Antiviral and virucidal activities against herpes simplex viruses of umesu phenolics extracted from Japanese apricot

Umesu phenolics were obtained from the salt extracts of Japanese apricot (Nanko‐mume cultivar of Prunus mume Sieb. et Zucc.) as purified phenolics. The antiviral activities of umesu phenolics obtained were then examined against herpes simplex virus type 1 (HSV‐1) and type 2 (HSV‐2), enveloped DNA viruses. The phenolics inhibited the multiplication of these viruses when added to the culture media of the infected cells. This inhibition occurred at phenolic concentrations at which they showed no severe cytotoxicity. One‐step growth experiments showed that the eclipse period in the HSV‐1 multiplication process was extended in the presence of umesu phenolics and that the addition of phenolics after the completion of viral DNA replication did not affect their multiplication. More drastic effects were observed on virucidal activities against HSV‐1 and HSV‐2; the infectivity decreased to 0.0001 when infected cells were incubated with 3 mg/ml phenolics at 30°C for 5 min. These results demonstrate the antiviral and virucidal activities of umesu phenolics and suggest a potential pharmacological use for these phenolics as a sanitizing or preventive medicine against superficial HSV infections.     

A viral RNA competitively inhibits the antiviral endoribonuclease domain of RNase L

Ribonuclease L (RNase L) is a latent endoribonuclease in an evolutionarily ancient interferon-regulated dsRNA-activated antiviral pathway. 2'-5' oligoadenylate (2-5A), the product of dsRNA-activated oligoadenylate synthetases (OASes), binds to ankyrin repeats near the amino terminus of RNase L, initiating a series of conformational changes that result in the activation of the endoribonuclease. A phylogenetically conserved RNA structure within group C enteroviruses inhibits the endoribonuclease activity of RNase L. In this study we report the mechanism by which group C enterovirus RNA inhibits RNase L. Viral RNA did not affect 2-5A binding to RNase L. Rather, the viral RNA inhibited the endoribonuclease domain. We used purified RNase L, purified 2-5A, and an RNA substrate with a 5' fluorophore and 3' quencher in FRET assays to measure inhibition of RNase L activity by the viral RNA. The group C enterovirus RNA was a competitive inhibitor of the endoribonuclease with a K(i) of 34 nM. Consistent with the kinetic profile of a competitive inhibitor, the viral RNA inhibited the constitutively active endoribonuclease domain of RNase L. We call this viral RNA the RNase L competitive inhibitor RNA (RNase L ciRNA).     

Carnosine exhibits significant antiviral activity against Dengue and Zika virus

Dengue virus (DENV) and Zika virus (ZIKV) are flaviviruses transmitted to humans by their common vector, Aedes mosquitoes. DENV infection represents one of the most widely spread mosquito‐borne diseases whereas ZIKV infection occasionally re‐emerged in the past causing outbreaks. Although there have been considerable advances in understanding the pathophysiology of these viruses, no effective vaccines or antiviral drugs are currently available. In this study, we evaluated the antiviral activity of carnosine, an endogenous dipeptide (β‐alanyl‐l ‐histidine), against DENV serotype 2 (DENV2) and ZIKV infection in human liver cells (Huh7). Computational studies were performed to predict the potential interactions between carnosine and viral proteins. Biochemical and cell‐based assays were performed to validate the computational results. Mode‐of‐inhibition, plaque reduction, and immunostaining assays were performed to determine the antiviral activity of carnosine. Exogenous carnosine showed minimal cytotoxicity in Huh7 cells and rescued the viability of infected cells with EC₅₀ values of 52.3 and 59.5 μM for DENV2 and ZIKV infection, respectively. Based on the mode‐of‐inhibition assays, carnosine inhibited DENV2 mainly by inhibiting viral genome replication and interfering with virus entry. Carnosine antiviral activity was verified with immunostaining assay where carnosine treatment diminished viral fluorescence signal. In conclusion, carnosine exhibited significant inhibitory effects against DENV2 and ZIKV replication in human liver cells and could be utilized as a lead peptide for the development of effective and safe antiviral agents against DENV and ZIKV.     

