Isolation and characterization of H5Nx highly pathogenic avian influenza viruses of clade 2.3.4.4 in Russia.

In 2016-2017, several subtypes of the highly pathogenic avian influenza (HPAI) virus were isolated on the territory of Russia. In the autumn of 2016, during the avian influenza virus surveillance in the territory of the Kamchatka region of Russia the HPAI A(H5N5) influenza virus was isolated. Then, during 2016-2017, multiple outbreaks among wild birds and poultry caused by HPAI A(H5N8) avian influenza virus were recorded in European part of Russia. At the end of 2017, an outbreak among poultry caused by HPAI A(H5N2) influenza virus was recorded in the European part of Russia. Phylogenetic analysis of HA of the A(H5N5), A(H5N8), A(H5N2) showed the strains belong to the clade 2.3.4.4 b. All isolated strains were antigenically closely related to candidate vaccine viruses of clade 2.3.4.4 and showed high virulence in mice. Genetic analysis revealed presence of genetic markers potentially related to high virulence in mice in all studied viruses.

[ANALYSIS OF POPULATION IMMUNITYAGAINST INFLUENZA PRIOR TO 2014 AND 2015 EPIDEMIC SEASONS].

AIM: Control for the population herd immunity against seasonal influenza viruses as well as for emergence of antibodies against influenza with pandemic potential in human blood sera. MATERIALS AND METHODS: HAI reaction against vaccine and epidemic influenza viruses as well as HPAI viruses A/rook/Chany/32/2015 (H5N1) (clade 2.3.2. lc.) andA/Anhui/01/2013 (H7N9). RESULTS: Among all the sera samples collected in the autumn of 2014 and 2015, none had reacted in HAI against A(H5N 1) and A(H7N9) antigens even at 1:10 dilution. Among samples collected in autumn 2014, 41% were positive to A/California/07/09(H1Nlpdm9) virus, 36% - A/Texas/50/2012 (H3N2), 40% - B/Brisbane/60/2008 (Vict.lin.) and 47% reacted in HAI against the B/Massachusetts/2/2012 (Yam.lin.) strain. 22% of all the samples had a titer of at least 40 against all the antigens and only 10% in HAI had a titer of 40 or more against all the vaccine strains. Among the samples collected in autumn 2015, the number of seropositive against A/California/07/09(HlNlpdmO9) varied from 31% in the Urals FD to 46% in the Southern FD. The amount of seropositive against A/Switzerland/9715293/13 (H3N2) strain was at the level of 4 - 13% in all the FDs except Urals, where this parameter was slightly above 30%. The amount of seropositive against vaccine influenza B viruses varied from 23 to 76%. Only 2% of sera had titers in HAI of 40 or above against all the vaccine strains, 29% of all the samples were seronegative. CONCLUSION: Population immunity in Russia against influenza A(H3N2) is at a very low level, thus socially significant consequences of influenza epidemics in many aspects will depend on the vaccination campaign of autumn 2016.

Fatal Cases of Seasonal Influenza in Russia in 2015-2016.

The influenza epidemic in 2015-2016 in Russia is characterized by a sharp increase of influenza cases (beginning from the second week of 2016) with increased fatalities. Influenza was confirmed in 20 fatal cases registered among children (0-10 years), in 5 cases among pregnant women, and in 173 cases among elderly people (60 years and older). Two hundred and ninety nine people died from influenza were patients with some chronic problems. The overwhelming majority among the deceased (more than 98%) were not vaccinated against influenza. We isolated 109 influenza A(H1N1)pdm09 and one A(H3N2) virus strains from 501 autopsy material samples. The antigenic features of the strains were similar to the vaccine strains. A phylogenic analysis of hemagglutinin revealed that influenza A(H1N1)pdm09 virus strains belonged to 6B genetic group that had two main dominant subgroups during the 2015-2016 season. In Russia strains of the first group predominated. We registered an increased proportion of strains with D222G mutation in receptor-binding site. A herd immunity analysis carried out immediately prior to the epidemic showed that 34.4% blood sera samples collected in different regions of Russia were positive to A/California/07/09(H1N1)pdm09. We came to a conclusion that public awareness enhancement is necessary to reduce unreasonable refusals of vaccination.

Highly pathogenic influenza H5N1 virus of clade 2.3.2.1c in Western Siberia.

In the spring of 2015, avian influenza virus surveillance in Western Siberia resulted in isolation of several influenza H5N1 virus strains. The strains were isolated from several wild bird species. Investigation of biological features of those strains demonstrated their high pathogenicity for mammals. Phylogenetic analysis of the HA gene showed that the strains belong to clade 2.3.2.1c.

Molecular characterization and phylogenetics of a reassortant H13N8 influenza virus isolated from gulls in Mongolia.

