SARS-CoV-2 papain-like protease inhibits ISGylation of the viral nucleocapsid protein to evade host anti-viral immunity.

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection causes mild-to-severe respiratory symptoms, including acute respiratory distress. Despite remarkable efforts to investigate the virological and pathological impacts of SARS-CoV-2, many of the characteristics of SARS-CoV-2 infection still remain unknown. The interferon-inducible ubiquitin-like protein ISG15 is covalently conjugated to several viral proteins to suppress their functions. It was reported that SARS-CoV-2 utilizes its papain-like protease (PLpro) to impede ISG15 conjugation, ISGylation. However, the role of ISGylation in SARS-CoV-2 infection remains unclear. We aimed to elucidate the role of ISGylation in SARS-CoV-2 replication. We observed that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation in cultured cells. Site-directed mutagenesis reveals that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation, alongside conserved lysine residue in MERS-CoV (K372) and SARS-CoV (K375). We also observed that the nucleocapsid-ISGylation results in the disruption of nucleocapsid oligomerization, thereby inhibiting viral replication. Knockdown of ISG15 mRNA enhanced SARS-CoV-2 replication in the SARS-CoV-2 reporter replicon cells, while exogenous expression of ISGylation components partially hampered SARS-CoV-2 replication. Taken together, these results suggest that SARS-CoV-2 PLpro inhibits ISGylation of the nucleocapsid protein to promote viral replication by evading ISGylation-mediated disruption of the nucleocapsid oligomerization.IMPORTANCEISG15 is an interferon-inducible ubiquitin-like protein that is covalently conjugated to the viral protein via specific Lys residues and suppresses viral functions and viral propagation in many viruses. However, the role of ISGylation in SARS-CoV-2 infection remains largely unclear. Here, we demonstrated that the SARS-CoV-2 nucleocapsid protein is a target protein for the HERC5 E3 ligase-mediated ISGylation. We also found that the residue K374 within the C-terminal spacer B-N3 (SB/N3) domain is required for nucleocapsid-ISGylation. We obtained evidence suggesting that nucleocapsid-ISGylation results in the disruption of nucleocapsid-oligomerization, thereby suppressing SARS-CoV-2 replication. We discovered that SARS-CoV-2 papain-like protease inhibits ISG15 conjugation of nucleocapsid protein via its de-conjugating enzyme activity. The present study may contribute to gaining new insight into the roles of ISGylation-mediated anti-viral function in SARS-CoV-2 infection and may lead to the development of more potent and selective inhibitors targeted to SARS-CoV-2 nucleocapsid protein.

Alocasia odora-mediated synthesis of silver nanoparticles, their cytotoxicity, and virucidal potential.

Silver nanoparticles (AgNPs) have various applications in the biomedical field and are considered excellent microbicidal agents. Moreover, biological synthesis of AgNPs using medicinal plants further improves the medicinal applicability of these plants. In this study, the aqueous extract of Alocasia odora rhizome (RE) and Alocasia odora stem (SE) were used to synthesize stem aqueous extract-AgNPs (SNP) and rhizome aqueous extract-AgNPs (RNP). Furthermore, RNP and SNP were evaluated for their virucidal potential. The synthesis of SNP and RNP was monitored using a UV spectrophotometer by observing their surface plasmon resonance peak. In addition, scanning electron microscopy (SEM) gave further insight into their morphology and particle size, whereas energy-dispersive X-ray spectroscopy (EDX) confirmed the presence of silver ions. Interestingly, Fourier-transform infrared spectroscopy (FTIR) analysis of AgNPs revealed that phytomolecules acted as capping and stabilizing agents for SNP and RNP. The in vitro cytotoxicity of SNP and RNP was further analyzed using MTT assay on the U87-MG human glioblastoma cancer cell line and SNP found to be the most cytotoxic (43.40 µg/ml) among all. Besides that, SNP has also found to show the maximum cytopathic effects (CPE) against dengue virus type 2 (DENV-2) on Huh-7 cell line. As a result of the observations, it can be concluded that they can become a promising antiviral drug candidate and thus merit further testing. KEY POINTS: • AgNPs were successfully synthesized through Alocasia odora aqueous extract. • AgNPs were more cytotoxic on the U87-MG cell line than the extract alone. • AgNPs have shown significant reduction in the dengue viral infection than the extract alone.

