# Genome sequences of human coronavirus NL63 diagnosed in southern France

Houmadi Hikmat, Céline Boschi, Sarah Aherfi, Aurélie Morand, Bernard La Scola, Philippe Colson

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Houmadi Hikmat, Céline Boschi, Sarah Aherfi, Aurélie Morand, Bernard La Scola, et al.. Genome sequences of human coronavirus NL63 diagnosed in southern France. Microbiology Resource Announcements, 2025, Dec 16, pp.e0084125. 10.1128/mra.00841-25. hal-05455971

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# Genome sequences of human coronavirus NL63 diagnosed in southern France

Houmadi Hikmat,¹,² Céline Boschi,¹,²,³ Sarah Aherfi,¹,² Aurélie Morand,⁴,⁵ Bernard La Scola,¹,²,³ Philippe Colson¹,²,³

**AUTHOR AFFILIATIONS** See affiliation list on p. 5.

**ABSTRACT** We report here 17 human coronavirus NL63 genomes from France. They were obtained from residues of respiratory samples collected from patients for diagnostics in southern France, using an in-house multiplex PCR amplification system followed by next-generation sequencing with Illumina technology. Sixteen genomes belong to subgenotype C2 and one to subgenotype B1.

**KEYWORDS** coronavirus, human coronavirus-NL63, genome, phylogenomics, genomic surveillance

Human coronavirus NL63 (HCoV-NL63), discovered in 2003 (1), belongs to the genus *Alphacoronavirus* (2) and likely has a zoonotic origin from bats (3). It has a single-stranded positive-sense RNA genome of ≈28 kilobases that encodes 16, 4, and 1 nonstructural, structural (spike, envelope, matrix, and nucleocapsid), and accessory proteins, respectively (4). Its cell receptor is angiotensin-converting enzyme 2 (5). HCoV-NL63 is ubiquitous worldwide (6, 7), causing respiratory tract infections, mostly in children, usually mild but potentially severe and life-threatening (8–12). A total of 345 genomes (>90% coverage of KY073745.1) were released in GenBank (https://www.ncbi.nlm.nih.gov/genbank/) as of 17 July 2025, obtained from patients sampled between 1983 and 2025, in 24% of the cases since 2020, mostly from the USA (31%), UK (28%), and China (21%). Three genotypes A–C and eight subgenotypes were delineated (12–14).

Here, HCoV-NL63 genomes were obtained from residues of HCoV-NL63 RNA-positive respiratory samples collected in 2021 from patients for routine diagnostic by multiplex qPCR (15). RNA was extracted with the MagMAX Viral/Pathogen Nucleic Acid Isolation kit on a KingFisher Flex system (Thermo Fisher Scientific). Thirty-five PCR primer pairs (Table 1) were designed with GEMI (16) and then used to pre-amplify HCoV-NL63 genomes with the SuperScript III One-Step RT-PCR kit with Platinum Taq DNA polymerase (Thermo Fisher Scientific), as previously described (15). Next-generation sequencing was performed as previously described (15) using Illumina technology and the COVIDSeq protocol (Illumina Inc.), with replacement of ARTIC COVID-19 primers with primers designed here. Library preparation and sequencing of 2 × 250 paired-end reads on a MiSeq instrument (Illumina Inc.) were performed following the manufacturer's instructions. There were, on average, 7,353,541 ± 5,020,276 reads generated per sample (range, 264,636–17,879,700). The Genome Detective web application (https://www.genomedeceptive.com/) (17) performed a trimming and quality control of reads, then the assembly, annotation, and taxonomic classification of viral genomes with default settings. Phylogeny was performed using IQ-TREE 2 (18).

Seventeen HCoV-NL63 genomes, 27,322–27,465 nucleotides long, were obtained. Mean coverage relative to genome NC_005831.2 dating back to 2003 was 98.0% (range, 90.0–99.9%). Coverage <100% was due to amplification defects of some regions, mostly 5' and 3' genome ends. Phylogeny identified subgenotypes C2 and B1 in 16 and one

**Editor** Simon Roux, DOE Joint Genome Institute, Berkeley, California, USA

Address correspondence to Bernard La Scola, bernard.la-scola@univ-amu.fr, or Philippe Colson, philippe.colson@univ-amu.fr.

