Development, production and characterization of SARS-CoV-2 virus-like particles (Coronaviridae: Orthocoronavirinae: Betacoronavirus: Sarbecovirus).

INTRODUCTION: The COVID-19 pandemic caused by SARS-CoV-2 has created serious health problems worldwide. The most effective way to prevent the occurrence of new epidemic outbreaks is vaccination. One of the modern and effective approaches to vaccine development is the use of virus-like particles (VLPs). The aim of the study is to develop a technology for production of VLP based on recombinant SARS-CoV-2 proteins (E, M, N and S) in insect cells. MATERIALS AND METHODS: Synthetic genes encoding coronavirus proteins E, M, N and S were used. VLP with various surface proteins of strains similar to the Wuhan virus, Delta, Alpha and Omicron were developed and cloned into the pFastBac plasmid. The proteins were synthesized in the baculovirus expression system and assembled into VLP in the portable Trichoplusia ni cell. The presence of insertion in the baculovirus genome was determined by PCR. ELISA and immunoblotting were used to study the antigenic activity of VLP. VLP purification was performed by ultracentrifugation using 20% sucrose. Morphology was assessed using electron microscopy and dynamic light scattering. RESULTS: VLPs consisting of recombinant SARS-CoV-2 proteins (S, M, E and N) were obtained and characterized. The specific binding of antigenic determinants in synthesized VLPs with antibodies to SARS-CoV-2 proteins has been demonstrated. The immunogenic properties of VLPs have been studied. CONCLUSION: The production and purification of recombinant VLPs consisting of full-length SARS-CoV-2 proteins with a universal set of surface antigens have been developed and optimized. Self-assembling particles that mimic the coronavirus virion induce a specific immune response against SARS-CoV-2.

[Antiviral effect of «Kagocel¼ substance in vitro on influenza viruses H1N1, H1N1pdm09 and H3N2.]

INTRODUCTION: Active circulation of pandemic influenza and new variants of influenza H3N2 strains requires monitoring of antiviral efficacy of drugs permitted for influenza therapy in the Russian Federation. PURPOSE: Assessment of antiviral efficacy of «Kagocel¼ substance against influenza viruses H1N1, H1N1pdm09 and H3N2 in vitro. MATERIAL AND METHODS: Cytotoxic effect of «Kagocel¼ substance on MDCK cells had been determined by stained with MTS. Antiviral efficacy of «Kagocel¼ substance against influenza infection has been studied in vitro in the culture of MDCK cells infected with influenza virus strains: A/Puerto Rico/8/34 (H1N1), А/California/7/2009 (H1N1)pdm09, А/Hong Kong/1/68 (H3N2) and А/ Hong Kong/4801/2014 (H3N2). The antiviral activity of «Kagocel¼ substance was tested by its effect on the infectious titer of the influenza viruses and on its impact on the expression level of viral antigens in the enzyme immunoassay test system. RESULTS: «Kagocel¼ substance had low toxicity for MDCK cells. «Kagocel¼ inhibited the infection titer of influenza virus strains A/Puerto Rico/8/34 (H1N1), А/California/7/2009 (H1N1)pdm09, А/Hong Kong/1/68 (H3N2) and А/ Hong Kong /4801/2014 (H3N2) in the MDCK cell culture with equal efficacy. Study of the impact of «Kagocel¼ substance on the expression level of viral antigens by ELISA also revealed its antiviral efficacy for all tested strains. Dose dependence was observed from concentration of substance and from infective dose of virus. DISCUSSION: Effective suppression of the reproduction of influenza virus strains A(H1N1), A(Н1N1)pdm09 and A(H3N2) in the different sublines of MDCK cells with «Kagocel¼ was shown by the different methods. These results give the possibility to suggest that along with the ability to induce interferons, «Kagocel¼ can impact on the reproduction of influenza virus, but the further research is needed. CONCLUSION: «Kagocel¼ substance effectively inhibits the reproduction of influenza virus strains A(H1N1), A(Н1N1)pdm09 and A(H3N2) in vitro. At the same time, the selectivity index is quite high.

[Detection of conservative and variable epitopes of the pandemic influenza virus A(H1N1)pdm09 hemagglutinin using monoclonal antibodies].

