Our study conducted a selection analysis on specific codons within the ToBRFV genome to investigate episodic positive selection and evolutionary dynamics. Utilizing a likelihood ratio test (LRT), we identified episodic positive selection primarily in the MP gene, with significant findings at codons 123 and 192 (Fig. 4). These findings suggest that certain codons within the ToBRFV genome are subject to episodic positive selection, which may be indicative of adaptive changes in response to host immune pressures or other environmental factors. The detection of these sites is crucial for understanding the evolutionary dynamics of the virus and could have implications for antiviral strategies.
For codon 123 (GTt > ACt), belonging to the first set of codons analyzed, we observed a non-synonymous rate (beta) of 1568.25 with a weight of 1.00, indicating strong positive selection. The LRT value of 8.712 (p = 0.0057) confirmed episodic selection at this site. Similarly, codon 192 exhibited a significant non-synonymous rate of 228.83, with an LRT value of 4.490 (p = 0.0491), signifying episodic positive selection (Table 4).
These findings suggest that certain codons in the ToBRFV genome undergo adaptive changes in response to environmental factors or host pressures, which are crucial for understanding the virus’s evolutionary dynamics and have implications for vaccine design and antiviral strategies (Table 4).
Comparing our results to previous studies, Güller et al. (2023) and Esmaeilzadeh et al. (2023) reported strong purifying (negative) selection on the MP and CP gene domains of ToBRFV. Our study corroborates these findings, indicating that negative selection is the predominant force shaping the evolution of the ToBRFV genome. Additionally, Çelik et al. (2022) highlighted strong negative evolutionary constraints on the ORFs of ToBRFV (Çelik et al., 2022; Esmaeilzadeh et al., 2023; Güller et al., 2023) but we found two coding region of MP with positive selective pressures.
Codon Usage Bias Analysis of Tomato Brown Rugose Fruit Virus Genome Analysis
The comparative analysis of the codon usage bias in the MP, CP, and RdRp genes of ToBRFV revealed distinct patterns of codon usage bias and adaptation to the host’s translational machinery (Fig. 5, Supplementary Table 1–3). The codon usage patterns observed in our study align with previous research findings in plant viruses (Adams & Antoniw, 2003; Cardinale et al., 2013). Our results indicate that there are significant differences in codon preferences among the genes, reflecting the functional importance and evolutionary pressures experienced by each gene.
In our study, the GC content in the CP gene was centered around 55%, suggesting a moderate GC bias that could influence codon usage and protein structure (Fig. 5). The Effective Number of Codons (ENC) values for CP indicated a moderate level of codon bias, possibly balancing mutational pressure and natural selection. The Codon Adaptation Index (CAI) peaks in CP at around 0.7, indicating a moderate level of adaptation to the host’s translational machinery (He et al., 2023).
For the MP gene, the GC content peaked at about 50%, slightly lower than CP, potentially impacting the amino acid composition of the protein. The ENC distribution for MP suggested a similar level of codon bias as CP, while the CAI peaks at around 0.8 indicated a higher adaptation to the host’s translational efficiency compared to CP (He et al., 2023).
In the RdRp gene, the GC content peaked slightly above MP, hinting at a potential for a more stable RNA structure. The broader distribution of ENC values for RdRp suggested less codon usage bias, indicating a more diverse set of codons used for encoding amino acids. The CAI for RdRp, although similar to MP, was slightly lower, suggesting a lesser degree of adaptation to the host’s translational machinery compared to MP (He et al., 2023).
Our findings support the idea that codon usage patterns are influenced by a combination of factors, including mutational bias and translational selection, rather than solely translational selection (Adams & Antoniw, 2003; Cardinale et al., 2013). The observed differences in codon preferences among genes within the same genome may be attributed to varying evolutionary pressures and functional constraints.
Understanding these patterns of codon usage bias and adaptation to the host’s translational machinery in ToBRFV genes can provide valuable insights into the virus’s evolution and host-virus interactions, ultimately aiding in the development of effective management strategies for ToBRFV.
