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Introduction: Previously unknown paramyxovirus strains were isolated from wild birds in 2013-2014 in Kazakhstan and subsequently identified as representatives of the novel Avian avulavirus 20 species. The aims and tasks were molecular genetic characterization of novel avulaviruses and investigation of their phylogenetic relationships., Material and Methods: Embryonated chicken eggs were inoculated with cloacal and tracheal swabs from wild birds with subsequent virus isolation. The complete nucleotide sequences of viral genomes were obtained by massive parallel sequencing with subsequent bioinformatics processing., Results: By initial infection of chicken embryos with samples from 179 wild birds belonging to the Anatidae, Laridae, Scolopacidae and Charadriidae families, 19 hemagglutinating agents were isolated, and five of them were identified as representatives of new viral species. The study of their sequenced genomes revealed their similarity in size, but there was a significant genetic variability within the species. 2,640 nucleotide substitutions were identified and 273 of them were nonsynonymous, influencing the protein structure of viruses. It was shown that isolates Avian avulavirus 20/black-headed gull/Balkhash/5844/2013 and Avian avulavirus 20 /great black-headed gull/Atyrau/5541/2013 were 86% and 95% respectively identical to the previously described reference strain, indicating a significant evolutionary divergence within species., Discussion: The authors suggest the existence of two independent lineages - the Caspian, represented by the reference strain Aktau/5976 and Atyrau/5541, as well as the second, geographically significantly distant Balkhash lineage., Conclusion: The study confirms the role of the birds of the Laridae family as the main reservoir of Avian avulavirus 20 in the avifauna that plays a key role in maintaining viruses of the genus Avulavirus in the biosphere and is a potential natural source for the emergence of new viral variants. Continuous surveillance of them in the wild is one of the most important tasks in ensuring the safety of the poultry industry., Competing Interests: The authors declare no conflict of interest.

Four H5N2 experimental vaccine strains and the apathogenic wild duck H5N3 influenza virus A/duck/ Moscow/4182/2010 (dk/4182) were tested as a live poultry vaccine. Experimental strains had the hemagglutinin of the A/Vietnam/1203/04 strain lacking the polybasic HA cleavage site or the hemagglutinin from attenuated virus (Ku/ at) that was derived from the highly pathogenic influenza virus A/chicken/Kurgan/3/2005 (H5N1). The hemagglutinin of the Ku-at has the amino acid substitutions Asp54/Asn and Lys222/Thr in HA1 and Val48/Ile and Lys131/Thr in HA2, while maintaining the polybasic HA cleavage site at an invariable level. The other genes of these experimental strains were from the H2N2 cold-adapted master strain A/Leningrad/134/17/57 (VN-Len and Ku-Len) or from the apathogenic H6N2 virus A/gull/Moscow/3100/2006 (VN-Gull and Ku-Gull). A single immunization of mice with all tested strains elicited a high level of serum antibodies and provided complete protection against the challenge with the lethal dose of A/chicken/Kurgan/3/05. The pathogenicity indexes of the Ku-at and the other strains for chicken were virtually zero, whereas the index of the parent H5N1 virus A/chicken/Kurgan/3/2005 was 2.98. Intravenous, intranasal, and aerosol routes of vaccination were compared. It was shown that the strain dk/4182 was totally apathogenic for one-day-old chicken and provided complete protection against the highly pathogenic H5N1 virus.

Reverse genetics was applied to engineering of the reassortantvaccine candidate strain against highly pathogenic avian influenza viruses (HPAIVs) of the H5 subtype. The new strain recPR8-H5N1 contains the HA gene from the Russian HPAIV A/Kurgan/05/2005 (H5N1), the NA and internal genes from A/Puerto Rico/8/34 (H1N1). The strain recPR8-H5N1 demonstrated the antigenic specificity (H5), high proliferation rate in 12 days chicken embryos, and was lethal for the embryos in 36 hours. An inactivated emulsified vaccine based on the strain recPR8-H5N1 elicited high antibody titers and protected 6-week-old chickens from lethal challenge with the HPAIV A/Kurgan/05/2005 (H5N1) on day 21 after single immunization. Infection of non-vaccinated birds with the strain recPR8-H5N1 did not cause any pathology, and the virus was not detected using PCR in blood and cloacal swabs on day 7 p.i. Specific weak seroconversion caused by infection with the strain recPR8-H5N1 was detected on day 14 p.i. As a result, a new influenza virus strain was obtained with modified properties.

