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While upregulation of 2,3-dioxygenase (IDO) accompanied by degradation of tryptophan along the kynurenine pathway have been reported to exert antimicrobial effects against a wide range of infectious agents, its role in the replication of influenza A virus remains uncertain. We performed experiments using influenza A/WSN/33 virus infection of mouse fibroblast cell-line (NIH-3T3) to study the effects of IDO on viral replication. Influenza infection resulted in prominent elevations of transcripts encoding IDO, interferon (IFN)-β, and segment 8 of the virus in NIH-3T3 cells. Introduction of siRNA targeted against IDO followed by infection resulted in further increased levels of viral RNA without altering IFN-β expression. Inhibition of IDO during the infection also resulted in reduction of virus-driven upregulation of 3-hydroxyanthranilate 3,4-dioxygenase (HAAO), but not kynurenine 3-monooxygenase (KMO), which are enzymes downstream in the kynurenine pathway. Thus, induction of IDO appears to contribute to limiting replication of the WSN/33 strain of influenza A virus in murine NIH-3T3 cells.

Eupatorium fortunei (EF) has long been used as herbal medicine in Korea, China, and Asian countries to treat a variety of diseases. Recent studies have reported that EF has anti-metastatic, anti-angiogenic, anti-bacterial, and anti-oxidant activities, as well as activities against malignant metastatic human cancers. The effect of EF and its components on viruses has not been reported. In the present study, the antiviral activity and mechanism of action of an aqueous extract of EF (WEF) and its components were evaluated in vitro . We found that pretreatment with WEF markedly reduced viral replication, as evaluated using a green fluorescent protein (GFP)-tagged virus (influenza A virus, Newcastle disease virus, and vesicular stomatitis virus) in murine RAW 264.7 macrophage cells. We demonstrated that WEF induces the production of type I IFN including pro-inflammatory cytokines. Additionally, we identified the active anti-viral components of WEF as quercetin, psoralen, and quercitrin. Thus, WEF and its active components are immunomodulators of the innate immune response in murine macrophages, a finding that is potentially useful to developing prophylactic or therapeutic treatments against a range of viruses.

Enterovirus 71 (EV71) is one of the causative pathogens of hand, foot and mouth disease (HFMD), especially the form associated with fatal neurological disorders. Sustained outbreaks of EV71 infections remain a serious health threat worldwide. However, no antiviral agent against EV71 for clinical therapy has been approved. Retro-2 cycl and Retro-2.1 are inhibitors of several pathogens specifically targeting the intracellular vesicle transport, which also participates in the EV71 lifecycle processes including progeny virus release. Here, we reported that Retro-2 cycl and Retro-2.1, respectively, could inhibit EV71 infection with 50% effective concentrations of 12.56 μM and 0.05 μM in a cytopathic effect inhibition assay and showed relatively low cytotoxicity with 50% cytotoxicity concentrations of more than 500 μM and 267.80 μM. Preliminary mechanism studies revealed that Retro-2 cycl and Retro-2.1 did not inhibit EV71 protein synthesis or RNA replication but could block progeny EV71 release specifically. Furthermore, administration of Retro-2 cycl at the dose of 10 mg/kg significantly protected 90% of newborn mice from lethal EV71 challenge. Consequently, our results for the first time identified Retro-2 cycl and Retro-2.1 as effective inhibitors of EV71 as well as lead compounds, which would contribute to anti-EV71 drug development. We also identified progeny virus release and the intracellular vesicle transport as antiviral targets for EV71., (Copyright © 2017 Elsevier B.V. All rights reserved.)

A review of the pharmacological applications of lignins provides evidence of their protective role against the development of different diseases. In many cases, the effects of lignins could be explained by their antioxidant capacity. Here, we present a systematic review of the literature from the period 2010-2016 which provides information concerning new applications of lignins derived from recent research. The most promising findings are reported, including the methodologies employed and results obtained with lignins or their derivatives which may improve human health. We highlight potential applications in the treatment of obesity, diabetes, thrombosis, viral infections and cancer. Moreover, we report both that lignins can be used in the preparation of nanoparticles to deliver different drugs and also their use in photoprotection., Competing Interests: The authors declare no conflict of interest.

