Genomic characterization of a new enamovirus infecting common bean

A novel enamovirus was identified in common bean plants with disease symptoms. Its genome of 5,781 nucleotides (nt) contains five open reading frames. This virus and other members of the genus Enamovirus share 50.4-68.4% nucleotide sequence identity in the complete genome and 19.9-51.9% amino acid sequence identity in the P0 protein, 24.9-52.5% in P1, 33.4-62.9% in P1-P2, 30.6-81.1% in P3, and 32.3-74.2% in P3-P5. Phylogenetic analysis showed that the virus is most closely related to alfalfa enamovirus 1 and pea enation mosaic virus 1 in the genus Enamovirus of the family Solemoviridae. These results suggest that this virus, tentatively named “bean enamovirus 1”, should be classified as a member of a new species in the genus Enamovirus.

Common bean (Phaseolus vulgaris L.), also called French bean, is a herbaceous annual crop belonging to the family Fabaceae. It is native to Mesoamerica and was introduced into China in the 15th century [1]. Young pods of common bean are used as vegetables, and the dried seeds are one of the major staple foods in countries of Africa, Latin America, the Caribbean, and Asia. According to statistics from the Food and Agriculture Organization of the United Nations (FAO) in 2018, China is the fifth-largest producer of common bean, with an annual planting area of about 100 million hm 2 and an output of nearly 1.3 million tons. However, common bean production is vulnerable to many diseases, including those caused by viruses. At least 43 viruses have been reported to infect common bean and cause a decline in production [2][3][4].
The genus Enamovirus was originally part of the family Luteoviridae, but recently, it was moved to the family Solemoviridae, which also includes the genera Polemovirus, Polerovirus, and Sobemovirus [5]. Enamoviruses have a single-stranded RNA genome of 5.7-6.0 kb in length that contains five open reading frames (ORF). They have a narrow host range and are transmitted by aphids. In this study, a novel enamovirus was identified in common bean and molecularly characterized. A survey of multiple crops showed that the virus infects several other legume crops.
In September 2020, common bean samples with symptoms of mild mottle were collected from fields in Kunming, Yunnan, China (Fig. 1A). To identify potential pathogens, five symptomatic samples were pooled for preparation of a total RNA sample that was subjected to high-throughput sequencing (HTS). The rRNA-depleted sample was used for construction of an RNA-seq library for sequencing on an Illumina HiSeq X Ten platform (Origingene Bio-pharm Technology, Shanghai, China). The RNA-seq data were analyzed using CLC Genomic Workbench 20.0 (QIAGEN). A total of 48,792,184 paired-end reads were obtained, and de novo assembly of the reads generated 138,508 contigs with more than 200 nucleotides (nt). A BLASTx search of the GenBank database revealed that about 0.1% of the total reads corresponded to bean yellow mosaic virus, tomato spotted wilt virus, or alfalfa mosaic virus, but there was an additional large contig of 5,374 nt with average coverage To determine the complete genome sequence of the novel enamovirus by HTS, six pairs of overlapping primers were designed based on the large contig covering most of the virus genome (Supplementary Table S1). Total RNA was extracted from individual plant samples using TRIpure Reagent (Bioteke Corporation, Beijing, China) and tested by RT-PCR for the presence of the new virus. An extract from an infected plant was used for amplification of fragments of the viral genome by RT-PCR. The PCR products were cloned into the vector pMD19-T (TaKaRa Biotechnology Co., Ltd, Dalian, China), and at least three clones were sequenced for each amplicon (BGI, Guangzhou, China). The 5'-and 3'-terminal sequences were amplified using a SMARTer ® RACE 5'/3' Kit as described by the manufacturer (TaKaRa Biotechnology, Dalian, China). The RT-PCR amplification, cloning, and sequence analysis were conducted as described by Lan et al. [6].
The complete genome of the new enamovirus consists of 5,781 nt (GenBank accession no. MZ361924) and contains five open reading frames (ORF). Its genomic organization is typical of members of the genus Enamovirus (Fig. 1C). The 5' and 3' untranslated regions (UTR) are 174 nt and 233 nt long, respectively, and the intergenic region (IR) between ORFs 2 and 3 is 188 nt in length. ORF0, starting at nt 175 and ending at nt 1,086, encodes a putative P0 protein of 303 amino acid (aa) residues that contains the sequence 213 LPxxL 217 , the putative F-box-like motif, which is conserved among enamoviruses [7]. ORF1 is predicted to be expressed by a ribosomal leaky scanning mechanism, encoding a putative P1 protein of 766 aa residues. The conserved sequence 390 H(x 29 )D(x 67 )GxSG 591 of the S39 serine protease is present in this region [8]. ORF2 is predicted to be translated by a -1 ribosomal frameshift at a putative slippery heptamer, 2039 GGG AAA T 2045 , within ORF1 to produce a fusion protein of P1-P2 with a length of 1188 aa residues. P1-P2 might be involved in replication of enamoviruses, as it contains the highly conserved motif 1,085 GDD 1,087 in the viral RNA-dependent RNA polymerase (RdRp) [8]. After a short IR, ORF3 starts at nt 4,022 and ends at nt 4,594, encoding a coat protein (CP) of 190 aa residues. ORF5 is predicted to be expressed by a putative in-frame readthrough from ORF3, producing a fusion protein of 508 aa residues (P3-P5). P3-P5 is thought to be an aphid-transmission subunit in members of the genus Enamovirus [9].
Pairwise comparisons of the genome sequences and the predicted aa sequences of the individual proteins of this virus and other members of the genus were conducted, and the results show that this new virus shares 50.4-68.4% nt sequence identity with enamoviruses at the whole-genome  Table S2). These values are below the species demarcation threshold of 10% aa sequence identity in any gene product for the genus Enamovirus [10]. It is worth noting that the CP is the most conserved protein, whereas ORF0 is the most divergent among the enamoviruses. A maximum-likelihood tree based on deduced P1-P2 fusion protein sequences of this virus and members of the genera Enamovirus, Polemovirus, Sobemovirus, and Polerovirus of the family Solemoviridae places the new virus together with AEV-1 [11] and pea enation mosaic virus 1 (PEMV-1) [12] in a legume clade (Fig. 2). The results of these analyses suggest that the new virus, tentatively named "bean enamovirus 1" (BEnV-1), should be considered a new member of the genus Enamovirus.
To investigate the distribution and potential natural host species of BEnV-1 in the field, a total of 378 leaf samples were collected from different crops and weeds with or without symptoms in Yunnan. RT-PCR was performed using the specific primers BEnVdF and BEnVdR (Supplementary Table S1), and five out of 25 vetch plants (21.7%), three out of 10 alfalfa plants (30.0%), and five out of 59 common bean plants (8.5%) were found to be infected with BEnV-1. The main symptom associated with BEnV-1 in the three infected legume plants was mild mottle (Fig. 1A-C). The virus was not detected in cowpea, soybean, pea, faba bean, pepper, tomato, potato, cucurbit, cucumber, and passion fruit. Common bean and alfalfa are important food and forage crops throughout the world. The effects of BEnV-1 on its hosts still needs to be assessed.

Conflict of interest As the corresponding authors, Ruhui Li and Fan
Li declare no conflict of interest involving the authors of this paper.