Randomized Clinical Trial of Hybrid vs. Surgical vs. Percutaneous Multivessel Coronary Revascularization: 5­year Follow-up of HREVS Trial.

Aim      To evaluate 5-year results of the HREVS (Hybrid REvascularization Versus Standarts) study.Material and methods  The study included 155 consecutive patients with multivessel coronary artery disease who were randomized into 3 groups: coronary artery bypass grafting (CABG) (n=50), hybrid coronary revascularization (HCR) (n=52) and percutaneous coronary intervention (PCI) (n=53) according to the consensus of the cardiology team on the technical and clinical feasibility of each of the three coronary revascularization strategies. The primary endpoint of the study was residual ischemia 12 months after revascularization according to data of single-photon emission computed tomography (SPECT). Secondary endpoints were major adverse cardiac and cerebrovascular events (MACCE) over 5 years of follow-up, which included all-cause death, myocardial infarction, stroke, and clinically determined repeat myocardial revascularization.Results Baseline characteristics of patients did not differ between study groups. Median residual ischemia determined by SPECT data after 12 months was not statistically significantly different in the CABG, HCR and PCI groups: 6.7 [4.6; 8.8]%, 6.4 [4.3; 8.5]% and 7.9 [5.9; 9.8]%, respectively (p=0.45). Mean follow-up period was 76.5 months (at least 60 months). There were no statistically significant differences in all-cause mortality between the CABG, HCR and PCI groups, 10.6, 12.8 and 8.2 %, respectively (p=0.23). Statistically significant differences between the groups of CABG, HCR and PCI in the incidence of myocardial infarction (12.8; 8.5 and 16.3 %; p=0.12), stroke (4.2; 6.4 and 10.2 % ; p=0.13), repeat revascularization for clinical indications (23.4; 23.4 and 34.7 %; p=0.11) were not observed either. However, the cumulative 5-year MACCE value was similar in the HCR group and the CABG group but significantly lower than in the PCI group (51.1, 51.1 and 69.4 %, respectively; p = 0.03).Conclusion      HCR that combines advantages of PCI and CABG is a promising strategy for coronary revascularization in multivessel coronary artery disease. HCR demonstrates satisfactory long-term results comparable to those of CABG but superior to PCI. To confirm the safety and efficacy of HCR, a large multicenter study is required that would have a sufficient power to evaluate clinical endpoints.

[Latex Agglutination as an Alternative to the Hemagglutination Reaction of Influenza Viruses].

As an alternative to the classical method of erythrocyte hemagglutination, a latex agglutination assay based on the interaction of influenza viruses with the sialoglycoprotein fetuin immobilized on the surface of polystyrene microspheres has been developed. Twelve influenza A virus strains of different subtypes and two influenza B viruses of different lines were tested. Simultaneous titration of viruses using the classical hemagglutination test and the proposed latex agglutination assay showed similar sensitivity and a high degree of correlation (R = 0.94). The obtained microspheres can be used for titration of viruses that recognize and bind sialylated glycans as receptors. In particular, latex aggregation was also induced by the Newcastle disease virus.

[Correction of enteral distress syndrome in acute intestinal obstruction]. / Korrektsiya enteral'nogo distress-sindroma pri ostroi kishechnoi neprokhodimosti.