Coptidis Rhizoma is derived from the dried rhizome of Ranunculaceous plants and is a commonly used traditional Chinese medicine. Although Coptidis Rhizoma is commonly used for its many therapeutic effects, antiviral activity against respiratory syncytial virus (RSV) has not been reported in detail. In this study, we evaluated the antiviral activities of Coptidis Rhizoma extract (CRE) against RSV in human respiratory tract cell line (HEp2) and BALB/c mice. An effective dose of CRE significantly reduces the replication of RSV in HEp2 cells and reduces the RSV-induced cell death. This antiviral activity against RSV was through the induction of type I interferon-related signaling and the antiviral state in HEp2 cells. More importantly, oral administration of CRE exhibited prophylactic effects in BALB/c mice against RSV. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we confirmed that palmatine was related to the antiviral properties and immunemodulation effect. Taken together, an extract of Coptidis Rhizoma and its components play roles as immunomodulators and could be a potential source as promising natural antivirals that can confer protection to RSV. These outcomes should encourage further allied studies in other natural products.

Although nucleot(s)ide analogues and pegylated interferon alpha 2a (PEG-IFN-α2a) can suppress hepatitis B virus (HBV) replication, it is difficult to achieve complete HBV elimination from hepatocytes. A novel site-specific pegylated recombinant human IFN-β (TRK-560) was recently developed. In the present study, we evaluated the antiviral effects of TRK-560 on HBV replication in vitro and in vivo. In vitro and in vivo HBV replication models were treated with antivirals including TRK-560, and changes in HBV markers were evaluated. To analyze antiviral mechanisms, cDNA microarray analysis and an enzyme-linked immunoassay (ELISA) were performed. TRK-560 significantly suppressed the production of intracellular HBV replication intermediates and extracellular HBV surface antigen (HBsAg) ( P < 0.001 and P < 0.001, respectively), and the antiviral effects of TRK-560 were enhanced in combination with nucleot(s)ide analogues, such as entecavir and tenofovir disoproxil fumarate. The reduction in HBV DNA levels by TRK-560 treatment was significantly higher than that by PEG-IFN-α2a treatment both in vitro and in vivo ( P = 0.004 and P = 0.046, respectively), and intracellular HBV covalently closed circular DNA (cccDNA) reduction by TRK-560 treatment was also significantly higher than that by PEG-IFN-α2a treatment in vivo ( P = 0.0495). cDNA microarrays and ELISA for CXCL10 production revealed significant differences between TRK-560 and PEG-IFN-α2a in the induction potency of interferon-stimulated genes. TRK-560 shows a stronger antiviral potency via higher induction of interferon-stimulated genes and stronger stimulation of immune cell chemotaxis than PEG-IFN-α2a. As HBsAg loss and HBV cccDNA eradication are important clinical goals, these results suggest a potential role for TRK-560 in the development of more effective treatment for chronic hepatitis B infection., (Copyright © 2017 American Society for Microbiology.)

Interferons α and β (IFNα and IFNβ) are type I interferons produced by the host to control pathogen propagation. However, only a minority of chronic hepatitis B (CHB) patients generate a sustained response after treatment with recombinant IFNα. The anti-HBV effect of IFNβ and the underlying mechanism are not well-understood. Here, we compared the antiviral activities of IFNα and IFNβ by application of IFNα or IFNβ expression plasmids using the well-established HBV hydrodynamic injection (HI) mouse model. Injection of IFNα expression plasmid could significantly reduce HBV serum markers including HBsAg, HBeAg and HBV DNA as well as the number of HBcAg positive cells in the liver, while IFNβ showed only a weak inhibition of HBV replication. In contrast to IFNβ, IFNα resulted in elevated expression levels of IFN stimulated genes (ISGs) as well as the proinflammatory cytokine interleukin 6 (IL6) in the liver. Moreover, IFNβ treated mice showed higher expression levels of the anti-inflammatory cytokines IL10 and TGFβ in the liver compared to IFNα. Our results demonstrated that both IFNα and IFNβ exert antiviral activities against HBV in HI mouse model, but IFNα is more effective than IFNβ., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

