(1) Background: The highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b is an emerging threat that poses a great risk to the poultry industry. A few human cases have been linked to the infection with this clade in many parts of the world, including the USA. Unfortunately, there are no specific vaccines or antiviral drugs that could help prevent and treat the infection caused by this virus in birds. Our major objective is to identify/repurpose some (novel/known) antiviral compounds that may inhibit viral replication by targeting some key viral proteins. (2) Methods: We used state-of-the-art machine learning tools such as molecular docking and MD-simulation methods from Biovia Discovery Studio (v24.1.0.321712). The key target proteins such as hemagglutinin (HA), neuraminidase (NA), Matrix-2 protein (M2), and the cap-binding domain of PB2 (PB2/CBD) homology models were validated through structural assessment via DOPE scores, Ramachandran plots, and Verify-3D metrics, ensuring reliable structural representations, confirming their reliability for subsequent in silico approaches. These approaches include molecular docking followed by molecular dynamics simulation for 50 nanoseconds (ns), highlighting the structural stability and compactness of the docked complexes. (3) Results: Molecular docking revealed strong binding affinities for both sofosbuvir and GS441524, particularly with the NA and PB2/CBD protein targets. Among them, GS441524 exhibited superior interaction scores and a greater number of hydrogen bonds with key functional residues of NA and PB2/CBD. The MM-GBSA binding free energy calculations further supported these findings, as GS441524 displayed more favorable binding energies compared to several known standard inhibitors, including F0045S for HA, Zanamivir for NA, Rimantadine and Amantadine for M2, and PB2-39 for PB2/CBD. Additionally, 50 ns molecular dynamics simulations highlighted the structural stability and compactness of the GS441524-PB2/CBD complex, further supporting its potential as a promising antiviral candidate. Furthermore, hydrogen bond monitor analysis over the 50 ns simulation confirmed persistent and specific interactions between the ligand and proteins, suggesting that GS441524 may effectively inhibit the NA, and PB2/CBD might potentially disrupt PB2-mediated RNA synthesis. (4) Conclusions: Our findings are consistent with previous evidence supporting the antiviral activity of certain nucleoside analog inhibitors, including GS441524, against various coronaviruses. These results further support the potential repurposing of GS441524 as a promising therapeutic candidate against H5N1 avian influenza clade 2.3.4.4b. However, further functional studies are required to validate these in silico predictions and support the inhibitory action of GS441524 against the targeted proteins of H5N1, specifically clade 2.3.4.4b.

The presently used vaccines do not offer solid immunity/protection against the currently circulating strains of the H5N1 viruses. We aim to design a pan-H5N1 vaccine that protects birds against the presently circulating clade 2.3.4.4b in chickens. We used AI tools, including epitope mapping, molecular docking, and immune simulation, to design a multiepitope DNA vaccine including the top-ranked B and T cell epitopes within four major proteins (HA, NA, NP, and M2) of H5N1 clade 2.3.4.4b. We selected the top-ranked 12 epitopes and linked them together using linkers. The designed vaccine is linked to IL-18 as an adjuvant. The molecular docking results showed a high binding affinity of those predicted epitopes from the MHC I and MHC II classes of molecules with chicken alleles. The immune simulation results showed that the designed vaccine has the potential to stimulate the host immune response, including antibody and cell-mediated immunity in chickens and other birds. We believe this vaccine is going to be a universal vaccine that offers good protection against HPAI-H5N1 clade 2.3.4.4b. We are reporting the successful molecular cloning of a recombinant multiepitope-based vaccine spanning some key epitopes within some key proteins of the currently circulating H5N1 clade 2.3.4.4b. These designed vaccines could be a great positive impact on the protection of birds and various species of animals, as well as humans, against the HP-H5N1 influenza virus. Further studies are required to validate this vaccine candidate in chickens.