Table 5
Relative Synonymous Codon Usage (RSCU) Analysis for the Coat Protein (CP) Gene of Tomato Brown Rugose Fruit Virus (ToBRFV). The table provides an in-depth look at the Relative Synonymous Codon Usage (RSCU) values for the CP gene of the ToBRFV. RSCU is a measure of codon bias that compares the observed frequency of codons to the expected frequency if all synonymous codons for the same amino acid were used equally. An RSCU value of 1 indicates no bias, values greater than 1 indicate a positive bias and values less than 1 indicate a negative bias.
| aa_code | amino_acid | codon | subfam | cts | rscu | w_cai | RSCU |
1 | F | Phe | TTT | Phe_TT | 6 | 0.7 | 1 | 1.4 |
2 | F | Phe | TTC | Phe_TT | 2 | 0.3 | 0.428571 | 0.6 |
3 | L | Leu | TTA | Leu_TT | 6 | 0.583333 | 1 | 1.166667 |
4 | L | Leu | TTG | Leu_TT | 4 | 0.416667 | 0.714286 | 0.833333 |
5 | S | Ser | TCT | Ser_TC | 3 | 0.307692 | 1 | 1.230769 |
6 | S | Ser | TCC | Ser_TC | 2 | 0.230769 | 0.75 | 0.923077 |
7 | S | Ser | TCA | Ser_TC | 3 | 0.307692 | 1 | 1.230769 |
8 | S | Ser | TCG | Ser_TC | 1 | 0.153846 | 0.5 | 0.615385 |
9 | Y | Tyr | TAT | Tyr_TA | 1 | 0.333333 | 0.5 | 0.666667 |
10 | Y | Tyr | TAC | Tyr_TA | 3 | 0.666667 | 1 | 1.333333 |
11 | C | Cys | TGT | Cys_TG | 1 | 0.666667 | 1 | 1.333333 |
12 | C | Cys | TGC | Cys_TG | 0 | 0.333333 | 0.5 | 0.666667 |
13 | W | Trp | TGG | Trp_TG | 3 | 1 | 1 | 1 |
14 | L | Leu | CTT | Leu_CT | 0 | 0.142857 | 0.25 | 0.571429 |
15 | L | Leu | CTC | Leu_CT | 0 | 0.142857 | 0.25 | 0.571429 |
16 | L | Leu | CTA | Leu_CT | 3 | 0.571429 | 1 | 2.285714 |
17 | L | Leu | CTG | Leu_CT | 0 | 0.142857 | 0.25 | 0.571429 |
18 | P | Pro | CCT | Pro_CC | 6 | 0.00406 | 0.006629 | 0.016241 |
19 | P | Pro | CCC | Pro_CC | 432 | 0.25116 | 0.410038 | 1.00464 |
20 | P | Pro | CCA | Pro_CC | 227 | 0.132251 | 0.215909 | 0.529002 |
21 | P | Pro | CCG | Pro_CC | 1055 | 0.612529 | 1 | 2.450116 |
22 | H | His | CAT | His_CA | 0 | 0.002179 | 0.002183 | 0.004357 |
23 | H | His | CAC | His_CA | 457 | 0.997821 | 1 | 1.995643 |
24 | Q | Gln | CAA | Gln_CA | 1311 | 0.654691 | 1 | 1.309381 |
25 | Q | Gln | CAG | Gln_CA | 691 | 0.345309 | 0.527439 | 0.690619 |
26 | R | Arg | CGT | Arg_CG | 1 | 0.000864 | 0.001601 | 0.003454 |
27 | R | Arg | CGC | Arg_CG | 1248 | 0.539292 | 1 | 2.157168 |
28 | R | Arg | CGA | Arg_CG | 840 | 0.363126 | 0.673339 | 1.452504 |
29 | R | Arg | CGG | Arg_CG | 223 | 0.096718 | 0.179343 | 0.386874 |
30 | I | Ile | ATT | Ile_AT | 1 | 0.2 | 0.333333 | 0.6 |
31 | I | Ile | ATC | Ile_AT | 1 | 0.2 | 0.333333 | 0.6 |
32 | I | Ile | ATA | Ile_AT | 5 | 0.6 | 1 | 1.8 |
33 | M | Met | ATG | Met_AT | 1 | 1 | 1 | 1 |
34 | T | Thr | ACT | Thr_AC | 6 | 0.002291 | 0.00413 | 0.009165 |
35 | T | Thr | ACC | Thr_AC | 884 | 0.289689 | 0.522124 | 1.158756 |