The goal of this work was to analyze the antigenic structure of the hemagglutinin (HA) of the pandemic influenza virus A(H1N1)pdm09 using monoclonal antibodies (MAbs) and to develop a sandwich ELISA for identification of pandemic strains. Competitive ELISA demonstrated that 6 MAbs against HA of the pandemic influenza A/ IIV-Moscow/01/2009 (H1N1)pdm09 virus identified six epitopes. Binding of MAbs with 22 strains circulating in Russian Federation during 2009-2012 was analyzed in the hemagglutination-inhibition test (HI). The MAbs differed considerably in their ability to decrease the HI activity of these strains. MAb 5F7 identified all examined strains; MAbs 3A3 and 10G2 reacted with the majority of them. A highly sensitive sandwich ELISA was constructed based on these three MAbs that can differentiate the pandemic influenza strains from the seasonal influenza virus. The constancy of the HA epitope that reacts with MAb 5F7 provides its use for identification of the pandemic influenza strains in HI test. MAbs 3D9, 6A3 and 1E7 are directed against the variable HA epitopes, being sensitive to several amino acid changes in Sa, Sb, and Ca2 antigenic sites and in receptor binding site. These MAbs can be used to detect differences in HA structure and to study the antigenic drift of the pandemic influenza virus A(H1N1)pdm09.

[The peculiarities of the influenza epidemics in some areas of Russia during 2012-2013 season. The influenza A (H1N1) pdm09 virus domination in European countries].

The peculiarities of the influenza viruses circulation in 2012-2013 are discussed. The results were obtained in 10 cities of Russia, where basic laboratories of the Influenza Ecology and Epidemics Center of on the basis of Ivanovsky Institute of Virology, Ministry of Health of the Russian Federation, are situated. The increasing rate of the ARD morbidity caused by influenza viruses was observed in January-March 2013. The highest indices of the morbidity were detected during 6-7 weeks with the following decreasing rate till threshold levels to week 14. The influenza A (H1N1) pdm09, A (H3N2), and B viruses were the cause of the epidemic, but their activity differed over areas of Russia. The results of study of the antigenic and genetic properties of the influenza strains demonstrated closed relatives with respect to vaccine strains. In addition, some heterogeneity of the circulating strains and their drift variants were found as well. All tested strains were sensitive to oseltamivir (excluding one A (H1N1) pdm09 strain), zanamivir, arbidol, and remained resistant to rimantadine. The ratio of the ARD viruses was comparable with the last epidemic seasons.

[Etiology of fatal pneumonia caused by influenza A(H1N1)pdm2009 virus during the pandemic in Russia].

The results of the study of the autopsy materials from 61 patients with the diagnosis of pneumonia received by virological and genetic methods are reviewed. The materials were studied at the Influenza Etiology and Epidemiology Center of the Ivanovsky Institute of Virology, Ministry of Health and Social Development of the Russian Federation, during epidemic seasons 2009-2010 and 2010-2011. The data were analyzed with respect to age, sex, comorbidity diseases and identified on the groups of the risk of severe forms of the disease. The presence of the pandemic influenza virus strain RNA was confirmed in 70.5% of materials; RNA of influenza B was detected in 1.2% cases. The co-infections caused by the bocavirus, adenovirus, parainfluenza virus type 2 and 4, rhinovirus, and streptococcus were detected only in 19.7%. In most cases, the influenza virus was the etiologic agent of lethal pneumonia, which justifies the necessity of the early etiological diagnosis and treatment with antiviral drugs.

[Correlation between the receptor specificity of pandemic influenza A (H1N1)pdm09 virus strains isolated in 2009-2011 and the structure of the receptor-binding site and the probability of fatal primary viral pneumonia].

The receptor specificity (RS) of pandemic influenza A(H1N1) pdm09 virus strains deposited into the State Collection of Viruses of the Russian Federation, D. I. Ivanovsky Research Institute of Virology, Ministry of Health and Social Development of Russia, in the 2009-2010 and 2010-2011 epidemic seasons to a panel of 9 sialoglycopolymers (SGP). The strains were divided into 3 groups according to the W(3/6) index proposed by the authors, which was equal to the amount of reactivities to unbranched alpha2-3-SGP to that of reactivities to unbranched alphal-6-SGP: W(3/6) < or = 1.0; 1.0 < W(3/6) < or = 1.5. The W(3/6) < or = 1.5 group showed a predominance of a2-3-RS, attended by the high incidence of fatal primary viral pneumonias (FPVP) (60.0%) and amino acid replacements in the HA1 receptor-binding site (RBS) (80.0%): D222{G, N} and Q223R. The 1.0 < W(3/6) < or = 1.5 group was characterized by mixed alpha2-3/alpha2-6-RS with the incidence of FPVP (29.7%) and amino acid replacements in the HA1 RBS (40.5%) (D222{G, N, V} and Q223), respectively. In the W(3/6) < or = 1.0 group, alpha2-6-RS was prevalent, FPVPs were absent and amino acid replacements in HA1 RBS (D222{G, E}) were seen only in 6.0% of cases. The number of strains with increased specificity to alpha2-3-sialosides increased in the 2010-2011 epidemic season as compared to the previous season. With their further spread among the population, there may be a rise in cases of severe primary viral pneumonias with possible fatal outcomes, which can be, however, accompanied by a decrease in the capacity of mutants to air-dropwise transmission.

