A Novel Antiviral Strategy against MERS-CoV and HCoV-229E Using Binase to Target Viral Genome Replication

© 2016, Springer Science+Business Media New York.RNA viruses cause most of the dangerous communicable diseases. Due to their high mutation rates, RNA viruses quickly evade selective pressures and can adapt to a new host. Therefore, new antiviral approaches are urgently needed, which target more than one specific virus variant and which would optimally prevent development of viral resistance. Among the family of coronaviruses (CoV), several human pathogenic strains (HCoV) are known to cause respiratory diseases and are implied in enteric diseases. While most strains contribute to common cold-like illnesses, others lead to severe infections. One of these viruses is the newly emerged (2012), highly pathogenic Middle East respiratory syndrome coronavirus (MERS-CoV) of zoonotic origin. MERS-CoV causes a severe respiratory infection with a high mortality rate of 35 %. There is no specific treatment or infection prevention available. Here, we show that the bacterial ribonuclease Binase is able to inhibit the replication of MERS-CoV and of the low-pathogenic human coronavirus 229E (HCoV-229E) in cell culture. We demonstrate that at non-toxic concentrations, Binase decreased the titers of MERS-CoV and HCoV-229E. On a molecular level, Binase treatment reduced (i) the viral subgenomic RNAs and (ii) the viral nucleocapsidprotein (N) and non-structural protein 13 (nsp13) accumulation. Furthermore, we show that the quantity of the replication/transcription complexes within the infected cells is diminished. Thus, the data obtained might allow further development of new anti-coronaviral approaches affecting viral replication, independent of the specific virus strain

Inhibitory activity of a standardized elderberry liquid extract against clinically-relevant human respiratory bacterial pathogens and influenza A and B viruses

<p>Abstract</p> <p>Background</p> <p>Black elderberries (<it>Sambucus nigra </it>L.) are well known as supportive agents against common cold and influenza. It is further known that bacterial super-infection during an influenza virus (IV) infection can lead to severe pneumonia. We have analyzed a standardized elderberry extract (Rubini, BerryPharma AG) for its antimicrobial and antiviral activity using the microtitre broth micro-dilution assay against three Gram-positive bacteria and one Gram-negative bacteria responsible for infections of the upper respiratory tract, as well as cell culture experiments for two different strains of influenza virus.</p> <p>Methods</p> <p>The antimicrobial activity of the elderberry extract was determined by bacterial growth experiments in liquid cultures using the extract at concentrations of 5%, 10%, 15% and 20%. The inhibitory effects were determined by plating the bacteria on agar plates. In addition, the inhibitory potential of the extract on the propagation of human pathogenic H5N1-type influenza A virus isolated from a patient and an influenza B virus strain was investigated using MTT and focus assays.</p> <p>Results</p> <p>For the first time, it was shown that a standardized elderberry liquid extract possesses antimicrobial activity against both Gram-positive bacteria of <it>Streptococcus pyogenes </it>and group C and G <it>Streptococci</it>, and the Gram-negative bacterium <it>Branhamella catarrhalis </it>in liquid cultures. The liquid extract also displays an inhibitory effect on the propagation of human pathogenic influenza viruses.</p> <p>Conclusion</p> <p>Rubini elderberry liquid extract is active against human pathogenic bacteria as well as influenza viruses. The activities shown suggest that additional and alternative approaches to combat infections might be provided by this natural product.</p

Ribonuclease from bacillus acts as an antiviral agent against negative- and positive-sense single stranded human respiratory RNA viruses