Double reassortant H13N8 influenza A virus was isolated from gull in Mongolia. The basic virological characteristics were studied. Complete genome sequence analysis indicated the complicated evolutionary history. The PA gene belongs to classical Avian-like lineage and more likely originated from non-gull avian virus pool. Data confirm the state of extensive geographic mosaicism in AIV from gulls in the Northern Hemisphere.

[Analysis of population immunity of residents of Russian Federation regions against seasonal and highly pathogenic strains of influenza virus].

AIM: Determine the level of antibodies against socially significant types/serotypes of influenza virus in sera of individuals residing in various regions of Russia. MATERIALS AND METHODS: 1525 samples of blood sera collected in August-December 2013 in 8 regions of Russian Federation were studied in hemagglutination inhibition reaction (HAI) with antigens obtained from A/California/07/09, (H1N1)pdm09, A/Victoria/361/2011(H3N2), B/Brisbane/60/2008 (Victoria line), B/Massachusetts/2/2012 (Yamagata line), A/Commongull/Chany/2006 (H5N1), A/Anhui/01/2013 (H7N9) influenza virus strains. RESULTS: None of the blood sera samples had significant HAI titers against A/H5 and A/H7 antigens. Of all the 1525 samples, 788 (52%) were positive with A(H1N1)pdm09 antigen; 734 (48%) reacted with A(H3N2) antigen; 1010 (66%) samples were positive with B/Victoria antigen and 602 (39%) samples were positive with B/Yamagata antigen. CONCLUSION: Healthcare institutions should pay attention to the correction of population immunity profile in regions for the reduction of social-economic losses from seasonal influenza epidemics.

Monitoring of influenza viruses in Western Siberia in 2008-2012.

Western Siberia is of great importance in ecology and epidemiology of influenza. This territory is nesting area for great amount of bird species. Territorial relations of Western Siberian birds that are established during seasonal migration are extremely wide since this region is an intersection point of bird migration flows wintering in different regions of the world: Europe, Africa, Middle East, Central Asia, Hindustan, and South East Asia. Reassortant influenza viruses that can cause outbreak among population may emerge in Western Siberia with high probability. Thus, it is extremely important to carry out widespread study of circulated viruses, their molecular biological properties, phylogenetic links in this region, as well as herd immunity to influenza virus serotypes with epidemic potential.

[Molecular-biological and antigenic features of H5N1 subtype highly pathogenic influenza virus strains isolated in southern Siberia in 2005-2009].

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.

[Genetical features of influenza virus A (H1N1) strain that caused the 2009 pandemic].

Genetical features of the A(H1N1) influenza virus strain that caused the 2009 pandemic are analyzed in the review. Mutations typical for this strain, unique and similar to influenza viruses of swine, avian and seasonal types, and phenotypic (pathologic) features associated with them, that are experimentally confirmed, are described. A possibility of reassortation of avian and swine influenza viruses and possible epidemiologic consequences are discussed.

[Studies of properties of pandemic influenza virus A/H1N1 circulated in Russian Federation in 2009 - 2010].

AIM: Studies of cultural, virologic, antigenic properties of 89 samples of pandemic influenza A(H1N1) 2009 virus isolated in Russian Federation from May 2009 to March 2010. MATERIALS AND METHODS: Properties of isolated samples were compared with those of the reference strain A/ California/04/2009 (H1N1). RESULTS: Studies of biological properties and analysis of genome nucleotide sequences of the isolated samples showed that those strains are closely related to the reference strain. CONCLUSION: Monitoring of genetic, virologic and antigenic properties of pandemic influenza A(H1N1) 2009 virus isolates carried out from May 2009 to March 2010 did not reveal significant changes in the abovementioned properties of the virus or emergence of mutations that can lead to such changes.

Influenza A/H1N1pdm virus in Russian Asia in 2009-2010.

In total 3566 blood sera samples were collected in the Russian Far East and Central and Western Siberia in 2009-2010. The presence of antibodies to influenza A/H1N1pdm, seasonal H1N1 and H3N2 influenza viruses in the sera was tested in the hemagglutination inhibition test. 29.5% of samples from the Far East were positive to pandemic influenza, this value for Central and Western Siberia is 2-fold lower (12.8% and 11%, respectively). Fifty-six influenza A/H1N1pdm viruses were isolated during 2009-2010 from samples collected in Central and Western Siberia and the Russian Far East. Molecular and genetic properties of 23 strains were studied. Nucleotide and amino-acid sequences of pandemic influenza virus strains were identical (99% identity or more) to reference strain A/California/04/2009. Sporadic substitutions in antigenic sites were detected but did not change antigenic characteristics of strains.

[Pandemic influenza virus A (H1N1) in Amur region in autumn 2009].

