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To study the pathogenicity factors of the pandemic A(H1N1) influenza virus, a number of mutant variants of the A/Hamburg/5/2009 (H1N1)pdm09 strain were obtained through passage in chicken embryos, mouse lungs, and MDCK cell culture. After 17 lung-to-lung passages of the A/Hamburg/5/2009 in mice, the minimum lethal dose of the derived variant decreased by five orders of magnitude compared to that of the parental virus. This variant differed from the original virus by nine amino acid residues in the following viral proteins: hemagglutinin (HA), neuraminidase (NA), and components of the polymerase complex. Additional passaging of the intermediate variants and cloning made it possible to obtain pairs of strains that differed by a single amino acid substitution. Comparative analysis of replicative activity, receptor specificity, and virulence of these variants revealed two mechanisms responsible for increased pathogenicity of the virus for mice. Thus, (1) substitutions in HA (Asp225Gly or Gln226Arg) and compensatory mutation decreasing the charge of HA (Lys123Asn, Lys157Asn, Gly158Glu, Asn159Asp, or Lys212Met) altered viral receptor-binding specificity and restored the functional balance between HA and NA; (2) Phe35Leu substitution in the PA protein increased viral polymerase activity.

Aim: Study the possibility of obtaining attenuated variants of influenza virus by including specially selected site-specific mutations into a conservative sequence of PA-gene (terminal segment of COOH-domain of the PA-gene) of a virulent strain., Materials and Methods: A/ WSN/33 - a virulent strain of influenza virus was used in the study. Inclusion of site-specif- ic mutations into PA-gene of the A/WSN/33 virulent strain was carried out using a two-step mutation PCR. Cloning was carried out using GoldenGate reaction. 8-plasmid transfection system based on pHW2000 vector was used. Transformation was carried out in rubidium competent bacterial cells of DH5(α strain. Transfection was done using Lipofectamine LTX (Invitrogen) reagentin a 293T and MDCK cells' co-culture., Results: Transfectants with F658A substitution in the COOH-domain of the PA-gene were shown to acquire ts-phenotype and sharply reduce the ability to reproduce in mice lungs. Introduction of F658A substitution into COOH-domain of the PA-gene in combination with introduction of ts-mutations from ca influenzavirus strains into the genome ofthe virulent strain resulted in obtaining transfectants that have phenotypic characteristics typical for live influenza vaccine candidates., Conclusion: The ability to obtain attenuated variants of influenza viruses by introducing spe- cially selected site-specific mutations into conservative sequence of the PA-gene is shown.

Shallot virus X is a typical representative of Allexiviruses. The transcription levels of principal genes involved in the RNA silencing in healthy and shallot virus X-infected plants have been quantified by real-time polymerase chain reaction. There is a negative correlation between the reproduction rates of RNA virus and the levels of RNA-dependent RNA polymerase and DCL proteins in roots and leaves of infected plants. These observations indicate that Shallot X virus employs noncanonical ways of overcoming the antiviral defense of the plant by systemic RNA silencing.

The vaccine strains for live attenuated influenza vaccines (LAIVs) have cold-adapted, temperature-sensitive, and attenuated phenotypes, which are guaranteed by the presence of specific mutations from the master donor virus in their internal genes. In this study, we used mutant viruses of the pathogenic A/Puerto Rico/8/34 (H1N1) that contained ts-mutations in PB1 (K265N, V591I), PB2 (V478L), and PA (L28P, V341L) genes along and/or in different combinations to evaluate the impact of these mutations in the immune responses. Sequential addition of tested mutations resulted in the stepwise decrease in virus-specific serum and, to a lesser extent, mucosal antibody levels. We demonstrated strong positive correlation between virus attenuation (virus titer in lung) and antibody titers. The ts-mutations in PB1, PB2, and PA genes are mostly involved in the modulation of the humoral immunity, but also have a moderate effect on the cellular adaptive immune response.

