Preclinical Studies of Immunogenity, Protectivity, and Safety of the Combined Vector Vaccine for Prevention of the Middle East Respiratory Syndrome.

The Middle East Respiratory Syndrome (MERS) is an acute inflammatory disease of the respiratory system caused by the MERS-CoV coronavirus. The mortality rate for MERS is about 34.5%. Due to its high mortality rate, the lack of therapeutic and prophylactic agents, and the continuing threat of the spread of MERS beyond its current confines, developing a vaccine is a pressing task, because vaccination would help limit the spread of MERS and reduce its death toll. We have developed a combined vector vaccine for the prevention of MERS based on recombinant human adenovirus serotypes 26 and 5. Studies of its immunogenicity have shown that vaccination of animals (mice and primates) induces a robust humoral immune response that lasts for at least six months. Studies of the cellular immune response in mice after vaccination showed the emergence of a specific CD4+ and CD8+ T cell response. A study of the vaccine protectivity conducted in a model of transgenic mice carrying the human DPP4 receptor gene showed that our vaccination protected 100% of the animals from the lethal infection caused by the MERS-CoV virus (MERS-CoV EMC/2012, 100LD50 per mouse). Studies of the safety and tolerability of the developed vaccine in rodents, rabbits, and primates showed a good safety profile and tolerance in animals; they revealed no contraindications for clinical testing.

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.

Experimental Study of Flakozid Activity in Viral Hepatitis C In Vitro.

Antiviral activity of a Russian drug flakozid towards infection caused by a cytopathogenic variant of hepatitis C virus in SPEV cells is studied. Flakozid is an individual natural flavonoid glycoside 7-O-ß-D-glucopyranoside-8-(methyl-but)-2-enyl)-5,4'-dioxyflavanolol), isolated from the leaves of Phellodendron amurense Rupr. and Ph. amurense var. lavallei (Dode) Sprague, Rutaceae family. High antiviral activity of flakozid manifests in response to its addition into the monolayer of SPEV cells infected by hepatitis C virus during all periods of experiment. Flakozid exhibits weak cytotoxicity and no viricidal activity towards hepatitis C virus, which is comparable to activity of ribavirin (antiviral drug). Chemotherapeutic index of flakozid is 15-25 times higher than that of the reference drug ribavirin.

[Assessment of the antiviral activity of 2HCl*H-His-Rim compound compared to the anti-influenza drug Arbidol for influenza caused by A/duck/Novosibirsk/56/05 (H5N1) (Influenza A virus, Alphainfluenzavirus, Orthomyxoviridae).]

INTRODUCTION: The emergence of influenza virus strains with drug resistance to antiviral drugs requires finding new compounds, potential direct-acting inhibitors. Аdamantane compounds drugs used since the 1960s have lost their activity the resulting due to resistance. Only neuraminidase inhibitors such as zanamivir and oseltamivir have been approved by WHO for influenza treatment. The Russian pharmaceutical drug Arbidol (Umifenovirum) is actively used in Russia. This drug is used to treat influenza in Russia, China and most post-Soviet republics. This work presents a new derivative of aminoadamantane - dichlorohydrate L-histidyl-1-adamantayl ethylamine (2HCl*H-His-Rim), which showed a high level of inhibition of strains of influenza virus A in vitro. OBJECTIVES: Comparison of antiviral properties of the new synthetic low-molecular inhibitor of influenza A virus replication and Arbidol drug pharmacy. METHODS: The compound 2HCl*H-His-Rim was obtained by classical peptide synthesis methods. It was identified by methods of mass spectrometry, infrared spectroscopy (IR) and nuclear magnetic resonance spectroscopy (NMR). Its antiviral properties have been studied in vitro for monolayer of cells Vero-E6 infected with a high-virulent strain of A/duck/Novosibirsk/56/06 (H5N1) influenza virus at various injection schemes of the investigated compounds. THE RESULTS: The antiviral activity of the 2HCl*H-His-Rim compound against the highly pathogenic strain of the influenza A/H5N1 virus was slightly higher than for the known pharmacy drug arbidol. DISCUSSION: The difference in antiviral activity of these two compounds is explained by different mechanisms of action on the viral particle. CONCLUSION: The 2HCl*H-His-Rim compound can be recommended as a candidate for preclinical and clinical trials in order to obtain an etiotropic antiviral drug based on it, due to its high efficacy and economic and synthetic availability. The synthetic compound 2HCl*H-His-Rim acts on influenza A virus variants resistant to Rimantadine and Amantadine.

