Anti-rheumatic colchicine phytochemical exhibits potent antiviral activities against avian and seasonal Influenza A viruses (IAVs) via targeting different stages of IAV replication cycle.

BACKGROUND: The continuous evolution of drug-resistant influenza viruses highlights the necessity for repurposing naturally-derived and safe phytochemicals with anti-influenza activity as novel broad-spectrum anti-influenza medications. METHODS: In this study, nitrogenous alkaloids were tested for their viral inhibitory activity against influenza A/H1N1 and A/H5N1 viruses. The cytotoxicity of tested alkaloids on MDCK showed a high safety range (CC50 > 200 µg/ml), permitting the screening for their anti-influenza potential. RESULTS: Herein, atropine sulphate, pilocarpine hydrochloride and colchicine displayed anti-H5N1 activities with IC50 values of 2.300, 0.210 and 0.111 µg/ml, respectively. Validation of the IC50 values was further depicted by testing the three highly effective alkaloids, based on their potent IC50 values against seasonal influenza A/H1N1 virus, showing comparable IC50 values of 0.204, 0.637 and 0.326 µg/ml, respectively. Further investigation suggests that colchicine could suppress viral infection by primarily interfering with IAV replication and inhibiting viral adsorption, while atropine sulphate and pilocarpine hydrochloride could directly affect the virus in a cell-free virucidal effect. Interestingly, the in silico molecular docking studies suggest the abilities of atropine, pilocarpine, and colchicine to bind correctly inside the active sites of the neuraminidases of both influenza A/H1N1 and A/H5N1 viruses. The three alkaloids exhibited good binding energies as well as excellent binding modes that were similar to the co-crystallized ligands. On the other hand, consistent with in vitro results, only colchicine could bind correctly against the M2-proton channel of influenza A viruses (IAVs). This might explicate the in vitro antiviral activity of colchicine at the replication stage of the virus replication cycle. CONCLUSION: This study highlighted the anti-influenza efficacy of biologically active alkaloids including colchicine. Therefore, these alkaloids should be further characterized in vivo (preclinical and clinical studies) to be developed as anti-IAV agents.

Vaccination and Antiviral Treatment against Avian Influenza H5Nx Viruses: A Harbinger of Virus Control or Evolution.

Despite the panzootic nature of emergent highly pathogenic avian influenza H5Nx viruses in wild migratory birds and domestic poultry, only a limited number of human infections with H5Nx viruses have been identified since its emergence in 1996. Few countries with endemic avian influenza viruses (AIVs) have implemented vaccination as a control strategy, while most of the countries have adopted a culling strategy for the infected flocks. To date, China and Egypt are the two major sites where vaccination has been adopted to control avian influenza H5Nx infections, especially with the widespread circulation of clade 2.3.4.4b H5N1 viruses. This virus is currently circulating among birds and poultry, with occasional spillovers to mammals, including humans. Herein, we will discuss the history of AIVs in Egypt as one of the hotspots for infections and the improper implementation of prophylactic and therapeutic control strategies, leading to continuous flock outbreaks with remarkable virus evolution scenarios. Along with current pre-pandemic preparedness efforts, comprehensive surveillance of H5Nx viruses in wild birds, domestic poultry, and mammals, including humans, in endemic areas is critical to explore the public health risk of the newly emerging immune-evasive or drug-resistant H5Nx variants.

Phytoestrogen ß-Sitosterol Exhibits Potent In Vitro Antiviral Activity against Influenza A Viruses.