OBJECTIVE: On the basis of a comprehensive assessment of the functional state of the intestine in acute mechanical small bowel obstruction, to justify adequate schemes of its protection. MATERIAL AND METHODS: A clinical and laboratory study of 48 patients with acute small bowel obstruction developed against the background of abdominal adhesions, strangulated abdominal hernia was conducted. The first group (n=25) of patients who underwent laparotomy, removal of intestinal obstruction (adhesiolysis and/or herniation, hernial gate plastic surgery), intestinal intubation, standardized therapy after surgery. The second group (n=23) - patients, therapy included Remaxol (Polysan Pharmaceutical Plant, Petersburg): 400.0 enterally intraoperatively through a probe after nasointestinal intubation, evacuation of stagnant contents and intestinal lavage with isotonic saline solution; 400.0 - intravenously for 5 days. A number of indicators of homeostasis (endogenous intoxication, oxidative stress), structural and functional state of the intestine were evaluated. RESULTS: It was found that the inclusion of remaxol in complex therapy (intraoperatively and in the early postoperative period) leads to the optimization of the treatment process of patients with acute intestinal obstruction. The number of complications according to the Clavien-Dindo classification decreased from 17 (first) up to 5 (second group) (χ2=3.988, p=0.046). Hospital stay decreased from 12.8±1.1 to 10.1±0.8 bed days (p<0.05). The effectiveness of the developed scheme is based on its ability to correct the phenomena of enteral distress syndrome relatively quickly, which was confirmed by laboratory and instrumental methods. The most important manifestation of this was a significant decrease in the phenomena of endogenous intoxica tion against the background of a significant decrease in the activity of peroxidation of membrane lipids - triggers of catabolic intestinal lesions. CONCLUSION: Studies document the effectiveness of the developed treatment regimen for patients with acute intestinal obstruction. The inclusion of remaxol parenterally and enterally makes it possible to significantly optimize the course of the early postoperative period. One of the main objects of its implementation was the relatively rapid restoration of intestinal function, reduction of manifestations of enteral distress syndrome. This provided rapid relief of endogenous intoxication and, as a result, prevented the progression of the systemic inflammatory response syndrome, which together determined the optimization of the early postoperative period.

Mechanism of Vascular Injury in Transcatheter Aortic Valve Replacement.

The aim of the study was to determine the potential mechanism of vascular complications due to "catheter-vascular wall" interaction in transcatheter aortic valve replacement using experimental and numerical analysis. MATERIALS AND METHODS: A series of full-scale bench tests and numerical simulations were carried out using the CoreValve commercial transfemoral delivery system for aortic valve bioprosthesis (Medtronic Inc., USA). Full-scale tests were carried out using a phantom of the vascular system (a polymeric silicone model of Transcatheter Aortic Valve; Trandomed 3D Inc., China) with simulation of all stages of delivery system movement along the vascular bed. They involved introduction into the common femoral artery, movement along the abdominal and thoracic parts of the aorta, the aortic arch, and positioning the system to the implantation site. The force arising from the passage of the delivery system was assessed using sensors of a Z50 universal testing machine (Zwick/Roell, Germany). Numerical simulation of transcatheter valve replacement procedure was carried out in a similar way with allowance for the patient-specific anatomy of the recipient's aorta using the finite element method in the Abaqus/CAE environment (Dassault Systèmes, France). RESULTS: It was found that in the process of the delivery system passing through the vascular system, there occurred force fluctuations associated with catheter bending and its interaction with the aortic wall in the region of its arch. For example, in the initial straight portions, the pushing force was 3.8-7.9 N; the force increased to the maximum (11.1 and 14.4 N with and without the prosthesis) with bending of the distal portion of the catheter. A similar increase was observed when performing numerical simulation with high-quality graphic visualization of stress on the "spots" of contact between the catheter and the vascular wall with an increase in stress to 0.8 MPa. CONCLUSION: Numerical and full-scale bench tests prove the significant effect of the properties of delivery system catheter for transcatheter aortic valve replacement on the interaction with the aortic walls.

[The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses].

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

Variability of nonpathogenic influenza virus H5N3 under immune pressure.

