Antiviral Substances Research Articles

Inhibition of Zika Virus Replication by Silvestrol.

The Zika virus (ZIKV) outbreak in 2016 in South America with specific pathogenic outcomes highlighted the need for new antiviral substances with broad-spectrum activities to react quickly to unexpected outbreaks of emerging viral pathogens. Very recently, the natural compound silvestrol isolated from the plant Aglaia foveolata was found to have very potent antiviral effects against the (−)-strand RNA-virus Ebola virus as well as against Corona- and Picornaviruses with a (+)-strand RNA-genome. This antiviral activity is based on the impaired translation of viral RNA by the inhibition of the DEAD-box RNA helicase eukaryotic initiation factor-4A (eIF4A) which is required to unwind structured 5´-untranslated regions (5′-UTRs) of several proto-oncogenes and thereby facilitate their translation. Zika virus is a flavivirus with a positive-stranded RNA-genome harboring a 5′-capped UTR with distinct secondary structure elements. Therefore, we investigated the effects of silvestrol on ZIKV replication in A549 cells and primary human hepatocytes. Two different ZIKV strains were used. In both infected A549 cells and primary human hepatocytes, silvestrol has the potential to exert a significant inhibition of ZIKV replication for both analyzed strains, even though the ancestor strain from Uganda is less sensitive to silvestrol. Our data might contribute to identify host factors involved in the control of ZIKV infection and help to develop antiviral concepts that can be used to treat a variety of viral infections without the risk of resistances because a host protein is targeted.

Time-of-addition and Temperature-shift Assays to Determine Particular Step(s) in the Viral Life Cycle that is Blocked by Antiviral Substance(s).

Viruses infect their host cells to produce progeny virus particles through the sequential steps of the viral life cycle, such as viral attachment, entry, penetration and post-entry events. This protocol describes time-of-addition and temperature-shift assays that are employed to explore which step(s) in the viral life cycle is blocked by an antiviral substance(s).

Evaluation of an inhibitory effect of edible mushroom extracts against rotavirus infection

Background According to recent statistics by the WHO, the annual mortality rate associated with diarrhea is 30 deaths per 100 000 among Egyptian children younger than 5 years. Nearly 3.9% of the reported deaths are because of rotavirus infection. It is necessary to look for alternative treatment against rotavirus. Aim and methods The aim of this study is to investigate the antiviral activity of aqueous (AqE) and ethanol (EtOHE) extracts of the fruiting bodies of Agaricus spp and Pleurotus ostreatus against rotavirus infection using cell culture-based MTT assay. Results The tested extracts demonstrated significant inhibition effects against rotavirus infection up to 96.7, 90.6, 86.3, and 83.1% at concentration of 1000 μg/ml of P. ostreatus EtOHE, P. ostreatus AqE, Agaricus spp EtOHE, and Agaricus spp AqE, respectively, when added at zero time of the infection. Nothing was observed when extracts were added after viral infection. The synergistic activity was observed when different extracts were combined. Our results exhibited an inhibitory effect against different phases of rotavirus infection. Conclusion The use of edible mushrooms as a potential antiviral substance might be an alternative treatment against rotavirus infection. Nonetheless, more investigations are requiring for studying the efficacy in vivo and for segregating their fractions, which might clarify the mechanism of the inhibitory effect.

Virucidal and Neutralizing Activity Tests for Antiviral Substances and Antibodies.

In a narrow definition, virucidal activity represents the activity by which to interact with and physically disrupt viral particles. In a broad definition, it includes the activity by which to functionally inhibit (neutralize) viral infectivity without apparent morphological alterations of the viral particles. The viral infectivity can be measured in cell culture system by means of plaque assay, infectious focus assay, 50% tissue culture infectious dose (TCID50) assay, etc. Morphologically, disruption of viral particles can be demonstrated by negative staining electron microscopic analysis of viral particles. In this article, we describe methods to assess virucidal activity in a broad definition.