36 | T | Thr | ACA | Thr_AC | 1694 | 0.554828 | 1 | 2.219313 |
37 | T | Thr | ACG | Thr_AC | 467 | 0.153191 | 0.276106 | 0.612766 |
38 | N | Asn | AAT | Asn_AA | 9 | 0.00579 | 0.005824 | 0.011581 |
39 | N | Asn | AAC | Asn_AA | 1716 | 0.99421 | 1 | 1.988419 |
40 | K | Lys | AAA | Lys_AA | 2110 | 0.562184 | 1 | 1.124368 |
41 | K | Lys | AAG | Lys_AA | 1643 | 0.437816 | 0.778778 | 0.875632 |
42 | S | Ser | AGT | Ser_AG | 2 | 0.010309 | 0.010417 | 0.020619 |
43 | S | Ser | AGC | Ser_AG | 287 | 0.989691 | 1 | 1.979381 |
44 | R | Arg | AGA | Arg_AG | 1440 | 0.805478 | 1 | 1.610956 |
45 | R | Arg | AGG | Arg_AG | 347 | 0.194522 | 0.241499 | 0.389044 |
46 | V | Val | GTT | Val_GT | 3 | 0.210526 | 0.8 | 0.842105 |
47 | V | Val | GTC | Val_GT | 4 | 0.263158 | 1 | 1.052632 |
48 | V | Val | GTA | Val_GT | 4 | 0.263158 | 1 | 1.052632 |
49 | V | Val | GTG | Val_GT | 4 | 0.263158 | 1 | 1.052632 |
50 | A | Ala | GCT | Ala_GC | 6 | 0.003091 | 0.00791 | 0.012362 |
51 | A | Ala | GCC | Ala_GC | 562 | 0.248565 | 0.636158 | 0.99426 |
52 | A | Ala | GCA | Ala_GC | 884 | 0.390728 | 1 | 1.562914 |
53 | A | Ala | GCG | Ala_GC | 809 | 0.357616 | 0.915254 | 1.430464 |
54 | D | Asp | GAT | Asp_GA | 3 | 0.005874 | 0.005908 | 0.011747 |
55 | D | Asp | GAC | Asp_GA | 676 | 0.994126 | 1 | 1.988253 |
56 | E | Glu | GAA | Glu_GA | 900 | 0.465152 | 0.869691 | 0.930305 |
57 | E | Glu | GAG | Glu_GA | 1035 | 0.534848 | 1 | 1.069695 |
58 | G | Gly | GGT | Gly_GG | 4 | 0.001699 | 0.004023 | 0.006796 |
59 | G | Gly | GGC | Gly_GG | 1051 | 0.357458 | 0.84634 | 1.429834 |
60 | G | Gly | GGA | Gly_GG | 1242 | 0.422358 | 1 | 1.689433 |
61 | G | Gly | GGG | Gly_GG | 642 | 0.218485 | 0.517297 | 0.873938 |
The Relative Synonymous Codon Usage (RSCU) values for the CP gene of ToBRFV were meticulously examined, revealing intriguing insights into codon bias patterns. RSCU serves as a metric for comparing the observed frequency of codons to the expected frequency under equal usage of synonymous codons. Notably, codons associated with Phenylalanine (Phe), Leucine (Leu), Serine (Ser), and Proline (Pro) exhibited varying degrees of bias towards specific codons, as evidenced by their RSCU values. For instance, the Proline codons CCC and CCG demonstrated substantial bias with RSCU values of 1.00464 and 2.450116, respectively, indicating a distinct preference for these codons. Similarly, Histidine (His) and Glutamine (Gln) codons displayed a pronounced bias towards CAC and CAA, respectively, with RSCU values approximating 2, suggestive of their preferential usage within the CP gene. Moreover, the Arginine (Arg) codons, particularly CGC and AGA, manifested noticeable bias, underscored by their elevated RSCU values, indicative of a predilection for these codons within the CP gene of ToBRFV (Table 5).