1-(4-Phenoxybenzyl) 5-Aminouracil Derivatives and Their Analogues - Novel Inhibitors of Human Adenovirus Replication.

Adenovirus infections are characterized by widespread distribution. The lack of causal therapy, which is effective in treating this group of diseases, explains the need for new therapeutic drugs. Notably, anti-adenoviral activity of [4-(phenoxy)benzyl]-5-(phenylamino)-6-azauracil, 1-[4-(phenoxy)benzyl]-5-(morpholino) uracil, 1-[4-(4-chlorophenoxy)benzyl]-5-(morpholino) uracil, and 1-[4-(4-fluorophenoxy)-benzyl]-5-(morpholino) uracil was observed.

DRIFT OF INFLUENA A(H3N2) VIRUS: BIOLOGICAL, ANTIGENIC AND GENETIC PROPERTIES IN EPIDEMIC SEASON 2016-2017 IN RUSSIA AND COUNTRIES OF THE NOTHERN HEMYSPHERE.

The article presents the features of the influenza virus circulation for the period from October 2016 to May 2017 in some territories of Russia collaborating with the D.I. Ivanovsky Institute of Virology, Federal State Budgetary Institution "N.F. Gamaleya Federal Research Centre for Epidemiology and Microbiology", Ministry of Health of the Russian Federation. One of the 2016-2017 season's peculiarities in Russia and countries of the Northern hemisphere was the earlier start of an increase in ARD morbidity with peak indexes reached towards the end of December 2016 - January 2017. First, influenza A(H3N2) virus was predominant; then, it was followed by influenza B virus activity observed until the end of the season. The indexes of morbidity were higher than in the previous season, while the rates of hospitalization and mortality were lower, lethal cases being detected in persons 65 years old and older. Epidemic strains of influenza A(H3N2) virus belonged to 3c.2a genetic group, reference strain A/Hong Hong/4408/2014, and its subgroup 3c.2a1, reference A/Bolzano/7/2016, that are antigenically similar. Strains of influenza B virus were antigenically similar to the B/Brisbane/60/2008 vaccine virus. Strains were sensitive to oseltamivir and zanamivir. The share participation of non-influenza ARI viruses was similar to preliminary epidemic seasons. WHO has issued recommendations for influenza virus vaccines composition for 2017-2018 for the Northern hemisphere.

[Antiviral activity of phosprenyl and gamapren against infection caused by influenza a (H5N1) virus in cell culture].

The antiviral activity of Phosprenyl and Gamapren in vitro against highly pathogenic strain of avian influenza H5N1 virus was studied. Inoculation of the virus to the susceptible cell culture led to development of the cytopathogenic effect. Preliminary introduction of Phosprenyl and Gamapren an hour prior to infecting the cells with virus 10.0 TCID50 dose completely inhibited the cytopathogenic activity of the virus. At higher doses of virus (100.0 TCID50) significant inhibition of the infectious activity of the virus was observed: 70% of infected cells survived under the action of Phosprenyl, and 90% under the action of Gamapren. With the introduction of the preparations simultaneously with the infection of cells with virus at a dose of 10.0 TCID50 virtually 100% of infected cells survived, while in control cultures death of 100% of the cells occurred. After infection with the virus at a dose of 100.0 TCID50 Phosprenyl and Gamapren caused 50% protection of the cells. The antiviral effect of the drugs Phosprenyl and Gamapren may be associated not only with their virulicidal, but with anti-viral activity as well.

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).

Genetic diversity of viruses of Chenuda virus species (Orbivirus, Reoviridae) circulating in Central Asia.

Chenuda virus (CNUV) (Orbivirus, Reoviridae) is the only known orbivirus associated with argas (Argasidae) ticks. Scientific study of this group is necessary for understanding of Orbivirus genus evolution patterns. We conducted a comparative analysis of full genomes of five different viruses of Chenuda virus species, including Baku virus strains (BAKV) circulating in a rather limited area in the Central Asia and Transcaucasia. It was shown that VP4(OC1) and VP6(Hel) proteins variability greatly exceeds the variability of other proteins. The divergence between CNUV and BAKV in this proteins is about 50%. Even in closely related strains isolated from the same geographical region, the conservative genes of which are 90-95% identical, the VP4(OC1) and VP6(Hel) divergence reaches values that would usually be indicative of different serotypes (74.1-82.2%).

New Carbocyclic Amino Acid Derivatives Inhibit Infection Caused by Highly Pathogenic Influenza A Virus Strain (H5N1).