Chicken anemia virus gene encoding apoptin, a selective killer of cancer cells was synthesized and inserted into vaccinia virus (strain L-IVP) genome. The insertion has replaced major part of the viral C11R gene encoding viral growth factor (VGF), which is important for the virulence. The recombinant virus VVdGF-ApoS24/2 was obtained through the transient dominant selection technique with the use of puromycin resistance gene as the selective marker. The expression apoptin gene from a synthetic early-late promoter of vaccinia virus effectively provides accumulation of the protein in the cells infected with the VVdGF-ApoS24/2 virus. Despite the presence of virus growth factor signal peptide at apoptin N-terminal secretion of the recombinant protein into culture medium did not occur. The recombinant virus VVdGF-ApoS24/2 was found to have a significantly greater selective lyticactivity on human cancer cell lines (A549, A431, U87MG, RD and MCF7) as compared with the parent strain L-IVP and its variant VVdGF2/6 with the deletion of the C11R gene. The results suggest that the use of apoptin represents a promising approach for improving the natural anticancer activities of vaccinia virus.

Aim: Study of molecular-biological and antigenic features of H5N1 subtype virus strains isolated in southern Siberia in 2005-2009., Materials and Methods: Study was performed by using standard procedures according to WHO recommendations., Results: Hemagglutinin gene of H5N1 subtype virus strain isolated in Siberia belongs to 2 genetical lineages: 2.2 and 2.3.2. Strains of these 2 lineages have antigenic differences. All of the strains are highly pathogenic for chicken and white mice., Conclusion: H5N1 subtype highly pathogenic influenza virus variants of 2 different antigenic lineages that have antigenic differences circulated in Siberia in 2005-2009. A possible role of antigenic drift in evolution of H5N1 subtype influenza virus is discussed.

The frequent disease outbreaks caused by avian influenza virus not only affect the poultry industry but also pose a threat to human safety. To address the problem, RNA interference (RNAi) has recently been widely used as a potential antiviral approach. Transgenesis in combination with RNAi to specifically inhibit avian enza virus gene expression has been proposed to make chickens resistant to the infection. For the transgenic breeding, screening in vitro efficient siRNAs as the candidate genes is one of the most important tasks. Here, we combined an online search tool and a series of bioinformatics programs with a set of rules for designing siRNAs targeted towards different mRNA regions of H5N1 avian influenza virus. Five rational siRNAs were chosen by this method, five U6 promoter-driven shRNA expression plasmids containing the siRNA genes were constructed and used for producing stably transfected MDCK cells. The data obtained by virus titration, IFA, PI-stained flow cytometry, real-time quantitative RT-PCR, and DAS-ELISA analyses showed that all five stably transfected cell lines we re resistant to virusreplication when exposed to 100 CCID50 of avian influenza virus H5N1. Finally, most effective plasmids (pSi-604i and pSi-1597i) as the candidates for making the transgenic chickens were chosen. These findings provide baseline information on use of RNAi technique for breeding transgenic chickens resistant to avian influenza virus.

The paper presents the results of classifying 20 Russian isolates of serotype 1 Marek's disease viruses (MDV) by the adapted "best fit" method (ADOL, USA). The native SPF-chickens of the Shchelkovo line have enough susceptibility for pathotyping of MDV. The Russian isolates were classified by the pathotype in comparison with the reference strains of varying virulence; 8 of them belonged to the vv+ pathotype, 11 to the vv pathotype, and 1 to the v pathotype.