Furthermore, the analysis of the w_cai column, reflecting the relative adaptiveness of each codon, unveiled crucial insights into the codon usage patterns of ToBRFV. Noteworthy, values closer to 1 in the w_cai column signify higher adaptiveness, thus delineating the efficiency of gene expression and protein synthesis within ToBRFV. This comprehensive analysis not only enhances our understanding of codon usage dynamics in ToBRFV but also bears significant implications for gene expression regulation and the formulation of targeted control strategies against the virus.
Comparing our findings with those of He et al. (2022), who investigated the codon usage patterns of Narcissus late season yellows virus (NLSYV) and Narcissus yellow stripe virus (NYSV) and Narcissus degeneration virus (NDV) CP genes, a recurring preference for A/U in the third codon position across narcissus viruses was observed (He et al., 2022). This comparative analysis sheds light on the shared characteristics and distinctive features of codon usage among different viruses, providing valuable insights into the evolutionary mechanisms and selective pressures governing codon bias in viral genomes.
Therefore, the meticulous analysis of RSCU values and codon adaptation index in the CP gene of ToBRFV offers the essential groundwork for deciphering the intricate interplay between codon bias, gene expression, and viral evolution. These findings not only enrich our understanding of viral genome dynamics but also hold promise for informing future antiviral strategies and therapeutic interventions.
Table 6
Relative Synonymous Codon Usage (RSCU) Analysis for the Movement Protein (MP) Gene of Tomato Brown Rugose Fruit Virus (ToBRFV). The table provides a comprehensive Relative Synonymous Codon Usage (RSCU) analysis for the MP gene of the ToBRFV. RSCU is a measure that indicates the relative frequency of synonymous codons used for encoding each amino acid. An RSCU value of 1 suggests no bias, above 1 indicates a preference for that codon, and below 1 indicates avoidance.
| aa_code | amino_acid | codon | subfam | cts | rscu | w_cai | RSCU |
1 | F | Phe | TTT | Phe_TT | 5 | 0.545455 | 1 | 1.090909 |
2 | F | Phe | TTC | Phe_TT | 4 | 0.454545 | 0.833333 | 0.909091 |
3 | L | Leu | TTA | Leu_TT | 4 | 0.384615 | 0.625 | 0.769231 |
4 | L | Leu | TTG | Leu_TT | 7 | 0.615385 | 1 | 1.230769 |
5 | S | Ser | TCT | Ser_TC | 3 | 0.222222 | 0.5 | 0.888889 |
6 | S | Ser | TCC | Ser_TC | 2 | 0.166667 | 0.375 | 0.666667 |