A new promising candidate to overcome drug resistant herpes simplex virus infections

Infections with Herpes simplex viruses (HSV) belong to the most common human diseases worldwide, resulting in symptoms ranging from painful, but commonly self-limiting lesions of the orofacial or genital tract to severe infections of the eye or life-threatening generalized infections. Frequent HSV-reactivations at the eye may lead to the development of herpetic stromal keratitis, which is one of the major causes of infectious blindness in developed countries. The vast majority of life-threatening generalized infections occur in immunocompromised individuals, such as transplant recipients or patients suffering from advanced human immunodeficiency virus (HIV) infection with concurrent HSV-reactivation. Over the past decades, Acyclovir (ACV) became the golden standard for the treatment of HSV infections. However, long-term antiviral treatment, as it is required mainly in immunocompromised patients, led to the emergence of resistances towards ACV and other antivirals. Therefore, there is a clear need for the development of new potent antivirals which combine good oral bioavailability and tolerability with low side effects. In the current study we present SC93305 as a novel potent antiviral substance that proved to be highly effective not only against different HSV-1 and HSV-2 strains but also towards ACV- and multi-resistant HSV-1 and HSV-2 isolates. SC93305 shows comparable antiviral activity as reported for ACV and very importantly it does not interfere with the activation of specific immune cells. Here we report that SC93305 does not affect the biological function of dendritic cells (DC), the most potent antigen presenting cells of the immune system to induce antiviral immune responses, nor T cell stimulation or the release of inflammatory cytokines. Thus, SC93305 is a new and promising candidate for the treatment of HSV-1 and HSV-2 infections and in particular also for the inhibition of drug-resistant HSV-1/2 strains.

Knock-in Vector for Expression of Insulin-like Growth Factor 1 on the Bovine β-casein Gene Locus

The production of therapeutic protein and improve of productivity of the domestic animal from transgenic animal is the major technology of biotechnology. Lactoferrin has its highest content in the colostrum, and is known as the antiviral substance. Insulin like growth factor1 is known to play an important role in the growth of the animal. The objective of this study is construction of knock-in vector that insulin like growth factor1 gene is inserted into the β-casein gene locus for expression of bovine insulin like growth factor1 (bIGF1) on the bovine β-casein gene. The knock-in vector consists of 5’ arm region (1.02 kb), insulin like growth factor 1 cDNA, CMV-EGFP, and 3’ arm region (1.83 kb). To express bIGF1 gene as transgene, the F2A sequence was fused to the 5’ terminal of bIGF1 gene and inserted into exon 7 of the β-casein gene. As a result, the knock-in vector is confirmed that the amino acids are synthesized without termination from the β-casein exon 7 region to the bIGF1 gene. These knock-in vectors may help to create transgenic dairy cattle expressing bovine IGF1 protein in the mammary gland via the expression system of the bovine β-casein gene.

Synthesis and antiviral study of 4-(7,7-dimethyl-4-(piperazin-1-yl)-5,6,7,8-tetrahydroquinazolin-2-yl) morpholine derivatives

A series of 4-(7,7-dimethyl-4-(piperazin-1-yl)-5,6,7,8-tetrahydroquinazolin-2-yl)morpholine substituted sulfonamide and urea derivatives has been synthesized and characterized using spectral techniques. The antiviral activity of these compounds against an avian paramyxo virus (AMPV-1) was screened using MTT assay and found that one of the sulfonamide derivatives (2d) show three-fold higher antiviral activity than Ribavirin, a commercial antiviral drug substance.

Proteomic analysis of rat peripheral nerves

Biodiversity forms the basis for a large pool of potential products and productive organisms offered by terrestrial cyanobacteria. They are stuck together by EPS (extracellular polymeric substances) that can obtain antiviral, antibacterial or anti-inflammatory substances. Most stress conditions, e.g. drought, induce the production of protective EPS or biotechnological-products for pharmaceutical application. However, the growth of a phototrophic biofilm is limited under submerged conditions. Therefore, a semi-continuous process to produce EPS by cyanobacteria was developed in an aerosol-based ePBR (emerse photobioreactor) that imitates the natural habitat of terrestrial cyanobacteria.The process consists of a growth-phase (biomass production), followed by a dry-phase (EPS-production) and a consecutive extraction. The EPS-productivities of Trichocoleus sociatus (ranging from 0.03 to 0.04 g L−1 d−1) were 32 times higher than described in topic-related literature. In comparison to submerge cultivations in shaking flasks, the EPS-productivities were sevenfold higher. To ensure that the extraction solvent has no influence on cell viability, a cell-vitality-test was performed. However, no statistically significant difference between the amount of living and dead cells before and after the extraction was detected. A bioactivity assay was then performed to identify antimicrobial activity within EPS extracts from emerse and submerge cultivations. The EPS revealed an antibacterial effect against gram-negative bacteria (E. coli) which was two times higher than EPS from a submerged cultivation.