Fluorinated 2-arylchroman-4-ones and their derivatives: synthesis, structure and antiviral activity.

A number of new biologically interesting fluorinated 2-arylchroman-4-ones and their 3-arylidene derivatives were synthesized based on the p-toluenesulfonic acid-catalyzed one-pot reaction of 2-hydroxyacetophenones with benzaldehydes. It was found that obtained (E)-3-arylidene-2-aryl-chroman-4-ones reacted with malononitrile under base conditions to form 4,5-diaryl-4H,5H-pyrano[3,2-c]chromenes. The structures of the synthesized fluorinated compounds were confirmed by 1H, 19F, and 13C NMR spectral data, and for some representatives of heterocycles also using NOESY spectra and X-ray diffraction analysis. A large series of obtained flavanone derivatives as well as products of their modification (35 examples) containing from 1 to 12 fluorine atoms in the structure was tested in vitro for cytotoxicity in MDCK cell line and for antiviral activity against influenza A virus. Among the studied heterocycles 6,8-difluoro-2-(4-(trifluoromethyl)phenyl)chroman-4-one (IC50 = 6 µM, SI = 150) exhibited the greatest activity against influenza A/Puerto Rico/8/34 (H1N1) virus. Moreover, this compound appeared active against phylogenetically distinct influenza viruses, A(H5N2) and influenza B (SI's of 53 and 42, correspondingly). The data obtained suggest that the fluorinated derivatives of 2-arylchroman-4-ones are prospective scaffolds for further development of potent anti-influenza antivirals.

Novel [1,2,4]triazolo[3,4-b][1,3,4]thiadiazine and [1,2,4]triazolo[3,4-b][1,3,4]thiadiazepine Derivatives: Synthesis, Anti-Viral In Vitro Study and Target Validation Activity.

This study of the interaction system of binucleophilic 3-substituted 4-amino-4H-1,2,4-triazole-5-thiols and 3-phenyl-2-propynal made it possible to develop a new approach to synthesis of such isomeric classes as 7-benzylidene-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazine and 8-phenyl-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazepine. Among the 20 compounds studied in vitro against influenza A/Puerto Rico/8/34 (H1N1) virus, half of them demonstrated selectivity index (SI) of 10 or higher and one of them (4-((3-phenylprop-2-yn-1-yl)amino)-4H-1,2,4-triazole-3-thiol) possessed the highest (SI > 300). Docking results and values showed that the preferred interactant for our ligands was M2 proton channel of the influenza A virus. Protein-ligand interactions modeling showed that the aliphatic moiety of ligands could negatively regulate target activity level.

Synthesis, Antiviral, and Antibacterial Activity of the Glycyrrhizic Acid and Glycyrrhetinic Acid Derivatives.

Glycyrrhizic acid and its primary metabolite glycyrrhetinic acid, are the main active ingredients in the licorice roots (glycyrrhiza species), which are widely used in several countries of the world, especially in east asian countries (China, Japan). These ingredients and their derivatives play an important role in treating many diseases, especially infectious diseases such as COVID-19 and hepatic infections. This review aims to summarize the different ways of synthesising the amide derivatives of glycyrrhizic acid and the main ways to synthesize the glycyrrhitinic acid derivatives. Also, to determine the main biological and pharmacological activity for these compounds from the previous studies to provide essential data to researchers for future studies. Supplementary Information: The online version contains supplementary material available at 10.1134/S1068162022050132.

Investigative study into whether an insect repellent has virucidal activity against SARS-CoV-2.