Bernard La Scola and Philippe Colson are scientific advisors of BioSellal and Triber companies. The other authors have no conflicts of interest to declare.

See the funding table on p. 5.

**Received** 1 September 2025

**Accepted** 24 October 2025

**Published** 16 December 2025

Copyright © 2025 Hikmat et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
<figure><img src="image_2.png" /><figcaption>FIG 1 Phylogeny reconstruction based on HCoV-NL63 genomes recovered in the present study or available from GenBank. Sequence alignment was performed using the MAFFT v.7 web application (https://mafft.cbrc.jp/alignment/server/index.html) with automatic parameter optimization and multithreading to improve speed and accuracy. Phylogeny reconstruction was performed using IQ-TREE 2 (18) using the maximum likelihood method according to the TN + FO + I model, with branch support assessed using 1,000 bootstrap replicates. The phylogenetic tree was visualized using iTOL (https://itol.embl.de). HCoV-NL63 genomes incorporated in the phylogenetic analysis included genomes from the different previously delineated lineages or sublineages and the five best BLAST hits in GenBank for each of the genomes obtained here. Blue circles indicate bootstrap values >80%. Genomes obtained here are indicated by a black star.</figcaption></figure>
<table><caption>TABLE 1 In-house designed PCR primer pairs used for HCoV-NL63 genome pre-amplification</caption><thead><tr><th>Primer name</th><th>Sequence (5'-3' orientation)</th><th>Pool</th><th>Primer concentration in pool (µM)</th></tr></thead><tbody><tr><td>NL63_F1</td><td>ARGTTTGTAAACTGGTTAGGC</td><td>1</td><td>10</td></tr><tr><td>NL63_R1</td><td>ACACGCCATAAAGACACACC</td><td></td><td>10</td></tr><tr><td>NL63_F2</td><td>ACTGGTGTGTCTTTATGGCG</td><td>2</td><td>10</td></tr><tr><td>NL63_R2</td><td>GAAGTAAGTGTAAGCTTAAGAG</td><td></td><td>10</td></tr><tr><td>NL63_F3</td><td>AGTATTGATGATGGCCRTTTAT</td><td>1</td><td>10</td></tr><tr><td>NL63_R3</td><td>CTGTACAAACACACCAGTAC</td><td></td><td>10</td></tr><tr><td>NL63_F4</td><td>GGTACTGGTGTGTTTGTACAG</td><td>2</td><td>15</td></tr><tr><td>NL63_R4</td><td>AGAAACACAGTTTAYAAGGTGT</td><td></td><td>15</td></tr><tr><td>NL63_F5</td><td>TTCACACCTTRTAAACTGTGTTTTC</td><td>1</td><td>10</td></tr><tr><td>NL63_R5</td><td>ATCCTTACACCAAAAATACCAC</td><td></td><td>10</td></tr><tr><td>NL63_F6</td><td>AATGGTATTCCACTTATGCCTC</td><td>2</td><td>12.5</td></tr><tr><td>NL63_R6</td><td>CTGCCTGTTGCAAAATACCAT</td><td></td><td>12.5</td></tr><tr><td>NL63_F7</td><td>AATGGTATTTTGCAACAGGCA</td><td>1</td><td>12.5</td></tr><tr><td>NL63_R7</td><td>TTCTTAAAACAGTCGTTAACATCC</td><td></td><td>12.5</td></tr><tr><td>NL63_F8</td><td>AATCTTGGTCGTTGTGTGCG</td><td>2</td><td>12.5</td></tr><tr><td>NL63_R8</td><td>CATTAGATGYTACACAACCAC</td><td></td><td>12.5</td></tr><tr><td>NL63_F9</td><td>TGTCTACTYTAGCTGTTACAGC</td><td>1</td><td>12.5</td></tr><tr><td>NL63_R9</td><td>TAATATAGGATCRCGAATGTGC</td><td></td><td>12.5</td></tr><tr><td>NL63_F10</td><td>TGGAAGCACATTCGYGATCC</td><td>2</td><td>10</td></tr><tr><td>NL63_R10</td><td>ATAAYATATGCAGGAGCTGTG</td><td></td><td>10</td></tr><tr><td>NL63_F11</td><td>GGCCTGGTAAYACTTTTATTAA</td><td>1</td><td>10</td></tr><tr><td>NL63_R11</td><td>ACCAACAACTATAGGACATTTATC</td><td></td><td>10</td></tr><tr><td>NL63_F12ALT</td><td>GGCATGAGGCTAAGTTTGGT</td><td>2</td><td>10</td></tr><tr><td>NL63_R12ALT</td><td>AGCCTCACTAGCACTACCA</td><td></td><td>10</td></tr><tr><td>NL63_F13</td><td>GGTACTTTTGAGAGTGCTGC</td><td>1</td><td>10</td></tr><tr><td>NL63_R13</td><td>CAAGCTCCATTTATAAAAGAAC</td><td></td><td>10</td></tr><tr><td>NL63_F14</td><td>GGAGCTTGTGGTTCTCCTG</td><td>2</td><td>10</td></tr><tr><td>NL63_R14</td><td>AGGTGTAAGTATMACAGGGTT</td><td></td><td>10</td></tr><tr><td>NL63_F15</td><td>TCTTCACAATGTTTTGGGCAG</td><td>1</td><td>10</td></tr><tr><td>NL63_R15</td><td>TAAATTCAGCAGCACTMACCT</td><td></td><td>10</td></tr><tr><td>NL63_F16</td><td>GTTTGTACTTATTGGGGCATT</td><td>2</td><td>10</td></tr><tr><td>NL63_R16</td><td>CAAATGATGAAGCAACACTCT</td><td></td><td>10</td></tr><tr><td>NL63_F17</td><td>GATTTTGGTCTTGATGGCCTT</td><td>1</td><td>10</td></tr><tr><td>NL63_R17</td><td>CAAATACTTCACTTGAGGACC</td><td></td><td>10</td></tr><tr><td>NL63_F18</td><td>GTGGGAGTATGAGGGTGG</td><td>2</td><td>12.5</td></tr><tr><td>NL63_R18</td><td>ATTACCATAAATGACTCTGCC</td><td></td><td>12.5</td></tr><tr><td>NL63_F19</td><td>CTGGTGCTTTGGCTGAGC</td><td>1</td><td>10</td></tr><tr><td>NL63_R19</td><td>AACCCATAAYAGGCATCATATA</td><td></td><td>10</td></tr><tr><td>NL63_F20</td><td>CCTAATATGGGTGTTCCCTG</td><td>2</td><td>10</td></tr><tr><td>NL63_R20</td><td>ACACCATCRATTAAAGTACGC</td><td></td><td>10</td></tr><tr><td>NL63_F21</td><td>ACACGCAATGCYACTGTTGT</td><td>1</td><td>8.75</td></tr><tr><td>NL63_R21</td><td>CGGTGTTATAGCCAAAATAAAC</td><td></td><td>8.75</td></tr><tr><td>NL63_F22</td><td>TTGTGGTGATTGTYTGCGTAA</td><td>2</td><td>12.5</td></tr><tr><td>NL63_R22</td><td>GTAATCATTACTCTAGGTGCA</td><td></td><td>12.5</td></tr><tr><td>NL63_F23</td><td>GGTGAYCCACAACAACTTCC</td><td>1</td><td>8.75</td></tr><tr><td>NL63_R23</td><td>CACAAAATATACCYTTCTTAGC</td><td></td><td>8.75</td></tr><tr><td>NL63_F24</td><td>ACTTCTGACACTGCACATGC</td><td>2</td><td>8.75</td></tr><tr><td>NL63_R24</td><td>GTCCTGGACATTACGCC</td><td></td><td>8.75</td></tr><tr><td>NL63_F25 ALT</td><td>TCATAGAAACGAGCATGATGC</td><td>1</td><td>10</td></tr><tr><td>NL63_R25 ALT</td><td>GTCAAAACAAACACAAACATCC</td><td></td><td>10</td></tr><tr><td>NL63_F26</td><td>ATTCAGGGTTCGTATGAGCG</td><td>2</td><td>12.5</td></tr></tbody></table>

