Combined Effect of Basic Antiherpetic Drugs with a New Inhibitor of the Terminase Complex of Herpes Simplex Virus Type 1 in Vero Cell Cultures.

Abstract-Carriers of herpes simplex virus type 1 (HSV-1) account for more than 90% of the global population. Infection manifests itself in the formation of blisters and ulcers on the face or genitals and can cause blindness, encephalitis, and generalized infection. All first- and second-line modern antiherpetic drugs selectively inhibit viral DNA polymerase. The purine-benzoxazine conjugate LAS-131 ((S)-4-[6-(purin-6-yl)aminohexanoyl]-7,8-difluoro-3,4-dihydro-3-methyl-2H-[1,4]benzoxazine), which we have described earlier, uses the large subunit of the HSV-1 terminase complex as a biotarget and selectively inhibits HSV-1 reproduction in vitro. Basically new results were for the first time obtained to characterize the combined effect on human herpesvirus infection for LAS-131 used in combination with practically significant antiviral compounds, including the nucleoside analogs acyclovir (ACV), penciclovir (PCV), ganciclovir (GCV), brivudine (BVdU), iododeoxyuridine (IdU), and adenine arabinoside (Ara-A); the nucleoside phosphonate analog cidofovir (CDV); and the pyrophosphate analog foscarnet (FOS). A cytopathic effect (CPE) inhibition assay showed that the drug concentration that inhibited the virus-induced CPE by 50% decreased by a factor of 2 (an additive effect, FOS) or more (a synergistic effect; ACV, PCV, GCV, IdU, BVdU, Ara-A, and CDV) when the drugs were used in combination with LAS-131. Nonpermissive conditions for HSV-1 reproduction were thus created at lower drug concentrations, opening up new real possibilities to control human herpesvirus infection.

The Mechanism of the Stimforte Effect on the Activation of Target Cell Defense against Viral Infection.

Stimforte in a wide range of concentrations (15-225 µg/mL) totally inhibits the cytopathic activity of hepatitis C virus (HCV) in the Vero-V cell culture. Interferons (IFN) play the most important role in the suppression of infection when the drug is introduced into the culture before the infection. When Stimforte is introduced after the infection, the mechanism of action seems to be different. The activators of IFN production are mainly (or exclusively) the ligands of receptor complexes TLR-4 and NOD-2 contained in the drug. The action of these substances is probably synergistic, similar to the action of LPS and MDP in Vero-V cells.

Different effects of the immunostimulatory drug Stimforte on infections of hepatitis C virus and herpes simplex virus type 1.

Stimforte, an immune response-stimulating preparation, is active with respect to hepatitis C virus (HCV) and herpes simplex virus type I (HSV-1). The effects of Stimforte in animals infected with either HCV or HSV-1 are fundamentally different. In mice with acute herpes virus infection, Stimforte administration leads to a higher activity of natural killer cells and cytotoxic lymphocytes, and the amount of interferon (IFN) λ grows. In mice infected with HCV, Stimforte administration results in a significant increase in IFN-ß but not IFN-λ in blood and affected organs. Stimforte has been found to affect directly HCV reproduction that causes the infected cell death, but it does not affect HSV-1 reproduction in the Vero cells (V).

Analysis of Antiviral Properties of Hexoral In Vitro against Some Viruses that Cause Acute Respiratory Infections and Herpes.

Antiviral properties of Hexoral (0.1% solution and 0.2% aerosol for local application) and its constituent hexetidine against viruses causing human respiratory tract infections and herpes virus were studied in vitro. It was found that non-cytotoxic concentrations of hexetidine (alone and as a component of Hexoral) attenuated infectious properties of highly virulent influenza virus A/H5N1, pandemic influenza virus A/H1N1pdm, respiratory syncytial virus, and herpes simplex virus type 1 after a short-term exposure (30 sec) by 100 or more times. It was found that hexidine mostly contributes to the virucidal effect of Hexoral.

[Interaction of Dystamycin Dimeric Analog with Poly(dA) x poly(dT), Poly[d(A-T)] x poly[d(A-T)] and Duplex O23 at Origin of Replication of the Herpes Simplex Virus].