Bovine coronavirus (BCoV) exhibits dual tissue tropism, infecting both the respiratory and enteric tracts of cattle. Viral entry into host cells requires a coordinated interaction between viral and host proteins. However, the specific cellular receptors and co-receptors facilitating BCoV entry remain poorly understood. Similarly, the roles of host proteases such as Furin, TMPRSS2, and Cathepsin-L (CTS-L), known to assist in the replication of other coronaviruses, have not been extensively explored for BCoV. This study aims to identify novel BCoV receptors and host proteases that modulate viral replication and tissue tropism. Bovine cell lines were infected with BCoV isolates from enteric and respiratory origins, and the host cell gene expression profiles post-infection were analyzed using next-generation sequencing (NGS). Differentially expressed genes encoding potential receptors and proteases were further assessed using in-silico prediction and molecular docking analysis. These analyses focused on known coronavirus receptors, including ACE2, NRP1, DPP4, APN, AXL, and CEACAM1, to identify their potential roles in BCoV infection. Validation of these findings was performed using the qRT-PCR assays targeting individual genes. We confirmed the gene expression profiles of these receptors and enzymes in some BCoV (+/−) lung tissues. Results revealed high binding affinities of 9-O-acetylated sialic acid and NRP1 to BCoV spike (S) and hemagglutinin-esterase (HE) proteins compared to ACE2, DPP4, and CEACAM1. Additionally, Furin and TMPRSS2 were predicted to interact with the BCoV-S polybasic cleavage site (RRSRR|A), suggesting their roles in S glycoprotein activation. This is the first study to explore the interactions of BCoV with multiple host receptors and proteases. Functional studies are recommended to confirm their roles in BCoV infection and replication.

(1) Background: The highly pathogenic avian influenzas virus H5N1 clade 2.3.4.4b is an emerging threat that poses great risk to the poultry industry. Few human cases have been linked to the infection with this clade in many parts of the world, including the USA. Unfortunately, there are no specific vaccines or antiviral drugs that could help prevent and treat the infection of this virus in birds. Our major objective is to identify/repurpose some (novel/known) antiviral compounds that may inhibit viral replication by targeting some key viral proteins. (2) Methods: We used state-of-the-art machine learning tools such as molecular docking and MD-simulation methods from Bio-via Discovery Studio (v24.1.0.321712). The target proteins homology models were validated through structural assessment via DOPE scores, Ramachandran plots, and Ver-ify-3D metrics, ensuring reliable structural representations, confirming their reliability for subsequent in silico approaches, including molecular docking and molecular dynamics simulation for 50 ns highlighted the structural stability and compactness of the docked complexes. (3) Results: Molecular docking revealed strong binding affinities of both compounds, particularly the GS441524, which demonstrated superior interaction energy and hydrogen bonding with critical functional residues of (HA, NA, M2, and PB2/CBD). The MM-GBSA binding free energy calculations supported these findings, with GS441524 showing more favorable energies than several known standard inhibitors for HA (F0045S), NA (Zanamivir), M2 (Rimantadine and Amantadine) and PB2/CBD (Pb2-39), especially in its interaction with NA and PB2/CBD. Molecular dynamics simulations over 50 ns highlighted the structural stability and compactness of the GS441524 complex with PB2/CBD. Hydrogen bond analysis further confirmed persistent and specific interactions, suggesting that GS441524 may effectively disrupt PB2-mediated RNA synthesis. (4) Conclusions: Our findings are consistent with previous evidence supporting the antiviral activity of certain nucleoside analog inhibitors, including GS441524, against various coronaviruses. These results further support the potential repurposing of GS441524 as a promising therapeutic candidate against H5N1 avian influenza clade 2.3.4.4b. However, additional functional studies are necessary to validate these in silico predictions of its anti-H5N1 activity.