Mutations arising in influenza viruses that have undergone immune pressure may promote a successful spread of mutants in nature. In order to evaluate the variability of nonpathogenic influenza virus A/duck/Moscow/4182-C/2010(H5N3) and to determine the common epitopes between it and highly pathogenic H5N1 avian influenza viruses (HPAIV), a set of escape mutants was selected due to action of MABs specific against A/chicken/Pennsylvania/8125/83(H5N2), A/Vietnam/1203/04(H5N1) and A/duck/Novosibirsk/56/05(H5N1) viruses. The complete genomes of escape mutants were sequenced and amino acid point mutations were determined in HA, NA, PA, PB1, PB2, M1, M2, and NP proteins. Comprehensive analysis of the acquired mutations was performed using the Influenza Research Database (https://www.fludb.org) and revealed that all mutations were located inside short linear epitopes, in positions characterized by polymorphisms. Most of the mutations found were characterized as substitutions by predominant or alternative amino acids existing in nature. Antigenic changes depended only on substitutions at positions 126, 129, 131, 145 and 156 of HA (H3 numbering). The positions 126, 145 and 156 were common for HA/H5 of different phylogenetic lineages of H5N1 HPAIV (arisen from A/goose/Guangdong/1/96) and low pathogenic American and Eurasian viruses. Additionally, mutation S145P increased the temperature of HA heat inactivation, compared to wild-type, as was proved by reverse genetics. Moreover, nonpathogenic A/duck/Moscow/4182-C/2010(H5N3) and H5N1 HPAI viruses have the same structure of short linear epitopes in HA (145-157) and internal proteins (PB2: 186-200, 406-411; PB1: 135-143, 538-546; PA: 515-523; NP: 61-68; M1: 76-84; M2: 45-53). These facts may indicate that H5 wild duck nonpathogenic virus could be used as vaccine against H5N1 HPAIV. Keywords: avian influenza virus; H5 hemagglutinin; escape mutants; genetic analysis; phenotypic properties; site-specific mutagenesis.

[Adiponectin gene expression in local fat depots in patients with coronary heart disease depending on the degree of coronary lesion].

AIM: To determine the dependence of adiponectin gene expression by subcutaneous, epicardial and perivascular adipocytes on the degree of coronary lesion in coronary heart disease. MATERIALS AND METHODS: 84 patients with coronary artery disease were examined. Of these, 39 people showed a moderate degree of atherosclerotic lesion of the coronary bed (less than or equal to 22 points) on the SYNTAX Score scale, 20 severe (2231 points), and 25 extremely severe (more than 32 points). Upon admission to the hospital, all patients underwent an echocardiographic study (Echocardiography, Acuson, Germany) with the calculation of the ejection fraction (EF) of the left ventricle (LV) to assess its systolic function. During a planned surgical intervention (coronary bypass surgery, CABG), adipocytes of subcutaneous, epicardial (EAT) and perivascular adipose tissue (PVAT) were taken. Adiponectin gene expression was evaluated by polymerase chain reaction (real-time PCR) using TaqMan probes. Statistical analysis was performed using Statistica 9.0. RESULTS: The maximum level of adiponectin expression was detected in adipocytes of PVAT, and the minimum EAT. With an increase in the degree of atherosclerotic lesion of the coronary bed, the expression of the adiponectin gene in adipocytes of local depots significantly decreases r=-0.82; p=0.023. Moreover, the low level of gene expression in EAT correlated with a decrease in LV EF by r=0.73; p=0.03. In adipocytes of subcutaneous and especially PVAT, gene expression was the highest in patients with a moderate degree of coronary lesion. CONCLUSIONS: Low adiponectin gene expression in EAT is associated with an increase in the degree of atherosclerotic lesion of the coronary bed and a decrease in LV EF.

The Effect of I155T, K156Q, K156E and N186K Mutations in Hemagglutinin on the Virulence and Reproduction of Influenza A/H5N1 Viruses.

The continued circulation of influenza A virus subtype H5 may cause the emergence of new potential pandemic virus variants, which can be transmitted from person to person. The occurrence of such variants is mainly related to mutations in hemagglutinin (HA). Previously we discovered mutations in H5N1 influenza virus hemagglutinin, which contributes to virus immune evasion. The purpose of this work was to study the role of these mutations in changing other, non-antigenic properties of the virus and the possibility of their maintenance in the viral population. Mutations were introduced into the HA gene of a recombinant H5N1 influenza A virus (VNH5N1-PR8/CDC-RG) using site-specific mutagenesis. The "variant" viruses were investigated and compared with respect to replication kinetics in chicken embryos, thermostability, reproductive activity at different temperatures (33, 37 and 40°C), and virulence for mice. Amino acid substitutions I155T, K156Q, K156E+V138A, N186K led to a decrease in thermal stability, replication activity of the mutant viruses in chicken embryos, and virulence for mice, although these effects differed between the variants. The K156Q and N186K mutations reduced viral reproduction at elevated temperature (40°C). The analysis of the frequency of these mutations in natural isolates of H5N1 influenza viruses indicated that the K156E/Q and N186K mutations have little chance to gain a foothold during evolution, in contrast to the I155T mutation, which is the most responsible for antigenic drift. The A138V and N186K mutations seem to be adaptive in mammalian viruses.