AIM: Isolation and study of molecular genetic characteristics of pandemic influenza virus A (H1N1) circulated in Amur region in autumn 2009 as well as testing of serum samples taken from citizens of this region during November- December 2009 in order to measure levels of antibodies to socially significant serotypes of influenza A virus. MATERIALS AND METHODS: Strain of pandemic influenza virus A/Blagoveschensk/01/2009 (H1N1) was isolated on MDCK cell culture and nucleotide sequences of all eight segments of viral genome were determined. Five hundred seventy-six serum samples taken in Amur region in autumn 2009 were tested by hemagglutination inhibition assay. RESULTS: Nucleotide sequence of A/Blagovechensk/01/2009 (H1N1) strain was 99.7% identical to reference influenza virus strain A/California/04/2009. Diagnostically significant titers of antibodies to pandemic influenza virus were observed in 46.3% of persons younger 30 years old and in 20.1% older persons. Antibodies to seasonal influenza virus H1N1 and H3N2 were detected in 39.5 and 29.8% of persons respectively. CONCLUSION: Final seroepidemiological picture of distribution of pandemic virus in Amur region matches with the one for seasonal influenza virus A (H1N1): > 60% of seropositive persons were registered in age group < 18 years old, and this proportion increases with increasing age.

[Characterization of the H5N1 influenza virus isolated during an outbreak among wild birds in Russia (Tuva Republic) in 2010].

The study of basic biological properties of H5N1 subtype strain isolated during an outbreak among wild birds in Russia in 2010 was presented. The study was carried out using conventional methods according to the WHO recommendations. H5N1 influenza virus isolated in Siberia belonged to clade 2.3.2 of the hemagglutinin gene; the phylogenetic analysis was performed. The antigenic characteristics and the basic genetic markers of biological properties were studied. It was shown that all strains were highly pathogenic for chickens and white mice. Thus, it was shown that in Russia in the 2010 H5N1 virus phylogenetically closely related to Asian variants caused epizootic among wild birds. The potential danger of this variant of the virus for humans was confirmed by different methods. We discussed the possibility of formation of H5N1 influenza natural focus.

[Detection of antibodies to A/H5N1 influenza virus in citizens of Russian Federation].

AIM: To determine levels of antibodies to influenza virus A/H5N1 in serum samples of people living in different regions of Russia in order to assess the risk of infection with avian influenza H5N1. MATERIALS AND METHODS: Two thousand one hundred sixty-eight serum samples were tested by hemagglutination inhibition assay for the presence of antibodies to influenza virus A/H5N1. RESULTS: Twenty-six serum samples obtained from residents of Khanty-Mansiysk Autonomous Area and 2 samples from residents of Novosibirsk region were positive for antibodies to serotype A/H5. There were no clinical cases of avain influenza A/H5N1 infection in medical history of studied persons. CONCLUSION: Since cases of asymptomatic carriage of A/H5N1 influenza virus in water birds are described and ability of the virus to survive in water environment for a long time is shown, it seems logical to detect antibodies to influenza virus A/H5 in sera of subjects living in Russian Federation taking into account that influenza virus A/H5N1 is isolated from wild fowl and poultry since 2005.

[Characteristic of high pathogenic avian influenza virus subtype H5N1 isolated from common gull (Larus canus)].

AIM: To study biological characteristics of H5N1 influenza virus isolated from common gull on south of West Siberia in 2006. MATERIALS AND METHODS: Isolation and characterization of biological characteristics performed according to recommendations of World Health Organization. RESULTS: Influenza virus A (H5N1) was first isolated from common gull (Larus canus) in Russia. Antigen of isolated virus had significant affinity to polyclonal sera obtained against high pathogenic avian influenza viruses H5N1 circulating in South-East Asia. Phylogenetic analysis of isolated strain revealed its belonging to group of Qinghai-related variants of H5N1 influenza virus. Aminoacid structure of hemagglutinin proteolytic cleavage site is characteristic for type A high pathogenicity avian influenza viruses. Experimental infection of chickens demonstrated high pathogenicity of the isolated virus. CONCLUSION: Involvement of common gulls in circulation of subtype H5N1 influenza virus is demonstrated for the first time. Important role of species from Laridae family in unprecedented spreading of H5N1 influenza virus started in 2005 is discussed.

[Monitoring for influenza in population of Western Siberia in 2007-2009].