New amino acid derivatives with carbocycles of adamantine and quinaldic acid were synthesized and their in vitro antiviral activity against influenza A/H5N1 virus was evaluated. Experiments on cultured embryonic porcine kidney epithelial cells showed that amino acid derivatives suppressed viral replication. Tret-butyloxycarbonyl-DL-methionylsulfonyl-1-adamantayl ethylamine and benzyloxycarbonyl-L-trypthophanyl-1-adamantayl ethylamine compounds demonstrated high activity in all in vitro experiments. Moreover, some compounds showed virucidal activity against influenza A/H5N1 virus.

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.

Virological, epidemiological, clinic, and molecular genetic features of the influenza epidemic in 2015-2016: prevailing of the influenza A(H1N1)09 pdm virus in Russia and countries of the Northern hemisphere.

This work describes the specific features of the influenza virus circulating in the period from October 2015 to March 2016 in 10 cities of Russia, the basic laboratories of CEEI at the D.I. Ivanovsky Institute of Virology "Federal Research Centre of Epidemilogy and Microbiology named after the honorary academician N.F. Gamaleya" of the Ministry of Health of the Russian Federation. The increase in the morbidity caused by influenza viruses was detected in January-February 2016. The duration of the morbidity peak was 4-5 weeks. The most vulnerable group included children at the age from 3 to 6; a high rate of hospitalization was also detected among people at the age of 15-64 (65%). In clinic symptoms there were middle and severe forms with high frequency of hospitalization as compared with the season of 2009-2010, but much higher in comparison with the season of 2014-2015. Some of the hospitalized patients had virus pneumonias, half of which were bilateral. Among these patients, 10% were children; 30%, adults. The mortality in the intensive care unit of the hospital was 46%. Almost all lethal cases were among unvaccinated patients in the case of late hospitalization and without early antiviral therapy. The predominance of the influenza A(H1N1)09pdm virus both in the Russian Federation and the major part of the countries in the Northern hemisphere was noted. The results of the study of the antigenic properties of influenza strains of A(H1N1)pdm09 virus did not reveal any differences with respect to the vaccine virus. The sequencing data showed the amino acid substitutions in hemagglutinin (receptor binding and Sa sites) and in genes encoding internal proteins (PA, NP, M1, NS1). Strains were sensitive to oseltamivir and zanamivir and maintained resistance to rimantadine. The participation of non-influenza ARI viruses was comparable to that in preliminary epidemic seasons.

Amino acid polymorphism at residue 222 of the receptor-binding site of the hemagglutinin of the pandemic influenza A(H1N1)pdm09 from patients 166 with lethal virus pneumonia in 2012-2014.

Survey data from autopsy specimens from patients who died from pneumonia caused by the influenza A(H1N1) pdm09 in 2012-2014 and mutant forms of influenza virus in these patients (position 222 in the receptor-binding region of hemagglutinin) were presented. In total, according to aggregate data, obtained with three different methods (sequencing, next-generation sequencing (NGS), virus isolation) mutant viruses were detected in 17 (41%) from 41 patients. The proportion of the mutant forms in viral populations ranged from 1% to 69.2%. The most frequent mixture was the wild type (D222) and mutant (D222G), with proportion of mutant type ranged from 3.3% to 69.2% in the viral population. Mutation D222N (from 1.1% to 5.5%) was found rarely. Composition of the viral population from one patient is extremely heterogeneous: in left lung there was only wild type D222, meantime in right lung - mixture of mutant forms 222D/N/G (65.4/32.5/1.1%), in trachea - mixture 222D/G/Y/A (61.8/35.6/1.2/1.4%, respectively), and in bronchi compound of 222D/G/N/A (64.3/33.7/1/1%, respectively) were detected. The obtained data indicate that the process of adaptation of the virus in the lower respiratory tract is coupled with the appearance of different virus variants with mutations in the receptor-binding region. Mutant forms of the virus are observed in the lower respiratory tract of the majority of patients with lethal viral pneumonia. However, if they are a minor part of the population, they cannot be detected by the method of conventional sequencing. They can be identified using the NGS methods.

Evolution of influ- 245 enza A/H5N1 virus (1996-2016).