Prognostic Model of Typical Complications Caused by Transcatheter Aortic Valve Replacement.

The aim of the study was to develop a prognostic model based on statistical discriminant analysis to assess the risk of postoperative disturbance of cardiac conduction and paraprosthetic regurgitation after transcatheter aortic valve replacement. MATERIALS AND METHODS: Clinical data of 10 patients implanted with CoreValveTM prostheses (Medtronic Inc., USA) were used to develop prognostic models. To that end, we analyzed changes in hemodynamic and functional parameters provided by echocardiography in the pre- and postoperative periods. RESULTS: We observed significant positive changes in the severity of left ventricular myocardial hypertrophy; on the contrary, volume indicators did not significantly change, which might be associated with the concentric type of left ventricular hypertrophy. The discriminant analysis made it possible to determine major (preoperative) morphological and functional indicators associated with the two most common complications of the procedure: left bundle branch block and paraprosthetic regurgitation. Left ventricular posterior wall thickness, interventricular septal thickness, left atrium dimension, and myocardial mass are the critical factors that determine the development of these complications. CONCLUSION: In the prognostic model, the proposed weighting coefficients allow one to assess the risk of postoperative complications; however, the presence of false-positive results requires further refinement of these coefficients within the linear equation.

[Immediate results of three methods of surgical myocardial revascularization in multivessel lesion of the coronary bed]. / Neposredstvennye rezul'taty trekh metodov khirurgicheskoi revaskuliarizatsii miokarda pri mnogososudistom porazhenii koronarnogo rusla.

INTRODUCTION: Percutaneous coronary intervention (PTI) and coronary artery bypass grafting (CABG) are currently the most commonly used techniques of myocardial revascularization. However, each of the methods has its own advantages and disadvantages. The creation of hybrid coronary revascularization (HCR) was based on an attempt to combine the benefits of CABG and PTI. AIM: The study was aimed at assessing the immediate results of three methods of surgical myocardial revascularization in patients with multivessel lesions of the coronary bed in stable ischaemic heart disease. PATIENTS AND METHODS: The study enrolled a total of 155 patients randomized into three groups of myocardial revascularization: HCR, CABG and PTI. In the HCR group, the first stage consisted in minimally invasive myocardial revascularization with the anterior descending artery (ADA) followed by PTI (within 1-3 days) with implantation of drug-eluting second-generation stents Xience to other coronary vessels. In the CABG and PTI groups we performed CABG and PTI, respectively, using the Xience stents. In all three groups we assessed the procedural success, frequency of major adverse cardiovascular events and bleeding. RESULTS: Full myocardial revascularization was achieved in all three groups in more than 90% of cases. No statistically significant differences in either the procedural success rate or frequency of major adverse cardiovascular events between the CABG, PTI and HCR were revealed. The PTI group was characterized by the lowest frequency of bleeding, need for rehabilitation, and length of hospital stay. CONCLUSION: Hybrid revascularization with the use of minimally invasive direct myocardial revascularization with the ADA followed by PTI with second-generation drug-eluting stents to other coronary arteries is a method of choice in treatment of patients with multivessel lesions of coronary arteries.

[Change of phenotypic properties of escape mutants and readaptants of influenza virus A (H1N1)pdm09 under the influence of selected mutations in the molecule of hemagglutinin.]