The binding of distamycin dimeric analog (Pt-bis-Dst) to poly[d(A-T)] x poly[d(A-T)1, poly(dA) x poly(dT) and duplex O23 with the sequence 5'-GCCAATATATATATATTATTAGG-3' which is present at the origin of replication of herpes simplex virus OriS is investigated with the use of UV and CD spectroscopy. The distinction of the synthetic polyamide from a natural antibiotic lies in the fact that in the synthetic polyamide there are two distamycin moieties bound via a glycine cis-diamino platinum group. It was shown that the binding of Pt-bis-Dst to poly[d(A-T)] x poly[d(A-T)] and poly(dA) x poly(dT) reaches saturation if one molecule of the ligand occurs at approximately every 8 bp. With further increase in the ratio of the added ligand to the base pairs in CD spectra of complexes with poly[d(A-T)] x poly[d(A-T)], we observed that the maximum wavelength band tend to be shifted towards longer wavelengths, while in the spectral region of 290-310 nm a "shoulder", that was absent in the spectra of the complexes obtained at low polymer coverages by the ligand, appeared. At high molar concentration ratios of ligand to oligonucleotide Pt-bis-Dst can bind to poly[d(A-T)] x poly[d(A-T)] in the form of hairpins or may form associates by the interaction between the distamycin moieties of neighboring molecules of Pt-bis-Dst. The structure of the complexes is stabilized by interactions between pirrolcarboxamide moieties of two molecules of Pt-bis-Dst adsorbed on adjacent overlapping binding sites. These interactions are probably also responsible for the concentration-dependent spectral changes observed during the formation of a complex between Pt-bis-Dst and poly[d(A-T)] x poly[d(A-T)]. Spectral changes are almost absent in binding of Pt-bis-Dst to poly(dA) x poly(dT). Binding of Pt-bis-Dst to duplex O23 reaches saturation if two ligand molecules occur in a duplex that contains a cluster of 18 AT pairs. With increasing the molar concentration ratio of the ligand to the duplex CD spectra undergo concentration-dependent changes similar to those observed during binding of Pt-bis-Dst to poly [d(A-T)] x poly[d(A-T)]. Testing for antiviral efficacy of Pt-bis-Dst showed that the concentration, at which the cytopathic effect produced by the herpes simplex virus in cell culture Vero E6 halved, is equal to 1.5 µg/ml and the selectivity index for evaluating antiviral activity is 65 at a relatively low cytotoxicity. The concentration of Pt-bis-Dst, at which approximately half the cells are killed, is equal to 100 µg/ml.

[Research of suppression of the herpes simplex virus reproduction with drug resistance using a combination 15-Lys-bis-Nt with some antiherpetic drugs].

The antiviral effect of combinations of netropsin derivative 15-Lys-bis-Nt with the known antiherpetic compounds, whose activity does not depend on viral TK and which are able to inhibit replication of HSV in most cases, including strains resistant to acyclovir and pencyclovir, was studied. The combinations evoking additive, synergistic and significant synergistic effects of interaction of tested compounds were observed. The results obtained in this work indicated the possibility of significant reduction of concentrations of high toxic compounds in case of the combined use.

[Research of suppression of the herpes simplex virus reproduction with drug resistance by combination phosphite of acycloguanosine with some antiherpetic drugs].

The activity of the phosphite of acycloguanosine (P-ACG) and six antivirals was tested individually and in double and triple combinations on two strains of the herpes simplex virus (HSV) type 1 (sensitive to acyclovir and resistant to acyclovir) using the CPE inhibition method in the Vero E6 cell microcultures. These are: phosphite of acycloguanosine (P-ACG), Ara-A, cidofovir (CDV), ribavirin (Rib), phosphonoformate (PFA), glycyrrhizic acid (GLN) and alpha-interferon (alpha-IFN). All studied double combinations and triple combinations including P-ACV inhibited replication of both HSV strains more effectively than any drug alone. Various types of interactions depending on the virus type were observed in both viral models: synergistic (double combinations P-ACG with PFA, CDV, Rib, alpha-IFN and triple combinations P-ACG with alpha-IFN +PFA, alpha-IFN +AraA, alpha-IFN +CDV, PFA+CDV, PFA+Rib, CDV+AraA, CDV+Rib, CDV+GLN,PFA+AraA) and additive (P-ACG with AraA and PFA+GLN). Neither antagonism nor interference was noted for any combinations. Adduced results suggest that these combinations might be clinically useful for the treatment of certain herpes simplex virus type 1 infections.

[The suppression of a herpes simplex virus reproduction with drug resistance by combination 15lys-bis-nt and phosphate of acycloguanosine with some antiherpetic drugs].

