Complete genome sequence of a putative novel ilarvirus isolated from Eleocharis dulcis

The complete genomic sequence of a novel ilarvirus from Eleocharis dulcis, tentatively named "water chestnut virus A" (WCVA), was determined using next-generation sequencing (NGS) combined with reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) PCR. The three genomic RNA components of WCVA were 3578 (RNA1), 2873 (RNA2), and 2073 (RNA3) nucleotides long, with four predicted open reading frames containing conserved domains and motifs typical of ilarviruses. Phylogenetic analysis of each predicted protein consistently placed WCVA in subgroup 4 of the genus Ilarvirus, together with prune dwarf virus, viola white distortion associated virus, Fragaria chiloensis latent virus, and potato yellowing virus. The genetic distances and lack of serological reaction to antisera against other ilarviruses suggest that WCVA is a novel member of the genus.

Water chestnut (Eleocharis dulcis (Burm. f.) Trin. is a perennial aquatic plant of the genus Eleocharis (Cyperaceae) that occurs naturally in China, Japan, the Ryukyu Islands, Nanyang Island, and India [1]. Its underground corms can be eaten raw or cooked and are one of the characteristic vegetables in China. There are about 50,000 ha of cultivated water chestnut in China, including 20,000 ha in Guangxi province, particularly around Hezhou and Lipu, and there is also significant cultivation in Tuanfeng and Shayang counties of Hubei province [2,3].
Because the plants are propagated vegetatively, viruses are easily transmitted to the next generation when germplasm is exchanged, causing a decline in crop yields and significant economic losses. Several viruses have been reported previously in water chestnuts. Cucumber mosaic virus (CMV) causes chlorosis, dwarfing, and deformity and was first identified in water chestnut plants in 2014 [4], while a double-stranded DNA virus named "water chestnut soymovirus-1" was discovered by high-throughput sequencing in 2019 [2].
Over the period 2016-2020, it was noticed that the quality of corms of a local water chestnut variety in Guangxi had declined; the corms were smaller and the leaves had chlorotic spots. Diseased leaf samples were collected in July 2020 and sent to Zhejiang Academy of Agricultural Sciences (Hangzhou, China) for next-generation sequencing (NGS). Total RNA was extracted using TRIzol Reagent (Invitrogen, Carlsbad, USA), and RNA integrity was checked using an Agilent 2100 Bioanalyzer (Agilent Technologies). A cDNA library was constructed using a TruSeq RNA Sample Preparation Kit (Illumina) according to the manufacturer's instructions. An Illumina NovaSeq 6000 platform with PE150bp and CLC Genomic Workbench 11 (QIAGEN) were used for sequencing and data analysis with default parameters. A total of 36,026,984 paired-end reads were obtained, and 12,675 contigs (903-18,749 bp) were generated de novo and compared with sequences in the GenBank database using BLASTn or BLASTx. Three of the contigs (3858 nt, 2861 nt and 2144 nt) were clearly related to the three genomic RNAs  [5][6][7]. Their genomes encode four or five proteins on three RNA strands, and they have mostly been classified into four phylogenetic subgroups [8].
To determine the complete genomic RNA sequence of the water chestnut virus, specific primer sets were designed (Supplementary Table S1) to amplify each of the RNAs in two overlapping segments for cloning and splicing. Total RNA was extracted from the infected water chestnut sample using an EASYspin RNA Plant Mini Kit (Aidlab Biotechnologies Co., Ltd, China). The RNA was polyadenylated using poly(A) polymerase (TAKARA, Japan) and then reverse transcribed using the primer M4-T and a First Strand cDNA Synthesis Kit (Toyobo, Osaka, Japan) [9]. Rapid amplification of cDNA ends (RACE) (Tiosbio, Beijing, China) was used to ensure that the complete sequences were obtained. Each PCR product was inserted into the pEASY-T5 Zero Cloning Vector (TransGen Biotech, Beijing), and more than five clones per reaction were sequenced (Ykang, Hangzhou, China). The 5' and 3'RACE fragments amplified using the corresponding primers were then assembled using DNA-MAN 8.0 software (Lynnon Biosoft, Canada).
To determine the relationship between WCVA and other known ilarviruses, phylogenetic trees were constructed for each of the ORFs using representative sequences of all previous reported ilarviruses (Supplementary Table S2), with CMV (genus Cucumovirus) included as an outgroup. Analysis was conducted in MEGA X [11] using the maximum-likelihood method with 1000 bootstrap replicates. In all ORFs, WCVA clustered in subgroup 4 with PDV, viola white distortion associated virus (VWDAV), Fragaria chiloensis latent virus (FCILV), and PYV, but the exact clustering pattern among these viruses differed slightly (Fig. 2). In addition, the P2 protein of WCVA showed the closest relationship to that of the GFAIV. However, the RNA2 is the only sequence of GFAIV that has been reported so far. Inspection of the trees suggests that three unclassified viruses -surrounding legume associated ilarvirus (SLAIV), grapevine associated ilarvirus (GAIV), and Solanum nigrum ilarvirus 1 (SNIV-1) -are very closely related and are probably members of a single (new) species.
Total protein extracted from leaves of infected water chestnut were tested in DAS-ELISA with antiserum to the ilarviruses american plum line pattern virus (APLPV), tobacco streak virus (TSV), PDV, and prunus necrotic ringspot virus (PNRSV) (Adgia), but there were no positive reactions.
Sequence similarity criteria for demarcation of species within the genus Ilarvirus have not been defined [12], but the low amino acid sequence identity to known ilarviruses in all of the predicted proteins (< 62%) and the serological results suggest that WCVA is a distinct novel member of the genus. Further research will focus on the incidence of this virus in different water chestnut varieties and its host range and pathogenicity.