INTRODUCTION: After the emergence and spread of pandemic H1N1 viruses in 2009, antigenic epitopes recognized by neutralizing antibodies against the hemagglutinin of influenza A/Moscow/01/09(H1N1)pdm09 viruses were studied. PURPOSE: The purpose of the study was to obtain readapted variants of the virus from a low-virulent escapemutant that has an increased affinity of the avian and the human types cellular receptors compared to the wild type and the comparative study of their antigenic and receptor specificity. MATERIAL AND METHODS: Viruses were accumulated in 10-day-old chicken embryos. The MAB panel against HA of influenza virus strain A/IIV-Moscow/01/09(H1N1)sw1 was used in the form of ascites fluids from mice. Immunization of mice, HI testing, elution of viruses from chicken erythrocytes, PCR and sequencing of readapted variants were performed by standard methods. RESULTS: The amino acid substitution A198E acquired in the process of readaptation leads to changes in the antigenic specificity. A correlation was found between a decrease in virulence of a low-virulent escape mutant associated with the substitution D190N in the hemagglutinin molecule and an increase in the hemagglutinating titer to inhibitors in normal mouse serum. Viruses with low affinity of cellular receptor analogs and carrying amino acid substitutions have an increased ability to elute from chicken erythrocytes. DISCUSSION: The results discuss the effect of mutations in the HA molecule of the influenza A(H1N1) pdm09 virus to the change in antigen specificity; virulence for mice, adsorption-elution at cellular receptors. CONCLUSION: A comparative study of the antigenic specificity and receptor-binding activity of the escape mutants was conducted for the hemagglutinin of the influenza virus A/Moscow/01/2009 (H1N1)swl, and the readapted variants obtained for one of the escape mutants with reduced virulence for mouse. Monitoring the pleiotropic effect of mutations in the hemagglutinin H1 molecule is necessary to predict variants of the virus with pandemic potential.

[Evolution of transcatheter aortic valve implantation: from planning to robotic systems]. / Évoliutsiia transkateternoi implantatsii aortal'nogo klapana.

Since its introduction in 2002, transcatheter aortic valve implantation (TAVI) has evolved dramatically and is now standard of care for intermediate risk patients with aortic stenosis. The development of innovative transcatheter heart valves and refinement of technical skills have contributed to the decrease in complication rates associated with TAVI. Increased experience, smaller sheaths, rigorous pre-procedural planning and improved vascular closing techniques have resulted in markedly lower rates of vascular complications. The next step was the simplification of the procedure, which contributed to a further decrease in complications, reduced procedural time, and shorter hospital stay. Change-over from general anaesthesia to conscious sedation, refusal from predilatation, and use of the radial approach instead of the contralateral femoral approach are all instrumental in achieving optimal results. Prospects for development include visual assist systems and robotic systems that can potentially optimize the transcatheter aortic valve implantation process, improve safety and effectiveness of the procedure.

[Vulnerable atherosclerotic plaques of coronary arteries in patients with stable coronary artery disease: 12-months follow-up].

RELEVANCE: A key objective of modern cardiology is the assessment of acute coronary syndrome (ACS) risk in patients with coronary artery disease (CAD) to develop preventive measures and choose optimal treatment strategies. OBJECTIVE: Detect vulnerable plaques of non-target coronary arteries in patients with stable CAD during routine percutaneous coronary intervention using virtual-histology intravascular ultrasound (VH-IVUS) and view their morphology over time. MATERIALS AND METHODS:  The prospective observational cohort study included 58 patients with stable CAD. After stenting of a target vessel, VH-IVUS was carried out in proximal and middle segments (6-8 cm) of a non-target coronary artery with no significant stenosis according to coronary angiography. Twelve months later, all patients underwent coronary angiography with re-IVUS of previously detected lesions. Death, myocardial infarction, rehospitalization, and unplanned myocardial revascularization due to vulnerable plaques were the endpoints of the study. RESULTS: IVUS with virtual histology revealed 58 lesions of non-target coronary arteries in 56 (96.5 %) patients. Two patients had no lesions in non-target coronary arteries. A large necrotic core with thin cap (thin-cap fibroatheroma) was detected in 12 (20.7 %) plaques, six of which had additional ACS risk criteria (stenosis area >70 % and / or lumen area <4 mm2). Within the 12­month follow-up period, three patients (one with a vulnerable plaque in IVUS) were hospitalized with a clinical picture of ACS. One cardiac death was registered in a patient with the IVUS vulnerable plaque. 7 of 12 vulnerable plaques stabilized in 12 months. CONCLUSION: 1) The data presented indicate a high rate (20.7 %) of vulnerable plaques of non-target coronary arteries in patients with stable CAD who underwent stenting; 2) Two (16.6 %) patients with vulnerable plaques reached endpoints (death and rehospitalization) within the 12­month follow-up period; 3) An analysis of atherosclerotic plaques in non-target coronary arteries over time showed that vulnerable plaques stabilized and did not cause ACS in more than half of cases (7 of 12); 4) Plaques that were not vulnerable according to IVUS were not likely to destabilize within the 12­month follow-up period.