[The specific features of the cocirculation of influenza viruses in the 2010-2011 postpandemic period according to the results of activities of the D. I. Ivanovsky Research Institute of Virology, Ministry of Health and Social Development of Russia].

The paper gives the results of monitoring the circulation of influenza viruses in the 2010-2011 season, that covers the second year of circulation of pandemic A(H1N1)v virus strains, and their interaction with seasonal A (H3N2) and B strains. Unlike the previous season, the beginning of an increase in morbidity was recorded in January 2011; its peak in the most of contiguous areas was noted at 5-7 weeks of 2011, with its further decline to threshold levels at week 11 of 2011. Preschool and school children were most involved in the epidemic process. Three influenza virus strains (A(H1N1)v, A(H3N2), and B) were found to circulate. Differences were found in the level of participation of the isolated strains in individual areas of the Russian Federation. Detailed typing of the isolated strains determined the compliance of the vast majority of them with vaccine viruses. The pandemic influenza A(H1N1)v virus strains retained their susceptibility to oseltamivir and were resistant to rimantadine. The participation of non-influenza acute respiratory viral infection pathogens was estimated as follows: 11.9% for parainfluenza viruses, 5.9% for adenoviruses, and 3.5% for PC viruses, and 0.7% for pneumonia Mycoplasma, which was comparable with the previous epidemic seasons.

[Susceptibility of pandemic influenza virus A 2009 H1N1 and highly pathogenic avian influenza virus A H5N1 to antiinfluenza agents in cell culture].

The data on cytotoxicity and antiviral activity of commercial antivirals, such as Remantadine, Oseltamivir, Arbidol and Ribavirin in the MDCK cell culture infected with highly pathogenic (H5N1) and pandemic 2009 (H1N1) influenza A viruses are presented. The study of the antiviral activity of antivirals in the MDCK cells culture demonstrated that Arbidol, Rimantadine and Ribavirin efficiently inhibited reproduction of the highly pathogenic H5N1 influenza viruses isolated from sick birds. Arbidol and Oseltamivir carboxylate selectively inhibited reproduction of the pandemic 2009 H1N1 influenza A viruses with changed specificity to the cell receptors, causing severe influenza in men, while remantadine had no effect on their reproduction.

[Taxonomic structure of Orthomyxoviridae: current views and immediate prospects].

Analysis of taxonomic structure of Orthomyxoviridae was undertaken in view of its anticipated evolution. Four concepts of circulation of influenza A viruses in the biosphere are discussed, viz. anthrponose, zooanthroponose, metastrongilose, and protozoan. All of them may be considered in the framework of the general zooantroponose concept. Influenza B and C viruses can not be regarded as strictly anthroponose. Comparative molecular-genetic analysis of the genus Thogotovirus provides a basis for the designation of Thogoto and Batken-Dhori as independent geni. It is speculated that t he proof of transmission of Isaviruses by copepods Caligus elongates and Lepeophtheirus salmonis (Crustacea: Copepoda) may open up a new line of developments in arborvirology since crustacean vectors of viruses have never been described before.

[Etiotropic therapy of influenza: lessons from the last pandemic].

Analysis of the experience gained during the last pandemic of 'swine' influenza A (H1N1) sw1 is presented with reference to clinical studies and etiotropic therapy. The mechanism of development of severe pneumonia as a result of mutations at the binding site of hemagglutinin receptor enhancing a2'-3'-sialoside specificity and pneumotropism of the virus is described. The data on the efficiency of Ingavirin, a new Russian antiviral for the treatment of influenza, are reported.

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.

[A possible association of fatal pneumonia with mutations of pandemic influenza A/H1N1 sw1 virus in the receptor-binding site of the HA1 subunit].