A small-scale study with Mosi-guard Natural spray, an insect repellent containing Citriodiol, was performed to determine if it has virucidal activity against SARS-CoV-2. A liquid test examined the activity of the insect repellent and the individual components for virucidal activity. A surface contact test looked at the activity of the insect repellent when impregnated on a latex surface as a synthetic skin for potential topical prophylactic application. Both Mosi-guard Natural spray and Citriodiol, as well as other components of the repellent, had virucidal activity in the liquid contact test. On a latex surface used to simulate treated skin, the titre of SARS-CoV-2 was less over time on the Mosi-guard Natural-treated surface but virus was still recovered.

Long non-coding RNA LNC_000641 regulates pseudorabies virus replication.

Long non-coding RNAs (lncRNAs) are a new arm of gene regulatory mechanism as discovered by sequencing techniques and follow-up functional studies. The lncRNAs regulation of pseudorabies virus (PRV) infection has rarely been reported so far. Using RNA sequencing analysis, 225 lncRNAs with significant altered expressions in 3D4/21 cells infected with PRV (ZJ01) were identified. Five lncRNAs upregulated in PRV-infected cells were verified in cells infected with different PRV strains by qRT-PCR. By down- and up-regulation of lnc641, the accelerating effect of lnc641 on PRV replication was confirmed. Furthermore, we found that lnc641 regulated PRV replication by inhibiting the JAK-STAT1 pathway. This study suggests that lnc641 could be a new host factor target for developing antiviral therapies against PRV infection.

Soybean-associated endophytic fungi as potential source for anti-COVID-19 metabolites supported by docking analysis.

AIMS: To identify the metabolites produced by the endophytic fungus, Aspergillus terreus and to explore the anti-viral activity of the identified metabolites against the pandemic disease COVID-19 in-silico. METHODS AND RESULTS: Herein, we reported the isolation of A. terreus, the endophytic fungus associated with soybean roots, which is then subcultured using OSMAC approach in five different culture media. Analytical analysis of media ethylacetate extracts using liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) was carried out. Furthermore, the obtained LC-MS data were statistically processed with MetaboAnalyst 4.0. Molecular docking studies were performed for the dereplicated metabolites against COVID-19 main protease (Mpro ). Metabolomic profiling revealed the presence of 18 compounds belonging to different chemical classes. Quinones, polyketides and isocoumarins were the most abundant classes. Multivariate analysis revealed that potato dextrose broth and modified potato dextrose broth are the optimal media for metabolites production. Molecular docking studies declared that the metabolites, Aspergillide B1 and 3a-Hydroxy-3, 5-dihydromonacolin L showed the highest binding energy scores towards COVID-19 main protease (Mpro ) (-9·473) and (-9·386), respectively, and they interact strongly with the catalytic dyad (His41 and Cys145) amino acid residues of Mpro . CONCLUSIONS: A combination of metabolomics and in-silico approaches have allowed a shorter route to search for anti-COVID-19 natural products in a shorter time. The dereplicated metabolites, aspergillide B1 and 3α-Hydroxy-3, 5-dihydromonacolin L were found to be potent anti-COVID-19 drug candidates in the molecular docking study. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed that the endophytic fungus, A. terreus can be considered as a potential source of natural bioactive products. In addition to, the possibility of developing the metabolites, aspergillide B1 and 3α-Hydroxy-3, 5-dihydromonacolin L to be used as phytopharmaceuticals for the management of COVID-19.

Pharmacological Potential and Chemical Composition of Crocus sativus Leaf Extracts.

