATC 27553 754
6F TGAGGATGTTTGTGTTTGTTTTGAC 18864 1,710
6R GTCAGGAACACCTAATTGTAACATA 20573 1,710
7F TGCGTGGTTGGTTGGGTATGGATGT 17234 1,682
7R ACGCTCATACGAACCCTGAATACTA 18915 1,682
8F ATTCAGCAACTGGTTCCTTAGATGT 15479 1,808
8R GTTATCGCCACAAACATGAGCACTT 17286 1,808
9F CTCCCTACTATGACACAGCTGAATC 14005 1,596
9R AGCCGCAAAGAGTCTAAGTGTATCT 15600 1,596
10F GACCGTACAACTATTCAAAGTGTTG 12398 1,659
10R GTTCTTTACCACTAATAGCATACTT 14056 1,659
11F GGGCTATGGCTAATGGTTATACAAG 9801 1,435
11R TTTGCGATATTCATGGCACGCTTCA 11235 1,435
12F ACCCTTCAGAGTGTTGCTTCATCAT 11090 1,378
12R AGTCGAGCTGCACTAGAACCCCTTG 12467 1,378
13F CAACCACTGTAACTAGCTTTCATGG 7758 2,103
13R CTGCCAAAATAGAATAGCACTCAAC 9860 2,103
14F GTCAAAAGGGTGATGCTGAAGAGGC 5424 2,394
14R TCAACTGACCATTCTCAATGTACTT 7817 2,394
15F TAGAGATGAATTGGGTGTTCGTGTT 3424 2,043
15R GGTCCAACATCACCTGTAACAAATT 5466 2,043
16F GCAGATGTTCCAGATGCTTTTCAAT 1637 1,864
16R GCAACTGTACAAGTGTGGTACTAAT 3500 1,864
17F CAGCAATTATGTTCTTCAGGACTTT 565 1,118
17R GTGTAAATGTGCGATAAACTGATTG 1682 1,118
18F CTTAAAGAATTTTTCTATCTATAGA 1 1,056
18R CATGCACCAACACTCCAACTCTCAG 1056 1,056
GSP 1 CGAAGAGCCTGTTGTTGGTATAGTC 3′ RACEa Unknown
AP GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 3′ RACE Unknown
GSP 2 CCAGGGCTGATAAGCCTTCTCAGTT Nested PCR Unknown
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
AAP GGCCACGCGTCGACTAGTACGGGGGGGGGG 5′ RACE Unknown
GSP 1 GTGTAAATGTGCGATAAACTGATTG 5′ RACE Unknown
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
GSP 2 CATGCACCAACACTCCAACTCTCAG Nested PCR Unknown
GSP3 CCATGGCCAAAAACAACATCAAAGT Nested PCR Unknown
a RACE, rapid amplification of cDNA ends.