牛γ-干扰素在重组杆状病毒中的表达及其抗病毒活性的测定

研究利用Bac-To-Bac杆状病毒表达系统构建含有牛γ-干扰素(Bovine interferon-γ,BoIFN-γ)完整开放阅读框的供体质粒pFastBacTM1-BoIFN-γ,转化DH10Bac感受态细胞获得重组穿梭质粒rBacmid-BoIFN-γ,转染sf9昆虫细胞救获表达BoIFN-γ的重组杆状病毒rBac-BoIFN-γ。采用抗BoIFN-γ单克隆抗体作为一抗进行间接免疫荧光(IFA)及间接ELISA检测,表明BoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得正确表达。利用VSV*GFP-MDBK细胞系统测定rBoIFN-γ抗病毒活性,重组杆状病毒表达重组BoIFN-γ(rBoIFN-γ)能有效抑制水疱性口炎病毒(VSV)在牛肾细胞(MDBK)上的复制,rBac-BoIFN-γ感染sf9昆虫细胞上清抗病毒活性为2×105IU/mL,而且其抗病毒活性可以被鼠抗原核表达重组BoIFN-γ免疫血清阻断。结果表明:rBoIFN-γ在重组杆状病毒rBac-BoIFN-γ感染的sf9昆虫细胞中获得良好表达,并具有高效抗病毒活性。     

RNA viruses as vectors for the expression of heterologous proteins

RNA viruses comprise a wide variety of infectious agents, some of which are the cause of disease in humans, animals, and plants. Recombinant DNA technology is now making it feasible to modify these genomes and engineer them to express heterologous proteins. Several different schemes are being employed that depend on the genome organization of the virus and on the strategy of replication of the particular virus. Several different examples are illustrated and potential uses as well as possible problems are discussed. In the future reverse genetics may convert some of these viruses from agents of disease to agents of cure.     

Antiviral Activity of Trichothecene Mycotoxins (Deoxynivalenol,Fusarenon-X,and Nivalenol) against Herpes Simplex Virus Types 1 and 2

The effect of trichothecene mycotoxins, deoxynivalenol (DON), fusarenon-X (FX) and nivalenol (NIV), on plaque formation of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) in HEp-2 cells was examined. The 50% effective concentrations (EC₅₀) of DON, FX, and NIV for HSV-1 plaque formation were 160, 56, and 120 ng/ml, respectively. Those for HSV-2 plaque formation were 94, 26, and 50 ng/ml, respectively. These three mycotoxins showed about 2-fold higher selectivity to HSV-2 than to HSV-1. Plaque formation of HSV-1 was not inhibited with trichothecenes at concentrations completely inhibiting plaque formation when cells were treated during virus adsorption period or 15 hr before infection. These results indicate that trichothecenes affect replication of HSV-1 after virus adsorption, but not before or during virus adsorption to the host cells.     

Broad-spectrum antiviral effect of Agrimonia pilosa extract on influenza viruses

Influenza virus continues to emerge and re-emerge, posing new threats for humans. Here we tested various Korean medicinal plant extracts for potential antiviral activity against influenza viruses. Among them, an extract of Agrimonia pilosa was shown to be highly effective against all three subtypes of human influenza viruses including H1N1 and H3N2 influenza A subtypes and influenza B virus. The EC₅₀ value against influenza A virus, as tested by the plaque reduction assay on MDCK cells, was 14–23 μg/ml. The extract also exhibited a virucidal effect at a concentration of 160–570 ng/ml against influenza A and B viruses when the viruses were treated with the extract prior to plaque assay. In addition, when tested in embryonated chicken eggs the extract exhibited a strong inhibitory effect in ovo on the H9N2 avian influenza virus at a concentration of 280 ng/ml. Quantitative RT-PCR analysis data showed that the extract, to some degree, suppressed viral RNA synthesis in MDCK cells. HI and inhibition of neuraminidase were observed only at high concentrations of the extract. And yet, the extract's antiviral activity required direct contact between it and the virus, suggesting that its antiviral action is mediated by the viral membrane, but does not involve the two major surface antigens, HA and NA, of the virus. The broad-spectrum antiviral activity of Agrimonia pilosa extract on various subtypes of influenza viruses merits further investigation as it may provide a means of managing avian influenza infections in poultry farms and potential avian-human transmission.     