Copyright © 2017 Raihan Shah Mahmud et al.Bacillus pumilus ribonuclease (binase) was shown to be a promising antiviral agent in animal models and cell cultures. However, the mode of its antiviral action remains unknown. To assess the binase effect on intracellular viral RNA we have selected single stranded negative- and positive-sense RNA viruses, influenza virus, and rhinovirus, respectively, which annually cause respiratory illnesses and are characterized by high contagious nature, mutation rate, and antigen variability. We have shown that binase exerts an antiviral effect on both viruses at the same concentration, which does not alter the spectrum of A549 cellular proteins and expression of housekeeping genes. The titers of influenza A (H1N1pdm) virus and human rhinovirus serotype 1A were reduced by 40% and 65%, respectively. A preincubation of influenza virus with binase before infection significantly reduced viral titer after single-cycle replication of the virus. Using influenza A virus mini genome system we showed that binase reduced GFP reporter signaling indicating a binase action on the expression of viral mRNA. Binase reduced the level of H1N1pdm viral NP mRNA accumulation in A549 cells by 20%. Since the viral mRNA is a possible target for binase this agent could be potentially applied in the antiviral therapy against both negative- and positive-sense RNA viruses

Human annexin A6 interacts with influenza a virus protein M2 and negatively modulates infection

Copyright © 2012, American Society for Microbiology. All Rights ReservedThe influenza A virus M2 ion channel protein has the longest cytoplasmic tail (CT) among the three viral envelope proteins and is well conserved between different viral strains. It is accessible to the host cellular machinery after fusion with the endosomal membrane and during the trafficking, assembly, and budding processes. We hypothesized that identification of host cellular interactants of M2 CT could help us to better understand the molecular mechanisms regulating the M2-dependent stages of the virus life cycle. Using yeast two-hybrid screening with M2 CT as bait, a novel interaction with the human annexin A6 (AnxA6) protein was identified, and their physical interaction was confirmed by coimmunoprecipitation assay and a colocalization study of virus-infected human cells. We found that small interfering RNA (siRNA)-mediated knockdown of AnxA6 expression significantly increased virus production, while its overexpression could reduce the titer of virus progeny, suggesting a negative regulatory role for AnxA6 during influenza A virus infection. Further characterization revealed that AnxA6 depletion or overexpression had no effect on the early stages of the virus life cycle or on viral RNA replication but impaired the release of progeny virus, as suggested by delayed or defective budding events observed at the plasma membrane of virus-infected cells by transmission electron microscopy. Collectively, this work identifies AnxA6 as a novel cellular regulator that targets and impairs the virus budding and release stages of the influenza A virus life cycle.This work was supported by the Research Fund for the Control of Infectious Disease (project 09080892) of the Hong Kong Government, the Area of Excellence Scheme of the University Grants Committee (grant AoE/M-12/-06 of the Hong Kong Special Administrative Region, China), the French Ministry of Health, the RESPARI Pasteur Network

Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism

Although the immunomodulatory and cytoprotective properties of itaconate have been studied extensively, it is not known whether its naturally occurring isomers mesaconate and citraconate have similar properties. Here, we show that itaconate is partially converted to mesaconate intracellularly and that mesaconate accumulation in macrophage activation depends on prior itaconate synthesis. When added to human cells in supraphysiological concentrations, all three isomers reduce lactate levels, whereas itaconate is the strongest succinate dehydrogenase (SDH) inhibitor. In cells infected with influenza A virus (IAV), all three isomers profoundly alter amino acid metabolism, modulate cytokine/chemokine release and reduce interferon signalling, oxidative stress and the release of viral particles. Of the three isomers, citraconate is the strongest electrophile and nuclear factor-erythroid 2-related factor 2 (NRF2) agonist. Only citraconate inhibits catalysis of itaconate by cis-aconitate decarboxylase (ACOD1), probably by competitive binding to the substrate-binding site. These results reveal mesaconate and citraconate as immunomodulatory, anti-oxidative and antiviral compounds, and citraconate as the first naturally occurring ACOD1 inhibitor

Citraconate inhibits ACOD1 (IRG1) catalysis, reduces interferon responses and oxidative stress, and modulates inflammation and cell metabolism