7 | S | Ser | TCA | Ser_TC | 7 | 0.444444 | 1 | 1.777778 |
8 | S | Ser | TCG | Ser_TC | 2 | 0.166667 | 0.375 | 0.666667 |
9 | Y | Tyr | TAT | Tyr_TA | 4 | 0.5 | 1 | 1 |
10 | Y | Tyr | TAC | Tyr_TA | 4 | 0.5 | 1 | 1 |
11 | C | Cys | TGT | Cys_TG | 4 | 0.833333 | 1 | 1.666667 |
12 | C | Cys | TGC | Cys_TG | 0 | 0.166667 | 0.2 | 0.333333 |
13 | W | Trp | TGG | Trp_TG | 2 | 1 | 1 | 1 |
14 | L | Leu | CTT | Leu_CT | 5 | 0.375 | 1 | 1.5 |
15 | L | Leu | CTC | Leu_CT | 4 | 0.3125 | 0.833333 | 1.25 |
16 | L | Leu | CTA | Leu_CT | 2 | 0.1875 | 0.5 | 0.75 |
17 | L | Leu | CTG | Leu_CT | 1 | 0.125 | 0.333333 | 0.5 |
18 | P | Pro | CCT | Pro_CC | 1 | 0.001021 | 0.001828 | 0.004086 |
19 | P | Pro | CCC | Pro_CC | 110 | 0.056691 | 0.101463 | 0.226762 |
20 | P | Pro | CCA | Pro_CC | 750 | 0.383555 | 0.686472 | 1.534219 |
21 | P | Pro | CCG | Pro_CC | 1093 | 0.558733 | 1 | 2.234934 |
22 | H | His | CAT | His_CA | 2 | 0.005348 | 0.005376 | 0.010695 |
23 | H | His | CAC | His_CA | 557 | 0.994652 | 1 | 1.989305 |
24 | Q | Gln | CAA | Gln_CA | 1966 | 0.486159 | 0.946128 | 0.972318 |
25 | Q | Gln | CAG | Gln_CA | 2078 | 0.513841 | 1 | 1.027682 |
26 | R | Arg | CGT | Arg_CG | 2 | 0.001029 | 0.002086 | 0.004117 |
27 | R | Arg | CGC | Arg_CG | 1437 | 0.49331 | 1 | 1.973242 |
28 | R | Arg | CGA | Arg_CG | 788 | 0.270669 | 0.548679 | 1.082676 |
29 | R | Arg | CGG | Arg_CG | 684 | 0.234991 | 0.476356 | 0.939966 |
30 | I | Ile | ATT | Ile_AT | 8 | 0.5625 | 1 | 1.6875 |
31 | I | Ile | ATC | Ile_AT | 2 | 0.1875 | 0.333333 | 0.5625 |
32 | I | Ile | ATA | Ile_AT | 3 | 0.25 | 0.444444 | 0.75 |
33 | M | Met | ATG | Met_AT | 7 | 1 | 1 | 1 |
34 | T | Thr | ACT | Thr_AC | 3 | 0.001523 | 0.00385 | 0.006091 |
35 | T | Thr | ACC | Thr_AC | 1038 | 0.395508 | 1 | 1.582033 |
36 | T | Thr | ACA | Thr_AC | 638 | 0.243243 | 0.615014 | 0.972973 |
37 | T | Thr | ACG | Thr_AC | 944 | 0.359726 | 0.909528 | 1.438904 |
38 | N | Asn | AAT | Asn_AA | 15 | 0.010936 | 0.011057 | 0.021873 |
39 | N | Asn | AAC | Asn_AA | 1446 | 0.989064 | 1 | 1.978127 |
40 | K | Lys | AAA | Lys_AA | 4647 | 0.572273 | 1 | 1.144546 |
41 | K | Lys | AAG | Lys_AA | 3473 | 0.427727 | 0.747418 | 0.855454 |
42 | S | Ser | AGT | Ser_AG | 6 | 0.009576 | 0.009669 | 0.019152 |
43 | S | Ser | AGC | Ser_AG | 723 | 0.990424 | 1 | 1.980848 |
44 | R | Arg | AGA | Arg_AG | 2698 | 0.545253 | 1 | 1.090505 |
45 | R | Arg | AGG | Arg_AG | 2250 | 0.454747 | 0.834013 | 0.909495 |
46 | V | Val | GTT | Val_GT | 13 | 0.4 | 1 | 1.6 |