(Continued on next page)
<table><caption>TABLE 1 In-house designed PCR primer pairs used for HCoV-NL63 genome pre-amplification (Continued)</caption><thead><tr><th>Primer name</th><th>Sequence (5'-3' orientation)</th><th>Pool</th><th>Primer concentration in pool (µM)</th></tr></thead><tbody><tr><td>NL63_R26</td><td>CTTCAAGRTAAACAGTAGCAC</td><td>12.5</td><td></td></tr><tr><td>NL63_F27</td><td>TCCACARTTGCAGTCTGCTG</td><td>1</td><td>15</td></tr><tr><td>NL63_R27</td><td>TTCACCMGATATAGTTATRC</td><td></td><td>15</td></tr><tr><td>NL63_F28</td><td>CAGAACAGTTAGGTGCGCC</td><td>2</td><td>15</td></tr><tr><td>NL63_R28</td><td>CACAAAAGTGCTAACATGAGT</td><td></td><td>15</td></tr><tr><td>NL63_F29</td><td>AACACYACTCATGTTAGCACT</td><td>1</td><td>10</td></tr><tr><td>NL63_R29</td><td>CTCACGAATACCAGARACAG</td><td></td><td>10</td></tr><tr><td>NL63_F30</td><td>ATACTATTGTTGGTGCTTTGTAT</td><td>2</td><td>12.5</td></tr><tr><td>NL63_R30</td><td>AGTACCCAAACCAGATGTAACA</td><td></td><td>12.5</td></tr><tr><td>NL63_F31_ALT</td><td>TGCCTTACGACTTAGTGCTC</td><td>1</td><td>15</td></tr><tr><td>NL63_R31_ALT</td><td>ATTAACATCAACGTAGTCAGGT</td><td></td><td>15</td></tr><tr><td>NL63_F32</td><td>CTGTCATACCTGACTACGTT</td><td>2</td><td>15</td></tr><tr><td>NL63_R32</td><td>ATCGAAGGAACATCTTCGTATA</td><td></td><td>15</td></tr><tr><td>NL63_F33</td><td>GCACCTGTTCCAGCTGAAG</td><td>1</td><td>8.75</td></tr><tr><td>NL63_R33</td><td>CCCCCTRCGCATACGCC</td><td></td><td>8.75</td></tr><tr><td>NL63_F34</td><td>GGCGTATGCGYAGGGGG</td><td>2</td><td>10</td></tr><tr><td>NL63_R34</td><td>GAGTCTCGTGAGTTGTTACG</td><td></td><td>10</td></tr><tr><td>NL63_F35</td><td>TCTGTTGTTGAGTTYGAGGAT</td><td>1</td><td>15</td></tr><tr><td>NL63_R35</td><td>TTACACTTTACTATCACTGGC</td><td></td><td>15</td></tr></tbody></table>

case, respectively (Fig. 1). Subgenotype C2 genomes exhibited a similarity of 98.3% on average between each other and of 90.5% with the subgenotype B1 genome. The closest relatives according to BLAST searches into GenBank and to phylogeny were obtained from the USA, Japan, UK, China, and Switzerland between 2017–2024; mean similarity was 97.3% with the best hits. Substitution I507L, located in the spike receptor binding domain and suspected to promote viral entry into host cells (12), was present in all subgenotype C2 genomes but absent in the subgenotype B1 genome.

In summary, the HCoV-NL63 genomes provided here from France account for around a fifth of those available worldwide since 2020. They evidence that at least subgenotypes C2 and B1 circulated in our geographical area. Prior evidence of expanding diversity and of frequent recombinations (19, 20) warrants intensifying HCoV-NL63 genome sequencing retrospectively and prospectively to get a more detailed picture of the epidemiology, diversity, and evolution of this virus.