The paper gives the results of sequence analysis of 150 positive samples in real-time RT-PCR, including 47 autopsy materials from patients (including 10 pregnant women), who died from fatal pneumonia mainly in November-December 2009, in whom the lifetime etiological diagnosis had not been made and hence no early etiotropic therapy performed. 70% of the primary materials from the deceased patients were found to have pandemic influenza A(H1N1) v mutants in the lung tissue with D222G (15%), D222N (15%), D222E (2%) substitutions, as well as a mixture of mutants (38%). Nasopharyngeal lavages from 3 Chukotka deceased patients exhibited only consensus (nonmutant) D222 virus variants; there was a mixture of consensus and mutant virus variants in the trachea and a mixture of mutant ones in the lung. Preliminary data from the study of the interaction of the hemagglutinin of two strains having D222G and D222N mutations with 9 oligosaccharides imitating the variants of cell receptors for influenza A virus suggest that there is a double receptor specificity for alpha2'-3' and alpha2'-6'-sialosides with a preponderance of alpha2'-3'-specificity. Further spread of the mutants that have acquired a high virulence and preserved their capacity for the respiratory route of human infection may lead to the situation similar to that seen in the 1918-1919 pandemic. Another scenario for evolution of the virus is to preserve its receptor specificity for alpha2'-3'-sialosides and high virulence with losses of alpha2'-6' specificity and capacity for aerosol transmission, by damping the pandemic.

[Spread of new pandemic influenza A(H1N1)v virus in Russia].

The paper presents the results of the investigations of the development of a influenza A(H1N1)v pandemic, conducted by the D. I. Ivanovsky Research Institute of Virology, Russian Academy of Medical Sciences, and collaborating laboratories in the European part of Russia, in the Urals, Siberia, and in the Far East. In the prepandemic period (April 27 - June 11, 2009) its first diagnosis was established on May 21, 2009; the first strain was isolated on May 24, 2009; the data on complete genome sequencing were sent to the GenBank; the sensitivity of the strain to commercial antiviral commercial agents was studied. In the early pandemic period (June 11 - August 15), 73 patients who had come from 14 countries of Europe, America, and Asia were identified; 19 virus strains (partially or completely sequenced) were isolated. The pandemic period (August 15 - December 1) was marked by absolute dominance of pandemic influenza virus virtually in the absence of seasonal influenza; the first death caused by pandemic influenza was detected in late August; 3053 subjects were infected with the pandemic strain, as shown by polymerase chain reaction diagnosis; 202 strains were identified.

[Detection of amino acid substitutions of asparaginic acid for glycine and asparagine at the receptor-binding site of hemagglutinin in the variants of pandemic influenza A/H1N1 virus from patients with fatal outcome and moderate form of the disease].

The paper analyzes the amino acid sequence of the receptor-binding site of hemagglutinin (HA) in the variants of pandemic influenza A/H1N1 swl from 18 patients with moderate (n=1) and fatal (n=17) forms of the disease in 2009. Nine samples contained asparaginic acid at position 222 of HA1 (D). This site exhibited mutations in 9 samples: D222G (n=3), D222N (n=3), and D222G/D222N (n=3). In one patient with the moderate form of the disease, D222G mutation was revealed after the second passage in the developing chick embryos; this mutation was not found in the primary sample from the patient. The findings suggest the mutant variants of the virus start to circulate among the population, which requires, firstly, continuation of molecular virological monitoring of the pandemic situation and, secondly, further study of the impact of amino acid substitutions at the receptor-binding site of HA1 on the increased virulence of influenza A virus.

[Study of the antiviral activity of Russian anti-influenza agents in cell culture and animal models].

The study of the antiviral activity of Russian anti-influenza agents in the cultured MDCK cells demonstrated that arbidol and ribavirin inhibited the reproduction of various influenza A virus strains, including rimantadine- and ozeltamivir-resistant variants, as well as influenza B viruses (IC50 2-8.5 microg/ml). Rimantadine at concentrations of 1-5 microg/ml completely inhibited the reproduction of reference and ozeltamivir-resistant influenza A virus strains, and it had no effect on the reproduction of influenza B viruses and rimantadine-resistant influenza A viruses. Arbidol and ribavirin also inhibited the reproduction of pandemic influenza A/California/04/2009(H1N1), A/California/07/2009(H1N1), and A/Moscow/01/2009(H1N1)swl viruses in the cultured MDCK cells (IC50 = 1.5-4.0 microg/ml) while rimantadine had no effect on their reproduction. The cultured cells showed no significant antiviral activity of ingavirin at nontoxic concentrations (up to 200 microg/ml) against all study strains of influenza A and B viruses, including pandemic A(H1N1) influenza virus strains. The activity of rimantadine, arbidol, and ingavirin was found on a model of Influenza pneumonia in mice infected with their adopted influenza A/Aichi/2/69(H3N2) virus. The preventive efficacy of the three test agents was similar and most pronounced when they were used 96 hours before infection, by preventing 40-50% death in the animals and their body weight loss and by increasing their survival by 1.3-1.5 times. Arbidol and rimantadine were more effective when used for treatment and prophylaxis in doses of 30 and 10 mg/kg/day, respectively, by protecting the infected animals from 60-80% death, increasing their survival by 1.7-2 times, and preventing their body weight loss as compared with the control. The same experiments with ingavirin showed that this agent was less effective than arbidol and rimantadine. Thus, arbidol and rimantadine have a pronounced antiviral infection in both cell culture and a model of influenza pneumonia. The found efficacy of ingavirin on an integral model of murine influenza pneumonia without its activity in the cultured cells is likely to be due to other pharmacological properties of the drug rather than its direct virus-specific action.

