# Orsay virus variants isolated from wild Caenorhabditis elegans nematodes, France

Chika Fujii, Susan Vernon, Marie-Anne Félix, David Wang

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Chika Fujii, Susan Vernon, Marie-Anne Félix, David Wang. Orsay virus variants isolated from wild Caenorhabditis elegans nematodes, France. Microbiology Resource Announcements, 2025, 14 (10), pp.00550-25. 10.1128/mra.00550-25. hal-05383681

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Submitted on 26 Nov 2025

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Orsay virus variants isolated from wild *Caenorhabditis elegans* nematodes, France

Chika Fujii,¹ Susan Vernon,¹ Marie-Anne Félix,² David Wang¹,³

**AUTHOR AFFILIATIONS** See affiliation list on p. 3.

**ABSTRACT** Four new variants of Orsay virus were identified from wild isolates of *Caenorhabditis elegans* nematodes collected from decaying plant matter in France. Near-complete genomes of the viruses were determined by metagenomic sequencing. The four genomes share 96.1–98.9% nucleotide identity with the reference Orsay virus sequence JUv1580.

**KEYWORDS** Orsay virus, *Caenorhabditis elegans*

Orsay virus is a single-stranded, positive-sense, and bi-segmented RNA virus that infects *Caenorhabditis elegans* (*C. elegans*) nematodes (1). The virus has yet to be formally classified by the International Committee on Taxonomy of Viruses, but it is closely related to viruses in the *Nodaviridae* family (1). The originally discovered wild isolate JUv1580 has provided molecular biological insights into the Orsay virus life cycle, and its RNA1 (HM030970.2) and RNA2 (HM030971.2) segment sequences have served as a reference genome. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) (2), whereas RNA2 contains two open reading frames encoding the alpha and delta proteins. The alpha protein forms the viral capsid (3), while the delta protein is implicated in viral exit (4). The alpha and delta proteins also exist in a fused form (5). Here, we report the sequences of four new Orsay virus variants, discovered in nematodes dwelling in decaying plant matter in France. The four Orsay virus variants, JUv2572, JUv2807, JUv2815, and LUAv001, were isolated from sites in Ivry (GPS 48.8092, 2.3862), Santeuil (GPS 49.12165, 1.95101), Orsay (GPS 48.7015, 2.1725), and Santeuil (GPS 49.121, 1.951), respectively. Viruses in infected nematodes were maintained as described previously (1) on agar plates seeded with *Escherichia coli* strain OP50. For isolation of RNA, infected nematodes were homogenized in TRIzol (Invitrogen), followed by extraction of the total RNA using the Direct-zol RNA extraction Kit (ZYMO). The TruSeq Stranded Total RNA Kit (Illumina) was used for library preparation. The library was subjected to paired-end sequencing on either the Illumina MiSeq (2x250 bp; JUv2572, JUv2815, LUAv001) or the NovaSeq (2x150 bp; JUv2807) platforms. Raw sequencing reads were processed in the CZID platform (6), a metagenomics analysis pipeline for detecting virus sequences in the NCBI database. Within the CZID platform, quality control was performed with Trimmomatic (adapter trimming) and STAR/Bowtie2 (host read removal), assembly was performed using SPAdes, and the assembled contigs were assessed for matches to NCBI's nucleotide databases. The *C. elegans* host genome was specified to be subtracted in the CZID metagenomics analysis pipeline. The number of reads, average depth of coverage, percentage of GC within the coding-complete genome, percent identity compared to the reference genome (JUv1580), the total lengths of the genomes, and the percent amino acid identities relative to JUv1580 of RdRp, alpha, delta, and alpha-delta fusion proteins are summarized in Table 1. Clustal Omega (7) was used to determine the percent identities relative to the JUv1580 reference. To visualize the relationships between the RNA1 and RNA2 among the variants, a phylogenetic tree was constructed (Fig. 1). The

**Editor** Jelle Matthijnssens, Katholieke Universiteit Leuven, Leuven, Belgium

Address correspondence to David Wang, davewang@wustl.edu.

The authors declare no conflict of interest.