47 | V | Val | GTC | Val_GT | 10 | 0.314286 | 0.785714 | 1.257143 |
48 | V | Val | GTA | Val_GT | 4 | 0.142857 | 0.357143 | 0.571429 |
49 | V | Val | GTG | Val_GT | 4 | 0.142857 | 0.357143 | 0.571429 |
50 | A | Ala | GCT | Ala_GC | 5 | 0.002223 | 0.003859 | 0.008892 |
51 | A | Ala | GCC | Ala_GC | 269 | 0.100037 | 0.173633 | 0.400148 |
52 | A | Ala | GCA | Ala_GC | 1554 | 0.576139 | 1 | 2.304557 |
53 | A | Ala | GCG | Ala_GC | 867 | 0.321601 | 0.558199 | 1.286402 |
54 | D | Asp | GAT | Asp_GA | 11 | 0.013393 | 0.013575 | 0.026786 |
55 | D | Asp | GAC | Asp_GA | 883 | 0.986607 | 1 | 1.973214 |
56 | E | Glu | GAA | Glu_GA | 2709 | 0.56096 | 1 | 1.121921 |
57 | E | Glu | GAG | Glu_GA | 2120 | 0.43904 | 0.782657 | 0.878079 |
58 | G | Gly | GGT | Gly_GG | 11 | 0.002415 | 0.004601 | 0.009662 |
59 | G | Gly | GGC | Gly_GG | 969 | 0.19525 | 0.371933 | 0.780998 |
60 | G | Gly | GGA | Gly_GG | 2607 | 0.52496 | 1 | 2.099839 |
61 | G | Gly | GGG | Gly_GG | 1377 | 0.277375 | 0.528374 | 1.109501 |
In this study, we conducted a comprehensive Relative Synonymous Codon Usage (RSCU) analysis for the MP and RdRp genes of ToBRFV. RSCU provides valuable insights into the preferences and biases in codon usage, shedding light on the genetic characteristics of the virus. The RSCU values, presented in Tables 6 and 7, reveal distinct patterns of codon usage in both genes, which could have significant implications for gene expression efficiency, protein synthesis, and the development of control strategies against ToBRFV.
Analyzing the MP gene of ToBRFV, our results indicate specific codon preferences. Notably, Phenylalanine (Phe) codons TTT and TTC exhibit a slight preference for TTT (RSCU = 1.090909), whereas Leucine (Leu) codons favor TTG over TTA (RSCU = 1.230769 and 0.769231, respectively). Additionally, Serine (Ser) codons demonstrate a strong preference for TCA (RSCU = 1.777778), and Proline (Pro) codons display a significant bias towards CCG (RSCU = 2.234934). Furthermore, Histidine (His), Glutamine (Gln), and Arginine (Arg) codons show notable biases towards CAC, CAG, and CGC, respectively, with RSCU values close to 2.
Table 7
Relative Synonymous Codon Usage (RSCU) Analysis for the RNA-Dependent RNA Polymerase (RdRp) Gene of Tomato Brown Rugose Fruit Virus (ToBRFV). The table presents the RSCU values for the RdRp gene of ToBRFV, offering insights into codon usage preferences. RSCU values greater than 1 indicate a bias towards a particular codon, while values less than 1 suggest avoidance.