Crocus sativus L. (saffron) has been traditionally used as a food coloring or flavoring agent, but recent research has shown its potent pharmacological activity to tackle several health-related conditions. Crocus sp. leaves, and petals are the by-products of saffron production and are not usually used in the medicine or food industries. The present study was designed to determine the chemical composition of the water and ethanolic extracts of C. sativus leaves and test their cytotoxic activity against melanoma (IGR39) and triple-negative breast cancer (MDA-MB-231) cell lines by MTT assay. We also determined their anti-allergic, anti-inflammatory, and anti-viral activities. HPLC fingerprint analysis showed the presence of 16 compounds, including hydroxycinnamic acids, xanthones, flavonoids, and isoflavonoids, which could contribute to the extracts' biological activities. For the first time, compounds such as tectoridin, iristectorigenin B, nigricin, and irigenin were identified in Crocus leaf extracts. The results showed that mangiferin (up to 2 mg/g dry weight) and isoorientin (8.5 mg/g dry weight) were the major active ingredients in the leaf extracts. The ethanolic extract reduced the viability of IGR39 and MDA-MB-231 cancer cells with EC50 = 410 ± 100 and 330 ± 40 µg/mL, respectively. It was more active than the aqueous extract. Kaempferol and quercetin were identified as the most active compounds. Our results showed that Crocus leaves contain secondary metabolites with potent cytotoxic and antioxidant activities.

Structure-based glycoengineering of interferon lambda 4 enhances its productivity and anti-viral potency.

Interferon lambda 4 (IFNλ4) has been recently known and studied for its role in hepatitis C virus (HCV) infection, but its clinical potential is significantly hampered due to its poor expression in vitro. Our study reports the successful production of IFNλ4 from a mammalian cell line through a glycoengineering and structure-based approach. We introduced de novo N-glycosylation of IFNλ4, guided by structural analysis, and produced IFNλ4 variants in Expi293F that displayed improved expression and potency. To preserve the structure and functionality of IFNλ4, the model structure of the IFNλ4 signaling complex was analyzed and the N-glycosylation candidate sites were selected. The receptor binding activity of engineered IFNλ4 variants and their receptor-mediated signaling pathway were similar to the E. coli version of IFNλ4 (eIFNλ4), while the antiviral activity and induction levels of interferon-stimulated gene (ISG) were all more robust in our variants. Our engineered IFNλ4 variants may be further developed for clinical applications and utilized in basic research to decipher the immunological roles of IFNλ4.

Novel cyclohexanone compound as a potential ligand against SARS-CoV-2 main-protease.

No commercially available drug candidate has yet been devised which is unique to and not repurposed against SARS-CoV-2 and has high efficacy or safe toxicity profile or both. Taking curcumin as a reference compound, we identified a new commercially available cyclohexanone compound, ZINC07333416 with binding energy (-8.72 kcal/mol) better than that of popularly devised anti-Covid-19 drugs like viral protease inhibitor Lopinavir, nucleoside analogue Remdesivir and the repurposed drug hydroxychloroquine when targeted to the active-site of SARS-CoV-2 Main protease (Mpro) through docking studies. The ligand ZINC07333416 exhibits crucial interactions with major active site residues of SARS-CoV-2 Mpro viz. Cys145 and His41 involving in the protease activity; as well as GLU-166 and ASN-142 which plays the pivotal role in the protein-dimerization. The protein-ligand stable interaction was further confirmed with molecular dynamics simulation (MDS) studies. Based on virtual assessment, ZINC07333416 also have significant values in terms of medicinal chemistry, pharmacokinetics, synthetic accessibility and anti-viral activity that encourage its experimental applications against COVID-19.

Melittin: a venom-derived peptide with promising anti-viral properties.

Despite tremendous advances in the development of anti-viral therapeutics, viral infections remain a chief culprit accounting for ongoing morbidity and mortality worldwide. Natural products, in particular animal venoms, embody a veritable cornucopia of exotic constituents, suggesting an immensurable source of anti-infective drugs. In this context, melittin, the principal constituent in the venom of the European honeybee Apis mellifera, has been demonstrated to exert anti-cancer, anti-inflammatory, anti-diabetic, anti-infective, and adjuvant properties. To our knowledge, there is no review appertaining to effects of melittin against viruses, prompting us to synopsize experimental investigations on its anti-viral activity throughout the past decades. Accumulating evidence indicates that melittin curbs infectivity of a diverse array of viruses including coxsackievirus, enterovirus, influenza A viruses, human immunodeficiency virus (HIV), herpes simplex virus (HSV), Junín virus (JV), respiratory syncytial virus (RSV), vesicular stomatitis virus (VSV), and tobacco mosaic virus (TMV). However, medication safety, different routes of administrations, and molecular mechanisms behind the anti-viral activity of melittin should be scrutinized in future studies.