Although the immunomodulatory and cytoprotective properties of itaconate have been studied extensively, it is not known whether its naturally occurring isomers mesaconate and citraconate have similar properties. Here, we show that itaconate is partially converted to mesaconate intracellularly and that mesaconate accumulation in macrophage activation depends on prior itaconate synthesis. When added to human cells in supraphysiological concentrations, all three isomers reduce lactate levels, whereas itaconate is the strongest succinate dehydrogenase (SDH) inhibitor. In cells infected with influenza A virus (IAV), all three isomers profoundly alter amino acid metabolism, modulate cytokine/chemokine release and reduce interferon signalling, oxidative stress and the release of viral particles. Of the three isomers, citraconate is the strongest electrophile and nuclear factor-erythroid 2-related factor 2 (NRF2) agonist. Only citraconate inhibits catalysis of itaconate by cis-aconitate decarboxylase (ACOD1), probably by competitive binding to the substrate-binding site. These results reveal mesaconate and citraconate as immunomodulatory, anti-oxidative and antiviral compounds, and citraconate as the first naturally occurring ACOD1 inhibitor. [Image: see text

Protective essential oil attenuates influenza virus infection: An in vitro study in MDCK cells

<p>Abstract</p> <p>Background</p> <p>Influenza is a significant cause of morbidity and mortality. The recent pandemic of a novel H1N1 influenza virus has stressed the importance of the search for effective treatments for this disease. Essential oils from aromatic plants have been used for a wide variety of applications, such as personal hygiene, therapeutic massage and even medical practice. In this paper, we investigate the potential role of an essential oil in antiviral activity.</p> <p>Methods</p> <p>We studied a commercial essential oil blend, On Guard™, and evaluated its ability in modulating influenza virus, A/PR8/34 (PR8), infection in Madin-Darby canine kidney (MDCK) cells. Influenza virus was first incubated with the essential oil and infectivity in MDCK cells was quantified by fluorescent focus assay (FFA). In order to determine the mechanism of effects of essential oil in viral infection inhibition, we measured hemagglutination (HA) activity, binding and internalization of untreated and oil-treated virus in MDCK cells by flow cytometry and immunofluorescence microscopy. In addition, the effect of oil treatment on viral transcription and translation were assayed by relative end-point RT-PCR and western blot analysis.</p> <p>Results</p> <p>Influenza virus infectivity was suppressed by essential oil treatment in a dose-dependent manner; the number of nascent viral particles released from MDCK cells was reduced by 90% and by 40% when virus was treated with 1:4,000 and 1:6,000 dilutions of the oil, respectively. Oil treatment of the virus also decreased direct infection of the cells as the number of infected MDCK cells decreased by 90% and 45% when virus was treated with 1:2,000 and 1:3,000 dilutions of the oil, respectively. This was not due to a decrease in HA activity, as HA was preserved despite oil treatment. In addition, oil treatment did not affect virus binding or internalization in MDCK cells. These effects did not appear to be due to cytotoxicity of the oil as MDCK cell viability was only seen with concentrations of oil that were 2 to 6 times greater than the doses that inhibited viral infectivity. RT-PCR and western blotting demonstrated that oil treatment of the virus inhibited viral NP and NS1 protein, but not mRNA expression.</p> <p>Conclusions</p> <p>An essential oil blend significantly attenuates influenza virus PR8 infectivity <it>in vitro </it>without affecting viral binding or cellular internalization in MDCK cells. Oil treated virus continued to express viral mRNAs but had minimal expression of viral proteins, suggesting that the antiviral effect may be due to inhibition of viral protein translation.</p

Up-Regulation of Hepatitis C Virus Replication and Production by Inhibition of MEK/ERK Signaling