| aa_code | amino_acid | codon | subfam | cts | rscu | w_cai | RSCU |
1 | F | Phe | TTT | Phe_TT | 45 | 0.589744 | 1 | 1.179487 |
2 | F | Phe | TTC | Phe_TT | 31 | 0.410256 | 0.695652 | 0.820513 |
3 | L | Leu | TTA | Leu_TT | 38 | 0.5 | 1 | 1 |
4 | L | Leu | TTG | Leu_TT | 38 | 0.5 | 1 | 1 |
5 | S | Ser | TCT | Ser_TC | 36 | 0.377551 | 1 | 1.510204 |
6 | S | Ser | TCC | Ser_TC | 19 | 0.204082 | 0.540541 | 0.816327 |
7 | S | Ser | TCA | Ser_TC | 21 | 0.22449 | 0.594595 | 0.897959 |
8 | S | Ser | TCG | Ser_TC | 18 | 0.193878 | 0.513514 | 0.77551 |
9 | Y | Tyr | TAT | Tyr_TA | 34 | 0.514706 | 1 | 1.029412 |
10 | Y | Tyr | TAC | Tyr_TA | 32 | 0.485294 | 0.942857 | 0.970588 |
11 | C | Cys | TGT | Cys_TG | 24 | 0.757576 | 1 | 1.515152 |
12 | C | Cys | TGC | Cys_TG | 7 | 0.242424 | 0.32 | 0.484848 |
13 | W | Trp | TGG | Trp_TG | 14 | 1 | 1 | 1 |
14 | L | Leu | CTT | Leu_CT | 35 | 0.409091 | 1 | 1.636364 |
15 | L | Leu | CTC | Leu_CT | 16 | 0.193182 | 0.472222 | 0.772727 |
16 | L | Leu | CTA | Leu_CT | 16 | 0.193182 | 0.472222 | 0.772727 |
17 | L | Leu | CTG | Leu_CT | 17 | 0.204545 | 0.5 | 0.818182 |
18 | P | Pro | CCT | Pro_CC | 15 | 0.001005 | 0.00233 | 0.004021 |
19 | P | Pro | CCC | Pro_CC | 3854 | 0.242224 | 0.561381 | 0.968897 |
20 | P | Pro | CCA | Pro_CC | 6866 | 0.43148 | 1 | 1.725919 |
21 | P | Pro | CCG | Pro_CC | 5176 | 0.325291 | 0.753895 | 1.301162 |
22 | H | His | CAT | His_CA | 21 | 0.002775 | 0.002782 | 0.005549 |
23 | H | His | CAC | His_CA | 7906 | 0.997225 | 1 | 1.994451 |
24 | Q | Gln | CAA | Gln_CA | 11534 | 0.52942 | 1 | 1.05884 |
25 | Q | Gln | CAG | Gln_CA | 10252 | 0.47058 | 0.88886 | 0.94116 |
26 | R | Arg | CGT | Arg_CG | 8 | 0.000468 | 0.001349 | 0.001873 |
27 | R | Arg | CGC | Arg_CG | 6123 | 0.318626 | 0.918003 | 1.274506 |
28 | R | Arg | CGA | Arg_CG | 6670 | 0.347086 | 1 | 1.388345 |
29 | R | Arg | CGG | Arg_CG | 6415 | 0.333819 | 0.961775 | 1.335276 |
30 | I | Ile | ATT | Ile_AT | 37 | 0.431818 | 1 | 1.295455 |
31 | I | Ile | ATC | Ile_AT | 23 | 0.272727 | 0.631579 | 0.818182 |
32 | I | Ile | ATA | Ile_AT | 25 | 0.295455 | 0.684211 | 0.886364 |
33 | M | Met | ATG | Met_AT | 45 | 1 | 1 | 1 |
34 | T | Thr | ACT | Thr_AC | 32 | 0.001208 | 0.002819 | 0.004834 |
35 | T | Thr | ACC | Thr_AC | 7244 | 0.265317 | 0.618966 | 1.061266 |
36 | T | Thr | ACA | Thr_AC | 11704 | 0.428645 | 1 | 1.714579 |
37 | T | Thr | ACG | Thr_AC | 8323 | 0.30483 | 0.711149 | 1.219321 |
38 | N | Asn | AAT | Asn_AA | 39 | 0.003893 | 0.003909 | 0.007787 |
39 | N | Asn | AAC | Asn_AA | 10233 | 0.996107 | 1 | 1.992213 |
40 | K | Lys | AAA | Lys_AA | 15986 | 0.535811 | 1 | 1.071622 |
41 | K | Lys | AAG | Lys_AA | 13849 | 0.464189 | 0.866329 | 0.928378 |
42 | S | Ser | AGT | Ser_AG | 24 | 0.002665 | 0.002672 | 0.005329 |
43 | S | Ser | AGC | Ser_AG | 9356 | 0.997335 | 1 | 1.994671 |
44 | R | Arg | AGA | Arg_AG | 15354 | 0.572905 | 1 | 1.14581 |
45 | R | Arg | AGG | Arg_AG | 11446 | 0.427095 | 0.74549 | 0.85419 |
46 | V | Val | GTT | Val_GT | 41 | 0.311111 | 1 | 1.244444 |