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label
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TABLE 1
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caption
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Primers used for the genome sequencing of HCoV-NL63
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p
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Primers used for the genome sequencing of HCoV-NL63
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table
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Primer Sequence (5′–3′) Target (nucleotide position) or amplification method Size (bp)
1F CCTGGCCTCTTGCTTTTTCACATGT 20504 1,686
1R ACTTCGACGGTTGAGAAACAAATAG 22189 1,686
2F CGCGTTAAGAGTGGTTCACCAGGTG 22059 1,623
2R CAAAGCTGCAAGCCGTCCAGTAATT 23681 1,623
3F TTCAATTCAAGCCGATCAACAAGTT 23624 1,600
3R GTCATCAATTAATCGAAGGAACATC 25223 1,600
4F CGAAGAGCCTGTTGTTGGTATAGTC 25153 1,690
4R AACACGCTTCCAACGAGGTTTCTTC 26849 1,690
5F CCAGGGCTGATAAGCCTTCTCAGTT 26800 754
5R GTGTATCCATATCAAAAACAATATC 27553 754
6F TGAGGATGTTTGTGTTTGTTTTGAC 18864 1,710
6R GTCAGGAACACCTAATTGTAACATA 20573 1,710
7F TGCGTGGTTGGTTGGGTATGGATGT 17234 1,682
7R ACGCTCATACGAACCCTGAATACTA 18915 1,682
8F ATTCAGCAACTGGTTCCTTAGATGT 15479 1,808
8R GTTATCGCCACAAACATGAGCACTT 17286 1,808
9F CTCCCTACTATGACACAGCTGAATC 14005 1,596
9R AGCCGCAAAGAGTCTAAGTGTATCT 15600 1,596
10F GACCGTACAACTATTCAAAGTGTTG 12398 1,659
10R GTTCTTTACCACTAATAGCATACTT 14056 1,659
11F GGGCTATGGCTAATGGTTATACAAG 9801 1,435
11R TTTGCGATATTCATGGCACGCTTCA 11235 1,435
12F ACCCTTCAGAGTGTTGCTTCATCAT 11090 1,378
12R AGTCGAGCTGCACTAGAACCCCTTG 12467 1,378
13F CAACCACTGTAACTAGCTTTCATGG 7758 2,103
13R CTGCCAAAATAGAATAGCACTCAAC 9860 2,103
14F GTCAAAAGGGTGATGCTGAAGAGGC 5424 2,394
14R TCAACTGACCATTCTCAATGTACTT 7817 2,394
15F TAGAGATGAATTGGGTGTTCGTGTT 3424 2,043
15R GGTCCAACATCACCTGTAACAAATT 5466 2,043
16F GCAGATGTTCCAGATGCTTTTCAAT 1637 1,864
16R GCAACTGTACAAGTGTGGTACTAAT 3500 1,864
17F CAGCAATTATGTTCTTCAGGACTTT 565 1,118
17R GTGTAAATGTGCGATAAACTGATTG 1682 1,118
18F CTTAAAGAATTTTTCTATCTATAGA 1 1,056
18R CATGCACCAACACTCCAACTCTCAG 1056 1,056
GSP 1 CGAAGAGCCTGTTGTTGGTATAGTC 3′ RACEa Unknown
AP GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 3′ RACE Unknown
GSP 2 CCAGGGCTGATAAGCCTTCTCAGTT Nested PCR Unknown
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
AAP GGCCACGCGTCGACTAGTACGGGGGGGGGG 5′ RACE Unknown
GSP 1 GTGTAAATGTGCGATAAACTGATTG 5′ RACE Unknown
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
GSP 2 CATGCACCAACACTCCAACTCTCAG Nested PCR Unknown
GSP3 CCATGGCCAAAAACAACATCAAAGT Nested PCR Unknown
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tr
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Primer Sequence (5′–3′) Target (nucleotide position) or amplification method Size (bp)
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|
th
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Primer
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|
th
|
Sequence (5′–3′)
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|
th
|
Target (nucleotide position) or amplification method
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|
th
|
Size (bp)
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|
