Molecular Characterization and Genetic Diversity of Broad Bean Wilt Virus 2 (BBWV-2) from

Abstract

Small RNA deep sequencing (sRNA-seq) is a powerful tool for detecting known and novel plant viruses by analyzing virus-derived small RNAs (vsRNAs). In this study, the complete genome of Broad Bean Wilt Virus 2 (BBWV-2) was recovered from leaf samples using sRNA-seq, followed by bioinformatics analysis. The Genome Detective Tool, an online bioinformatics platform, identified both RNA1 (5.952 kb) and RNA2 (3.509 kb) of the BBWV-2 form raw data. RNA1 and RNA2 showed 78.8% and 78.1% nucleotide identity, and 88.2% and 87.4% amino acid identity, respectively, with their corresponding reference sequences. Comparative sequence analysis identified multiple mutations, comprising 8 and 6 indels distributed across the full-length sequences of RNA1 and RNA2, respectively, along with 1027 and 690 single-nucleotide substitutions localized within their coding regions. Analysis of the 5' untranslated regions (UTRs) identified five distinct motifs from both RNAs. Structural modeling suggested that RNA2 exhibits greater flexibility than RNA1, which may influence its biological functions. Phylogenetic analysis placed the Indian isolate within Group I, clustering with isolates from South Korea, China, the USA, and Germany, without clear host or geographic clustering. This study provides the first complete sequences of BBWV-2 RNA1 and RNA2 from India and represents the first global report of its occurrence in . Further, these findings enhance our understanding of the molecular diversity and evolutionary dynamics of BBWV-2.

1

Schematic illustration depicting the organizational structure of the BBWV2 genome, characterized by a segmented, bipartite linear single-stranded positive strand (+)­ssRNA. RNA1 has a length of 5.952 kb, and RNA2 has a length of 3.509 kb. Each genomic segment possesses a VPg (viral protein genome-linked) to its 5′ end, and poly­(A) tailing at a 3′ end. The abbreviations used include NTPase-helicase (Hel), polyprotein major protease or peptidase C3 (Pro), RNA-dependent RNA polymerase (POL), movement protein (MP), large capsid protein (CPL), and small capsid protein (CPS).

2

Detection of BBWV2 by RT-PCT. Agarose gel electrophoresis showing amplification of 471 bp segment of RNA2 from infected samples (lanes 1, 2, 4, and 7). Lane M is the 100 bp DNA ladder (GeneDireX, Taiwan).

3

Pairwise sequence identity comparison between the Indian BBWV-2 isolate and other global isolates using the Sequence Demarcation Tool (SDT) v1.2. The color-coded matrix represents the percentage of sequence identity on nucleotide level between the Indian C. roseus isolate and reference sequences from the database. The Indian isolate is marked with a red rectangular box for identification.

4

Phylogenetic tree constructed via the rooted circular neighbor–joining method, which displays two separate clusters of sequences. This tree distinguishes two groups separately based on the full deduced nucleotide sequences of RNA1 and RNA2 from the Indian isolate CR8-JH (highlighted in red) of BBWV2 compared with other sequences (in black) from various corresponding RNA1 and RNA2 nucleotide sequences available in the NCBI GenBank database. The lengths of the branches on the tree represent the estimated number of substitutions that have occurred in each lineage. Additionally, red circles at the branching points denote bootstrap support values, which provide a statistical measure of confidence in the tree’s branching structure.

5

(A) Sequence logos of motifs identified in the 5′ UTRs of RNA1 and RNA2 of BBWV-2 Indian isolate using the MEME Suite. (B) Distribution of motif locations and corresponding average p values within the same 5′ UTR region.

6

Secondary structures of various motifs of 5′ UTR of RNA1 of BBWV-2 (Indian isolate). (A) MFE secondary structure. (B) Centroid secondary structure. The structures were predicted using RNAfold. The structure is color-coded by base-pairing probabilities. Red shows high probability of forming stable base pairs, green/yellow show intermediate probability whereas blue shows low probability or unpaired regions.

7

Secondary structures of various motifs of 5′ UTR of RNA2 of BBWV-2 (Indian isolate). (A) MFE secondary structure. (B) Centroid secondary structure. The structures were predicted using RNA fold. The structure is color-coded by base-pairing probabilities. Red shows high probability of forming stable base pairs, green/yellow show intermediate probability whereas blue shows low probability or unpaired regions.

8

Alignment of RNA-1 sequences from the reference genome (NC_003003.1) and the new Indian isolate (OR826802) highlighting indels (insertions and deletions) in the 5′ UTR, CDS, and 3′ UTR regions. Significant variations include insertions at positions 88, 90, 106, 566, and 5936 and deletions at positions 200, 5850, and 5873 are visualized by dash.

9

Alignment of RNA-2 sequences from the reference genome (NC_003004.1) and the new Indian isolate (OR826803) showing indels in the 5′ and 3′ UTR regions. Significant variations include insertions at positions 40, 183, and 198, and deletions at positions 152, 3418, and 3441. The figure uses dash to denote Indel whereas color-coded differences and annotations are used to visualize the genetic changes between the two sequences.

10

Stacked bar chart illustrating the distribution of mutation types in the CDS regions of RNA1 and RNA2 of BBWV-2. RNA1 exhibits a total of 1027 codon mutations, comprising 806 silent mutations and 221 missense mutations. The missense mutations include 118 conservative and 103 nonconservative substitutions. RNA2 displays a total of 690 codon mutations, with 436 silent mutations and 134 missense mutations, further categorized into 65 conservative and 69 nonconservative substitutions.