47 | V | Val | GTC | Val_GT | 27 | 0.207407 | 0.666667 | 0.82963 |
48 | V | Val | GTA | Val_GT | 28 | 0.214815 | 0.690476 | 0.859259 |
49 | V | Val | GTG | Val_GT | 35 | 0.266667 | 0.857143 | 1.066667 |
50 | A | Ala | GCT | Ala_GC | 29 | 0.001415 | 0.002957 | 0.005661 |
51 | A | Ala | GCC | Ala_GC | 5457 | 0.257501 | 0.537893 | 1.030006 |
52 | A | Ala | GCA | Ala_GC | 10146 | 0.478722 | 1 | 1.91489 |
53 | A | Ala | GCG | Ala_GC | 5560 | 0.262361 | 0.548044 | 1.049443 |
54 | D | Asp | GAT | Asp_GA | 67 | 0.006883 | 0.00693 | 0.013765 |
55 | D | Asp | GAC | Asp_GA | 9811 | 0.993117 | 1 | 1.986235 |
56 | E | Glu | GAA | Glu_GA | 12496 | 0.526633 | 1 | 1.053266 |
57 | E | Glu | GAG | Glu_GA | 11232 | 0.473367 | 0.898856 | 0.946734 |
58 | G | Gly | GGT | Gly_GG | 27 | 0.001094 | 0.002342 | 0.004375 |
59 | G | Gly | GGC | Gly_GG | 6962 | 0.271971 | 0.582337 | 1.087884 |
60 | G | Gly | GGA | Gly_GG | 11956 | 0.467034 | 1 | 1.868135 |
61 | G | Gly | GGG | Gly_GG | 6653 | 0.259902 | 0.556494 | 1.039606 |
For the RdRp gene of ToBRFV, our analysis reveals distinct codon usage preferences. Phenylalanine (Phe) codons favor TTT over TTC, while Leucine (Leu) codons exhibit no bias between TTA and TTG. Serine (Ser) codons display a moderate bias, with TCT showing the highest RSCU value. Proline (Pro) codons show a strong bias towards CCA and CCG, and Histidine (His) and Glutamine (Gln) codons exhibit a strong bias towards CAC and CAA, respectively. Moreover, Arginine (Arg) codons display notable biases, particularly for CGA and CGC.
Comparing our findings with previous studies on cucurbit-infecting tobamoviruses (He et al., 2023), we observe similarities in codon usage preferences, particularly in the preference of U over C in most synonymous third codon positions. This consistency across tobamoviruses underscores the potential for targeted interventions in viral gene control and attenuation. Strategies such as deoptimizing synonymous codons less used by both the virus and host may offer avenues for reducing viral gene expression and virulence. However, considerations of codon-specific biases, such as increasing codons ending with CpA to align with host preferences, must be carefully evaluated to avoid adverse effects on viral survival.
Moreover, our study contributes to understanding the mutation pressures shaping codon usage in ToBRFV genes. We found a preference for A/G in TuMV protein-coding regions, indicative of mutation pressures (Qin et al., 2022). This insight underscores the dynamic nature of codon usage and its implications for viral evolution and adaptation.
In conclusion, our analysis provides valuable insights into the codon usage patterns of ToBRFV, which could inform strategies for controlling viral gene expression and virulence. Further research into the functional implications of codon biases and their interplay with host factors is warranted to deepen our understanding of virus-host interactions and aid in the development of effective control measures against ToBRFV.