The Artashat virus (ARTSV) was originally isolated fom the Ornithodoros alactagalis Issaakjan, 1936 (Argasidae Koch, 1844), which were collected in the burrow of small five-toed jerboa (Allactaga elater Lichtenstein, 1825) in Armenia in 1972. Later, the ARTSV was isolated from the O. verrucosus Olenev, Sassuchin et Fenuk, 1934 collected in the burrows of Persian gerbil (Meriones persicus Blanford, 1875) in Azerbaijan. Based on the virion morphology, the ARTSV was assigned to the Bunyaviridae viruses. In this work, the ARTSV genome was partially sequenced (GenBank ID: KF801650) and it was shown that the ARTSV is a new member of the Nairovirus genus. ARTSV has from 42% (Issyk-Kul virus) to 58% (Raza virus, Hughes group) similarity with the nairoviruses for nucleotide sequence of part of RNA-dependent RNA-polymerase (RdRp). The similarity on the amino acid level is 65-70%. Low level of homology and the equidistant position of the ARTSV on phylogenetic tree indicate that the ARTSV is a new prototype species of the Nairovirus genus (Bunyaviridae) forming a separate phylogenetic branch.

Full-length genome of the Chim virus (CHIMV) (strain LEIV-858Uz) was sequenced using the next-generation sequencing approach (ID GenBank: KF801656). The CHIMV/LEIV-858Uz was isolated from the Ornithodoros tartakovskyi Olenev, 1931 ticks collected in the great gerbil (Rhombomys opimus Lichtenstein, 1823) burrow in Uzbekistan near Chim town (Kashkadarinsky region) in July of 1971. Later, four more CHIMV strains were isolated from the O. tartakovskyi, O. papillipes Birula, 1895, Rhipicephalus turanicus Pomerantsev, 1936 collected in the great gerbil burrows in Kashkadarinsky, Bukhara, and Syrdarya regions of Uzbekistan, and three strains--from the Hyalomma asiaticum Schulze et Schlottke, 1930 from the great gerbil burrows in Dzheskazgan region of Kazakhstan. The virus is a potential pathogen of humans and camels. The phylogenetic analysis revealed that the CHIMV is a novel member of the Nairovirus genus (Bunyaviridae) and closely related to the Qalyub virus (QYBV), which is prototype for the group of the same name. The amino acid homology between the CHIMV and QYBV is 87% for the RdRp catalytic center (L-segment) that is coincident with both QYBV and CHIMV associated with the Ornithodoros ticks and burrow of rodents as well. The CHIMV homologies with other nairoviruses are 30-40% for the amino acid sequences of precursor polyprotein GnGc (M-segment), whereas 50%--for the nucleocapsid N (S-segment). The data obtained permit to classify the CHIMV as a member of the QYBV group in the genus of Nairovirus (Bunyaviridae).

Aim: Genotyping of noroviruses that had circulated in the territory of Nizhny Novgorod during 6 epidemic seasons (2006 - 2012), detection of dominating genovariants and analysis of their change., Materials and Methods: Feces samples from children hospitalized in an intestinal infection department of one of the infectious disease hospitals of Nizhny Novgorod served as material for the study. Noroviruses were detected by reverse transcription polymerase chain reaction. Genotypes and gene variants were determined by analysis of nucleotide sequences of viral genome regions coding capsid protein and RNA-dependent RNA-polymerase., Results: During examination of 6589 children with an acute intestinal infection between July 2006 and June 2012 noroviruses were detected in 17.55% of cases. Nucleotide sequences of capsid and/or polymerase gene regions were determined for 114 norovirus isolates. Genotyping has shown that noroviruses of 8 various genotypes had circulated in the territory of Nizhny Novgorod--GII.1, GII.2, GII.3, GII.4, GII.6, GII.7, GII. 12, GII.13 with the domination of GII.4 noroviruses for the whole observation period. A dynamic of change of epidemic variants of genotype GII.4 noroviruses that had been accompanied by an increase of frequency of detection of norovirus in children hospitalized with acute intestinal infection similar to global was established. A short-term circulation of GII.4 2006b-NN 2008 norovirus subvariant in spring of 2008 and spread of genotype GII.12 norovirus during 2009, 2010 epidemic season were also shown., Conclusion: The data obtained give evidence to the necessity of norovirus circulation monitoring with the aim of early detection of novel virus variants that may determine an increase of norovirus infection morbidity.

The Khasan virus (KHAV) was originally isolated in Khasansky District and Maritime Territory in 1971 from the ticks Haemophysalis longicornis (Neumann, 1901) collected from the deers Cervus nippon (Temmink, 1838). Based on the biological properties and virion morphology, KHAV was identified as an unclassified member of the Bunyaviridae family. In order to elucidate the KHAV taxonomy in more detail, viral genome was partially sequenced using the next-generation sequencing technology. According to the phylogenetic analysis conducted for partial sequences of the three genome segments, KHAV was attributed to the genus Phlebovirus. KHAV is phylogenetically mostly related to the viruses of the Uukuniemi group and has up to 62% identity with them. The maximum identity level is observed for sequences of the RNA-dependent-RNA-polymerase (RdRp) gene. The KHAV homology level with the tick-borne Uukuniemi group viruses is 50 to 62%; however, for the mosquito-borne phleboviruses it does not exceed 30%.