Influenza is a contagious infection in humans that is caused frequently by low pathogenic seasonal influenza viruses and occasionally by pathogenic avian influenza viruses (AIV) of H5, H7, and H9 subtypes. Recently, the clinical sector in poultry and humans has been confronted with many challenges, including the limited number of antiviral drugs and the rapid evolution of drug-resistant variants. Herein, the anti-influenza activities of various plant-derived phytochemicals were investigated against highly pathogenic avian influenza A/H5N1 virus (HPAIV H5N1) and seasonal low pathogenic human influenza A/H1N1 virus (LPHIV H1N1). Out of the 22 tested phytochemicals, the steroid compounds ß-sitosterol and ß-sitosterol-O-glucoside have very potent activity against the predefined influenza A viruses (IAV). Both steroids could induce such activity by affecting multiple stages during IAV replication cycles, including viral adsorption and replication with a major and significant impact on the virus directly in a cell-free status "viricidal effect". On a molecular level, several molecular docking studies suggested that ß-sitosterol and ß-sitosterol-O-glucoside exhibited viricidal effects through blocking active binding sites of the hemagglutinin surface protein, as well as showing inhibitory effects against replication through the binding with influenza neuraminidase activity and blocking the active sites of the M2 proton channel activity. The phytoestrogen ß-sitosterol has structural similarity with the active form of the female sex hormone estradiol, and this similarity is likely one of the molecular determinants that enables the phytoestrogen ß-sitosterol and its derivative to control IAV infection in vitro. This promising anti-influenza activity of ß-sitosterol and its O-glycoside derivative, according to both in vitro and cheminformatics studies, recommend both phytochemicals for further studies going through preclinical and clinical phases as efficient anti-influenza drug candidates.

Antiviral activities of plant-derived indole and ß-carboline alkaloids against human and avian influenza viruses.

The persistent evolution of drug-resistant influenza strains represents a global concern. The innovation of new treatment approaches through drug screening strategies and investigating the antiviral potential of bioactive natural-based chemicals may address the issue. Herein, we screened the anti-influenza efficacy of some biologically active indole and ß-carboline (ßC) indole alkaloids against two different influenza A viruses (IAV) with varied host range ranges; seasonal influenza A/Egypt/NRC098/2019(H1N1) and avian influenza A/chicken/Egypt/N12640A/2016(H5N1). All compounds were first assessed for their half-maximal cytotoxic concentration (CC50) in MDCK cells and half-maximal inhibitory concentrations (IC50) against influenza A/H5N1. Intriguingly, Strychnine sulfate, Harmalol, Harmane, and Harmaline showed robust anti-H5N1 activities with IC50 values of 11.85, 0.02, 0.023, and 3.42 µg/ml, respectively, as compared to zanamivir and amantadine as control drugs (IC50 = 0.079 µg/ml and 17.59 µg/ml, respectively). The efficacy of the predefined phytochemicals was further confirmed against influenza A/H1N1 and they displayed potent anti-H1N1 activities compared to reference drugs. Based on SI values, the highly promising compounds were then evaluated for antiviral efficacy through plaque reduction assay and consistently they revealed high viral inhibition percentages at non-toxic concentrations. By studying the modes of antiviral action, Harmane and Harmalol could suppress viral infection via interfering mainly with the viral replication of the influenza A/H5N1 virus, whilst Harmaline exhibited a viricidal effect against the influenza A/H5N1 virus. Whereas, Strychnine sulfate elucidated its anti-influenza potency by interfering with viral adsorption into MDCK cells. Consistently, chemoinformatic studies showed that all studied phytochemicals illustrated HB formations with essential peptide cleft through the NH of indole moiety. Among active alkaloids, harmalol displayed the best lipophilicity metrics including ligand efficiency (LE) and ligand lipophilic efficiency (LLE) for both viruses. Compounds geometry and their ability to participate in HB formation are very crucial.

Robust Antiviral Activity of Santonica Flower Extract (Artemisia cina) against Avian and Human Influenza A Viruses: In Vitro and Chemoinformatic Studies.