tr
|
1F CCTGGCCTCTTGCTTTTTCACATGT 20504 1,686
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|
td
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1F
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|
td
|
CCTGGCCTCTTGCTTTTTCACATGT
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|
td
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20504
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|
td
|
1,686
|
|
tr
|
1R ACTTCGACGGTTGAGAAACAAATAG 22189 1,686
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|
td
|
1R
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|
td
|
ACTTCGACGGTTGAGAAACAAATAG
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|
td
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22189
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|
td
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1,686
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tr
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2F CGCGTTAAGAGTGGTTCACCAGGTG 22059 1,623
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|
td
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2F
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|
td
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CGCGTTAAGAGTGGTTCACCAGGTG
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|
td
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22059
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|
td
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1,623
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|
tr
|
2R CAAAGCTGCAAGCCGTCCAGTAATT 23681 1,623
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|
td
|
2R
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|
td
|
CAAAGCTGCAAGCCGTCCAGTAATT
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|
td
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23681
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|
td
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1,623
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|
tr
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3F TTCAATTCAAGCCGATCAACAAGTT 23624 1,600
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|
td
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3F
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|
td
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TTCAATTCAAGCCGATCAACAAGTT
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|
td
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23624
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|
td
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1,600
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tr
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3R GTCATCAATTAATCGAAGGAACATC 25223 1,600
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|
td
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3R
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|
td
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GTCATCAATTAATCGAAGGAACATC
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|
td
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25223
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|
td