[Effect of amino acid substitutions in small subunit of avian H5N2 influenza virus hemagglutinin on selection of the mutants resistant to neutralizing monoclonal antibodies].

Changes associated with the resistance to physical and chemical factors in the hemagglutinin (HA) of influenza A viruses may play an important role in the selection of different influenza variants during circulation in nature. Here, we studied the escape mutants of influenza virus A/mallard/Pennsylvania/10218/84 (H5N2) that were selected by the monoclonal antibody. The escape mutant m4F11(4) carried a single amino acid substitution in large subunit (HA1) of the HA, S145P1, and two ones, m4G10(10) and m4G10(6), had additional amino acid changes in the small subunit (HA2), namely: L124F2 and L124F2 + N79D2, respectively. As it has been found the substitutions appeared in the HA2 of m4G(10) and m4G(6) viruses compensated negative effect of the S145P1 mutation and provided a significant increase in the viral replication ability at the early stage of infection in embryonated chicken eggs as well as in HA thermostability in comparison with m4F11(4) mutant. Phenotypic properties that provide advantages in the process of virus replication can play a role of the positive selection factor in viral population.

[Prognostic value of SYNTAX score for outcomes and revascularization strategy choice in ST-segment elevation myocardial infarction patients with multivessel coronary artery disease].

AIM: To study the prognostic significance of the SYNTAX score in the evaluation of outcomes of primary percutaneous coronary interventions (PCIs) and revascularization strategy choice in patients with ST-elevation myocardial infarction (STEMI) with multivessel coronary artery disease. MATERIAL AND METHODS: The long-term outcomes of primary PCIs were analyzed in 163 patients with STEMI in terms of the objective assessment of the severity of the coronary bed lesion according to SYNTAX scores. RESULTS: In a cohort of STEMI patients who had undergone primary PCI, the SYNTAX score of > or = 23 (a severe lesion) was associated with the higher incidence of acute heart failure (Killip class II) and three-vessel coronary artery disease (odds ratio (OR) 2.8), with the higher risk of death (OR 7.5) and the higher rate of the combined endpoint of death, myocardial infarction, and target vessel revascularization (OR 2.8) as compared with patients with a SYNTAX score of < or = 22 (a moderate lesion). CONCLUSION: The SYNTAX score has a prognostic value in assessing the outcomes of primary PCIs in the cohort of STEMI patients with multivessel disease, which can find use in the differential choice of the optimal revascularization strategy and improve treatment results. In the group of patients with a SYNTAX score of > or = 23, the incomplete revascularization strategy shows the least favorable results as compared to multivessel stenting and staged revascularization.

Yields of virus reassortants containing the HA gene of pandemic influenza 2009 virus.

KEYWORDS: influenza virus; reassortment; virus yield; gene constellation.

[High-yield reassortant virus containing hemagglutinin and neuraminidase genes of pandemic influenza A/Moscowl/01/2009 (H1N1) virus].

