The notorious virus recorded to prompt 50-60% of disease incidence over Tamil Nadu in chilli. The characteristic symptoms of mild to severe mosaic, mosaic mottling, leaf filiformity (shoe string), chlorosis and stunted growth were observed (Fig. 1). The presence of CMV in the infected plant samples were confirmed through RT-PCR using CP gene specific primer. The products of 1200-bp were amplified, cloned and then sequenced (Figure S1). Therefore, five CMV isolates (MT396263; MT396265; MT395346; MZ093618; MZ093617) showed greater variation has been used in the study. Nucleotide sequences of CP (657nt) was compared and evaluated to comprehend genetic properties and variability in amino acids. Comparatively, TN isolates MT396263 (BTN1), MT396265 (BTN2) and MT395346 (BTN3) shared 98.28- 98.48% identity with MZ093618 (BTN4) and MZ093617 (BTN5). The nucleotide sequences of TN CMV isolates shared 97.65-98.92% homology with Indian chilli isolates (HM348786; KM272275; KJ645896; MT396265; KM272276; KJ645897; KX9611535). The heat map generated using SDTv1.2 explicated that TN isolates shared amino acid homology of 97-99% identity with subgroup IB, 93-95% identity with subgroup IA and 75-79% identity with subgroup II of CP respectively (Fig. 2). Moreover, TN isolate of BTN1 shared 99.5% identity with BTN2 and BTN3 isolates. Similarly, 96.7% identity with BTN4 and 97.2% identity with BTN5 of TN isolates. The pairwise percent identity of TN isolates with other members of CMV isolates were presented in Table 1.
Deduced amino acid sequence analysis of complete coat protein
Any variation in a single amino acid may catastrophically affect the virulence, transmission, symptomatology and stability of virus. The multiple alignments of deduced CP amino acid sequences explicate substitution, deletion and conserved regions among different strains of CMV. There are five subgroup IA strains, seven subgroup IB strains and five subgroup II representative strains were used for comparative sequence analysis (Fig. 3). Comparative amino acid sequence analysis depicted Phe99 amino acid to be conserved in subgroup IB strains, whereas subgroup IA and II strains possess Tyr99 amino acid respectively. The significant role of the amino acid was not well understood. Indeed, 30 amino acids were found to be conserved among subgroup I strains comparatively with subgroup II strains. Similarly, amino acids (Lys65 and Lys82) at the position of 65 and 82 observed to be conserved among subgroup IB and subgroup II strains. Besides, conserved basic ARM (Arginine Rich Motif) observed at 5 prime end (14-24 position) of CP in subgroup I and II strains. Therefore, deletion of amino acid threonine (Thr12) observed at 12th position of TN isolates (subgroup IB) subsequently in subgroup I strains as well. It suggests that Thr12 amino acid conserved for CMV subgroup I strains alone. Similarly, deletion was observed in 25th position of subgroup II strains. In contemplate, subgroup IA strains possess proline (Pro25) and subgroup IB strains possess serine (Ser25) at 25th position.
Notably, unique amino acid substitution observed at 129th position of CP (Pro129 and Ser129). Whereas some of isolates possess Pro129 (BTN1; BTN2; BTN3) and some of possess Ser129 (BTN4; BTN5). It depicts that CMV isolates possess Pro129 are mosaic inducing strains (BTN1; BTN2; BTN3) and thus substituted with Ser129 are chlorosis inducing stains (BTN4; BTN5). Hence it evinces the existence of chlorosis and mosaic inducing strains of CMV in chilli growing areas of Tamil Nadu. Indeed, mosaic and chlorosis symptom induction bestowed by conserved amino acid 129 (Ser129 and Pro129). Moreover, quaint amino acid substitution (Thr137) observed over 137th position in chlorosis inducing TN strains comparatively with other mosaic inducing strains of subgroup IB members (Ser137). Hence, the result implies that amino acids Ser129 and Thr137 to be conserved in CP of chlorosis inducing TN strains. In contemplate, amino acids of Pro129 and Ser137 to be conserved in mosaic inducing strains of CMV. Similarly, aphid vector transmission conferred by conserved alanine (Ala162) at the position of 162. Empirically, some of chlorosis inducing strains also showed amino acid substitution of Thr162 over conserved Ala162.