A semi-continuous process based on an ePBR for the production of EPS using Trichocoleus sociatus

Biodiversity forms the basis for a large pool of potential products and productive organisms offered by terrestrial cyanobacteria. They are stuck together by EPS (extracellular polymeric substances) that can obtain antiviral, antibacterial or anti-inflammatory substances. Most stress conditions, e.g. drought, induce the production of protective EPS or biotechnological-products for pharmaceutical application. However, the growth of a phototrophic biofilm is limited under submerged conditions. Therefore, a semi-continuous process to produce EPS by cyanobacteria was developed in an aerosol-based ePBR (emerse photobioreactor) that imitates the natural habitat of terrestrial cyanobacteria.The process consists of a growth-phase (biomass production), followed by a dry-phase (EPS-production) and a consecutive extraction. The EPS-productivities of Trichocoleus sociatus (ranging from 0.03 to 0.04gL−1d−1) were 32 times higher than described in topic-related literature. In comparison to submerge cultivations in shaking flasks, the EPS-productivities were sevenfold higher. To ensure that the extraction solvent has no influence on cell viability, a cell-vitality-test was performed. However, no statistically significant difference between the amount of living and dead cells before and after the extraction was detected. A bioactivity assay was then performed to identify antimicrobial activity within EPS extracts from emerse and submerge cultivations. The EPS revealed an antibacterial effect against gram-negative bacteria (E. coli) which was two times higher than EPS from a submerged cultivation.

Antiviral Activity of Aspalathus linearis against Human Influenza Virus

Influenza A viruses are responsible for annual epidemics and occasional pandemics, which cause significant morbidity and mortality. The limited protection offered by influenza vaccination, and the emergence of drug-resistant influenza strains, highlight the urgent need for the development of novel anti-influenza drugs. However, the search for antiviral substances from the library of low molecular weight chemical compounds is limited. Thus, because of their natural diversity and accessibility, plants or plant-derived materials are rapidly becoming valuable sources for the discovery and development of new antiviral drugs. In this study, crude extracts of Aspalathus linearis, a plant reported to have anti-HIV activity, were evaluated in vitro for their activity against the influenza A virus Of the extracts tested, an alkaline extract of Aspalathus linearis demonstrated the strongest inhibition against influenza A virus and could also inhibit different.types of influenza viruses, including Oseltamivir-resistant influenza viruses A and B. Our time course of addition studies indicated that the alkaline extract of Aspalathus linearis exerts its antiviral effect predominantly during the late stages of the influenza virus replication process.

A direct quantitative PCR-based measurement of herpes simplex virus susceptibility to antiviral drugs and neutralizing antibodies