AIM: To analyze influenza viruses isolated in the 2008-2009 autumn-winter season, and to test sera collected in the south of Western Siberia during the beginning and the end of the epidemic seasons from 2007 until the A/H1N1 pandemic. MATERIALS AND METHODS: Total 149 clinical samples were analyzed and 2190 blood sera were tested. During the 2008-2009 season 17 influenza viruses were isolated. 9 of these were A/H1N1, 5-were A/H3N2, and 3 were influenza B viruses. The nucleotide sequences and amino acid composition of influenza A virus hemagglutinin (HA) were compared with reference strains. RESULTS: Among A/H1N1 viruses circulating in Novosibirsk region three viruses contained four amino acid replacements in antigen sites Ca, Cb and Sb. In A/ H3N2 viruses from Novosibirsk, 2 amino acid substitutions were detected in antigen sites B and E. CONCLUSION: Based on genotyping influenzae epidemic on February to April of 2009 in the south of western Siberia was associated with influenza viruses A/H1N1, A/H3N2, and B. All A/H3N2 influenza virus isolates were variants of reference A/Brisbane/10/2007(H3N2) and A/ H1N1 influenza viruses isolates were similar to reference A/Brisbane/59/2007(H1N1).

[Investigation of susceptibility of influenza viruses A (H1N1), the cause of infection in humans in April-May 2009, to antivirals in MDCK cell culture].

Biological properties of influenza viruses A (H1N1), that were the cause of the infection in humans in April - May 2009, and the action of the Russian antivirals on their reproduction were studied in vitro. The nucleotide sequence in the viruses was determined and followed by detection of the mutations responsible for resistance to the antiinfluenza drugs. The experiments showned that arbidol and ribavirin had a selective inhibitory action on reproduction of the viruses in the MDCK cell culture while rimantadine had no affect on their reproduction. The data were confirmed by the results of the genome analysis in influenza viruses A/California/04/2009(H1N1), A/California/07/2009(H1N1) and A/Moscow/01/2009(H1N1)swl, that revealed no replacements defining the resistance to arbidol while the viruses contained a mutation in position 31 of M2 protein, responsible for the resistance to adamantans.

[Hemagglutination inhibition test for retrospective diagnosis of avian influenza in mammals].

AIM: To compare usage of native and formalinized erythrocytes from different animal species in hemagglutination inhibition (HI) test for detection of level of specific antibodies to H5N1 influenza virus in sera of mammals. MATERIALS AND METHODS: Level of anti-H5 antibodies to influenza H5 control antigen and to influenza viruses A/Common gull/Chany/2006 (H5N1), A/duck/Tuva/01/06 (H5N1), A/Anas platyrhynchos/Chany Lake/9/03 (H5N3) was determined by hemagglutination inhibition test in two influenza A (H5) reference antisera as well as in ferret antisera to native strains of avian influenza virus. Equine, rhesus macaque, sheep, guinea pig, goose, and chicken erythrocytes were used. RESULTS: Using reference antisera, H5 hemagglutinin was detected in all tested antigens with all used erythrocytes. While testing ferret antisera in HI test with reference antigen, anti-H5 antibodies were not detected or detected in extremely low titre (1/80) and only with equine erythrocytes. In most cases, titers of anti-H5 antibodies in HI test with formalinized erythrocytes were higher than with native ones. CONCLUSION: During monitoring for antibodies to H5N1 avian influenza virus in human population it is necessary to use native strains of A/H5N1 along with reference antigen. It is possible to use formalinized equine, rhesus macaque, goose, and chicken erythrocytes.

[Theoretical and experimental study of the effect of membrane pores on spectra of amplitudes of polarization of a cell].

Spectra of amplitudes of polarization of erythrocytes, yeast cells, and latex particles in the range of 1-10 Hz were investigated by the method of dielectrophoresis. Positive dielectrophoresis of erythrocytes and yeast cells the frequency range of 60 - 100 Hz was revealed. The theoretically positive dielectrophoresis was evidenced by the occurrence of channels across the cell membrane and bacterial cell wall.

[Isolation and molecular characterization of the influenza virus A/H5N1 strains isolated during outbreak of avian influenza among birds in the European part of Russia in 2005: strain with ozeltamivir-resistance mutation was found].

Isolation and characterization of the influenza virus A/H5N1 strains, isolated from chicken in the Yandovka village (Tula Region) and from wild swan near the orifice of the Volga River that died during an outbreak of avian flu in autumn 2005, were carried out. Genetic and phylogenetic analyses were performed. The goals of the analysis were to determine possible geographical origin of the strain, genetic similarity of isolated strains to earlier sequenced isolates, epidemic potential, existence of pathogenicity markers, and resistance to antiviral drugs. It was shown that the isolated influenza virus belonged to highly pathogenic variants of China origin by a reassortment of viruses genotypes Z and V circulated in poultry and wild birds. A number of molecular markers of pathogenicity to gallinaceous birds and mammals were found out. Mutations in the hemagglutinin gene promoting potentially high rate of replication in humans as well as mutations causing the resistance to amantadine/rimantadine were not found. The strain isolated from wild swan had the mutation causing resistance to tamiflu/ozeltamivir.