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1,600
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|
tr
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4F CGAAGAGCCTGTTGTTGGTATAGTC 25153 1,690
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|
td
|
4F
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|
td
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CGAAGAGCCTGTTGTTGGTATAGTC
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|
td
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25153
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|
td
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1,690
|
|
tr
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4R AACACGCTTCCAACGAGGTTTCTTC 26849 1,690
|
|
td
|
4R
|
|
td
|
AACACGCTTCCAACGAGGTTTCTTC
|
|
td
|
26849
|
|
td
|
1,690
|
|
tr
|
5F CCAGGGCTGATAAGCCTTCTCAGTT 26800 754
|
|
td
|
5F
|
|
td
|
CCAGGGCTGATAAGCCTTCTCAGTT
|
|
td
|
26800
|
|
td
|
754
|
|
tr
|
5R GTGTATCCATATCAAAAACAATATC 27553 754
|
|
td
|
5R
|
|
td
|
GTGTATCCATATCAAAAACAATATC
|
|
td
|
27553
|
|
td
|
754
|
|
tr
|
6F TGAGGATGTTTGTGTTTGTTTTGAC 18864 1,710
|
|
td
|
6F
|
|
td
|
TGAGGATGTTTGTGTTTGTTTTGAC
|
|
td
|
18864
|
|
td
|
1,710
|
|
tr
|
6R GTCAGGAACACCTAATTGTAACATA 20573 1,710
|
|
td
|
6R
|
|
td
|
GTCAGGAACACCTAATTGTAACATA
|
|
td
|
20573
|
|
td
|
1,710
|
|
tr
|
7F TGCGTGGTTGGTTGGGTATGGATGT 17234 1,682
|
|
td
|
7F
|
|
td
|
TGCGTGGTTGGTTGGGTATGGATGT
|
|
td
|
17234
|
|
td
|
1,682
|
|
tr
|
7R ACGCTCATACGAACCCTGAATACTA 18915 1,682
|
|
td
|
7R
|
|
td
|
ACGCTCATACGAACCCTGAATACTA
|
|
td
|
18915
|
|
td
|
1,682
|
|
tr
|
8F ATTCAGCAACTGGTTCCTTAGATGT 15479 1,808
|
|
td
|
8F
|
|
td
|
ATTCAGCAACTGGTTCCTTAGATGT
|
|
td
|
15479
|
|
td
|
1,808
|
|
tr
|
8R GTTATCGCCACAAACATGAGCACTT 17286 1,808
|
|
td
|
8R
|
|
td
|
GTTATCGCCACAAACATGAGCACTT
|
|
td
|
17286
|
|
td
|
1,808
|
|
tr
|
9F CTCCCTACTATGACACAGCTGAATC 14005 1,596
|
|
td
|
9F
|
|
td
|
CTCCCTACTATGACACAGCTGAATC
|
|
td
|
14005
|
|
td
|
1,596
|
|
tr
|
9R AGCCGCAAAGAGTCTAAGTGTATCT 15600 1,596
|
|
td
|
9R
|
|
td
|
AGCCGCAAAGAGTCTAAGTGTATCT
|
|
td
|
15600
|
|
td
|
1,596
|
|
tr
|
10F GACCGTACAACTATTCAAAGTGTTG 12398 1,659
|
|
td
|
10F
|
|
td
|
GACCGTACAACTATTCAAAGTGTTG
|
|
td
|
12398
|
|
td
|
1,659
|
|
tr
|
10R GTTCTTTACCACTAATAGCATACTT 14056 1,659
|
|
td
|
10R
|
|
td
|
GTTCTTTACCACTAATAGCATACTT
|
|
td
|
14056
|
|
td
|
1,659
|
|
tr
|
11F GGGCTATGGCTAATGGTTATACAAG 9801 1,435
|
|
td
|
11F
|
|
td
|
GGGCTATGGCTAATGGTTATACAAG
|
|
td
|
9801
|
|
td
|
1,435
|
|
tr
|
11R TTTGCGATATTCATGGCACGCTTCA 11235 1,435
|
|
td
|
11R
|
|
td
|
TTTGCGATATTCATGGCACGCTTCA
|
|
td
|
11235
|
|
td
|
1,435
|
|
tr
|
12F ACCCTTCAGAGTGTTGCTTCATCAT 11090 1,378
|
|
td
|
12F
|
|
td
|
ACCCTTCAGAGTGTTGCTTCATCAT
|
|
td
|
11090
|
|
td
|
1,378
|
|
tr
|
12R AGTCGAGCTGCACTAGAACCCCTTG 12467 1,378
|
|
td
|
12R
|
|
td
|
AGTCGAGCTGCACTAGAACCCCTTG
|
|
td
|
12467
|
|
td
|
1,378
|
|
tr
|
13F CAACCACTGTAACTAGCTTTCATGG 7758 2,103
|
|
td
|
13F
|
|
td
|
CAACCACTGTAACTAGCTTTCATGG
|
|
td
|
7758
|
|
td
|
2,103
|
|
tr
|
13R CTGCCAAAATAGAATAGCACTCAAC 9860 2,103
|
|
td
|
13R
|
|
td
|
CTGCCAAAATAGAATAGCACTCAAC
|
|
td
|
9860
|
|
td
|
2,103
|
|
tr
|
14F GTCAAAAGGGTGATGCTGAAGAGGC 5424 2,394
|
|
td
|
14F
|
|
td
|
GTCAAAAGGGTGATGCTGAAGAGGC
|
|
td
|
5424
|
|
td
|
2,394
|
|
tr
|