Herpes simplex viruses (HSV) are common human pathogens that can cause painful but benign manifestations and recurrent complaints, but can also cause significant morbidity and mortality on infection of the eye or brain and with disseminated infection of an immunosuppressed patient or a neonate. HSV growth inhibition measurement by plaque or yield reduction is a key task in the development of novel antiviral compounds but the manual methods are very labour intensive. The sensitive and specific PCR technology could be an effective method for quantitation of HSV DNA related to virus replication; however the currently described PCR approaches have a major limitation, namely the requirement of purification of DNA from the infected cells. This limitation makes this approach unfeasible for high-throughput screenings. The monitoring of HSV specific antibody titre is essential in vaccination trials and in the improvement of HSV-based oncolytic virotherapy. Usually, conventional cytopathic effect-based and plaque reduction neutralization tests are applied to measure the neutralization titre, but these methods are also time-consuming. To overcome this, we developed a quantitative PCR (qPCR) method for the detection of HSV-2 DNA directly from the infected cells (direct qPCR) and the method was further adapted to measure the titre of HSV specific neutralizing antibody in human sera. The conditions of direct qPCR assay were optimized to measure the antiviral activity of known and novel antiviral substances. Using HSV-2 seronegative and seropositive patients’ sera, the validity of the direct qPCR neutralization test was compared to traditional cytopathic effect-based assay.The direct qPCR method was able to detect the HSV-2 DNA quantitatively between multiplicity of infection 1/64 and 1/4194304, indicating that the dynamic range of the detection was approximately 65,500 fold with high correlation between the biological and technical replicates. As a proof of the adaptability of the method, we applied the direct qPCR for antiviral inhibitory concentration 50 (IC50) measurements of known and novel antiviral compounds. The measured IC50 of acyclovir was ∼0.28μg/ml, similar to the previously published IC50 value. The IC50 of novel antiviral candidates was between 1.6–3.1μg/ml. The direct qPCR-based neutralization titres of HSV positive sera were 1:32–1:64, identical to the neutralization titres determined using a traditional neutralization assay. The negative sera did not inhibit the HSV-2 replication in either of the tests.Our direct qPCR method for the HSV-2 growth determination of antiviral IC50 and neutralization titre is less time-consuming, less subjective and a more accurate alternative to the traditional plaque titration and growth reduction assays.

Super Antibiotics: Part III. Hyperforin, Revision of the Relative and Absolute Stereochemistry Presented by Bystrov <i>et al</i>.

Hyperforin is an antibiotic, antibacterial, antiprotozoal, antiviral, anticancer, and immunomodulatory substance. It is possibly a new and potent tool for curative treatment of a broad array of infection diseases. Despite the medical usefulness of Hypericum perforatum L., which has been known since ancient times, its constituents were not well studied until the middle of the 20th century. Several pharmacological active substances (PASs) were then discovered as constituents of H. perforatum. Among these was the antibacterial substance, hyperforin. The chemistry and relative and absolute stereochemistry of hyperforin were studied at the Shemyakin Institute of Bioorganic Chemistry, USSR Academy of Sciences, Moscow, USSR, the Institute of Biochemistry and Physiology of Microorganisms, USSR Academy of Sciences, Moscow, USSR, and the Zabolotny Institute of Microbiology and Virology at the Ukrainian Academy of Sciences, Kiev, UkrSSR. In this paper, hyperforin’s relative and absolute stereochemistry, as reported in earlier published former USSR literature (cited herein), is discussed, as well as cited up to date in international literature. In 1979, the relative and absolute stereochemistry of hyperforin and some constituents of H. perforatum were studied at the University of Oslo, Oslo, Norway. In 1979, Brondz et al. challenged the correctness of the relative and absolute stereochemistry of hyperforin reported previously by Bystrov et al. The relative stereochemistry of a drug is the basis for possible partial or total synthesis and absolute stereochemistry is crucial for the action of drugs on enzymes. Correct molecular isometry (absolute stereochemistry) is key for high pharmacological activity versus harm/toxicity. Elucidation of the ste-reochemistry of an unknown molecule is of great academic and theoretical interest, and in the case of a drug, it also has great practical interest.

Antiviral and Antitumor Activity of Licorice Root Extracts.

In the search for anti-viral and antitumor substances from natural resources, antiviral and antitumor activities of licorice root extract and purified ingredients were investigated. Viability of cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method. Antiviral activity was quantified by the selectivity index, defined as the ratio of the 50% cytotoxic concentration (CC50) to the 50% effective concentration against human immunodeficiency virus (HIV) or herpes simplex virus (HSV)-infected cells (EC50). The tumor specificity was calculated by the ratio of CC50 against human normal oral cells to that against human oral squamous cell carcinoma cell lines. Licorice flavonoids and lower molecular polyphenols were subjected to quantitative structure-activity relationship analysis. Alkaline extract of licorice root had higher anti-HIV activity than did water extracts, confirming our previous reports. On the other hand, water extract, especially the flavonoid-rich fraction, had higher anti-HSV activity than did the alkaline extract. The flavonoid-rich fraction was more cytotoxic against human oral squamous cell carcinoma cell lines compared to normal oral cells, suggesting their tumor-specific cytotoxicity. The present study suggests that water and alkaline extracts of licorice root exert different mechanisms of actions against these two viruses. Physicochemical properties, rather than the category of compounds, may be important in determining their anti-HSV activity.