The epizootic etiologically associated with highly pathogenic avian influenza H5N1 genotype 2.3.2 that is new for Russia among wild and domestic birds in the south of the Primorye Territory during spring migration in April 2008 has been decoded. About 25% of the wild birds of a water complex, which include European teals (Anas crecca), mallard ducks (Anas platyrhynchos), great-crested grebes (Podiceps cristatus), are involved in viral circulation in the area of the Suifun-Khankai plain. Chicken embryos and the cell lines MDCK, SPEV, BHK-21, SW-13 were used to isolate 3 strains from recently deceased hens (A/chicken/Primorje/1/08, A/chicken/Primorje/11/08, and A/chicken/Primorje/12/08) and one strain from a European teal (A/Anas crecca/Primorje/8/08). The strains were deposited in the State Collection of Viruses of the Russian Federation, D. I. Ivanovsky Research Institute of Virology, Russian Academy of Medical Sciences. The nucleotide sequences of the full-sized genomes of A/chicken/Primorje/1/08 and A/Anas crecca/Primorje/8/08 were sent to the International databank GenBank. The strains from domestic and wild birds were shown to be identical. The isolated strains are most close to the strains Alchicken/Viet Nam/10/05, A/chicken/Guangdong/178/04, and A/duck/Viet Nam/12/05. Molecular genetic analysis has indicated that the strains isolated are susceptible to rimantadine and ozeltamivir and less adapted to mammalian cells (particularly, they contain E627 in RV2, which agrees with the biological properties of these strains in vitro). Penetration of the newly isolated virus into the Far East ecosystem provides in the foreseeable future a way for infecting the birds wintering in America and Australia in the nesting places, with further carriage of viral populations there in the period of autumn migrations.

The paper presents the results of interpreting the epizootic outbreak etiologically associated with high-virulent influenza virus A/H5N1 among domestic and wild birds in the Zernogradsky and Tselinsky districts of the Rostov Region. Epizooty was characterized by a high infection rate in the synanthropic birds of a ground-based complex. RT-PCT revealed influenza virus A/H5 in 60% of pigeons and crows and in around 20% of starlings, and in 10% of tree sparrows. Fifteen viral strains from chickens (Gallus gallus domesticus), Indian ducks (Cairina moschata), rooks (Corvus frugilegus), rock pigeons (Columba livia), tree sparrows (Passer montanus), common starlings (Sturnus vulgaris), and great white herons (Egretta alba) were isolated and deposited in the State Collection of Viruses of the Russian Federation. Full-sized genomes of 5 strains were sequenced and deposited in the international database GenBank. The isolated strains belong to the Quinhai-Siberian (2.2) genotype, an Iranian-Northern Caucasian subgroup, they are phylogenetically closest to the strain A/chicken/Moscow/2/2007 (inducing epizooty among poultry in the near-Moscow Region in February 2007) and have 13 unique amino acid replacements as the consensus of the Quinhai-Siberian genotypes in the proteins PB2, PA, HA, NP, NA, and M2, by preserving thereby 4 unique replacements first describes for the strain A/chicken/Moscow/2/2007. The findings are indicative of a different mechanism that is responsible for bringing the virus into the northeastern part of the Azov Sea area in September 2007 (during the fall migration of wild birds) and in December 2007 in the south-western Rostov Region where a human factor cannot be excluded. Mass infection of synanthropic birds endangers the further spread of epizooty, including that in the central regions of the Russian Federation in spring after near migrants return after wintering.

Work is devoted to studying of circulation of avian flu influenza A (H5N1) virus at poultry in territory of Novosibirsk region in preepizootic period. 1901 sample from clinically healthy poultry is collected and investigated. On chicken embryos it has been isolated 28 virions, including from poultry of the industrial maintenance--16, from the poultry contained in private farmsteads--12. At the analysis of isolates it has been shown, that among poultry in preepizootic period circulated various serovars of influenza viruses namely, HI, H4 and H3 serogroups. The percent of isolates from geese of the industrial maintenance increased to 0.6% depending on area. The percent of isolates from a bird contained in private farmsteads, increased to 7.5%. The greatest percent of isolates was observed from the samples taken from hens--77. Thus, it is shown, that among poultry circulation of not pathogenic influenza viruses of type A is the usual phenomenon, and asymptomatic carriers 7.5% of a livestock. Also it has been established, that in preepizootic period 2005 (January-June) at poultry it is not revealed influenza viruses of H5 serotype, caused epizootia at Summer of 2005 in territory of Western Siberia.

A field chick infectious bronchitis virus (IBV) was isolated from the pathological material on chick embryos. The nucleotide sequence of the S1 gene was determined and comparatively analyzed with some sequences of this gene of foreign and Russian vaccine strains and isolates. A cross-neutralization test using sera to various IBV seroptypes was performed. The isolate was shown to antigenically differ from the reference strains. Bioassay was carried out, by using one-day chicks and the immunogenic properties of the virus were investigated.

