Antiviral activity in vitro of two preparations of the herbal medicinal product Sinupret? against viruses causing respiratory infections

Sinupret(?), a herbal medicinal product made from Gentian root, Primula flower, Elder flower, Sorrel herb, and Verbena herb is frequently used in the treatment of acute and chronic rhinosinusitis and respiratory viral infections such as common cold. To date little is known about its potential antiviral activity. Therefore experiments have been performed to measure the antiviral activity of Sinupret(?) oral drops (hereinafter referred to as "oral drops") and Sinupret(?) dry extract (hereinafter referred to as "dry extract"), in vitro against a broad panel of both enveloped and non-enveloped human pathogenic RNA and DNA viruses known to cause infections of the upper respiratory tract: influenza A, Chile 1/83 (H1N1) virus (FluA), Porcine Influenza A/California/07/2009 (H1N1) virus (pFluA), parainfluenza type 3 virus (Para 3), respiratory syncytial virus, strain Long (RSV), human rhinovirus B subtype 14 (HRV 14), coxsackievirus subtype A9 (CA9), and adenovirus C subtype 5 (Adeno 5). Concentration-dependent antiviral activity (EC(50) between 13.8 and 124.8 μg/ml) of Sinupret(?) was observed against RNA as well as DNA viruses independent of a viral envelope. Remarkable antiviral activity was shown against Adeno 5, HRV 14 and RSV in which dry extract was significantly superior to oral drops. This could be ascertained with different assays as plaque-reduction assays in plaque forming units (PFU), the analyses of a cytopathogenic effect (CPE) and with enzyme immunoassays (ELISA) to determine the amount of newly synthesised virus. Our results demonstrate that Sinupret(?) shows a broad spectrum of antiviral activity in vitro against viruses commonly known to cause respiratory infections.     

Inhibition of enveloped virus infection of cultured cells by valproic acid

Valproic acid (VPA) is a short-chain fatty acid commonly used for treatment of neurological disorders. As VPA can interfere with cellular lipid metabolism, its effect on the infection of cultured cells by viruses of seven viral families relevant to human and animal health, including eight enveloped and four nonenveloped viruses, was analyzed. VPA drastically inhibited multiplication of all the enveloped viruses tested, including the zoonotic lymphocytic choriomeningitis virus and West Nile virus (WNV), while it did not affect infection by the nonenveloped viruses assayed. VPA reduced vesicular stomatitis virus infection yield without causing a major blockage of either viral RNA or protein synthesis. In contrast, VPA drastically abolished WNV RNA and protein synthesis, indicating that this drug can interfere the viral cycle at different steps of enveloped virus infection. Thus, VPA can contribute to an understanding of the crucial steps of viral maturation and to the development of future strategies against infections associated with enveloped viruses.     

Human astrovirus coat protein inhibits serum complement activation via C1, the first component of the classical pathway

Human astroviruses (HAstVs) belong to a family of nonenveloped, icosahedral RNA viruses that cause noninflammatory gastroenteritis, predominantly in infants. Eight HAstV serotypes have been identified, with a worldwide distribution. While the HAstVs represent a significant public health concern, very little is known about the pathogenesis of and host immune response to these viruses. Here we demonstrate that HAstV type 1 (HAstV-1) virions, specifically the viral coat protein (CP), suppress the complement system, a fundamental component of the innate immune response in vertebrates. HAstV-1 virions and purified CP both suppress hemolytic complement activity. Hemolytic assays utilizing sera depleted of individual complement factors as well as adding back purified factors demonstrated that HAstV CP suppresses classical pathway activation at the first component, C1. HAstV-1 CP bound the A chain of C1q and inhibited serum complement activation, resulting in decreased C4b, iC3b, and terminal C5b-9 formation. Inhibition of complement activation was also demonstrated for HAstV serotypes 2 to 4, suggesting that this phenomenon is a general feature of these human pathogens. Since complement is a major contributor to the initiation and amplification of inflammation, the observed CP-mediated inhibition of complement activity may contribute to the lack of inflammation associated with astrovirus-induced gastroenteritis. Although diverse mechanisms of inhibition of complement activation have been described for many enveloped animal viruses, this is the first report of a nonenveloped icosahedral virus CP inhibiting classical pathway activation at C1.     