表面活性素体外抗伪狂犬病毒活性研究

研究了表面活性素（surfactin）体外抗伪狂犬病毒（Pseudorabies Virus,PRV）效果。观察表面活性素的细胞毒性、对PRV直接灭活作用、抗PRV吸附作用及对PRV生物合成抑制作用。结果表明表面活性素对猪肾（porcinekidney,PK-15）细胞的TD50和TD0分别为31．25、4．03μg／mL;具有直接灭活PRV效果,不具有抗PRV吸附作用,对PRV生物合成无显著影响.     

猪I型与II型干扰素的克隆、表达及抗病毒活性比较

干扰素(IFN)是由多种细胞受病毒感染或其他生物诱导剂刺激而产生的天然蛋白质,主要功能为抗病毒增殖、调节免疫反应和激活免疫细胞等。本研究克隆并测序了猪干扰素(PoIFN)α、γ、αγ及ω基因。构建原核表达载体pET-His/PoIFN-α、pET-His/PoIFN-γ、pET-His/PoIFN-αγ和pET-His/PoIFN-ω,转化大肠杆菌Rosetta(DE3)进行表达,经纯化、复性得到具有生物学活性的蛋白。用细胞病变抑制法在Marc-145/PRRSV、Marc-145/VSV、PK-15/VSV、Vero/VSV、MDBK/VSV系统上进行抗病毒活性测定,结果表明猪α和αγ融合干扰素有较为显著的抗病毒活性,抗PRRSV活性高达108U/mg;猪γ干扰素活性效价约为α干扰素的1/2到1/3;猪ω干扰素几乎未检测到抗病毒活性,需进一步验证。本研究对干扰素在抗病毒、提高机体免疫方面的应用提供了理论依据。     

Complex of the herpes simplex virus type 1 origin binding protein UL9 with DNA as a platform for the design of a new type of antiviral drugs

The herpes simplex virus type 1 origin-binding protein, OBP, is a DNA helicase encoded by the UL9 gene. The protein binds in a sequence-specific manner to the viral origins of replication, two OriS sites and one OriL site. In order to search for efficient inhibitors of the OBP activity, we have obtained a recombinant origin-binding protein expressed in Escherichia coli cells. The UL9 gene has been amplified by PCR and inserted into a modified plasmid pET14 between NdeI and KpnI sites. The recombinant protein binds to Box I and Box II sequences and possesses helicase and ATPase activities. In the presence of ATP and viral protein ICP8 (single-strand DNA-binding protein), the initiator protein induces unwinding of the minimal OriS duplex (≈80?bp). The protein also binds to a single-stranded DNA (OriS^?) containing a stable Box I-Box III hairpin and an unstable AT-rich hairpin at the 3′-end. In the present work, new minor groove binding ligands have been synthesized which are capable to inhibit the development of virus-induced cytopathic effect in cultured Vero cells. Studies on binding of these compounds to DNA and synthetic oligonucleotides have been performed by fluorescence methods, gel mobility shift analysis and footprinting assays. Footprinting studies have revealed that Pt-bis-netropsin and related molecules exhibit preferences for binding to the AT-spacer in OriS. The drugs stabilize structure of the AT-rich region and inhibit the fluctuation opening of AT-base pairs which is a prerequisite to unwinding of DNA by OBP. Kinetics of ATP-dependent unwinding of OriS in the presence and absence of netropsin derivatives have been studied by measuring the efficiency of Forster resonance energy transfer (FRET) between fluorophores attached to 5′- and 3′- ends of an oligonucleotide in the minimal OriS duplex. The results are consistent with the suggestion that OBP is the DNA Holiday junction (HJ) binding helicase. The protein induces conformation changes (bending and partial melting) of OriS duplexes and stimulates HJ formation in the absence of ATP. The antiviral activity of bis-netropsins is coupled with their ability to inhibit the fluctuation opening of АТ base pairs in the А?+?Т cluster and their capacity to stabilize the structure of the АТ-rich hairpin in the single-stranded oligonucleotide corresponding to the upper chain in the minimal duplex OriS. The antiviral activities of bis-netropsins in cell culture and their therapeutic effects on HSV1-infected laboratory animals have been studied.     