Synthesis and biological evaluation of 6-nitro-1,2,4-triazoloazines containing polyphenol fragments possessing antioxidant and antiviral activity.

Stable σ-adducts of azolo[5,1-c]triazines and azolo[1,5-a]pyrimidines with different polyphenols were synthesized and their antioxidant and antiviral activity were investigated. Their affinity to viral hemagglutinin was assessed using molecular modelling. The phloroglucinol-modified azolo-azines possessed the highest virus-inhibiting activity. According to the results of the study of antioxidant properties of compounds, the most promising ones exhibiting highest antioxidant capacity were adducts containing in their structure pyrogallol and catechol residues and 6-nitro-triazolotriazin-7-ol scaffold. No correlation between antioxidant and virus-inhibiting activity of compounds studied was detected. The most active compounds demonstrated the ability to prevent binding of viral hemagglutinin with cellular receptor as shown in hemagglutination inhibition assay. Our results demonstrate that polyphenol-modified azolo-azines are prospective for further optimization as potential antivirals and that their action is directed against viral hemagglutinin.

[Anti-viral activity of enisamium iodide against viruses of influenza and ARVI's on different cell lines].

Influenza and ARVI represent the most numerous and dangerous group of causative agents of respiratory infections human. AIM: Characterization of the antiviral properties of enisamium iodide against human respiratory viruses in in vitro experiments. MATERIALS AND METHODS: In the course of experiments, the cytotoxic properties of enisamium iodide were studied against the cell lines Vero, MA-104, A549, L-41 and HEp-2. The antiviral activity of enisamium iodide was studied using virus yield reduction assay against influenza viruses, parainfluenza virus, respiratory syncytial virus, Coxsackie B3 and Coxsackie B4 viruses, as well as adenoviruses types 5 and 6. RESULTS: The most sensitive to the action of enisamium iodide was the human parainfluenza virus, whose activity decreased by 2.3 orders of magnitude under the action of the drug in A549 cells. Of the cell cultures used, enisamium iodide exhibited the maximum antiviral effect in human lung carcinoma cells A549, where, in its presence, the level of reproduction of adenoviruses of types 5 and 6, Coxsackie viruses B3 and B4, and human parainfluenza virus decreased by an order of magnitude or more. The antiviral activity of enisamium iodide was least manifested in Vero cells. CONCLUSION: According to the results of in vitro experiments, enisamium iodide can be considered as an antiviral drug with a wide spectrum of activity against human respiratory viruses.

Long non-coding RNA PSMB8-AS1 regulates influenza virus replication.

Long non-coding RNAs (lncRNAs) are a new arm of gene regulatory mechanism as discovered by sequencing techniques and follow-up functional studies. There are only few studies on lncRNAs as related to gene expression regulation and anti-viral activity during influenza virus infection. We sought to identify and characterize lncRNAs involved in influenza virus replication. Using RNA sequencing analysis, we found that 1,912 lncRNAs were significantly changed in human lung epithelial A549 cells infected with influenza A/Puerto Rico/8/34. Gene ontology analysis on neighboring genes of these lncRNAs revealed that the genes involved in type I interferon signaling and cellular response were highly enriched. Seven selected up-regulated lncRNAs (AC015849.2, RP-1-7H24.1, PSMB8-AS1, CTD-2639E6.9, PSOR1C3, AC007283.5 and RP11-670E13.5) were verified by real-time PCR. These lncRNAs were also induced by other two influenza H1N1 virus strains (A/WSN/1933 and A/Oklahoma/3052/09) and interferon ß1. Repression of PSMB8 antisense RNA 1 (PSMB8-AS1) using CRISPR interference reduced viral mRNA and protein levels as well as the release of progeny influenza virus particles. Our study suggests that lncRNA PSMB8-AS1 could be a new host factor target for developing antiviral therapy against influenza virus infection.