Aim: To identify the genes that are responsible for attenuation of donor viruses for live influenza vaccine. MATERIALS AND METHODS. Analysis of phenotypical properties of reassortants of wild type A and B influenza viruses with A/Leningrad/134/17/57 (H2N2) (A17) and B/USSR/60/69 (B60) master donor viruses was performed by comparison of their capability to grow at different temperatures in chicken eggs or/and MDCK cells., Results: Ts phenotype of 178 reassortants of A17 with current non-ts influenza A wild type viruses and 33 reassortants of B60 with current non-ts influenza B wild type viruses were evaluated. Reassortants inherited two polymerase genes PB2 and PA or PB 1 from A17 regularly demonstrated ts phenotype. The polymerase PA and PB2 gene segments of B60 independently controlled manifestation of ts phenotype of B60 based reassortants. The other nonpolymerase genes played no role in manifestation of ts phenotype of reassortants A17 and B60 viruses., Conclusion: The molecular basis for the development ts phenotype of both A and B influenza vaccine reassortant viruses determined by polymerase genes complex.

The pathogenesis of a disease caused by Qinghai-like H5N1 influenza virus in BALB/c mice was studied. Clinical, morphological, and immunological characteristics of the experimental infection caused by highly pathogenic A/duck/Tuva/01/06/ (H5N1) virus are described.

The authors examined a role of some mutated A/Leningrad/134/17/57(H2N2) virus genes in the realization of growth characteristics. The latter of single gene reassortants (SGRs) (PB2, PB1, PA, M, and NS), epidemic virus and attenuation donor were assessed by infecting MDCK cells and hen embryos at a low inoculation index. Viral replication in the hen embryos and cultured tissue was compared at 34 degrees C. The viruses and reassortants tested showed a high growth capacity in the hen embryos (9.5-10.5 Ig TCID50). The growth curves of viruses were studied on the cultured MDCK cells at a low inoculation index indicated that Len/17 and the single gene reassortants M and NS had the highest growth capacity. At the same time the growth of both PB1 and PB2 SGRs was less extensive. The reproduction of PB2 SGR was 100-1000 times less than that of other viruses tested. M, NS, and PA gene mutations did not affect viral growth in hen embryos and cultured tissue while PB2 gene mutation and its constellations with other genes caused a reduction in viral growth in the cultured tissue.

Molecular virological studies of the field material collected in the epicenter of epizooty with high mortality among mute swans (Cygnus olor) in the area of the lower estuary of the Volga River (November 2005) could establish the etiological role of highly pathogenic influenza A (HPAI) virus of the subtype H5N1. Ten HPAI/H5N1 strains deposited at the State Collection of Viruses of the Russian Federation with the priority dated December 1, 2005 were isolated from the cloacal/tracheal swabs and viscera of sick and freshly died mute swans. Complete nucleotide sequences of all fragments of the genome of 6 strains have been deposited in the Gene Bank. The paper discusses the molecular genetic characteristics of isolated strains.

The full-size genomes of 2 highly pathogenetic avian influenza (HPAI) virus strains isolated from a wild great-crested grebe (A/Grebe/Novosibirsk/29/05) and a domestic duck (A/Duck/Novosibirsk/56/05) in the tract of the Chany hollow, Barabino forest-steppe (Novosibirsk Region) during the epizootic outbreak in the summer of 2005. The reproductive properties of these strains successively increase in the series of cell lines BHK-2 --> LEH --> Vero-E6 --> MDCK --> PS. A/Grebe/Novosibirsk/29/05 and A/Duck/Novosibirsk/56/05 were shown to be genetically close in all genomic segments to both each other and a group of HPAI/H5N1 A/Qinghai 05 strains isolated from wild birds on the Kukunor Lake in the northwestern province of Tsinkhai, China, in May 2005. All the above strains have the HPAI/H5-specific amino acid sequence of a proteolytic cleavage site (PQGERRRKKRGLF) with Lys-627 in the protein PB2 (which is associated with increased virulence to mammalian cells), Glu-92 in the protein NS1 (that suppresses an antiviral response in the host), Ser-31 in M2 (that is a marker of rimantadine/amantadine sensitivity), 20-member amino acid deletion in the protein NA (positions 49-68) that is a marker of affiliation to the so-called genotype Z and of increased tropism to poultry.