14R TCAACTGACCATTCTCAATGTACTT 7817 2,394
|
|
td
|
14R
|
|
td
|
TCAACTGACCATTCTCAATGTACTT
|
|
td
|
7817
|
|
td
|
2,394
|
|
tr
|
15F TAGAGATGAATTGGGTGTTCGTGTT 3424 2,043
|
|
td
|
15F
|
|
td
|
TAGAGATGAATTGGGTGTTCGTGTT
|
|
td
|
3424
|
|
td
|
2,043
|
|
tr
|
15R GGTCCAACATCACCTGTAACAAATT 5466 2,043
|
|
td
|
15R
|
|
td
|
GGTCCAACATCACCTGTAACAAATT
|
|
td
|
5466
|
|
td
|
2,043
|
|
tr
|
16F GCAGATGTTCCAGATGCTTTTCAAT 1637 1,864
|
|
td
|
16F
|
|
td
|
GCAGATGTTCCAGATGCTTTTCAAT
|
|
td
|
1637
|
|
td
|
1,864
|
|
tr
|
16R GCAACTGTACAAGTGTGGTACTAAT 3500 1,864
|
|
td
|
16R
|
|
td
|
GCAACTGTACAAGTGTGGTACTAAT
|
|
td
|
3500
|
|
td
|
1,864
|
|
tr
|
17F CAGCAATTATGTTCTTCAGGACTTT 565 1,118
|
|
td
|
17F
|
|
td
|
CAGCAATTATGTTCTTCAGGACTTT
|
|
td
|
565
|
|
td
|
1,118
|
|
tr
|
17R GTGTAAATGTGCGATAAACTGATTG 1682 1,118
|
|
td
|
17R
|
|
td
|
GTGTAAATGTGCGATAAACTGATTG
|
|
td
|
1682
|
|
td
|
1,118
|
|
tr
|
18F CTTAAAGAATTTTTCTATCTATAGA 1 1,056
|
|
td
|
18F
|
|
td
|
CTTAAAGAATTTTTCTATCTATAGA
|
|
td
|
1
|
|
td
|
1,056
|
|
tr
|
18R CATGCACCAACACTCCAACTCTCAG 1056 1,056
|
|
td
|
18R
|
|
td
|
CATGCACCAACACTCCAACTCTCAG
|
|
td
|
1056
|
|
td
|
1,056
|
|
tr
|
GSP 1 CGAAGAGCCTGTTGTTGGTATAGTC 3′ RACEa Unknown
|
|
td
|
GSP 1
|
|
td
|
CGAAGAGCCTGTTGTTGGTATAGTC
|
|
td
|
3′ RACEa
|
|
td
|
Unknown
|
|
tr
|
AP GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT 3′ RACE Unknown
|
|
td
|
AP
|
|
td
|
GGCCACGCGTCGACTAGTACTTTTTTTTTTTTTTTTT
|
|
td
|
3′ RACE
|
|
td
|
Unknown
|
|
tr
|
GSP 2 CCAGGGCTGATAAGCCTTCTCAGTT Nested PCR Unknown
|
|
td
|
GSP 2
|
|
td
|
CCAGGGCTGATAAGCCTTCTCAGTT
|
|
td
|
Nested PCR
|
|
td
|
Unknown
|
|
tr
|
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
|
|
td
|
AUAP
|
|
td
|
GGCCACGCGTCGACTAGTAC
|
|
td
|
Nested PCR
|
|
td
|
Unknown
|
|
tr
|
AAP GGCCACGCGTCGACTAGTACGGGGGGGGGG 5′ RACE Unknown
|
|
td
|
AAP
|
|
td
|
GGCCACGCGTCGACTAGTACGGGGGGGGGG
|
|
td
|
5′ RACE
|
|
td
|
Unknown
|
|
tr
|
GSP 1 GTGTAAATGTGCGATAAACTGATTG 5′ RACE Unknown
|
|
td
|
GSP 1
|
|
td
|
GTGTAAATGTGCGATAAACTGATTG
|
|
td
|
5′ RACE
|
|
td
|
Unknown
|
|
tr
|
AUAP GGCCACGCGTCGACTAGTAC Nested PCR Unknown
|
|
td
|
AUAP
|
|
td
|
GGCCACGCGTCGACTAGTAC
|
|
td
|
Nested PCR
|
|
td
|
Unknown
|
|
tr
|
GSP 2 CATGCACCAACACTCCAACTCTCAG Nested PCR Unknown
|
|
td
|
GSP 2
|
|
td
|
CATGCACCAACACTCCAACTCTCAG
|
|
td
|
Nested PCR
|
|
td
|
Unknown
|
|
tr
|
GSP3 CCATGGCCAAAAACAACATCAAAGT Nested PCR Unknown
|
|
td
|
GSP3
|
|
td
|
CCATGGCCAAAAACAACATCAAAGT
|
|
td
|
Nested PCR
|
|
td
|
Unknown
|
|
table-wrap-foot
|
a RACE, rapid amplification of cDNA ends.
|
|
footnote
|
a RACE, rapid amplification of cDNA ends.
|
|
label
|
a
|
|
p
|
RACE, rapid amplification of cDNA ends.
|
|
figure
|
FIG 1 Phylogenetic analysis of HCoV-NL63 based on complete genomes. The complete genomes of 69 coronavirus references were obtained from the GenBank database, and multiple alignments were performed using MAFFT version 7.158 with default parameters. The phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstraps in MEGA version 5.10 with default parameters. The numbers at the nodes represent bootstrap support. Bootstrap values greater than 70% were considered statistically significant for grouping. The HCoV-NL63 strains presented in this study are marked with red circles.