A Direct and Versatile Assay Measuring Membrane Penetration of Adenovirus in Single Cells

Endocytosis is the most prevalent entry port for viruses into cells, but viruses must escape from the lumen of endosomes to ensure that viral genomes reach a site for replication and progeny formation. Endosomal escape also helps viruses bypass endolysosomal degradation and presentation to certain Toll-like intrinsic immunity receptors. The mechanisms for cytosolic delivery of nonenveloped viruses or nucleocapsids from enveloped viruses are poorly understood, in part because no quantitative assays are readily available which directly measure the penetration of viruses into the cytosol. Following uptake by clathrin-mediated endocytosis or macropinocytosis, the nonenveloped adenoviruses penetrate from endosomes to the cytosol, and they traffic with cellular motors on microtubules to the nucleus for replication. In this report, we present a novel single-cell imaging assay which quantitatively measures individual cytosolic viruses and distinguishes them from endosomal viruses or viruses at the plasma membrane. Using this assay, we showed that the penetration of human adenoviruses of the species C and B occurs rapidly after virus uptake. Efficient penetration does not require acidic pH in endosomes. This assay is versatile and can be adapted to other adenoviruses and members of other nonenveloped and enveloped virus families.     

Viperin抑制黄病毒属感染作用机制研究进展

Viperin是近年来发现的具有重要免疫活性的宿主蛋白之一,其在细胞内的表达在病毒感染或干扰素诱导后明显上升,显示出广泛的抗病毒活性。已证实它可以影响许多囊膜病毒在宿主细胞中的组装和释放,但在不同的病毒中所表现的具体抗病毒活性不同。黄病毒属病毒为单股正链具囊膜的RNA病毒,该种属病毒具有相似的结构特征。Viperin蛋白可以抑制多数黄病毒在细胞中的复制。就Viperin抗几种黄病毒属病毒作用机制进行综述,为相关研究提供参考。     

芽胞杆菌及其代谢物抑制包膜病毒感染的研究进展

包膜病毒指具有一层脂质双层膜的病毒,如流感病毒、冠状病毒等,这些包膜病毒每年在世界范围内导致许多严重的疾病,严重威胁人类健康。使用抗病毒药物是预防与治疗病毒感染的主要策略,芽胞杆菌(Bacillus)及其代谢物能够抑制多种包膜病毒的感染。本文综述了芽胞杆菌代谢的粗提物、肽、酶、胞外聚合物、小双链RNA和热灭活的枯草芽胞杆菌孢子在抗包膜病毒感染中发挥的重要作用,其机制是通过直接破坏病毒包膜、阻止膜融合、与病毒基因组RNA直接配对、催化裂解病毒RNA、激活天然免疫反应等对抗病毒,期望为包膜病毒的持续预防和治疗提供参考。     

Borna disease virus glycoprotein is required for viral dissemination in neurons

Borna disease virus (BDV) is a nonsegmented negative-strand RNA virus with a tropism for neurons. Infection with BDV causes neurological diseases in a wide variety of animal species. Although it is known that the virus spreads from neuron to neuron, assembled viral particles have never been visualized in the brains of infected animals. This has led to the hypothesis that BDV spreads as nonenveloped ribonucleoproteins (RNP) rather than as enveloped viral particles. We assessed whether the viral envelope glycoprotein (GP) is required for neuronal dissemination of BDV by using primary cultures of rat hippocampal neurons. We show that upon in vitro infection, BDV replicated and spread efficiently in this system. Despite rapid virus dissemination, very few infectious viral particles were detectable in the culture. However, neutralizing antibodies directed against BDV-GP inhibited BDV spread. In addition, interference with BDV-GP processing by inhibiting furin-mediated cleavage of the glycoprotein blocked virus spread. Finally, antisense treatment with peptide nucleic acids directed against BDV-GP mRNA inhibited BDV dissemination, marking BDV-GP as an attractive target for antiviral therapy against BDV. Together, our results demonstrate that the expression and correct processing of BDV-GP are necessary for BDV dissemination in primary cultures of rat hippocampal neurons, arguing against the hypothesis that the virus spreads from neuron to neuron in the form of nonenveloped RNP.     