Antiherpetic activity of the double and triple combinations, including original connections 15Lys-bis-Nt and phosphate of acycloguanosine (P-ACG), was studied in vitro. For the first time, it was demonstrated that in case of their combined use with known antiherpetic agents, whose activity does not depend on TK of HSV (PFA, AraA, CDV, Rib, GLN, αa-IFN), synergistic or additive effects of interaction was observed. The antiviral effect of the tested combinations was studied on the model of ACG-resistant viral strain. The tested combinations could be of interest for practical medicine.

[Substances from pyrolised tissues of reptile carcases differently modulate immunity of mammals of both sexes].

Substances with gender action on immunity were detected in water soluble hydrolised matter from reptile carcases. The gender action was shown on isolated blood neutrophils, whole blood and in vivo by the antiviral activity on experimental animals, contaminated with three types of viruses: Herpes simplex type 1, the virus of encephalomyocarditis and the virus of hepatitis of mice. The possible mechanism of the inhibitory action on the male immunity was associated with the protein kinase cascade, including protein kinase C, activated by phorbolmyristate in the cells of the immune system.

[Stimforte action on the main inflammation characteristics in experimental animals contaminated by Herpes simplex-1].

In the brain and lungs of the experimental animals contaminated by Herpes simplex-1 there were detected much higher levels of the thiobarbituric acid-stained lipid oxidation products and proteolytic activity, evident of the inflammation process. Stimforte lowered the inflammation indices to the level, close to that in the brain of the noninfected animals. Yet the drug provided lower titers of the virus in the brain, lungs and serum in the contaminated animals and arrested the infection process by stimulation of the immune system. The mechanism of the inflammation suppression is discussed.

[Molecular-genetic analysis of DNA pol and TK of HSV-1 population using NGS technology].

It was determined the ratio of viral DNA and DNA from Vero cells using the polymerase chain reaction in real time in Vero cell lysate, infected with L2 strain of the herpes simplex virus type 1. Copy number of the virus reached a maximum after 24 hours of incubation of infection. Total DNA was isolated and sequenced using NGS technology by Ion Torrent device. Nucleotide sequences of the thymidine kinase gene (UL23) and DNA polymerase (UL30) were determined for a population of HSV-1 strain L2. Comparison of the primary structure of these genes with the corresponding nucleotide sequences of known strains of HSV-1 KOS and 17 was conducted. Differences in the structure of genes UL23 and UL30 between strain L2 and reference strains KOS and 17 are not important, because changes are found in non-conservative regions.

[The arsenolysis reaction in the biotechnological method of synthesis of the ribavirin. Inhibition of reproduction of influenza A virus with the combination of ribavirin and ozeltamivir in experiments in vitro and in vivo].

Improved biotechnological method of receiving the antiviral drug ribavirin by the reaction of transglycosilation by addition of catalytic amounts of sodium arsenate in the reaction mixture. Such approach allows to hydrolyze the amount of the excess natural nucleoside donor--ribose and, as a consequence, to simplify the composition of the reaction mixture and the process of separation of ribavirin. The effect of ribavirin and ozeltamivir carboxylate and their combination on the reproduction of the virus of the influenza A in cell culture and in experiments on laboratory animals (mouse Balb/C). The greatest anti-influenza effect is observed when using a combination of drugs, as compared to each of them taken separately.

[Estimation of activity of bis-netropsin derivatives based on a model of an experimental cutaneous herpes simplex virus disease of guinea pigs].

Using the model of an experimental cutaneous infection of guinea pig males caused by herpes simple virus type 1, it is shown that application of dimerico derivatives of netropsin Lys-bis Nt and 15Lys-bis Nt in the form of polietilenglicol-based ointment suppresses viral infection more effectively than acyclovir.

[Inhibition of herpes simplex virus helicase UL9 by netropsin derivatives and antiviral activities of bis-netropsins].

Data obtained show that antiviral activities of bis-linked netropsin derivatives are targeted by specific complexes formed by helicase UL9 of herpes simplex virus type 1 with viral DNA replication origins, represented by two OriS sites and one OriL site. According to the results of footprinting studies bis-netropsins get bound selectively to an A+T-cluster which separates interaction sites I and II for helicase UL9 in OriS. Upon binding to DNA bis-netropsins stabilize a structure of the A+T-cluster and inhibit thermal fluctuation-induced opening of AT- base pairs which is needed for local unwinding of DNA by helicase UL9. Kinetics of ATP-dependent DNA unwinding in the presence and absence of Pt-bis-netropsin are studied by measuring the efficiency of Forster resonance energy transfer (FRET) between the fluorescent probes attached covalently to 3?- and 5?-ends of the oligonucleotides in the minimal OriS duplex. Pt-bis-netropsin and related molecules inhibit unwinding of OriS duplex by helicase UL9. Pt-bis-netropsin is also able to reduce the rate of unwinding of the AT- rich hairpin formed by the upper strand in the minimal OriS duplex. The antiviral activities and toxicity of bis-linked netropsin derivatives are studied in cell cultured experiments and experiments with animals infected by herpes virus.