Structural annotation of CP
The amino acid sequence based structural annotation has been carried out for CP of mosaic inducing (BTN1) and chlorosis inducing strain (BTN5) using Swiss model and validated by Ramachandran plot. The results postulate that CP has canonical topology comprised of 180 sub units dispersed as 60 copies in three (A, B and C) subunits with threefold symmetry. Moreover, subunit A possess pentameric capsomer whereas subunit B and C form hexameric capsomer. Speculatively, those three subunits observed to have identical amino acid sequences with little different conformations. A Notable modification in secondary structure of CP was detected in BTN5 (chlorosis inducing stain) when compared with BTN1 (mosaic inducing strain). Presumably, βB- βC loop positioned at 76-83 residues, βD-βE loop at 113-119 residues, βE- αEF loop at 129-136 residues, βF-βG loop at 155-163 residues and βH-HI loop at 191-199 resides in coat protein of CMV.Intriguingly, the super imposed structure of BTN1 and BTN5 vindicated the structural loop modification of βE-αEF (129 residue), βF-βG (162 residue) and βB-βC loops (78 residues) in coat protein of CMV (Fig. 4). The corresponding identical and non-similar amino acids in the superimposed protein structure based on dihedral angle were depicted in Ramachandaran plot (Fig. 5).
Furthermore, confound positional modification of helix domain was observed at 170-172 amino acid of BTN5 whereas BTN1 confer at 171-172 amino acids. Similarly, confound modification of β sheets observed at 106- 108 amino acid of BTN5 whereas BTN1 confer at 106-110 amino acids. Amicably, BTN5 flaunted disordered protein binding domain while compared with BTN1. The graphical representation of helix, β sheets, coil and disordered protein binding laid on amino acid position of BTN1 and BTN5 of CP have been depicted graphically (Fig. 6). Furthermore, amino acid sequence based analysis of domain identification algorithm explicated the presence of functional motif of glycosylation (NTSS) and phosphorylation sites (TcR, StK, TIK, SIK, StcE, SsiD and SviE) in CP (Table 2). Distinctly, upon PROSITE functional site search, Casein kinase II phosphorylation site also observed in CP which is purely conserved for CPs of all Cucumovirus.
Furthermore, diversity analysis of CP using DnaSP ver.5.10 explicates haplotype diversity of TN isolates as one and number of segregation sites observed as 449. Comparatively, greater synonymous and non-synonymous substitution of nucleotides observed in TN chlorosis inducing strains than mosaic inducing strains. Moreover, cumulatively significant synonymous substitutions were discerned in CP of TN isolates over non-synonymous substitution. Over all, it implies that chlorosis inducing strains having higher nucleotide diversity than of mosaic inducing strains of CMV (Table 3).
Phylogenetic relationship of CP of TN isolates evaluated with subgroup I and II members of CMV isolates reported from different parts of world. The phylogenetic tree generated with deduced CP amino acid sequences of CMV isolates using Mega 7 program with 1000 replicates of bootstrap values. Besides, Fny, M, Y and Tfn strains of CMV were taken as standard subgroup I reference strains and Trk7II strain taken as subgroup II reference strain. Alike, Peanut stunt virus (U15730) included as out group for rooting in the phylogeny. Phylogenetic analysis explicates the formation of two different cluster forsubgroup I and subgroup II (Fig. 7). Moreover, TN isolates shared high vicinity with members of subgroup I. Precisely, TN isolates clustered together with subgroup IB strains, well separated from subgroup IA. While, subgroup IA strains were clustered with Fny, M, Y and Tfn reference strains and subgroup II clustered with Trk7II reference strain of CMV. It clearly depicts that mosaic inducing and chlorosis inducing strains of TN CMV isolates were belonging to subgroup IB.