A dsRNA-binding protein of a complex invertebrate DNA virus suppresses the Drosophila RNAi response

Invertebrate RNA viruses are targets of the host RNA interference (RNAi) pathway, which limits virus infection by degrading viral RNA substrates. Several insect RNA viruses encode suppressor proteins to counteract this antiviral response. We recently demonstrated that the dsDNA virus Invertebrate iridescent virus 6 (IIV-6) induces an RNAi response in Drosophila. Here, we show that RNAi is suppressed in IIV-6-infected cells and we mapped RNAi suppressor activity to the viral protein 340R. Using biochemical assays, we reveal that 340R binds long dsRNA and prevents Dicer-2-mediated processing of long dsRNA into small interfering RNAs (siRNAs). We demonstrate that 340R additionally binds siRNAs and inhibits siRNA loading into the RNA-induced silencing complex. Finally, we show that 340R is able to rescue a Flock House virus replicon that lacks its viral suppressor of RNAi. Together, our findings indicate that, in analogy to RNA viruses, DNA viruses antagonize the antiviral RNAi response.     

Paramyxovirus assembly and budding: Building particles that transmit infections

The paramyxoviruses define a diverse group of enveloped RNA viruses that includes a number of important human and animal pathogens. Examples include human respiratory syncytial virus and the human parainfluenza viruses, which cause respiratory illnesses in young children and the elderly; measles and mumps viruses, which have caused recent resurgences of disease in developed countries; the zoonotic Hendra and Nipah viruses, which have caused several outbreaks of fatal disease in Australia and Asia; and Newcastle disease virus, which infects chickens and other avian species. Like other enveloped viruses, paramyxoviruses form particles that assemble and bud from cellular membranes, allowing the transmission of infections to new cells and hosts. Here, we review recent advances that have improved our understanding of events involved in paramyxovirus particle formation. Contributions of viral matrix proteins, glycoproteins, nucleocapsid proteins, and accessory proteins to particle formation are discussed, as well as the importance of host factor recruitment for efficient virus budding. Trafficking of viral structural components within infected cells is described, together with mechanisms that allow for the selection of specific sites on cellular membranes for the coalescence of viral proteins in preparation of bud formation and virion release.     

Interaction of infectious viral particles with a quaternary ammonium chlorid (QAC) surface

The antiviral activity of a surface-bonded quaternary ammonium chloride (QAC) was examined in this study. The mechanism of inactivation was elucidated by a combination of infectivity assay, radioactive labeling assay, and sedimentation analysis. Although the virions are still infectious when attached onto the chemically modified surface, we found these viruses are inactivated if they are eluted from the surface. The inactivation is caused by the disruption of the viral envelope with subsequent release of the nucleocapsid. No evidence indicates the released nucleocapsid is further disrupted. An enveloped virus shows a much higher affinity for the QAC-treated surface than a nonenveloped one due to hydrophobic interaction. The QAC-treated beads can effectively remove the enveloped viruses at low protein concentrations. The titer of herpes simplex virus was reduced by a factor of nearly 5 logarithm units in a 0.5 wt % bovine serum albumin solution with less that 10% protein loss. However, the presence of proteins in the solution reduced both the rate and capacity of this nonspecific adsorption-inactivation process. As a consequence, the removal efficiency is relatively poor in solutions with high protein content.     

Inhibition of Influenza A Virus Infection In Vitro by Peptides Designed In Silico

Influenza A viruses are enveloped, segmented negative single-stranded RNA viruses, capable of causing severe human respiratory infections. Currently, only two types of drugs are used to treat influenza A infections, the M2 H⁺ ion channel blockers (amantadine and rimantadine) and the neuraminidase inhibitors (NAI) (oseltamivir and zanamivir). Moreover, the emergence of drug-resistant influenza A virus strains has emphasized the need to develop new antiviral agents to complement or replace the existing drugs. Influenza A virus has on the surface a glycoprotein named hemagglutinin (HA) which due to its important role in the initial stage of infection: receptor binding and fusion activities of viral and endosomal membranes, is a potential target for new antiviral drugs. In this work we designed nine peptides using several bioinformatics tools. These peptides were derived from the HA1 and HA2 subunits of influenza A HA with the aim to inhibit influenza A virus infection. The peptides were synthetized and their antiviral activity was tested in vitro against several influenza A viral strains: Puerto Rico/916/34 (H1N1), (H1N1)pdm09, swine (H1N1) and avian (H5N2). We found these peptides were able to inhibit the influenza A viral strains tested, without showing any cytotoxic effect. By docking studies we found evidence that all the peptides were capable to bind to the viral HA, principally to important regions on the viral HA stalk, thus could prevent the HA conformational changes required to carry out its membranes fusion activity.     