[Trends in the spread of pandemic influenza A(H1N1) swl in the Far East in 2009].

The paper describes the trend in the spread of pandemic influenza A(H1N1) swl virus in the Far East, which started in this region 2-3 months later than that in the European part of Russia. By mid-October seasonal epidemic influenza was practically displaced by pandemic one.

[The 24 May, 2009 isolation of the first A/IIV-Moscow/01/2009 (H1N1)swl strain similar to swine A(H1N1) influenza virus from the first Moscow case detected on May 21, 2009, and its deposit in the state collection of viruses (SCV No. 2452 dated May 24, 2009)].

The paper presents the results of the first isolation of the new influenza virus in Moscow and the Russian Federation, which was similar to the swine A/IIV-Moscow/01/2009(H1N1)swl strain isolated on May 24, 2009 from a Russian arrived in Moscow from the USA on May 19, 2009. The antigenic, biological, and molecular genetic properties of this virus were studied. The virus was isolated on MDCK and chick embryos, the hemagglutination titers being 1:8-1:16 AE; the infectious titers being 6.51g of the tissue cytopathogenic infective dose (TCID50) and 7.01g of the common infective dose (CID50). The virus was sensitive to arbidol, ribavirin, oseltamivir, and resistant to rimantadine. The complete virus genome was sequenced; the data were accepted to the Gen Bank on May 28, 2009 under GQ219584-GQ219590 and GQ202724. The significant gene substitution of neuraminidase Asp for Gly in position 451, which has been undetectable in any other strain published in the Gen Bank by the present time is unique only to A/IIV-Moscow/01/2009 (H1N1)swl. The virus has been deposited in the State Collection of Viruses, D. I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, under No. 2452 dated May 24, 2009.

[Dinamics in virulence of highly pathogenic influenza A virus A/H5N1 genotype 2.2 strains isolated in Russia in 2005-2007].

In 2005 to 2007, there was a reduction in the virulence of highly pathogenic avian influenza A virus A/H5N1 genotype 2.2 strains deposited in the State Collection of Viruses of the Russian Federation. Decreased virulence was most marked in the strains from domestic birds, the least in those from wild birds. Comparison of phenotypical and molecular genetic data revealed point amino acid replacements that might affect reduced virulence.

[Monoclonal antibodies to highly pathogenic avian influenza A(H5N1) strain isolated in the Russian Federation: development and properties].

Highly pathogenic avian influenza (HPAI) virus subtype H5N1 has recently caused extensive epizootics in different regions of the world and presents a serious threat to man. Since 2005, HPAI virus subtype H5N1 strains have been circulating in Russia, which differ from the earlier isolated Southern Asia strains. A panel from 15 monoclonal antibodies (Mabs) to HPAI virus A/duck/Novosibirsk/56/05 (H5N1) was developed. Eleven Mabs interacted with the hemagglutinin molecule (HA), 4 with influenza A virus nucleoprotein (NP) in the Western blot assay. The bulk of the obtained Mabs interacted with homologous virus in ELISA and showed an antigen in the infected cells in the indirect immunofluorescence assay. Nine Mabs were active in the hemagglutination inhibition (HI) assay and 8 of them were capable to neutralize viral activity. The comparative analysis of the properties of Mabs in the HI assay using various influenza A strains showed that Mabs 2C6, 6F3, 4G10, 3G9, and 7B3 inhibited hemagglutination of study avian influenza viruses subtype H5, Mab 6F3 being most active. Mab 3B5 reacted only with the viruses isolated in the Russian Federation in 2005-2007 and failed to interact with the other study influenza A viruses subtype H5. The obtained panel of Mabs can be used to study the fine antigenic structure of hemagglutinin and to make a differential diagnosis of avian influenza viruses subtype A/H5N1. The high neutralizing activity of Mabs creates a prospect for preparing humanized antibodies for specific prevention and treatment.

[The first break-through of the genotype 2.3.2 of high-virulence influenza A virus A/HSN1, which is new for Russia, in the Far East].

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.