Hepatitis C virus antibody titers associated with cognitive dysfunction in an asymptomatic community-based sample

ABSTRACTBackground: Hepatitis C virus (HCV) infection is associated with cognitive dysfunction in clinic-based studies. The risk could be attributed to factors such as antiviral medications, substance abuse, or coincidental infection. Aim: The aim was to evaluate cognitive function in relation to HCV antibody titers in a community-based sample of asymptomatic individuals at low risk for substance abuse. Method: Adults were ascertained from a community in Mansoura, Egypt, where HCV is endemic (n = 258). Cognitive performance was evaluated using the Arabic version of the Penn Computerized Neurocognitive Battery. Substance abuse and psychopathology were also assessed. Antibodies to HCV and Toxoplasma gondii (TOX), a common protozoan that can affect cognition, were estimated using serological IgG assays. Results: The prevalence of HCV and TOX infection was 17.6% and 52.9%, respectively. HCV antibody titers were significantly associated with worse function in four cognitive tests for accuracy and three tests for speed, after adjusting for covariates (p < .05, beta coefficients, 2.1–3.2). TOX antibody titers were associated with impaired accuracy in one test. Conclusions: The association between HCV antibody titers and cognitive impairment is not mediated by antiviral treatment or substance abuse in this sample. Whether HCV has a causal role in the cognitive dysfunction should be investigated.

Scientific foundations for the creation of antiviral and antibacterial preparations

The results of the Ural scientific school’s organic chemists on the creation of antiviral and antibacterial, including antitubercular, chemopreparations are considered. Basic research data were generalized on the synthesis and study of antiviral activity and the establishment of the metabolism and mechanism of azoloazine series compounds—azaanalogs of adenine and guanine, as well as their nucleosides, which led to the creation of a new family of antiviral substances. One of them, triazavirin, has become a regular therapeutic fixture as an antiflu preparation. The results of synthetic and biological studies on substituted pyrimidines, nucleosides of the benzimidazole and purine series, and other biologically active azaheterocycles are also discussed.

Molecular Choreography Of An Antibiotic Assembly Line

Plants and microorganisms produce a vast array of structurally complex and diverse antibacterial, antiviral, antitumor and antimetabolite substances with wide‐ranging human and animal health applications. In fact the majority of compounds used clinically and agriculturally in the United States are either natural products or semi‐synthetic derivatives thereof. Polyketides are an important class of natural compounds that often constitute the core structures or complete chemical entities for many clinically approved therapeutic agents. Their biosynthetic machinery, represented by type I polyketide synthases, has an architecture in which successive modules catalyze two‐carbon linear extensions and keto group processing reactions on intermediates covalently tethered to carrier domains. We recently employed cryo‐electron microscopy (cryo‐EM) to visualize a full‐length module from the biosynthetic pathway that creates the antibiotic pikromycin. The cryo‐EM 3D reconstructions revealed a totally unexpected architecture compared to the homologous dimeric mammalian fatty acid synthase. The dimeric PKS module creates a single reaction chamber for the two acyl carrier protein (ACP) domains that carry building blocks and intermediates between acyltransferase (AT), ketosynthase (KS), and ketoreductase (KR) active sites. By trapping the PKS module in each of several biochemical states we obtained 3D maps that recapitulate its full enzymatic cycle. The ACP domains are differentially and precisely positioned after polyketide chain substrate loading on the active site of KS, after extension to the b‐keto‐intermediate, and after b‐hydroxy product generation. The structures reveal the ACP dynamics for sequential binding to catalytic domains within the reaction chamber, and for transferring the elongated and processed polyketide substrate to the next module in the PKS pathway. This study highlights the conformational transitions coupled to substrate transfer and modification in a way that facilitates high throughput unidirectional substrate processing, and establishes a new model for molecular dissection of these multifunctional enzyme systems.