Structural studies of algal lectins with anti-HIV activity

A number of antiviral lectins, small proteins that bind carbohydrates found on viral envelopes, are currently in pre-clinical trials as potential drugs for prevention of transmission of human immunodeficiency virus (HIV) and other enveloped viruses, such as the Ebola virus and the coronavirus responsible for severe acute respiratory syndrome (SARS). Lectins of algal origin whose antiviral properties make them candidate agents for prevention of viral transmission through topical applications include cyanovirin-N, Microcystis viridis lectin, scytovirin, and griffithsin. Although all these proteins exhibit significant antiviral activity, their structures are unrelated and their mode of binding of carbohydrates differs significantly. This review summarizes the current state of knowledge of the structures of algal lectins, their mode of binding of carbohydrates, and their potential medical applications.     

Rottlerin plays an antiviral role at early and late steps of Zika virus infection

Infection of Zika virus (ZIKV) may cause microcephaly and other neurological disorders, while no vaccines and drugs are available. Our study revealed that rottlerin confers a broad antiviral activity against several enveloped viruses, including ZIKV, vesicular stomatitis virus, and herpes simplex virus, but not against two naked viruses (enterovirus 71 and encephalomyocarditis virus). Rottlerin does not have a direct virucidal effect on the virions, and its antiviral effect is independent of its regulation on PKCδ or ATP. Both pretreatment and post-treatment of rottlerin effectively reduce the viral replication of ZIKV. The pretreatment of rottlerin disturbs the endocytosis of enveloped viruses, while the post-treatment of rottlerin acts at a late stage through disturbing the maturation of ZIKV. Importantly, administration of rottlerin in neonatal mice significantly decreased the ZIKV replication in vivo, and alleviated the neurological symptoms caused by ZIKV. Our work suggests that rottlerin exerts an antiviral activity at two distinct steps of viral infection, and can be potentially developed as a prophylactic and therapeutic agent.     

Targeting Membrane-Bound Viral RNA Synthesis Reveals Potent Inhibition of Diverse Coronaviruses Including the Middle East Respiratory Syndrome Virus

Coronaviruses raise serious concerns as emerging zoonotic viruses without specific antiviral drugs available. Here we screened a collection of 16671 diverse compounds for anti-human coronavirus 229E activity and identified an inhibitor, designated K22, that specifically targets membrane-bound coronaviral RNA synthesis. K22 exerts most potent antiviral activity after virus entry during an early step of the viral life cycle. Specifically, the formation of double membrane vesicles (DMVs), a hallmark of coronavirus replication, was greatly impaired upon K22 treatment accompanied by near-complete inhibition of viral RNA synthesis. K22-resistant viruses contained substitutions in non-structural protein 6 (nsp6), a membrane-spanning integral component of the viral replication complex implicated in DMV formation, corroborating that K22 targets membrane bound viral RNA synthesis. Besides K22 resistance, the nsp6 mutants induced a reduced number of DMVs, displayed decreased specific infectivity, while RNA synthesis was not affected. Importantly, K22 inhibits a broad range of coronaviruses, including Middle East respiratory syndrome coronavirus (MERS–CoV), and efficient inhibition was achieved in primary human epithelia cultures representing the entry port of human coronavirus infection. Collectively, this study proposes an evolutionary conserved step in the life cycle of positive-stranded RNA viruses, the recruitment of cellular membranes for viral replication, as vulnerable and, most importantly, druggable target for antiviral intervention. We expect this mode of action to serve as a paradigm for the development of potent antiviral drugs to combat many animal and human virus infections.     

Innate defense against influenza A virus: activity of human neutrophil defensins and interactions of defensins with surfactant protein D