Twenty years ago in the South Chinese province of Guangdong the epizooty of highly pathogenic avian influenza (HPAI) H5N1 virus, which has laid the foundation of the largest epizooty in the contemporary history, has flashed. Hemagglutinin of prototype A/goose/Guangdong/1/1996 (H5N1) changing many times and generating new genetic subgroups participated in various reassortations; it still exists today. The present review is devoted to the retrospective analysis of HPAI/H5N1evolution for the last twenty years in the territory of Eurasia, Africa and America. The basis for the discussion is ecological model according to which new genetic variants are formed in the migration pathways with close contacts between different bird populations and in the overwintering areas where the maximum values of the immune layer occur; amplification of virus variants occurs in nesting areas among juvenile populations. The updated system of designations of genetic groups introduced by WHO/OIE/FAO H5 Evolution Working Group in 2015 is used.

[The biological characteristic of the collection strains of viruses from the subgroup of Japanese encephalitis].

Perennial (since 1966) study of the biological properties of the viruses from the flavivirus subgroups of the Japanese encephalitis (JE) made it possible to collect and deposit in the State collection of JE virus strains JE virus strains isolated from natural foci in different geographic zones, as well as the JE virus strains selected in the laboratory. The collection of the flaviviruses strains of Japanese encephalitis complex, West Nile fever (West Nile virus and Usutu), which were studied and preserved, are listed. The data are provided on the origin of strains and their pathogenicity for laboratory animals.

Amino acid derivatives of adamantane carbocycle are capable of inhibiting replication of highly virulent avian influenza A/H5N1 virus.

We studied the capacity amino acid derivatives of adamantane to inhibit replication of highly virulent avian influenza A/duck/Novosibirsk/56/05 (H5N1) virus in cultures of swine embryonic kidney cells. Amino acid derivatives of adamantane H-His-Rem and Ad(CH2-Ser-OMe)2 were characterized by lower toxicity than remantadine previously used in the treatment of influenza. Histidine-containing adamantane derivative (H-His-Rem) was the most effective and low-toxic inhibitor of influenza А/H5N1 virus replication and can be recommended for clinical trials to produce a preparation for the treatment and prevention of influenza.

[Complete genome analysis of the Batai virus (BATV) and the new Anadyr virus (ANADV) of the Bunyamwera group (Bunyaviridae, Orthobunyavirus) isolated in Russia].

Almost complete nucleotide sequences for the S, M, and L segments were obtained for three strains of the Batai virus (Bunyamwera serogroup, genus Orthobunyavirus, Bunyaviridae family). Based on the results of the phylogenetic analysis conducted forthe three genomic segments LEIV Ast507 and LEIV-Ast528 strains were grouped with other European BATV isolates and were found to be almost identical to the strain 42 isolated from Volgograd Region, Russia, 2003. Surprisingly, LEIV-13395 strain isolated from the Aedes sp. mosquitos in Magadan Oblast, 1987, turned out to be a novel genotype inside Bunyamwera serogroup. The highest nucleotide identity levels of LEIV-13395 genomicsegments (86.9%, 80.8%, 79.7% for S, M and L segments respectively) were observed with corresponding segments of the Batai virus.

[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.

Development of monoclonal antibodies to highly pathogenic avian influenza H5N1 virus and their application to diagnostics, prophylaxis, and therapy.

A panel of 17 monoclonal antibodies (MAbs) against highly pathogenic avian influenza virus (HPAIV) A/Duck/Novosibirsk/56/05 A/H5N1 (subclade 2.2) isolated in Russian Federation was developed. Immunoblot analysis showed that 12 MAbs were specific for the hemagglutinin (HA) and 5 MAbs for nucleoprotein (NP). All anti-HA MAbs were reactive in ELISA and immunofluorescence (IF) test and 10 of them were reactive in hemagglutination-inhibition (HI) and neutralization tests. Quantitative competitive ELISA revealed that anti-HA MAbs recognized at least 4 non-overlapping antigenic determinants and anti-NP MAbs recognized at least 3 non-overlapping antigenic determinants. Four sandwich ELISA procedures were developed using the obtained MAbs. These procedures are useful for 1) identification of avian, human, and swine influenza A viruses, 2) differentiation of avian influenza virus (AIV) from human and swine influenza viruses, 3) differentiation of AIV H5 from other AIV subtypes, and 4) differentiation between 2.2 and 2.3.2 subclades of H5N1 influenza viruses. Prophylactic and therapeutic efficacy of anti-HA MAbs with high neutralization activity was tested in BALB/c mice. A complete protection was achieved by single injection of MAbs (20 mg/kg) 24 hrs before challenge with 10 LD50 of HPAIV H5N1. Therapeutic efficacy was 90% that was similar to those of Rimantadine and Tamiflu.