White spot syndrome virus (WSSV) is highly lethal and contagious in shrimps; its outbreaks causes an economic crisis for aquaculture. Several attempts have been made to treat this disease; however, to date, there is no effective cure. Because of their antimicrobial activities, silver nanoparticles (AgNPs) are the most studied nanomaterial. Although the antiviral properties of AgNPs have been studied, their antiviral effect against viral infection in aquaculture has not been reported. The AgNPs tested herein are coated with polyvinylpyrrolidone (PVP) and possess multiple international certifications for their use in veterinary and human applications. The aim of this work was to evaluate the survival rate of juvenile white shrimps (Litopenaeus vannamei) after the intramuscular administration of AgNPs. For this, different concentrations of metallic AgNPs and PVP alone were injected into the organisms. After 96 h of administration, shrimp survival was more than 90% for all treatments. The oxygen consumption routine rate and total hemocyte count remained unaltered after AgNP injection, reflecting no stress caused. We evaluated whether AgNPs had an antiviral effect in shrimps infected with WSSV. The results revealed that the survival rate of WSSV-infected shrimps after AgNP administration was 80%, whereas the survival rate of untreated organisms was only 10% 96 h after infection. These results open up the possibility to explore the potential use of AgNPs as antiviral agents for the treatment of diseases in aquaculture organisms, particularly the WSSV in shrimp culture., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Phytohemagglutinin (PHA) isolated from the family of Phaseolus vulgaris beans is a promising agent against viral infection; however, it has not yet been demonstrated in vivo. We herein investigated this issue using Drosophila as a host. Adult flies were fed lectin approximately 12 h before they were subjected to a systemic viral infection. After a fatal infection with Drosophila C virus, death was delayed and survival was longer in flies fed PHA-P, a mixture of L4, L3E1, and L2E2, than in control unfed flies. We then examined PHA-L4, anticipating subunit L as the active form, and confirmed the protective effects of this lectin at markedly lower concentrations than PHA-P. In both experiments, lectin feeding reduced the viral load prior to the onset of fly death. Furthermore, we found a dramatic increase in the levels of the mRNAs of phagocytosis receptors in flies after feeding with PHA-L4 while a change in the levels of the mRNAs of antimicrobial peptides was marginal. We concluded that P. vulgaris PHA protects Drosophila against viral infection by augmenting the level of host immunity.

Porcinereproductiveandrespiratorysyndromevirus (PRRSV) causes significant economic losses to the pork industry worldwide. Previously, we demonstrated that heme oxygenase-1 (HO-1) interferes with PRRSV replication. To elucidate the mechanisms involved, here we assess whether the HO-1 downstream metabolites biliverdin (BV) and/or iron mediate the HO-1 antiviral effect. We demonstrate a BV concentration-dependent suppression of PRRSV replication and show that virions are not directly inactivated by BV. Additionally, BV or N-acetyl cysteine (NAC) significantly reduced reactive oxygen species (ROS) in PRRSV-infected MARC-145 cells; however, because NAC did not reduce viral load, the BV antiviral effect is independent of decreased ROS levels. Moreover, a secondary metabolite of BV, bilirubin (BR), specifically mediates this anti-PRRSV activity via a nitric oxide (NO)-dependent cGMP/PKG signaling pathway. While increased iron via addition of FeCl 3 did not interfere with PRRSV replication, iron depletion by deferoxamine (DFO) after cobalt-protoporphyrin IX induction of HO-1 did not restore PRRSV replication. Collectively, our findings identify a HO-1-BV/BR-NO-cGMP/PKG cascade as a novel pathway underlying the host cell antiviral effect. These results provide a unique insight into the molecular mechanisms underlying the antiviral effects of the stress-responsive protein HO-1 during PRRSV infection., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Coxsackievirus B3 (CVB3) is the main cause of acute myocarditis and dilated cardiomyopathy. Plant extracts are considered as useful materials to develop new antiviral drugs. We had previously selected candidate plant extracts, which showed anti-inflammatory effects. We examined the antiviral effects by using a HeLa cell survival assay. Among these extracts, we chose the Amomi Cardamomi ( Amomi ) extract, which showed strong antiviral effect and preserved cell survival in CVB3 infection. We investigated the mechanisms underlying the ability of Amomi extract to inhibit CVB3 infection and replication. HeLa cells were infected by CVB3 with or without Amomi extract. Erk and Akt activities, and their correlation with virus replication were observed. Live virus titers in cell supernatants and viral positive- and negative-strand RNA amplification were measured. Amomi extract significantly increased HeLa cell survival in different concentrations (100-10 µg/ml). CVB3 capsid protein VP1 expression (76%) and viral protease 2A-induced eIF4G1 cleavage (70%) were significantly decreased in Amomi extract (100 µg/ml) treated cells. The levels of positive- (20%) and negative-strand (80%) RNA were dramatically decreased compared with the control, as revealed by reverse transcription-PCR. In addition, Amomi extract improved mice survival (51% vs 26%) and dramatically reduced heart inflammation in a CVB3-induced myocarditis mouse model. These results suggested that Amomi extract significantly inhibited Enterovirus replication and myocarditis damage. Amomi may be developed as a therapeutic drug for Enterovirus .