The review gives recent data on the structure of hepatitis C virus genome. Linear RNA comprises one translated and two nontranslated regions: 5'UTR and 3'UTR. Translation of viral RNA gives rise to a single polyprotein precursor that contains structural and nonstructural regions. The structural region comprises one nucleocapsid core-protein and two envelope proteins known as E1 and E2. E2 protein includes two hypervariable regions: HVR1 and HVR2. The nonstructural region consists of 7 proteins: p7 (NS1), NS (metalloproteinase), NS (serine protease/ helicase), NS4A (co-factor protease, co-factor RNA-dependent RNA polymerase), and NS4B, NS5A, NS5B (RNA-dependent RNA polymerase). NS5A includes ISDR (interferon-sensitivity-determining region).

Hybridization analysis of the double-strand RNA-elements isolated from the sugar beet of the Kyiv, Vinnytsya and Poltava Regions was carried out. A positive signal has been obtained with the dsRNA fragment, 1.9 kbp long, which codes the viral RNA- dependent RNA-polymerase having high level of homology with the analogous enzyme of mycoviruses from Partitiviridae family. Molecular identity of these dsRNA-elements in the sugar beet samples from the Vinnytsya and Poltava regions has been shown The RT-PCR test system has been developed for detecting the nondescribed mycovirus which is proposed to be called Helicobasidium purpureum partitivirus.

To determine the roles of the mutations that were earlier found in the structure of the attenuated tomato strain V-69 of tobacco mosaic virus (TMV), the nucleotide sequences of genomic RNAs of its four pathogenic revertants have been identified and analyzed. The comparison of the structures of viral genomes has demonstrated that all revertants studied have a single reverse mutation at position 1654 relative to the parental TMV strain V-69. This reversion has been introduced into the plasmid construction harboring a full-length cDNA copy of the TMV strain V-69 RNA, and the infective transcripts synthesized in vitro have been tested on TMV-sensitive tomato and tobacco plants. The tests have confirmed that the single reversion at position 1654, which changes the amino acid residue at position 528 of the 126-kDa and 183-kDa proteins of the viral replicase is responsible for the increase in the pathogenicity of revertants compared to the TMV V-69 strain. The mutation at position 1654 of the viral genome may be supposed to be the main factor of the attenuation of TMV strain V-69.

A total of 135 polioviruses (PV), including 25 wild and 110 vaccine-related, isolated in Belarus in 1957-1999 were studied by the analysis of the polymorphism of the restriction fragments lengths of two distal regions of the genome: the region (480 oligonucleotide pairs) coding the N-terminal fragment of capsid protein VP1 (RLFP-1) and the region (291 oligonucleotide pairs) coding the N-terminal fragment of nonstructural protein of 3D-polymerase (RLFP-3D1). The genetic analysis of the viruses made it possible to determine 3 epidemiologically different periods of PV circulation: (1) the prevaccination period (1957-1959) when wild PV of all 3 serotypes circulated on the territory of Belarus; (2) the early period of the use of Oral Poliomielytis Vaccine (1960-1966), characterized by simultaneous circulation of wild and vaccine PV, as well as vaccine/wild recombinant PV; (3) the period of the elimination of wild PV of indigenous origin and the circulation of vaccine-related viruses (1967-1999). The characteristic feature of wild PV was their pronounced genetic variability. 8 genetic variants of PV1, including 4 genetic groups, 2 genetic variants of PV2 and 1 genetic variant of PV3 were detected; 2 vaccine/wild recombinant PV were detected in 1960 and 1966. More than 40% of the vaccine-related PV under study had altered genetic characteristics (mutations and/or recombinations. Reverse variability, linked with the loss of a number of signs of attenuation, was shown to be characteristic of vaccine PV1. Recombinants occurred most frequently among PV3 (44.9%) and PV2 (40.0%), their recombinations being formed mainly with PV1. Recombinants PV2/PV1 and PV3/PV1 were found to have high frequency of reversion in the "PV1" fragment of the genome; this frequency exceeded that in PV1 with the homotypical genome (66.7 and 44.4% in contrast to 12.5%).