The method of chicken reovirus strain differentiation was worked out on the basis of RT-PCR and heteroduplex mobility assay (HMA). The S3 gene cDNA (633-896 b.p.) of some Russian and Italian chicken reovirus isolates was amplified by RT-PCR. The analysis of these cDNA samples was carried out by HMA. The relation between nucleotide differences and relative mobility of compared cDNA heteroduplex was reflected by the regression curve. The equation of linear regression was derived (y = 91.726-0.89x; where y is the level of nucleotide difference of compared cDNA (%), x is the relative mobility of compared cDNA heteroduplex (%)). This method made it possible to take correct results within 5-35% of nucleotide difference in heteroduplex sequences.

Six strains of influenza AH5N1 virus were isolated, by using PS and MDCK continuous cell lines from poultry and wild birds, which were collected on July 28, 2005 in the samples taken from 5 examines species of wild birds in the Novosibirsk region during the epizootic outbreak with a high mortality. The strains were identified by means of HIT, RT-PCR, and microchip-based techniques. Two strains, A/Grebe/Novosibirsk/29/05 (H5N1) and A/Duck/Novosibirsk/56/05 (H5N1), were deposited to the Russian State Collection of Viruses (Registration Nos. 2372 and 2371, respectively) with the priority date 08.08.2005. Positive results in RT-PCR for influenza A/H5N1 virus detec- tion were obtained in 100% of the samples from dead and sick poultry; 93% from the clinically healthy poultry kept together with sick one; positive results ranged from 0 to 36%. Sequencing established the identity of genetic characteristics of strains isolated for wild birds and poultry as well as their affiliation to high pathogenic avian influenza (HPAI). Phylogenetic analysis revealed a high homology of hemagglutining of West-Siberian strains and strains isoolated from bar-headed gooses (Eulabeia indica) on the Qinghai Lake (Western China) in the 2005 spring.

The nucleotide sequence of a part of the HindIII-D fragment (3300 b.p.) of adenovirus KR95 DNA has been determined. Analysis of the nucleotide sequence disclosed a continuous ORF for hexon gene (2814 b.p.) coding the 937 residue protein, part of ORF for the C-terminal region of pVI polypeptide, including 114 residues and the beginning of ORF coding 25 N-terminal residues for viral endoproteinase. Comparison of predicted KR95 hexon sequence and 8 mammalian and avian adenovirus hexon sequences revealed the highest homology between KR95 strain and avian adenoviruses FAV10 and FAV1 (91.1 and 80.1%, respectively). The results were used for creating a test system on the basis of the polymerase chain reaction. The system was used in analysis of fowl samples obtained from 12 poultry farms in Russia. The sequences of hexon gene amplified fragments in the isolated strains and similar fragments of other mammalian and avian adenoviruses have been compared.

Influenza virus A (H5N1) was isolated from the tracheal swab of a 3-year-old boy who died from influenza with the Raye syndrome in Hong Kong in May, 1997. Up to the present time, influenza viruses with hemagglutinin H5 were known to circulate only among birds. They caused a variety of diseases: from asymptomatic to epizootic with 100% mortality, particularly among chickens. The main difference between virulent and avirulent strains is as follows: virulent viruses are isolated from all tissues of an infected bird. A (H5) virus hemagglutinin, transformed into a virulent variant, becomes sensitive to cleavage by proteases of mammalian and avian cells. Intensive epidemiological surveillance of influenza in Hong Kong started by the WHO and Department of Public Health of Hong Kong in August-September, 1997, resulted in detection of 17 more cases with Influenza A (H5N1) in November-December 1997. all of the occurred before December 28, 1997 and were detected in hospitals and health centers of Hong Kong. Nine patients were children aged under 5 years. Six patients died as a result of complications (pneumonia) and exacerbations of concomitant chronic diseases. Virological and logical studies showed that the main route of infection transmission was from birds to humans. Human to human transmission is probable. Study of 7 influenza A (H5N1) viruses isolated from patients showed that they contained all 8 RNA gene segments of avian virus. There are no reports about new cases of influenza A (H5N1) in humans in January 1998, and we can hope that the outbreak of Influenza A (H5N1) in Hong Kong caused by avian virus will not develop into a new influenza pandemic, although an unfavorable course of events is probable.