The spiro compound 5,6-dimethyl-3H,3'H-spiro(benzofuran-2,1'-isobenzofuran)-3,3'-dione (KR-23502) has antiviral activity against influenza A and more potently B viruses. The aim of this study is to elucidate its mechanism of action. Subcellular localization and time-course expression of influenza B viral proteins, nucleoprotein (NP) and matrix protein 1 (M1), showed that KR-23502 reduced their amounts within 5 h post-infection. Early steps of virus life cycle, including virus entry, nuclear localization of NP and viral RNA-dependent RNA replication, were not affected by KR-23502. Instead it interrupted a later event corresponding to nuclear export of NP and M1 proteins. Delivery of viral ribonucleoprotein (vRNP)-M1 complex has been known to be mediated by the viral nuclear export protein (NEP) through interaction with cellular chromosomal maintenance 1 (CRM1) protein. In this study, we experimentally demonstrated that the compound targets the nuclear export of vRNP. Moreover, a single mutation (aspartate to glycine) at amino acid position 54 in M1 [M1(D54G)] was detected after 18 passages in the presence of KR-23502 with a 2-fold increase in 50% effective concentration indicating that this compound has a relatively high genetic barrier to resistance. Interestingly, it was observed that proteasome-mediated degradation of M1(D54G) was attenuated by KR-23502. In conclusion, we suggest that KR-23502 shows its anti-influenza activity by downregulating NEP/CRM1-mediated nuclear export of influenza vRNP and M1. KR-23502 provides a core chemical skeleton for further structure-based design of novel antivirals against influenza viruses., (Copyright © 2016 Elsevier B.V. All rights reserved.)

This study focuses on the antiviral activity of selected flavonoids against the Chikungunya virus (CHIKV), a mosquito-transmitted virus that can cause incapacitating arthritis in infected individuals. Based on the results of screening on Vero cells, the tested compounds were evaluated further with various assays, including cytotoxicity assay, virus yield assay by quantitative reverse transcription polymerase chain reaction (qRT-PCR), virus RNA replication assay with a CHIKV replicon cell line, Western blotting, and quantitative immunofluorescence assay. Baicalein, fisetin, and quercetagetin displayed potent inhibition of CHIKV infection, with 50% inhibitory concentrations [IC50] of 1.891 μg/ml (6.997 μM), 8.444 μg/ml (29.5 μM), and 13.85 μg/ml (43.52 μM), respectively, and with minimal cytotoxicity. The time-of-addition studies and various antiviral assays demonstrated that baicalein and quercetagetin mainly inhibited CHIKV binding to the Vero cells and displayed potent activity against extracellular CHIKV particles. The qRT-PCR, immunofluorescence assay, and Western blot analyses indicated that each of these flavonoids affects CHIKV RNA production and viral protein expression. These data provide the first evidence of the intracellular anti-CHIKV activity of baicalein, fisetin, and quercetagetin., (Copyright © 2016 Elsevier B.V. All rights reserved.)

In HBV-infected patients, therapies with nucleoside analogues or IFNα remain ineffective in eradicating the infection. Our aim was to re-analyze the anti-HBV activity of a large panel of IFNs and cytokines in vitro using non-transformed cultured hepatocytes infected with HBV, to identify new immune-therapeutic options. HepaRG cells and primary human hepatocytes were infected with HBV and, when infection was established, treated with various concentrations of different IFNs or inflammatory cytokines. Viral parameters were evaluated by quantifying HBV nucleic acids by qPCR and Southern Blot, and secreted HBV antigens were evaluated using ELISA. The cytokines tested were type-I IFNs, IFNγ, type-III IFNs, TNFα, IL-6, IL-1β, IL-18 as well as nucleos(t)ide analogues tenofovir and ribavirin. Cytokines and drugs, with the exception of IL-18 and ribavirin, exhibited a suppressive effect on HBV replication at least as strong as, but often stronger than, IFNα. The cytokine presenting the highest effect on HBV DNA was IL-1β, which exerted its inhibition within picomolar range. Importantly, we noticed differential effects on other parameters (HBV RNA, HBeAg, HBsAg) between both IFNs and inflammatory cytokines, thus suggesting different mechanisms of action. The combination of IL-1β and already used therapies, i.e. IFNα or tenofovir, demonstrated a stronger or similar anti-HBV activity. IL-1β was found to have a very potent antiviral effect against HBV in vitro. HBV was previously shown to promptly inhibit IL-1β production in Kupffer cells. Strategies aiming at unlocking this inhibition and restoring local production of IL-1β may help to further inhibit HBV replication in vivo., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Reovirus not only causes considerable economic loss in the swine industry of the United States and other countries, but also threatens the public health due to its zoonotic potential. According to previous reports, LiCl has antiviral activity against a number of viruses. The inhibitory effects of LiCl on reovirus life cycle in Vero cells were evaluated. The unpaired t-test and one-way ANOVA were used to analyze the differences between experimental groups. We first found that LiCl treatment significantly inhibited reovirus replication in a dose-dependent manner. Furthermore, we found that this antiviral activity of LiCl targets the early stage of viral replication. LiCl could be a potential drug against reovirus infection., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Aims: The aim of this study was to determine the antiviral activity of four probiotic metabolites (Lactobacillus and Bifidobacetrium species) against rotavirus in vitro infection monitored by the NSP4 protein production and Ca(2+) release., Methods and Results: The antiviral effect of the metabolites was performed due a comparison between a blocking model and an intracelullar model on MA104 cells, with the response of NSP4 production and Ca(2+) liberation measured by flow cytometry. Significant results were obtained with the metabolites of Lactobacillus casei, and Bifidobacterium adolescentis in the reduction of the protein production (P = 0·04 and P = 0·014) and Ca(2+) liberation (P = 0·094 and P = 0·020) in the intracellular model, which suggests a successful antiviral activity against RV infection., Conclusions: This study demonstrates that probiotic metabolites were able to interfere with the final amount of intracellular NSP4 protein and a successful Ca(2+) regulation, which suggests a new approach to the mechanism exerted by probiotics against the rotavirus infection., Significance and Impact of the Study: A novel anti-rotaviral effect exerted by probiotic metabolites monitored by the NSP4 protein during the RV in vitro infection and the effect on the Ca(2+) release is reported; suggesting a reduction on the impact of the infection by decreasing the damage of the cells preventing the electrolyte loss., (© 2016 The Society for Applied Microbiology.)

