To evaluate the stability and physical movement of atoms in docked complexes, we performed molecular dynamics simulations using the iMODS server ³⁴. The simulations were performed in NMA. The results of the apo-eIF4E, m7GTP-eIF4E and hesperetin-eIF4E complex are depicted in Fig. 8. The affine-model (Fig. 8A, large coloured arrows) representation and NMA mobility based colour scheme of eIF4E residue were utilized to observe the mobility of eIF4E regions in all three cases. ³⁴. As shown in Fig. 8A (upper panel) , the apo-eIF4E loop regions displayed a bathochromic shift of colors in its backbone (green, yellow and red shades). Besides, wider affine-model (colored arrows) and longer arrow fields (black arrows) also suggested higher mobility of these residues as compared to the eIF4E-m7GTP and eIF4E-hesperetin complexes (Fig. 8A) .

To gain further insights into the protein flexibility and mobility, we analysed the deformability and B factor plots, respectively, which were generated by the iMODS server (Fig 8B and Fig. 8C) . High peaks in the deformability plot of apo-eIF4E demonstrated flexible regions (Fig 8B, upper panel) which diminished after complexion with m7GTP and hesperetin (Fig 8B, middle and lower panel) . This was complimented by the B-factor plot (Fig 8C), which is proportional to the root mean square (RMS) and demonstrate the stabilities of m7GTP- and hesperetin-docked complexes. Additionally, high eigenvalues of 7.542249e^-05 and 7.541401e^-05 were observed for m7GTP- and hesperetin-docked complexes respectively (Fig 8D), which when compare with the eigenvalues of apo-eIF4E (3.114206e^-04) indicated a higher energy requirement for the deformation of docked complexes. This suggests the stability of docked complexes . The analysis of variance of individual modes (indicating their relative contribution to the equilibrium motions) (Fig. 8E) , covariance map (indicating coupling between correlated, uncorrelated and anti-correlated motions residues) (Fig. 8F) and elastic network model (correlating to the stiffness of the complex) (Fig. 8G ) inferred a close resemblance between the m7GTP-eIF4E and hesperetin-eIF4E binding. In contrast, apo-eIF4E, displayed high mobility and flexibility due to highly uncorrelated and elastic motion between atoms.

A wide variety of poxviruses, including BPXV, induce pock lesions on the CAM in embryonated chicken eggs ^38,39. To evaluate thein ovo antiviral efficacy of hesperetin against BPXV, we first determined the subcytotoxic concentration of hesperetin in specific pathogen free (SPF) embryonated chicken eggs. As shown in Fig. 9A, hesperetin at a dose rate of ≥4000 µg/egg induced mortality in the chicken embryos, whereas no mortality was observed at a dose rate of 1000 µg/egg. The LD₅₀ of hesperetin was determined to be 1080.74 µg/egg. For evaluation of the in ovo antiviral efficacy, the eggs were infected with BPXV at 100 EID₅₀ and treated with the indicated concentrations of hesperetin. As shown inFig. 9B, hesperetin provided protection from BPXV challenge infection in a dose-dependent manner. Moreover, the size of localized pock lesion was also suppressed with increasing concentrations of hesperetin (Fig. 9C) . The EC₅₀ was calculated to be 22.5 µg/egg and the therapeutic index 48.1.

We also evaluated the potential of hesperetin to select drug resistant virus mutants under the long-term selection pressure of hesperetin, wherein BPXV was sequentially passaged (P) in the presence of hesperetin or an equivalent volume of DMSO. The infectious virions collected in the supernatant were used for the next round of infection, and the process was repeated until P40. The resulting viruses were named BPXV-P40-hesperetin and BPXV-P40-DMSO. As shown in Fig. 10, the levels of suppression of the virus yield by hesperetin in Vero cells were comparable in both BPXV-P40-hesperetin and BPXV-P40-DMSO. This suggested that hesperetin does not select drug-resistant virus mutants.

In this study, we demonstrated the in vitro antiviral efficacy of hesperetin against multiple poxviruses, which include BPXV, VACV and LSDV. This, together with hesperetin-induced protection in chicken embryos against virulent BPXV, led to the conclusion that hesperetin may potentially be developed as a broad-spectrum antiviral drug against poxviruses. Previous studies have also identified in vitroantiviral activity of hesperetin against DENV ²⁰, CHIKV ²¹, ZIKV ²¹ and Sindbis virus²². The docking and molecular dynamics simulations displayed the effect of hesperetin on the protease 3D models of CHIKV and ZIKV ²⁰. However, the precise mechanism of the antiviral action of hesperetin remains unknown.

The incubation of cell free virions with hesperetin did not affect the residual infectivity of the virions, which seems to suggest that hesperetin may not be directly targeting viral factors in poxviruses, although this needs further in-depth investigation. We demonstrated that hesperetin treatment mainly suppresses BPXV protein synthesis with a marginal inhibitory effect on the levels of viral DNA and mRNA but without affecting other steps of the viral life cycle such as attachment, entry, and budding. The 5’-cap of mRNA is highly conserved and is an important structural modification critical for eIF4E-mRNA interaction. In order to effectively synthesize viral proteins and to circumvent the action of 5′-3′ exonucleases, viral capping mechanisms that generated mRNA capping identical to that of the host were selected during the co-evolution of viruses and hosts ⁴⁰. Previous studies have demonstrated that BPXV exploits cap-dependent mechanism of protein translation ^24,35,41. Therefore, we asked whether hesperetin blocks BPXV translation initiation by abrogating the interaction of the 5’ cap of viral mRNA and eIF4E. The CHIP assay suggested that hesperetin disrupts binding of the 5’ cap (m7GTP binds) of viral mRNA with eIF4E. The molecular docking and MD simulation studies, also confirmed stable binding of the hesperetin with the cap-binding pocket of eIF4E, in a similar conformation as m7GTP binds. This is a novel mechanism wherein hesperetin has been shown to exert its indirect antiviral action by targeting a cellular factor (eIF4E). Further, in agreement with the previous studies^24,41, we demonstrated that BPXV exploits ERK-MNK1-eIF4E signalling to effectively replicate in the target cells. Hesperetin targets this signalling axis by inhibiting the interaction of eIF4E with viral mRNA, which results in the shut off of viral protein translation.

One of the major limitations of antiviral drugs is that they rapidly induce the development of drug-resistant viral mutants⁴². However, as compared to the drugs that directly target viral factors, drugs based on targeting essential cellular factors are considered to have minimal or no tendency to induce antiviral drug resistance ²⁴. In our study, hesperetin-resistant BPXV mutants were not observed even when the virus was sequentially cultured 40 times in the presence of hesperetin. This seems to be due to the low genetic variability of the host factor (eIF4E), thereby imposing a higher genetic barrier to the generation of resistant viruses ^24,25,41.

Since hesperetin can alter cell metabolism, its long-term use could eventually result in cytotoxicity. Therefore, its further validation, long-term in vivo efficacy, and clinical trials will be essential before actually introducing it from research into clinical settings.

In conclusion, hesperetin was shown to exert a potent in vitroand in ovo antiviral efficacy against poxviruses. Mechanistically, hesperetin was shown to competitively inhibit binding of the viral mRNA with eIF4E (Fig. 11), thereby blocking viral protein translation. Most importantly, hesperetin was not shown to readily select drug-resistant viral mutants.