|
|
label
|
FIG 1
|
|
caption
|
Phylogenetic analysis of HCoV-NL63 based on complete genomes. The complete genomes of 69 coronavirus references were obtained from the GenBank database, and multiple alignments were performed using MAFFT version 7.158 with default parameters. The phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstraps in MEGA version 5.10 with default parameters. The numbers at the nodes represent bootstrap support. Bootstrap values greater than 70% were considered statistically significant for grouping. The HCoV-NL63 strains presented in this study are marked with red circles.
|
|
p
|
Phylogenetic analysis of HCoV-NL63 based on complete genomes. The complete genomes of 69 coronavirus references were obtained from the GenBank database, and multiple alignments were performed using MAFFT version 7.158 with default parameters. The phylogenetic tree was constructed using the neighbor-joining method with 1,000 bootstraps in MEGA version 5.10 with default parameters. The numbers at the nodes represent bootstrap support. Bootstrap values greater than 70% were considered statistically significant for grouping. The HCoV-NL63 strains presented in this study are marked with red circles.
|
|
p
|
Only two complete genome sequences of HCoV-NL63 associated with acute respiratory illness have been obtained and reported in China. The complete genome sequence data from our study will provide insight into the evolution and genetic diversity of HCoV-NL63 in China.
|
|
sec
|
Data availability.
The complete genome sequences of the five newly identified HCoV-NL63 strains have been deposited in GenBank under the accession numbers MK334043, MK334044, MK334045, MK334046, and MK334047. The sequencing reads are available in the SRA database under BioProject accession number PRJNA601331.
|
|
title
|
Data availability.
|
|
p
|
The complete genome sequences of the five newly identified HCoV-NL63 strains have been deposited in GenBank under the accession numbers MK334043, MK334044, MK334045, MK334046, and MK334047. The sequencing reads are available in the SRA database under BioProject accession number PRJNA601331.
|
|
back
|
ACKNOWLEDGMENTS
This research was supported by grants from the National Key Research and Development Program of China (2018YFC1200100), National Natural Science Foundation of China (NSFC 81702047, 81772191, 91842106, and 8181101118), State Key Laboratory of Respiratory Disease (SKLRD-QN-201715 and SKLRD-QN-201912), National Key Technology R&D Program (2018YFC1311900), Guangdong Science and Technology Foundation (2019B030316028), 2015 Thousand Talents Plan Award of China, China Postdoctoral Science Foundation, and the Ph.D. Start-up Fund of the Natural Science Foundation of Guangdong Province, China.
|
|
ack
|
ACKNOWLEDGMENTS
This research was supported by grants from the National Key Research and Development Program of China (2018YFC1200100), National Natural Science Foundation of China (NSFC 81702047, 81772191, 91842106, and 8181101118), State Key Laboratory of Respiratory Disease (SKLRD-QN-201715 and SKLRD-QN-201912), National Key Technology R&D Program (2018YFC1311900), Guangdong Science and Technology Foundation (2019B030316028), 2015 Thousand Talents Plan Award of China, China Postdoctoral Science Foundation, and the Ph.D. Start-up Fund of the Natural Science Foundation of Guangdong Province, China.
|
|
title
|
ACKNOWLEDGMENTS
|
|
p
|
This research was supported by grants from the National Key Research and Development Program of China (2018YFC1200100), National Natural Science Foundation of China (NSFC 81702047, 81772191, 91842106, and 8181101118), State Key Laboratory of Respiratory Disease (SKLRD-QN-201715 and SKLRD-QN-201912), National Key Technology R&D Program (2018YFC1311900), Guangdong Science and Technology Foundation (2019B030316028), 2015 Thousand Talents Plan Award of China, China Postdoctoral Science Foundation, and the Ph.D. Start-up Fund of the Natural Science Foundation of Guangdong Province, China.
|