Antiviral activity of fullerene C60 nanocrystals modified with derivatives of anionic antimicrobial peptide maximin H5

Many active antiviral substances come from natural sources. In this way, peptides, isolated from Asian toad Bombina maxima, called maximins, are very promising. Most of them have good antimicrobial activity; however, derivatives of anionic 20 amino acids-long maximin H5 show also promising antiviral activity. The effect can be enhanced by binding to suitable nanocarriers such as fullerenes. In the present study, six mutants of maximin H5 were designed where aspartic acid at position 11 was replaced by asparagine, histidine, tyrosine, alanine, glycine, or valine. The binding yield of each peptide to fullerene C60 nanocrystals was studied by derivatization with fluorescent reagent fluorescamine. The antiviral activity of these peptides and peptides bound to fullerene C60 nanocrystals was studied using bacteriophage λ as a model virus. All of the designed peptides had higher antiviral activity compared to maximin H5. The highest antiviral activity was observed in case of maximin variants H5N, H5V, or H5Y. Moreover, the antiviral activity was dependent on the amount of peptide bound on the surface of fullerene C60 nanocrystals, which was enhanced by trimesic acid (benzene-1,3,5-tricarboxylic acid) treated fullerene C60 nanocrystals.

Associated microflora of medicinal ferns: biotechnological potentials and possible applications

Plant associated microorganisms that colonize the upper and internal tissues of roots, stems, leaves and flowers of healthy plants without causing any visible harmful or negative effect on their host. Diversity of microbes have been extensively studied in a wide variety of vascular plants and shown to promote plant establishment, growth and development and impart resistance against pathogenic infections. Ferns and their associated microbes have also attracted the attention of the scientific communities as sources of novel bioactive secondary metabolites. The ferns and fern alleles, which are well adapted to diverse environmental conditions, produce various secondary metabolites such as flavonoids, steroids, alkaloids, phenols, triterpenoid compounds, variety of amino acids and fatty acids along with some unique metabolites as adaptive features and are traditionally used for human health and medicine. In this review attention has been focused to prepare a comprehensive account of ethnomedicinal properties of some common ferns and fern alleles. Association of bacteria and fungi in the rhizosphere, phyllosphere and endosphere of these medicinally important ferns and their interaction with the host plant has been emphasized keeping in view their possible biotechnological potentials and applications. The processes of host-microbe interaction leading to establishment and colonization of endophytes are less-well characterized in comparison to rhizospheric and phyllospheric microflora. However, the endophytes are possessing same characteristics as rhizospheric and phyllospheric to stimulate the in vivo synthesis as well as in vitro production of secondary metabolites with a wide range of biological activities such as plant growth promotion by production of phytohormones, siderophores, fixation of nitrogen, and phosphate solubilization. Synthesis of pharmaceutically important products such as anticancer compounds, antioxidants, antimicrobials, antiviral substances and hydrolytic enzymes could be some of the promising areas of research and commercial exploitation.

Super Antibiotics, Part II. Hyperforin, Mass Spectroscopy (MS) and Gas Chromatography-Mass Spectrometry (GC-MS), Evidence of Permeability of the Blood-Testis Barrier (BTB) and the Blood-Brain Barrier (BBB) to Hyperforin

In the first article of this series, we presented some evidence of hyperforin as an antibiotic, antiprotozoal, antiviral, anticancer, and immunomodulatory substance. In the present article, evidence of the permeability of the blood-testis barrier (BTB) and blood-brain barrier (BBB) to hyperforin and its distribution in other organs of the domestic pig (Sus scrofa domesticus) are revealed. Seven-month-old male boars with a body mass of 100 kg were fed a diet containing hyperforin. Organs were surgically removed under anesthesia. Organs were suitable prepared and extracted, and then analyzed using gas chromatography-mass spectrometry with supersonic molecular beams (GC-MS with SMB). The presence of hyperforin was recorded in all organs and body fluids. Special attention was paid to the evaluation of the presence of hyperforin in the brain and testes of experimental animals. The presence of hyperforin in the brain and testes of experimental animals was established by GC-MS with SMB. The results are of interest because penicillin and numerous other antibiotics cannot pass through the BTB or BBB if healthy or non-inflamed, which limits their use in patients with meningitis and gonorrhea. The findings are also of interest in cases of penicillin- and multi-antibiotic-resistant bacterial infections.

ПОЛЯРИЗОВАНЕ СВІТЛО У ФІЗІОТЕРАПІЇ

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