BACKGROUND: Viruses interact with and exploit the host cellular machinery for their multiplication and propagation. The MEK/ERK signaling pathway positively regulates replication of many RNA viruses. However, whether and how this signaling pathway affects hepatitis C virus (HCV) replication and production is not well understood. METHODS AND RESULTS: In this study, we took advantage of two well-characterized MEK/ERK inhibitors and MEK/ERK dominant negative mutants and investigated the roles of the MEK/ERK signaling pathway in HCV gene expression and replication. We showed that inhibition of MEK/ERK signaling enhanced HCV gene expression, plus- and minus-strand RNA synthesis, and virus production. In addition, we showed that this enhancement was independent of interferon-alpha (IFN-alpha) antiviral activity and did not require prior activation of the MEK/ERK signaling pathway. Furthermore, we showed that only MEK and ERK-2 but not ERK-1 was involved in HCV replication, likely through regulation of HCV RNA translation. CONCLUSIONS: Taken together, these results demonstrate a negative regulatory role of the MEK/ERK signaling pathway in HCV replication and suggest a potential risk in targeting this signaling pathway to treat and prevent neoplastic transformation of HCV-infected liver cells

Unexpected Inheritance: Multiple Integrations of Ancient Bornavirus and Ebolavirus/Marburgvirus Sequences in Vertebrate Genomes

Vertebrate genomes contain numerous copies of retroviral sequences, acquired over the course of evolution. Until recently they were thought to be the only type of RNA viruses to be so represented, because integration of a DNA copy of their genome is required for their replication. In this study, an extensive sequence comparison was conducted in which 5,666 viral genes from all known non-retroviral families with single-stranded RNA genomes were matched against the germline genomes of 48 vertebrate species, to determine if such viruses could also contribute to the vertebrate genetic heritage. In 19 of the tested vertebrate species, we discovered as many as 80 high-confidence examples of genomic DNA sequences that appear to be derived, as long ago as 40 million years, from ancestral members of 4 currently circulating virus families with single strand RNA genomes. Surprisingly, almost all of the sequences are related to only two families in the Order Mononegavirales: the Bornaviruses and the Filoviruses, which cause lethal neurological disease and hemorrhagic fevers, respectively. Based on signature landmarks some, and perhaps all, of the endogenous virus-like DNA sequences appear to be LINE element-facilitated integrations derived from viral mRNAs. The integrations represent genes that encode viral nucleocapsid, RNA-dependent-RNA-polymerase, matrix and, possibly, glycoproteins. Integrations are generally limited to one or very few copies of a related viral gene per species, suggesting that once the initial germline integration was obtained (or selected), later integrations failed or provided little advantage to the host. The conservation of relatively long open reading frames for several of the endogenous sequences, the virus-like protein regions represented, and a potential correlation between their presence and a species' resistance to the diseases caused by these pathogens, are consistent with the notion that their products provide some important biological advantage to the species. In addition, the viruses could also benefit, as some resistant species (e.g. bats) may serve as natural reservoirs for their persistence and transmission. Given the stringent limitations imposed in this informatics search, the examples described here should be considered a low estimate of the number of such integration events that have persisted over evolutionary time scales. Clearly, the sources of genetic information in vertebrate genomes are much more diverse than previously suspected

The Epidermal Growth Factor Receptor (EGFR) Promotes Uptake of Influenza A Viruses (IAV) into Host Cells

Influenza A viruses (IAV) bind to sialic-acids at cellular surfaces and enter cells by using endocytotic routes. There is evidence that this process does not occur constitutively but requires induction of specific cellular signals, including activation of PI3K that promotes virus internalization. This implies engagement of cellular signaling receptors during viral entry. Here, we present first indications for an interplay of IAV with receptor tyrosine kinases (RTKs). As representative RTK family-members the epidermal growth factor receptor (EGFR) and the c-Met receptor were studied. Modulation of expression or activity of both RTKs resulted in altered uptake of IAV, showing that these receptors transmit entry relevant signals upon virus binding. More detailed studies on EGFR function revealed that virus binding lead to clustering of lipid-rafts, suggesting that multivalent binding of IAV to cells induces a signaling platform leading to activation of EGFR and other RTKs that in turn facilitates IAV uptake