The nervous necrosis virus (NNV) is an aquatic virus that can infect more than 30 species including the grouper, which is a valuable fish species in Taiwan. NNV causes up to 90-100% mortality in the aquaculture industry. Interferons (IFNs) are a family of cytokines that stimulate the expression of numerous proteins to protect the host against viruses and possess very unique specific characteristics in fish. The cross-reactivity of heterologous IFNs on grouper cells and larvae has not been well-studied to date. To evaluate and compare the anti-NNV effect of different fish IFNs in grouper, we successfully synthesized, subcloned, expressed and purified several fish type I IFNs in the present study: grouper (gIFN), salmon (sIFN), seabass (sbIFN) and tilapia (tpIFN). The gIFN and sIFN proteins up-regulated myxovirus resistance protein (Mx) gene expression in grouper kidney (GK) cells, but similar effects were not observed for sbIFN and tpIFN. Following co- and pre-treatment with the 4 types of IFNs with NNV infection in GK cells, sIFN exhibited the strongest antiviral ability to suppress NNV gene replication (especially at 24 h) and significantly reduced the cytopathic effect (CPE) at 72 h, followed by gIFN. Unsurprisingly, sbIFN and tpIFN had no significant effect on CPE but slightly suppressed NNV gene replication. The cytotoxicity of these four fish IFNs on GK cells was also examined for the first time. In the in vivo test, we confirmed that gIFN and sIFN had a significant protective effect against NNV when administered by intraperitoneal (IP) injection and the oral route in Malabar grouper (Epinephelus malabaricus) larvae. This study compared the protective effects of IFNs from various fish species against NNV and demonstrated crosstalk between sIFN and grouper cells for the first time. These results provide information concerning the efficacy of fish IFNs for possible therapeutic applications., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A new class of phenolic lactones with trivial names of spiromastilactones A-M (1-13) was isolated from a deep-sea derived Spiromastix sp. fungus. Their structures featured by various chlorination at aromatic rings were determined on the basis of extensive spectroscopic analyses. An antiviral assay revealed that most of the tested compounds exert inhibitory activity against WSN influenza virus with low cytotoxicity, while the structure-activity relationships were discussed. Spiromastilactone D (4), a 5'-chloro-2'-methoxy substituted analogue, displayed the most potent to inhibit a panel of influenza A and B viruses in addition to drug-resistant clinical isolates. Mechanistic investigation resulted in that compound 4 bonded to hemagglutinin protein (HA), potentially at the spherical head, and disrupted the HA-sialic acid receptor interaction, that is essential for the attachment and entry of all influenza viruses. In addition, compound 4 also showed inhibitory effect toward viral genome replication via targeting viral RNP complex. The synergistic effects of 4 on both viral entry and replication assumed it to be a promising lead for the development of a new influenza inhibitor., (Copyright © 2015 Elsevier Masson SAS. All rights reserved.)