## ACKNOWLEDGMENTS

We are very grateful to the technical team of the next-generation sequencing platform of IHU Méditerranée Infection.

This work was supported by the French Government under the "Investments for the Future" program managed by the National Agency for Research (ANR), Méditerranée-Infection 10-IAHU-03. This work was also supported by the French Ministry of Higher Education, Research and Innovation (Ministère de l'Enseignement supérieur, de la Recherche et de l'Innovation) and the French Ministry of Solidarity and Health (Ministère des Solidarités et de la Santé). Funding sources had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; or preparation, review, or approval of the manuscript.

Conceived and designed the experiments: B.L.S., P.C. Contributed materials, analysis tools: All authors. Analyzed the data: H.H., C.B., B.L.S., P.C. Writing—original draft preparation: H.H., B.L.S., and P.C. Writing—review and editing: All authors. All authors have read and agreed to the published version of the manuscript.
## AUTHOR AFFILIATIONS

¹IHU Méditerranée Infection, Marseille, France

²Microbes Evolution Phylogeny and Infections (MEPHI), Aix-Marseille University (AMU),
Marseille, France

³Assistance Publique-Hôpitaux de Marseille (AP-HM), Marseille, France

⁴Service de Pédiatrie générale, Hôpital Timone, AP-HM, Marseille, France

⁵Service d'accueil des Urgences Pédiatriques, Hôpital Nord, AP-HM, Marseille, France

## AUTHOR ORCIDs

Bernard La Scola http://orcid.org/0000-0001-8006-7704

Philippe Colson http://orcid.org/0000-0001-6285-0308

## FUNDING

<table><thead><tr><th>Funder</th><th>Grant(s)</th><th>Author(s)</th></tr></thead><tbody><tr><td rowspan="6">Agence Nationale de la Recherche (ANR)</td><td rowspan="6">Méditerranée-Infection<br>10-IAHU-03</td><td>Houmadi Hikmat</td></tr><tr><td>Céline Boschi</td></tr><tr><td>Sarah Aherfi</td></tr><tr><td>Aurélie Morand</td></tr><tr><td>Bernard La Scola</td></tr><tr><td>Philippe Colson</td></tr><tr><td rowspan="3">French ministry of higher education,<br>research and innovation</td><td></td><td>Céline Boschi</td></tr><tr><td></td><td>Bernard La Scola</td></tr><tr><td></td><td>Philippe Colson</td></tr><tr><td rowspan="3">French ministry of solidarity and health</td><td></td><td>Céline Boschi</td></tr><tr><td></td><td>Bernard La Scola</td></tr><tr><td></td><td>Philippe Colson</td></tr></tbody></table>

## AUTHOR CONTRIBUTIONS

Houmadi Hikmat, Formal analysis, Methodology, Writing – original draft, Writing – review and editing | Céline Boschi, Methodology, Writing – review and editing | Sarah Aherfi, Methodology, Writing – review and editing | Aurélie Morand, Resources, Writing – review and editing | Bernard La Scola, Conceptualization, Formal analysis, Writing – original draft, Writing – review and editing | Philippe Colson, Conceptualization, Formal analysis, Methodology, Supervision, Writing – original draft, Writing – review and editing

## DATA AVAILABILITY

The HCoV-NL63 genomes analyzed here have been submitted to the GenBank sequence database (https://www.ncbi.nlm.nih.gov/genbank/) under accession no. PX111341 to PX111357. Next-generation sequencing raw data have been submitted to the NCBI Sequence Read Archive (SRA) (https://www.ncbi.nlm.nih.gov/sra) under BioProject accession no. PRJNA1308010 and SRA no. SRX30150765 to SRX30150781.

## ETHICS APPROVAL

The present study has been registered on the Health Data Access Portal of Marseille public and university hospitals (Assistance Publique-Hôpitaux de Marseille [AP-HM]) and was approved by the Ethics and Scientific Committee of AP-HM with no. PADS24-190 and CSE_PADS24-190, respectively.
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