Metabolites of Seaweeds as Potential Agents for the Prevention and Therapy of Influenza Infection.

CONTEXT: Seaweed metabolites (fucoidans, carrageenans, ulvans, lectins, and polyphenols) are biologically active compounds that target proteins or genes of the influenza virus and host components that are necessary for replication and reproduction of the virus. OBJECTIVE: This review gathers the information available in the literature regarding to the useful properties of seaweeds metabolites as potential agents for the prevention and therapy of influenza infection. MATERIALS AND METHODS: The sources of scientific literature were found in various electronic databases (i.e., PubMed, Web of Science, and ScienceDirect) and library search. The retrospective search depth is 25 years. RESULTS: Influenza is a serious medical and social problem for humanity. Recently developed drugs are quite effective against currently circulating influenza virus strains, but their use can lead to the selection of resistant viral strains. In this regard, new therapeutic approaches and drugs with a broad spectrum of activity are needed. Metabolites of seaweeds fulfill these requirements. This review presents the results of in vitro and in vivo experimental and clinical studies about the effectiveness of these compounds in combating influenza infection and explains the necessity of their use as a potential basis for the creation of new drugs with a broad spectrum of activity.

Dietary supplementation with olive mill wastewaters induces modifications on chicken jejunum epithelial cell transcriptome and modulates jejunum morphology.

BACKGROUND: The Mediterranean diet is considered one of the healthier food habits and olive oil is one of its key components. Olive oil polyphenols are known to induce beneficial effects in several pathological conditions, such as inflammatory bowel disease, and to contrast the proliferation of cancer cells or hypercholesterolemia. Polyphenols are also present in waste products derived from the olive industry: olive mill wastewaters (OMWW) are rich in polyphenols and there is an increasing interest in using OMWW in animal nutrition. OMWW are attributed with positive effects in promoting chicken performance and the quality of food-derived products. However, a tissue-specific transcriptome target analysis of chickens fed with OMWW has never been attempted. RESULTS: We explored the effect of dietary OMWW on the intestinal function in broilers. A morphological analysis of the jejunum revealed that OMWW reduced crypt depth, whereas no significant modifications were observed for villus height and the villus height/crypt depth ratio. An RNA Sequencing analysis was performed on isolated, intestinal, epithelial cells and 280 differentially expressed genes were found using a count-based approach. An enrichment analysis revealed that the majority of up regulated genes in the OMWW group were over-represented by the regulation of viral genome replication-related GO-Terms, whereas down regulated genes were mainly involved in cholesterol and lipid metabolism. CONCLUSIONS: Our study showed how an industrial waste product can be recycled as a feed additive with a positive relapse. OMWW dietary supplementation can be a nutritional strategy to improve chicken performance and health, prevent intestinal damage, enhance innate immunity and regulate cholesterol metabolism and fat deposition.

Inhibition of enterovirus 71 replication and viral 3C protease by quercetin.