Influenza virus belongs to orthomyxoviridae family. This virus is a major public health problems, with high rates of morbidity and mortality. Despite a wide range of pharmacotherapeutic choices inhibiting specific sequences of pathological process of influenza, developing more effective therapeutic options is an immediate challenge. In this paper, a comprehensively review of natural polyphenolic products used worldwide for the management of influenza infection is presented. Cellular and molecular mechanisms of the natural polyphenols on influenza infection including suppressing virus replication cycle, viral hemagglutination, viral adhesion and penetration into the host cells, also intracellular transductional signaling pathways have been discussed in detail. Based on cellular, animal, and human evidence obtained from several studies, the current paper demonstrates that natural polyphenolic compounds possess potential effects on both prevention and treatment of influenza, which can be used as adjuvant therapy with conventional chemical drugs for the management of influenza and its complications.

Angelica tenuissima Nakai is a widely used commodity in traditional medicine. Nevertheless, no study has been conducted on the antiviral and immune-modulatory properties of an aqueous extract of Angelica tenuissima Nakai. In the present study, we evaluated the antiviral activities and the mechanism of action of an aqueous extract of Angelica tenuissima Nakai both in vitro and in vivo. In vitro, an effective dose of Angelica tenuissima Nakai markedly inhibited the replication of Influenza A virus (PR8), Vesicular stomatitis virus (VSV), Herpes simplex virus (HSV), Coxsackie virus, and Enterovirus (EV-71) on epithelial (HEK293T/HeLa) and immune (RAW264.7) cells. Such inhibition can be described by the induction of the antiviral state in cells by antiviral, IFNrelated gene induction and secretion of IFNs and pro-inflammatory cytokines. In vivo, Angelica tenuissima Nakai treated BALB/c mice displayed higher survivability and lower lung viral titers when challenged with lethal doses of highly pathogenic influenza A subtypes (H1N1, H5N2, H7N3, and H9N2). We also found that Angelica tenuissima Nakai can induce the secretion of IL-6, IFN-λ, and local IgA in bronchoalveolar lavage fluid (BALF) of Angelica tenuissima Nakai treated mice, which correlating with the observed prophylactic effects. In HPLC analysis, we found the presence of several compounds in the aqueous fraction and among them; we evaluated antiviral properties of ferulic acid. Therefore, an extract of Angelica tenuissima Nakai and its components, including ferulic acid, play roles as immunomodulators and may be potential candidates for novel anti-viral/anti-influenza agents.

Viral infections are the major causes of morbidity and mortality in elderly people and young children throughout the world. The most common pathogens include coxsackie virus (CV) and respiratory syncytial virus (RSV). However, no antiviral agents with low toxicity and drug resistance are currently available in clinic therapy. The present study aimed to examine the antiviral activities of emodin (an ingredient of Rheum palmatum) against CVB5 and RSV infections, in an attempt to discover new antiviral agents for virus infection. The monomer emodin was extracted and isolated from Rheum palmatum. The antiviral activities of emodin on HEp-2 cells were evaluated, including virus replication inhibition, virucidal and anti-absorption effects, by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tet-razolium bromide (MTT) assay and plaque reduction assay (PRA). The kinetics of virus inhibition by emodin in a period of 14 h was further determined by plaque assay and quantitative real time PCR (qPCR). Cytokine (IFN-γ, TNF-α) mRNA expressions after emodin treatment (7.5, 15, 30 μmol/L) were also assessed by qPCR post-infection. The results showed that emodin had potent inhibitory activities against CVB5 and RSV, with the 50% effective concentration (EC50) ranging from 13.06 to 14.27 μmol/L and selectivity index (SI) being 5.38-6.41 μmol/L. However, emodin couldn't directly inactivate the viruses or block their absorption to cells. It acted as a biological synthesis inhibitor against CVB4 and RSV in a concentration- and time-dependent manner, especially during the first 0-4 h post-infection. Moreover, emodin could decrease the mRNA expression of IFN-α but enhance TNF-γ expression significantly compared to the viral controls in vitro. Our results provide a molecular basis for development of emodin as a novel and safe antiviral agent for human enterovirus and respiratory virus infection in the clinical therapy.

Caffeic acid phenethyl ester (CAPE) is found in a variety of plants and well-known the active ingredient of the honeybee propolis. CAPE showed anti-inflammatory, anticarcinogenic, antimitogenic, antiviral, and immunomodulatory properties in several studies. The beneficial effects of CAPE on different health issues attracted scientists to make more studies on CAPE. Specifically, the anti-viral effects of CAPE and its molecular mechanisms may reveal the important properties of virus-induced diseases. CAPE and its targets may have important roles to design new therapeutics and understand the molecular mechanisms of virus-related diseases. In this mini-review, we summarize the antiviral effects of CAPE under the light of medical and chemical literature.