The evolution of drug-resistant viral strains following natural acquisition of resistance mutations is a major obstacle to antiviral therapy. Besides the improper prescription of the currently licensed anti-influenza medications, M2-blockers and neuraminidase inhibitors, to control poultry outbreaks/infections potentiates the emergence of drug-resistant influenza variants. Therefore, there is always a necessity to find out new alternatives with potent activity and high safety. Plant extracts and plant-based chemicals represent a historical antiviral resource with remarkable safety in vitro and in vivo to control the emerging and remerging health threats caused by viral infections. Herein, a panel of purified plant extracts and subsequent plant-derived chemicals were evaluated for their anti-avian influenza activity against zoonotic highly pathogenic influenza A/H5N1 virus. Interestingly, santonica flower extract (Artemisia cina) showed the most promising anti-H5N1 activity with a highly safe half-maximal cytotoxic concentration 50 (CC50 > 10 mg/mL) and inhibitory concentration 50 (IC50 of 3.42 µg/mL). To confirm the anti-influenza activity, we assessed the anti-influenza activity of the selected plant extracts against seasonal human influenza A/H1N1 virus and we found that santonica flower extract showed a robust anti-influenza activity that was comparable to the activity against influenza A/H5N1. Furthermore, the mode of action for santonica flower extract with strong inhibitory activity on the abovementioned influenza strains was elucidated, showing a virucidal effect. To go deeper about the activity of the chemometric component of the extract, the major constituent, santonin, was further selected for in vitro screening against influenza A/H5N1 (IC50 = 1.701 µg/mL) and influenza A/H1N1 (IC50 = 2.91 µg/mL). The oxygen of carbonyl functionality in the cyclohexene ring succeeded to form a hydrogen bond with the neuraminidase active site. Despite the fact that santonin revealed similarity to both reference neuraminidase inhibitors in forming hydrogen bonds with essential amino acids, it illustrated shape alignment to oseltamivir more than zanamivir according to Tanimoto algorithms. This study highlights the applicability of santonica flower extract as a promising natural antiviral against low and highly pathogenic influenza A viruses.

Comparative study on the immunopotentiator effect of ISA 201, ISA 61, ISA 50, ISA 206 used in trivalent foot and mouth disease vaccine.

AIM: A comparison study was conducted to explore the best internationally available adjuvant that could be used in production of a highly potent foot and mouth disease (FMD) vaccine, that could stimulate a strong immune response and possibly give greater protection against FMD. MATERIALS AND METHODS: Four experimental batches of trivalent FMD vaccine were prepared with different available oil adjuvants which included Montanide ISA 201, 206, 61 and 50. RESULTS: The results indicated that vaccines emulsified using Montanide ISA 201 and Montanide ISA 206 adjuvants elicited a protective humoral immune response from the 2(nd) week postvaccination (WPV) as for ISA 201 with serum neutralization test (SNT) and enzyme-linked immune sorbent assay (ELISA) antibody titers of 1.62±0.047(a) and 1.8±0.049(a), 1.59±0.076(a) and 1.836±0.077(a), and 1.71±0.06(b) and 1.96±0.074(b) for serotypes O, A, SAT2, respectively, and for ISA 206 at SNT and ELISA antibody titers of 1.5±0.082(a) and 1.84±0.084(a), 1.56±0.037(a) and 1.818±0.052(a), and 1.5±0.106(a,b) and 1.81±0.104(a,b) for FMD virus serotypes O, A and SAT2, respectively. For ISA 61 and ISA 50, the protective antibody titer appeared in the 3(rd) WPV. In the ISA 61 FMD vaccine, SNT and ELISA titer were 1.59±0.076(a) and 1.9±0.094(a), 1.53±0.056(a) and 1.83±0.070(a), and 1.5±0.082(a) and 1.84±0.094(a) for serotypes O, A and SAT2, respectively, and in the case of ISA 50 FMD vaccine, the SNT, and ELISA titer were recorded for serotypes O, A and SAT2 respectively, 1.59±0.037(a) and 1.8±0.030(a), 1.68±0.056(a,b) and 1.916±0.065(a,b), and 1.65±0.082(a) and 1.9±0.09(a). On estimating the cellular immune response, the highest delta optical density levels for ISA 201 (0.395-0.460) and ISA 206 (0.375-0.428) were observed on 14 and 21 days post vaccination (DPV) respectively, while the highest levels of lymphoproliferation for ISA 61 (0.375-0.455) and ISA 50 (0.411-0.430) were on 21 and 28 DPV, respectively. CONCLUSION: The duration of immunity from Montanide ISA oils (201, 206, 61 and 50) FMD vaccines is a long-lived immunity which ranged between 32 and 38 weeks post vaccination but the Montanide ISA 201 FMD vaccine is superior to the others in the rapid cellular immune response of the vaccinated animals which showed its highest level within 14 days post vaccination.