BACKGROUND: Enterovirus 71 (EV71) is one of the major causative agents of hand, foot, and mouth disease (HFMD), which is sometimes associated with severe central nervous system disease in children. There is currently no specific medication for EV71 infection. Quercetin, one of the most widely distributed flavonoids in plants, has been demonstrated to inhibit various viral infections. However, investigation of the anti-EV71 mechanism has not been reported to date. METHODS: The anti-EV71 activity of quercetin was evaluated by phenotype screening, determining the cytopathic effect (CPE) and EV71-induced cells apoptosis. The effects on EV71 replication were evaluated further by determining virus yield, viral RNA synthesis and protein expression, respectively. The mechanism of action against EV71 was determined from the effective stage and time-of-addition assays. The possible inhibitory functions of quercetin via viral 2Apro, 3Cpro or 3Dpol were tested. The interaction between EV71 3Cpro and quercetin was predicted and calculated by molecular docking. RESULTS: Quercetin inhibited EV71-mediated cytopathogenic effects, reduced EV71 progeny yields, and prevented EV71-induced apoptosis with low cytotoxicity. Investigation of the underlying mechanism of action revealed that quercetin exhibited a preventive effect against EV71 infection and inhibited viral adsorption. Moreover, quercetin mediated its powerful therapeutic effects primarily by blocking the early post-attachment stage of viral infection. Further experiments demonstrated that quercetin potently inhibited the activity of the EV71 protease, 3Cpro, blocking viral replication, but not the activity of the protease, 2Apro, or the RNA polymerase, 3Dpol. Modeling of the molecular binding of the 3Cpro-quercetin complex revealed that quercetin was predicted to insert into the substrate-binding pocket of EV71 3Cpro, blocking substrate recognition and thereby inhibiting EV71 3Cpro activity. CONCLUSIONS: Quercetin can effectively prevent EV71-induced cell injury with low toxicity to host cells. Quercetin may act in more than one way to deter viral infection, exhibiting some preventive and a powerful therapeutic effect against EV71. Further, quercetin potently inhibits EV71 3Cpro activity, thereby blocking EV71 replication.

Bacterial ribonuclease binase exerts an intra-cellular anti-viral mode of action targeting viral RNAs in influenza a virus-infected MDCK-II cells.

BACKGROUND: Influenza is a severe contagious disease especially in children, elderly and immunocompromised patients. Beside vaccination, the discovery of new anti-viral agents represents an important strategy to encounter seasonal and pandemic influenza A virus (IAV) strains. The bacterial extra-cellular ribonuclease binase is a well-studied RNase from Bacillus pumilus. Treatment with binase was shown to improve survival of laboratory animals infected with different RNA viruses. Although binase reduced IAV titer in vitro and in vivo, the mode of action (MOA) of binase against IAV at the molecular level has yet not been studied in depth and remains elusive. METHODS: To analyze whether binase impairs virus replication by direct interaction with the viral particle we applied a hemagglutination inhibition assay and monitored the integrity of the viral RNA within the virus particle by RT-PCR. Furthermore, we used Western blot and confocal microscopy analysis to study whether binase can internalize into MDCK-II cells. By primer extension we examined the effect of binase on the integrity of viral RNAs within the cells and using a mini-genome system we explored the effect of binase on the viral expression. RESULTS: We show that (i) binase does not to attack IAV particle-protected viral RNA, (ii) internalized binase could be detected within the cytosol of MDCK-II cells and that (iii) binase impairs IAV replication by specifically degrading viral RNA species within the infected MDCK-II cells without obvious effect on cellular mRNAs. CONCLUSION: Our data provide novel evidence suggesting that binase is a potential anti-viral agent with specific intra-cellular MOA.

Study on the Rationality for Antiviral Activity of Flos Lonicerae Japonicae-Fructus Forsythiae Herb Chito-Oligosaccharide via Integral Pharmacokinetics.

In the present study, the rationality for the antiviral effect (H1N1 virus) of Flos Lonicerae Japonicae (FLJ, named JinYinHua)-Fructus forsythiae (FF, named LianQiao) herb couple preparations improved by chito-oligosaccharide (COS) was investigated. We found that the improvement of antiviral activity for four preparations attributed to the enhancement of bioavailability for the FLJ-FF herb couple in vivo, and that caffeic acid derivatives are the most important type of components for antiviral activity. The anti-Influenza virus activity-half maximal inhibitory concentration (IC50), not area under concentration (AUC) was considered as the weighting factor for integrating the pharmacokinetics of caffeic acid derivatives. It was found that the integral absorption, both in vitro and in vivo, especially that in Shuang-Huang-Lian, can be improved significantly by COS, an absorption enhancer based on tight junction. The results indicated that the antiviral activity in four preparations improved by COS was mainly attributed to the integral absorption enhancement of caffeic acid derivatives.