The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1) was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic RNA-hydrolyzing activity. Intranasal administration of 3D8 scFv (50 μg/day) for five days prior to infection demonstrated an antiviral activity (70% survival) against H1N1 infection. The antiviral ability of 3D8 scFv to penetrate into epithelial cells from bronchial cavity via the respiratory mucosal layer was confirmed by immunohistochemistry, qRT-PCR, and histopathological examination. The antiviral activity of 3D8 scFv against H1N1 virus infection was not due to host immune cytokines or chemokines, but rather to direct antiviral RNA-hydrolyzing activity of 3D8 scFv against the viral RNA genome. Taken together, our results suggest that the RNase activity of 3D8 scFv, coupled with its ability to penetrate epithelial cells through the respiratory mucosal layer, directly prevents H1N1 virus infection in a mouse model system.

Ribavirin is a nucleoside which is used as an antiviral agent against both RNA and DNA viruses. However, accumulation in erythrocytes causes hemolysis which limits its usefulness. To minimize ribavirin-induced hemolysis and increase its antiviral effect, considerable efforts have been made involving chemical prodrugs and various formulations. Combination with macromolecules to achieve better targeting and increased uptake is one of the most promising strategies. In addition, decreasing the association with RBCs through prodrugs and delivery systems is considered. This review summarizes prodrugs and delivery systems for ribavirin and, at the same time examines these different strategies. Moreover, a novel design of prodrug is proposed for further study., (Copyright © 2015 Elsevier B.V. All rights reserved.)