Surfactant protein D (SP-D) plays important roles in innate host defense against influenza A virus (IAV) infection, in part by modifying interactions with neutrophils. Human neutrophil defensins (HNPs) inhibit infectivity of enveloped viruses, including IAV. Our goal in this study was to characterize antiviral interactions between SP-D and HNPs. Recombinant and/or natural forms of SP-D and related collectins and HNPs were tested for antiviral activity against two different strains of IAV. HNPs 1 and 2 did not inhibit viral hemagglutination activity, but they interfered with the hemagglutination-inhibiting activity of SP-D. HNPs had significant viral neutralizing activity against divergent IAV strains. However, the HNPs generally had competitive effects when combined with SP-D in assays using an SP-D-sensitive IAV strain. In contrast, cooperative antiviral effects were noted in some instances when relatively SP-D-resistant strains were treated with SP-D and HNPs. HNPs were found to bind to the neck and/or carbohydrate recognition domain of SP-D. This binding was specific because no, or minimal, binding to other collectins was found. HNPs precipitated SP-D from bronchoalveolar lavage fluid and reduced the antiviral activity of bronchoalveolar lavage fluid. HNP-1 and -2 differed somewhat in their independent antiviral activity and their binding to SP-D. These results are relevant to the early phase of host defense against IAV, and suggest a complex interplay between SP-D and HNPs at sites of active inflammation.     

Inhibition of RNA Recruitment and Replication of an RNA Virus by Acridine Derivatives with Known Anti-Prion Activities

Background

Small molecule inhibitors of RNA virus replication are potent antiviral drugs and useful to dissect selected steps in the replication process. To identify antiviral compounds against Tomato bushy stunt virus (TBSV), a model positive stranded RNA virus, we tested acridine derivatives, such as chlorpromazine (CPZ) and quinacrine (QC), which are active against prion-based diseases.

Methodology/Principal Findings

Here, we report that CPZ and QC compounds inhibited TBSV RNA accumulation in plants and in protoplasts. In vitro assays revealed that the inhibitory effects of these compounds were manifested at different steps of TBSV replication. QC was shown to have an effect on multiple steps, including: (i) inhibition of the selective binding of the p33 replication protein to the viral RNA template, which is required for recruitment of viral RNA for replication; (ii) reduction of minus-strand synthesis by the tombusvirus replicase; and (iii) inhibition of translation of the uncapped TBSV genomic RNA. In contrast, CPZ was shown to inhibit the in vitro assembly of the TBSV replicase, likely due to binding of CPZ to intracellular membranes, which are important for RNA virus replication.

Conclusion/Significance

Since we found that CPZ was also an effective inhibitor of other plant viruses, including Tobacco mosaic virus and Turnip crinkle virus, it seems likely that CPZ has a broad range of antiviral activity. Thus, these inhibitors constitute effective tools to study similarities in replication strategies of various RNA viruses.     

Strategies for antiviral screening targeting early steps of virus infection

Viral infection begins with the entry of the virus into the host target cell and initiates replication.For this reason,the virus entry machinery is an excellent target for antiviral therapeutics.In general,a virus life cycle includes several major steps: cell-surface attachment,entry,replication,assembly,and egress,while some viruses involve another stage called latency.The early steps of the virus life cycle include virus attachment,receptor binding,and entry.These steps involve the initial interactions between a virus and the host cell and thus are major determinants of the tropism of the virus infection,the nature of the virus replication,and the diseases resulting from the infection.Owing to the pathological importance of these early steps in the progress of viral infectious diseases,the development of inhibitors against these steps has been the focus of the pharmaceutical industry.In this review,Herpes Simplex Virus(HSV),Hepatitis C Virus(HCV),and Human Enterovirus 71(EV71)were used as representatives of enveloped DNA,enveloped RNA,and non-enveloped viruses,respectively.The current mechanistic understanding of their attachment and entry,and the strategies for antagonist screenings are summarized herein.     

乳杆菌DM9811发酵液提取物中RNA组分对水泡性口炎病毒抑制作用研究

目的通过乳杆菌DM9811发酵液提取物中RNA组分对水泡性口炎病毒(VSV)抑制作用研究,探讨其在抵抗肠道病毒性感染性疾病方面的作用。方法应用50%细胞感染计量法(TCID50)、免疫荧光法、ELISA和MTT法探讨不同浓度的RNA组分对VSV的抑制作用。结果 RNA组分200μg/mL浓度时与对照比较,RNA组分具有竞争性抑制VSV感染作用,细胞存活率为(90.9±3.67)%,并具有阻断VSV侵入细胞作用,细胞存活率为(96.6±1.47)%。但RNA组分对病毒生物合成的抑制作用不明显。此外RNA组分具有诱导BALB/c小鼠脾细胞产生IFN-α作用,并呈现一定的剂量依赖关系。结论乳杆菌DM9811发酵液提取物中RNA组分对VSV具有明显抑制作用。