Antiviral activity of aqueous and ethanol extracts and of an isolated polysaccharide from Agaricus brasiliensis against poliovirus type 1

AIMS: Agaricus brasiliensis (previously named Agaricus blazei ss. Heinem), also known as the sun mushroom is native of Southeast Brazil, and is widely consumed, mainly in the form of tea, due to its nutritional and pharmacological properties. In this study, we tested aqueous (AqE) and ethanol (EtOHE) extracts and an isolated polysaccharide (PLS) from the fruiting body of A. brasiliensis, for antiviral activity against poliovirus type 1 in HEp-2 cells. METHODS AND RESULTS: The evaluation of the time of addition by plaque assay showed that when AqE, PLS and EtOHE were added, just after the virus inoculation (time 0 h), there was a concentration-dependent reduction in the number of plaques up to 50%, 67% and 88%, respectively, with a selectivity index (SI) of 5.4, 9.9 and 12.3, respectively. CONCLUSIONS: The test substances showed antiviral activity and were more effective when added during the poliovirus infection. As they had little effect on reducing viral adsorption and did not show any virucidal effect, we suggest that they act at the initial stage of the replication of poliovirus. SIGNIFICANCE AND IMPACT OF THE STUDY: These results corroborate that basidiomycetes can be a rich source of potential antiviral compounds.     

Sensitivity of influenza a viruses to human interferons in human diploid cells

Abstract The sensitivity of influenza virus to the action of natural human interferon (IFN)-α+β and -γ, and to the action of highly purified recombinant HuIFN-αB, -αD, and -αF, has been investigated. A plaque assay for the fowl-plague strain of influenza A virus has been established using human embryonic foreskin (HEF) cells. The sensitivity of influenza virus to all IFNs tested in this assay was comparable to that shown by vesicular stomatitis virus (VSV) which was taken as the reference standard. The high sensitivity to IFN action found for the fowl-plague strain was confirmed for the WSN strain of human origin in a yield reduction assay.     

Inactivation and elimination of human enteric viruses by Pacific oysters

Aims: To investigate the comparative elimination of three different human enterically transmitted viruses [i.e. hepatitis A virus (HAV), norovirus (NoV) and poliovirus (PV)] and inactivation of HAV and PV by Pacific oysters. Methods and Results: New Zealand grown Pacific oysters (Crassostrea gigas) were allowed to bioaccumulate HAV, NoV and PV. Samples of oyster gut, faeces and pseudofaeces were then analysed by using real‐time RT‐PCR to determine the amount of viral RNA and cell culture methods to identify changes in the number of plaque forming units. The results suggest that the majority of the PV present in the oyster gut and oyster faeces is noninfectious, while in contrast, most of the HAV detected in the oyster gut are infectious. Depuration experiments identified a large drop in the count of PV in the gut over a 23‐h cleansing period, whereas the levels of HAV and NoV did not significantly decrease. Conclusions: Human enterically transmitted viruses are eliminated and inactivated at different rates by Pacific oysters. Significance and Impact of Study: The research presented in this article has implications for risk management techniques that are used to improve the removal of infectious human enteric viruses from bivalve molluscs.     