**Received** 9 June 2025

**Accepted** 20 August 2025

**Published** 10 September 2025

Copyright © 2025 Fujii et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
<table><caption>TABLE 1 Summary of the sequencing information and features of the four new Orsay virus strains</caption><thead><tr><th rowspan="2">Virus</th><th rowspan="2">Year collected</th><th rowspan="2">Total no. of reads</th><th colspan="4">RNA1</th><th colspan="4">RNA2</th><th colspan="4">Alpha protein amino acid identity (%)</th><th colspan="2">Delta protein amino acid identity (%)</th><th colspan="2">Alpha-delta fusion protein amino acid identity (%)</th></tr><tr><th>No. of reads mapped to RNA1</th><th>Average coverage depth (x)</th><th>Coding-complete genome GC content (%)</th><th>Nucleotide identity compared to the JUv1580 reference (%)</th><th>Total length (bp)</th><th>RdRp protein amino acid identity (%)</th><th>No. of reads mapped to RNA2</th><th>Average coverage depth (x)</th><th>Coding-complete genome GC content (%)</th><th>Nucleotide identity compared to the JUv1580 reference (%)</th><th>Total length (bp)</th><th>RdRp protein amino acid identity (%)</th><th>Delta protein amino acid identity (%)</th><th>Alpha-delta fusion protein amino acid identity (%)</th></tr></thead><tbody><tr><td>JUV2572</td><td>2013</td><td>1,478,516</td><td>7,579</td><td>483</td><td>55.9</td><td>96.9</td><td>3,401</td><td>98.3</td><td>7,646</td><td>664</td><td>52.6</td><td>98.1</td><td>2,768</td><td>98.5</td><td>98.8</td><td>98.4</td></tr><tr><td>JUV2807</td><td>2014</td><td>38,124,337</td><td>113,997</td><td>4,869</td><td>55.7</td><td>96.9</td><td>3,417</td><td>98.4</td><td>343,566</td><td>19,411</td><td>52.7</td><td>96.1</td><td>2,573</td><td>99.2</td><td>96.5</td><td>97.8</td></tr><tr><td>JUV2815</td><td>2014</td><td>831,671</td><td>12,889</td><td>845</td><td>55.6</td><td>96.6</td><td>3,394</td><td>98.1</td><td>17,526</td><td>1,563</td><td>52.7</td><td>98.9</td><td>2,523</td><td>99.7</td><td>100.0</td><td>99.7</td></tr><tr><td>LUAv001</td><td>2017</td><td>3,047,176</td><td>9,257</td><td>562</td><td>55.8</td><td>96.3</td><td>3,396</td><td>98.1</td><td>13,354</td><td>1,088</td><td>52.9</td><td>96.5</td><td>2,560</td><td>98.5</td><td>96.0</td><td>97.1</td></tr></tbody></table>
<figure><img src="image_2.png" /><figcaption>RNA2</figcaption></figure>

<figure><img src="image_3.png" /><figcaption>FIG 1 Maximum likelihood phylogenetic trees of the Orsay virus RNA1 and RNA2 segments, constructed with the reference JUv1580 and the four newly identified virus strains. Sequences were aligned with Clustal Omega, and a maximum likelihood phylogenetic tree was constructed with MEGA11 (8) with 1,000 bootstrap replicates. JUv1580 was included in the analysis as the reference genome. The node values represent the probability with which the sequences clustered together in the bootstrap test with 1,000 replicates.</figcaption></figure>

availability of these virus isolates and their genome sequences will enable experimental
studies using different Orsay virus strains, as well as studies of viral evolution.

AUTHOR AFFILIATIONS

AUTHOR ORCIDs

Chika Fujii ID https://orcid.org/0000-0002-7494-5424

David Wang ID http://orcid.org/0000-0002-0827-196X

<footnote n="1">¹Department of Molecular Microbiology, School of Medicine, Washington University in St. Louis, St. Louis, Missouri, USA</footnote>

<footnote n="2">²Institute of Biology of the Ecole Normale Supérieure (IBENS), Paris, France</footnote>

<footnote n="3">³Department of Pathology & Immunology, School of Medicine, Washington University in St. Louis, St. Louis, Missouri, USA</footnote>
DATA AVAILABILITY

The raw sequencing results of this Orsay virus metagenomic sequencing project have been deposited in SRA under the SRR accession numbers SRR33389215 (JUv2572), SRR33389213 (JUv2807), SRR33389212 (JUv2815), and SRR33389211 (LUAv001). The coding-complete genomic sequences of the Orsay virus variants were deposited in GenBank under the accession numbers PV629397 (JUv2572_RNA1), PV629398 (JUv2572_RNA2), PV629401 (JUv2807_RNA1), PV629402 (JUv2807_RNA2), PV629403 (JUv2815_RNA1), PV629404 (JUv2815_RNA2), PV629405 (LUAv001_RNA1), and PV629406 (LUAv001_RNA2).

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