[Antiherpetic activity of netropsin derivatives as tested in experiments in laboratory animals].

Two dimeric netropsin derivatives (Lys-bis-Nt 15Lys-bis-Nt) were comprehensively tested for antiviral and toxic activity in cell cultures and laboratory animals. The two compounds were found to provide effective and selective inhibition of reproduction of herpes simplex I both in cell culture Vero E6 and in brain of infected white mice, thereby increasing the survival rate and mean life expectation of treated animals as compared to control.

[Enzymatic activity of thymidine kinase of herpes simlex virus strain resistant to H-phosphonates of Acv].

Cloned laboratory mutants of herpes simplex virus type I resistant to acycloguanosine H-phosphonate have been investigated. For all clones were shown that mutations resulted to increasing of sensitivity to acting of sidofovir. Thymidine kinase of mutant viruses partially preserves the ability to phosphorilate thymidine, but loses the ability to phosphorilate BVDU.

[Cloning, expression, isolation and properties of thymidine kinase herpes simplex virus, strain L2].

A thymidine kinase UL23 gene (EC 2.7.1.145) from an acyclovir-sensitive strain L2 of herpes simplex virus type 1 was cloned and expressed in E. coli. Enzyme was purified by chromatography to a homogeneous state controlled by PAG electrophoresis. The Michaelis constants for the reactions with thymidine and an acyclovir were determined. It was found that enzyme phosphorilate some modified nucleosides such as d2T, d4T, d2C, 3TC, FLT, BVDU, GCV. A comparison of the purified enzyme properties and properties ofthymidine kinase of other strains of herpes simplex virus, previously published was carried out. It is shown that enzyme is inhibited by acyclovir H-phosphonate.

[Effect of Zanamivir substance on infection induced by highly pathogenic avian influenza A/H5N1 in cell cultures].

In vitro experiments revealed that Zanamivir substance had high antiviral activity against infection induced by genotypes 2.2 and 2.3.2 of highly virulent influenza A/H5N1 virus in the porcine embryonic renal epithelial (PERE) cell cultures. Zanamivir at used concentrations (1.0 microg/ml or lower) had no cytotoxic properties and was equally highly effective when used for prevention (1 hour prior to cell inoculation), treatment-and-prevention (at the time of cell inoculation), and treatment (2 hours after inoculation). The high antiviral potential of Zanamivir was shown in the experiments dealing with its effect on the ability of infected PERE cells to produce an infectious virus in the first 24 hours after inoculation. The findings suggest that Zanamivir (Relenza) shows promise as an effective antiinfluenza agent.

[Antiherpesviral activity of acycloguanosine H-phosphonate in experiments using laboratory animals].

A study of the antiherpesviral activity of acycloguanosine (ACG) H-phosphonate (ACG-P) on a model of fatal herpesvirus infection in inbred BALB/c albino mice has established that ACG-P reduces death rates in the animals, considerably increases their average lifespan, and significantly decreases brain virus titers with both 60% mortality in the control and 92% mortality in the control group. There was also a significant inhibition of herpes simplex virus type 1 (HSV-1) replication in the brain tissue of animals receiving ACG-P on a model of ACG-resistant HSV-1/L2/RACG(TK-).

[Antiviral activity of recombinant interferon-alpha-2b in combination with certain antioxidant].

In vitro activity of interferon-alpha-2b in combination with various antioxidants against the influenza virus and Herpes simplex was studied. The standard strains and a clinical strain of Herpes simplex isolated from a patient with resistance to acyclovir were used. The in vitro studie showed that antioxidants, such as alpho-tocoferol acetate (vitamin E), Unithiol and ascorbic acid had a significant antiinfluenzae and antiherpetic action on the influenza virus A/H5N1 and Herpes simplex variants. They protected up to 100% of the cell monolayer from the virus cytopathic effect. The taurin solutions had no antiviral activity irrespective of the infection dose. Combinations of interferon-alpha-2b with alpha-tocopherol acetate (vitamin E), Unithiol or ascorbic acid showed a significant synergistic effect: the antiviral activity of interferon increased several times. The antiinfluenza activity of interferon-a-2b in the presence of various concentrations of taurin did not change.