Background: Melanin protects skin and hair from the effects of ultraviolet (UV) radiation damage, which contributes to all forms of skin cancer, including melanoma. Human melanocytes produce two main types of melanin: eumelanin provides effective photoprotection, and pheomelanin offers less protection against UV-induced skin damage. The agouti signaling protein (ASIP) antagonizes the melanocortin-1 receptor (MC1R), hinders melanocyte signaling, and shifts pigmentation toward pheomelanin, promoting UV vulnerability. In this study, we aim to discover compounds that inhibit ASIP–MC1R interaction and effectively preserve eumelanogenic signaling. Methods: The ASIP–MC1R interface-based pharmacophore model from ASIP is implicated in MC1R receptor protein engagement. We performed virtual screening with a validated pharmacophore model for ~4000 compounds curated from ZINCPharmer and applied drug-likeness filters, viz. ADMET and toxicity profiling tests. Further, the screened candidates were targeted for docking to the ASIP C-terminal domain corresponding to the MC1R-binding moiety. Top compounds underwent a 100-nanosecond (ns) run of molecular dynamics (MD) simulations to assess complex stability and persistence of key contacted residues. Results: Sequential triage, including pharmacophore, ADME–toxicity (ADMET), and docking/ΔG, yielded a focused group of candidates against ASIP antagonists with a favorable fit value. The MD run for 100 ns supported pose stability at the targeted pocket. Based on these predictions and analyses, compound ZINC14539068 was screened as a new potent inhibitor of ASIP to preserve α-MSH-mediated signaling of MC1R. Conclusions: Our in silico pipeline identifies ZINC14539068 as a potent inhibitor of ASIP at its C-terminal interface. This compound is predicted to disrupt ASIP–MC1R binding, thereby maintaining eumelanin-biased signaling. These findings motivate experimental validation in melanocytic models and in vivo studies to confirm pathway modulation and anti-melanoma potential.

Parvovirus infection causes severe diseases in both feline and canine species. It primarily affects adult cats and dogs but poses a higher risk to kittens and puppies. This virus is highly contagious and is easily transmitted through contaminated food, shared shelter environments, as well as the hands and clothing of people. The recovered species may continue to shed parvovirus in their feces for an extended period, leading to severe environmental contamination. There is no universal vaccine available that protects dogs and cats against parvovirus infections. The main goal of this study is to design a pan-parvovirus multiepitope-based vaccine that could be administered to dogs and cats. We utilized AI-machine learning-incorporated server tools such as IEDB and NetMHCpan to predict B-cell and T-cell epitopes. VaxiJen and ToxinPred were used to analyze immune characteristic features and docking with feline alleles using the HADDOCK server. Following this, the immune response and stability of the vaccine construct were confirmed with disulfide engineering, normal mode analysis, and molecular docking performed with toll-like receptors of both feline and canine (TLR4 and TLR5), and molecular dynamics simulation was performed for 10 ns. The triggered immune response was determined with immuno-simulation (ImmSim), and their activity in a biological environment was reinforced with in silico cloning. The B-cell epitopes (NS1-9, NS2-4, VP1-12 and VP2-9) predicted with the IEDB database were subjected to antigenicity prediction. MHC class I and IFN prediction and MHC class II and IL-4 prediction were performed with IEDB and NetMHCpan. The T-cell epitopes showed high binding affinities with the feline alleles. The final vaccine was designed by combining the top-ranked B-cell epitopes and T-cell epitopes, filtered for high antigenicity, non-allergic, non-toxic, and good solubility, and with the better binding affinity score of the structural and non-structural proteins (NS1, NS2, VP1, and VP2) of feline and canine parvoviruses through linkers and adjuvants. The disulfide bond prediction and normal mode analysis showed that our vaccine construct is stable and flexible. The molecular docking analysis was performed between the designed vaccine epitopes and the TLRs (TLR4–feline and TLR5–canine) with Biovia Discovery Studio using Zdock; it showed better binding interactions with a value of 22.26 (Zdock score), −47.409 (Zrank score) for feline and 16.54 (Zdock score), −134.295 (Zrank score) for canine. A pan-multi-epitope-based vaccine based on the two structural and non-structural proteins (NS1, NS2, VP1, and VP2) was designed and constructed to provide dual protection against parvovirus in both feline and canine species. The molecular docking and molecular dynamics simulation analysis showed higher binding affinities and stable conformations with canine (TLR5) and feline (TLR4) toll-like receptors. Although computational analysis supports the prediction of top-ranked epitopes and their immunogenic properties with greater precision, further experimental validation is required before they can be used against these viruses.