A series of novel isosteviol derivatives having C4-amide substituents were synthesized in order to test for antiviral effects against the hepatitis B virus (HBV) in vitro. Among them, IN-4 [N-(propylcarbonyl)-4α-amino-19-nor-ent-16-ketobeyeran] (5) exhibited inhibitory activity against secretion of HBsAg and HBeAg as well as inhibition of HBV DNA replication. Therefore, the mechanism of its antiviral activity was further analyzed using HBV-transfected Huh7 cells. Exposure to IN-4 produced minimal inhibitory effects on viral precore/pregenomic RNA expression. However, expression levels of the 2.4/2.1-kb preS/major S RNA of the viral surface gene significantly decreased, along with intracellular levels of HBV DNA. A promoter activity analysis demonstrated that IN-4 significantly inhibited viral X, S, and preS expression levels but not viral core promoter activities. In particular, IN-4 was observed to significantly inhibit HBV gene regulation by disrupting nuclear factor (NF)-κB-associated promoter activity. In addition, the nuclear expression of p65/p50 NF-κB member proteins was attenuated following IN-4 treatment, while cytoplasmic IκBα protein levels were enhanced. Meanwhile, IN-4 was observed to inhibit the binding activity of NF-κB to putative DNA elements. Furthermore, transfection of a p65 expression plasmid into Huh7 cells significantly reversed the inhibitory effect of IN-4 on HBV DNA levels, providing further evidence of the central role of NF-κB in its antiviral mechanism. It is therefore suggested that IN-4 inhibits HBV by interfering with the NF-κB signaling pathway, resulting in downregulation of viral gene expression and DNA replication., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The sterile alpha motif and HD domain 1 (SAMHD1) protein is a novel innate immunity restriction factor that inhibits HIV-1 infection in myeloid cells. Here, we cloned the full-length SAMHD1 complementary DNA (cDNA) from porcine peripheral blood lymphocytes. The porcine SAMHD1 cDNA was of 3951 bp with an open reading frame of 1884 bp, encoding a polypeptide of 627 amino acids. Porcine SAMHD1 mRNA was detected in all swine tissues examined, with the higher expression in the tonsil, lung, liver, and lymph node tissues. The SAMHD1 protein was localized to the nucleus. Overexpression of SAMHD1 blocked the proliferation of HuN4, a highly pathogenic strain of porcine reproductive and respiratory syndrome virus (HP-PRRSV), in MARC-145 cells, by inhibiting the synthesis of the HuN4 complement RNA. The antiviral effects of the simian SAMHD1 protein were nearly equivalent to those of porcine SAMHD1 in the HuN4-infected MARC-145 cells. Phosphorylation analysis of SAMHD1 showed that overexpressed SAMHD1 protein was in primarily an unphosphorylated state. SAMHD1 overexpression increased the transcript abundance of IFN-stimulated genes ISG15 and ISG56. The mRNA levels of SAMHD1 and ISGs were significantly increased in porcine alveolar macrophages infected with HP-PRRSV. SAMHD1 protein level was also elevated, and the protein was not phosphorylated during infection. Collectively, our data indicate that SAMHDI inhibits HP-PRRSV proliferation through inhibiting the replication of HP-PRRSV. SAMHD1 might be the protein participating in the IFN signaling and is thus an important immunoregulatory protein in innate immunity., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Several studies have reported that molecules extracted from invertebrates have activity against different viruses, even against those that do not infect these organisms in their environment. One of the main mechanisms against pathogens in these organisms is the production of antimicrobial peptides. The objective of this study was to determine whether the coelomic fluid (CF) of the sea urchin Tripneustes depressus has activity against Suid herpesvirus type 1 (SHV-1) and/or rabies virus (RV). We tested the antiviral activity of CF in neutralizing assays and observed 50% inhibition against SHV-1 lytic plaque formation using 33 μg of CF, whereas 21 μg CF was sufficient to obtain more than 90% inhibition for RV. Cytotoxicity to MDBK and BHK-21 cells was found with whole CF yet was eliminated by heating at 56 or 72 °C (even when using 50 μg of heat-inactivated CF supernatant [SN or thermostable fraction]), and SN retained the antiviral effect. In both cases, the antiviral effect was direct and thermostable (SN 56 and 72 °C), and the best inhibition was observed when CF + virus was incubated prior to the addition of the cells. Therefore, the coelomic fluid of T. depressus has antiviral activity against SHV-1 and RV that is direct and stable at 72 °C. We suggest that further assays should be performed using more accurate methods to characterize new molecules with antiviral activity that may result in new drugs., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The polymerase basic 2 (PB2) protein is one of four proteins that make up the influenza A virus replication complex, which is responsible for viral gene transcription and replication. To assess the antiviral potential of an anti-PB2 monoclonal antibody that inhibits RNA transcription of influenza A viruses, Mardin-Darby canine kidney (MDCK) cells were transformed with two transgenes that encode the light and heavy chains of the monoclonal antibody. The transformed cell lines expressing this monoclonal antibody displayed resistance to several subtypes of influenza A virus infection. In the transformed cell lines infected with influenza A virus, the level of viral RNA transcription was decreased and the effective nuclear transportation of the PB2 protein was also inhibited. These results demonstrate that the anti-PB2 intrabody is potentially able to interfere with the effective nuclear transportation of PB2 protein, resulting in the observed resistance to influenza A virus infection in vitro., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Interferon-inducible transmembrane (IFITM) protein family is novel viral restriction factors with representative transmembrane structure. These proteins also exist in fish, however, their roles in the innate immune response remain unknown. Here, we report a characterization of teleost IFITM1 from flounder Paralichthys olivaceus (PoIFITM1), which exhibits conserved structure characteristic of the IFITM family but comprises a relatively longer N-terminal region. The expression and promoter activity of PoIFITM1 are markedly induced by aquatic animal viruses: Rana grylio virus (RGV) and Scophthalmus maximus rhabdovirus (SMRV). Overexpression and siRNA-mediated knockdown demonstrate that PoIFITM1 exhibits strong antiviral effects against both DNA virus (RGV) and RNA virus (SMRV), expanding the spectrum of viruses inhibited by IFITM proteins. Further analysis shows that PoIFITM1 suppresses viral entry into host cells, confirming that the IFITM-mediated restriction is conserved from lower vertebrates to mammals. Deletion mutagenesis reveals that PoIFITM1 exerts antiviral activity by targeting to Golgi complex and the N-terminal region is required for its subcellular localization, which is not observed in other known IFITM family members. Our current data provide the first evidence that IFITM1 functions as a key effector of the innate immune to restrict virus replication in lower vertebrates, through the action of impeding viral entry., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Influenza A viruses present a significant threat to public health worldwide. High-affinity human scFv antibodies against a conserved epitope can potentially provide immunity to diverse viruses and protect against future pandemic viruses. A library of phage-displayed human scFv containing 6.0×10(8) members was generated from lymphocytes of H5N1 virus vaccinated individuals. Using the recombinant H5N1 virus hemagglutinin ectodomain (HA1), 4F5 scFv was identified with neutralizing activity against both clade 2 and 9 H5N1 viruses. In embryonated chicken eggs, the antiviral activity of 4F5 scFv conferred a 100% survival rate and at least a 62.5% survival rate against different clades of H5N1 viruses by pre-treatment and post-treatment, respectively. 4F5 scFv belongs to the VH-3-43 family according to the IMGT database, and a peptide (76)WLLGNP(81) containing half of an α-helix in HA1 was identified as the binding pocket. The conserved binding epitope of this novel broadly neutralizing scFv may become key in the design and implementation of vaccines or RNA interference against H5N1 viruses., (Copyright © 2013 Elsevier B.V. All rights reserved.)