Automatic solid-phase synthesis of 15-19-meric oligoribonucleotides was carried out using 5'-O-dimethoxytrityl-2'-O-(2-tetrahydropyranyl)- [or 2'-O-(2-tetrahydrofuranyl)-] N-acylribonucleoside-3'-O-(methyl-N, N-diisopropyl)phosphoramidite synthons and 1-H-tetrazole as an activator. Comparative analysis of the template activity of the oligoribonucleotides synthesized, which are models of the translation initiation region of the replicase gene of MS2 and fr phages, showed that the minimal active fragment of RNA is a 16-mer containing the initiation AUG codon of the gene, a short spacer, a Shine-Dalgarno domain, and the 5'-terminal AUGA sequence with a functionally important termination AUG codon.

A comparative analysis is presented of 24 known amino acid sequences of RNA-dependent RNA polymerases of positive strand RNA viruses infecting animals, plants and bacteria. Using a newly proposed methodology of group alignment for weakly similar sequences, evolutionary conserved fragments of all these proteins were unambiguously aligned. A unique pattern (consensus) of 7 invariant amino acid residues was revealed which is absent from the sequences of other RNA and DNA polymerases and is thought to unequivocally identify the RNA-dependent RNA polymerases of positive strand RNA viruses. Based on the obtained alignment a tentative phylogenetic tree of viral RNA polymerases was constructed for the first time. The RNA-dependent RNA polymerases of positive strand RNA viruses are concluded to comprise a distinct family of evolutionary related proteins.

The synthesis of RNA by polar coat protein mutants f2sus3 and Qbetaam12 under suppressor (Escherichia coli S26R1E, Su+-1; H12R8a Su+-3) and non-suppressor (E. coli AB259; S26) conditions was examined. It was demonstrated that the synthesis of viral RNA under non-suppressor conditions in the presence of rifamycin produced the same gaussian pattern of rates as the synthesis of RNA by wild type phage or non-polar coat protein mutants. However, the total amount of RNA was decreased approximately 10-fold and the peak of RNA synthesis was displaced 7--10 min later. The number of infective centers was reduced also 10-fold indicating that a certain time-lapse was required to overcome the polarity of the parental RNA, this process being of single occurrence, exclusively on the parental RNA, but not on the progeny strains. As a consequence, it was concluded that the initiation of translation at the replicase cistron starts on the nascent RNA chains within the replicative complexes and not on the fully-synthesized templates with their complete secondary structure. The data obtained are not in contradiction with the hypothesis concerning the role of the repressor complex II (replicase-RNA) to slow down the synthesis of replicase and RNA in the coat protein mutants. The polarity can not be responsible probably for the blocking of the replicase cistron on the nascent chain following the block of coat protein cistron. Therefore, it appears appropriate to assume the existence of two binding sites for the replicase as repressor which is in keeping with the conclusions of Weissmann and co-workers.

A number of ts-mutants with altered characteristics of RNA synthesis in non-suppressor cells at elevated temperatures have been obtained after HNO2-treatment of phage MS2am623 (amber mutation in site 50). The mutant ts130 is studied in detail: its peak of RNA synthesis is displaced 10 min. later and the total amount of RNA is reduced in 5-10 times. No RNA synthesis is observed in ts130-infected cell at 46 degrees C. Changes in the character of RNA synthesis are accompanied by the over-production of phage subunit of replicase: at 42 degrees C the replicase/RNA ratio in ts130 is 20 times higher than that of the original phage MS2am623. It is assumed that there is a decrease in the activity of replicase--RNA repressor complex in ts130 resulting in the loss of replicase control over its own synthesis while the control of coat protein synthesis remains unaffected.

Presumptive phylogenetic trees of evolutionary conserved fragments of RNA-dependent RNA polymerases of 26 positive strand RNA viruses were generated using a simple clustering procedure or a novel approach based on the so-called maximal topologic similarity principle. The latter methodology involves a quantitative measure of the degree of correspondence between the topology of generated trees and structure of the initial distance matrix. The algorithm for tree construction based on the maximal topologic similarity principle does not include the assumption of evolutionary rate constancy, as opposed to the clustering procedure. Nevertheless, it is demonstrated that the trees generated by the two methods are topologically similar, indicating that no drastic change of evolutionary rate had occurred in evolution of the positive strand RNA virus RNA polymerases. This in turn suggests that RNA-dependent RNA polymerases (or at least their evolutionary conserved core domains used for construction of the phylogenetic trees) are principally functionally equivalent in all positive strand RNA viruses.