The crossing of influenza A/Moscow/01/2009 (H1N1) virus and reassortant strain X31 (H3N2) containing the genes of internal and non-structural proteins of A/Puerto Rico/8/34 (H1N1) strain gave rise to reassortant virus ReM8. The reassortant contained hemagglutinin (HA) and neuraminidase (NA) genes of pandemic 2009 influenza virus and 6 genes of high-yield A/Puerto Rico/8/34 (H1N1) strain. The reassortant ReM8 produced higher yields in the embryonated chicken eggs than the parent pandemic virus, as suggested by infectivity and HA activity titration as well as by ELISA and the measurement of HA protein content by scanning electrophoresis in polyacrylamide gel slabs. High immunogenicity of ReM8 reassortant was demonstrated by immune protection studies in mice. The reassortant virus ReM8 is suitable as a candidate strain for the production of inactivated and subunit influenza vaccines.

[Amino acid substitutions in the hemagglutinin of H5 influenza virus changing the antigenic specificity and virus virulence].

In our earlier studies, we mapped the hemagglutinin antigenic epitopes of H5 influenza virus by selecting mutants resistant to the neutralizing effect of the antibody (escape mutants). Several escape mutants were shown to have a lowered virulence for mice. The readaptation of low-virulent escape mutants to mice resulted in the restoration of virulence. In the present communication. We present data on the assay of virulence of single-gene reassortants containing HA genes of the wild-type virus, low-virulent escape mutant, or re-adapted variant, and the other genes of a mouse-adapted H9N2 Influenza virus. The results demonstrate that the amino acid change S145F (H3 numbering) in the hemagglutinin ensuring the resistance to a monoclonal antibody can be deleterious to virulence, and that the damaging effect on virulence may be compensated for by additional amino acid changes in position 186 in the hemagglutinin arising in the course of virus passaging in mice. The data indicate that the compensational mutations restoring the pathogenic potential of antigenic variants may be regarded as an additional factor in the evolution of influenza virus hemagglutinin.

[Differential incorporation of genomic segments into the influenza A virus reassortants in mixed infection].

The gene composition of the viral population obtained via mixed infection of embryonated chick eggs with influenza viruses at a high multiplicity of infection was analyzed. In mixed infection caused by influenza A/WSN/33 (H1N1) and A/Duck/Czechoslovakia/56 (H4N6) viruses, the population showed a preponderance of the reassortants whose content of genomic segments originating from either of the parent virus deviated strongly from the random pattern: the hemagglutinin (HA) gene of A/WSN/33 (H1N1) virus and the NP gene of A/Duck/Czechoslovakia/56 (H4N6) virus were prevalent in the gene composition of the reassortants. The mixed infection produced by influenza A/Udorn/72 (H3N2) virus and the reassortant R8 containing the HA gene of A/Duck/Ho Chi Minh/014/78 (H5N3) virus, the population of reassortants contained mainly the HA gene of A/Udorn/72 (H3N2) virus and the NP gene of the reassortant R8. The findings are discussed due to the problem of specific recognition of gene segments when incorporated into the viral particles.

Deviation from the random distribution pattern of influenza A virus gene segments in reassortants produced under non-selective conditions.

High-frequency reassortment of gene segments is characteristic for influenza viruses, and it is considered to be of significance for the origin of pandemic influenza. In order to analyze whether the segregation of genes in the reassortants is random, or it deviates from the random pattern, we inoculated embryonated chicken eggs simultaneously with two influenza viruses, A/WSN/33 (H1N1) and A/Duck/ Czechoslovakia/56 (H4N6), at a high multiplicity of infection. The virus yield was used for plaque cloning, and the genetic content of plaque isolates was determined by analysis of the mobility of virus-induced proteins in polyacrylamide gel (for NP and NS genes), partial sequencing (for M gene) and polymerase chain reaction analysis with strain-specific primers for the other genes. Out of 37 isolates, 27 were reassortants. The majority of the reassortants contained the HA gene of A/WSN/33 (H1N1) virus and the NP gene of A/Duck/Czechoslovakia/56 (H4N6) virus. The data demonstrate the previously unrecognized phenomenon of segment-specific deviation from the random distribution of parent genes in the reassortants. The results are discussed in connection with the problem of differential competition between influenza A virus gene segments in mixed infection and random versus non-random reassortment of gene segments under non-selective conditions.