Susceptibility of endothelial cells derived from different blood vessels to common viruses

Summary We examined whether endothelial cells derived from different blood vessels vary in their susceptibility to viral infection. Five common viral pathogens of humans (herpes simplex 1, measles, mumps, echo 9, and coxsackie B4 viruses) were evaluated for growth in endothelial cells derived from bovine fetal pulmonary artery thoracic aorta, and vena cava. All five viruses replicated in each type of endothelial cell. There were apparent differences in the quantities of measles and mumps viruses produced in pulmonary artery endothelium compared with thoracic aorta and vena cava when endothelial cells were obtained from different animals. However when pulmonary artery endothelial cells were compared with vena cava cells from the same animal, growth of each virus was similar in the two cell types. Four of the viruses replicated in the various endothelial cells without producing appreciable changes in cell morphology. These results indicate that endothelial cells from different blood vessels are equally susceptible to the human viruses evaluated, and that viral replication can occur without major alteration in cell morphology. Endothelial cells could serve as permissive cells permitting viruses to leave the circulation and initiate infection in adjacent tissues, including subendothelial smooth muscle cells. This work was supported by Public Health Service grants HL28220, HL 29492, and HL 24914 from the National Heart, Lung and Blood Institute, Bethesda, MD.     

3-Trifluoromethylpyrazolones derived nucleosides: Synthesis and antiviral evaluation

Dengue (DENV) viral infection is a global public health problem that infrequently develops life threatening diseases such as dengue hemorrhagic fever (DFS) and dengue shock syndrome (DSS). Middle East respiratory syndrome coronavirus (MERS-CoV) is a highly pathogenic human corona virus with 38% fatality rate of infected patients. A series of 4-arylhydrazono-5-trifluoromethyl-pyrazolones, their ribofuranosyl, and 5′-deoxyribofuranosyl nucleosides were synthesized, geometry optimized using Density functional theory (DFT), and evaluated for their antiviral activity. 2-Nitrophenylhydrazonopyra-zolone derivative 5 showed significant activity against MERS-CoV (EC₅₀ = 4.6?μM). The nucleoside analog 8 showed moderate activity against DENV-2 (EC₅₀ = 10?μM), while the activity was abolished with the corresponding 5′-deoxyribonucleoside analogs. The identified hits in this study set this category of compounds for further future optimizations.     

Silencing of UBP43 by shRNA enhances the antiviral activity of interferon against hepatitis B virus

Previous studies have shown that expression of the interferon-sensitive gene (ISG)I5 protease UBP43 is increased in the liver biopsy specimens of patients who do not respond to interferon (IFN)-α therapy. We hypothesized that UBP43 might hinder the ability of IFN to inhibit HBV replication. In this study, we investigated whether vector-based siRNA promoted by Hi (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. UBP43 was specifically silenced using shRNA. In HepG2.2.15 cells, the HBeAg and HBV DNA levels were significantly reduced by IFN after transfection of shRNA, imphicated that vector-based siRNA promoted by HI (psiUBP43) could enhance IFN inhibiting HBV replication in cell culture. These data suggest that UBP43 modulates the anti-HBV type I IFN response, and is a possible therapeutic target for the treatment of HBV infection.     

Enzymatic and nonenzymatic functions of viral RNA-dependent RNA polymerases within oligomeric arrays

Few antivirals are effective against positive-strand RNA viruses, primarily because the high error rate during replication of these viruses leads to the rapid development of drug resistance. One of the favored current targets for the development of antiviral compounds is the active site of viral RNA-dependent RNA polymerases. However, like many subcellular processes, replication of the genomes of all positive-strand RNA viruses occurs in highly oligomeric complexes on the cytosolic surfaces of the intracellular membranes of infected host cells. In this study, catalytically inactive polymerases were shown to participate productively in functional oligomer formation and catalysis, as assayed by RNA template elongation. Direct protein transduction to introduce either active or inactive polymerases into cells infected with mutant virus confirmed the structural role for polymerase molecules during infection. Therefore, we suggest that targeting the active sites of polymerase molecules is not likely to be the best antiviral strategy, as inactivated polymerases do not inhibit replication of other viruses in the same cell and can, in fact, be useful in RNA replication complexes. On the other hand, polymerases that could not participate in functional RNA replication complexes were those that contained mutations in the amino terminus, leading to altered contacts in the folded polymerase and mutations in a known polymerase–polymerase interaction in the two-dimensional protein lattice. Thus, the functional nature of multimeric arrays of RNA-dependent RNA polymerase supplies a novel target for antiviral compounds and provides a new appreciation for enzymatic catalysis on membranous surfaces within cells.