Virological, epidemiological, clinic, and molecular genetic features of the influenza epidemic in 2015-2016: prevailing of the influenza A(H1N1)09 pdm virus in Russia and countries of the Northern hemisphere.

This work describes the specific features of the influenza virus circulating in the period from October 2015 to March 2016 in 10 cities of Russia, the basic laboratories of CEEI at the D.I. Ivanovsky Institute of Virology "Federal Research Centre of Epidemilogy and Microbiology named after the honorary academician N.F. Gamaleya" of the Ministry of Health of the Russian Federation. The increase in the morbidity caused by influenza viruses was detected in January-February 2016. The duration of the morbidity peak was 4-5 weeks. The most vulnerable group included children at the age from 3 to 6; a high rate of hospitalization was also detected among people at the age of 15-64 (65%). In clinic symptoms there were middle and severe forms with high frequency of hospitalization as compared with the season of 2009-2010, but much higher in comparison with the season of 2014-2015. Some of the hospitalized patients had virus pneumonias, half of which were bilateral. Among these patients, 10% were children; 30%, adults. The mortality in the intensive care unit of the hospital was 46%. Almost all lethal cases were among unvaccinated patients in the case of late hospitalization and without early antiviral therapy. The predominance of the influenza A(H1N1)09pdm virus both in the Russian Federation and the major part of the countries in the Northern hemisphere was noted. The results of the study of the antigenic properties of influenza strains of A(H1N1)pdm09 virus did not reveal any differences with respect to the vaccine virus. The sequencing data showed the amino acid substitutions in hemagglutinin (receptor binding and Sa sites) and in genes encoding internal proteins (PA, NP, M1, NS1). Strains were sensitive to oseltamivir and zanamivir and maintained resistance to rimantadine. The participation of non-influenza ARI viruses was comparable to that in preliminary epidemic seasons.

Amino acid polymorphism at residue 222 of the receptor-binding site of the hemagglutinin of the pandemic influenza A(H1N1)pdm09 from patients 166 with lethal virus pneumonia in 2012-2014.

Survey data from autopsy specimens from patients who died from pneumonia caused by the influenza A(H1N1) pdm09 in 2012-2014 and mutant forms of influenza virus in these patients (position 222 in the receptor-binding region of hemagglutinin) were presented. In total, according to aggregate data, obtained with three different methods (sequencing, next-generation sequencing (NGS), virus isolation) mutant viruses were detected in 17 (41%) from 41 patients. The proportion of the mutant forms in viral populations ranged from 1% to 69.2%. The most frequent mixture was the wild type (D222) and mutant (D222G), with proportion of mutant type ranged from 3.3% to 69.2% in the viral population. Mutation D222N (from 1.1% to 5.5%) was found rarely. Composition of the viral population from one patient is extremely heterogeneous: in left lung there was only wild type D222, meantime in right lung - mixture of mutant forms 222D/N/G (65.4/32.5/1.1%), in trachea - mixture 222D/G/Y/A (61.8/35.6/1.2/1.4%, respectively), and in bronchi compound of 222D/G/N/A (64.3/33.7/1/1%, respectively) were detected. The obtained data indicate that the process of adaptation of the virus in the lower respiratory tract is coupled with the appearance of different virus variants with mutations in the receptor-binding region. Mutant forms of the virus are observed in the lower respiratory tract of the majority of patients with lethal viral pneumonia. However, if they are a minor part of the population, they cannot be detected by the method of conventional sequencing. They can be identified using the NGS methods.

[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.

[Efficiency of the influenza A and B viruses isolation from nasopharyngeal swabs taken in the test tubes Sigma-Virocult (M40 Compliant, Sigma Virocult) and Virocult (M40 Compliant, Virocult) in 2010-2011 epidemic season].

The goal of this work was to compare the efficiency of the influenza A and B viruses Isolated during 2010-2011 epidemic season. The clinical samples were taken in the test tubes with the transport medium on the.basis of the medium EMEM and commercial test tubes Sigma-Virocult (M40 Compliant, Sigma Virocult) and Virocult (M40 Compliant, Virocult). The results of this work demonstrated higher efficiency of influenza A and B viruses isolation from nasopharyngeal swabs of the patients taken in the test tubes Sigma-Virocult (M40 Compliant, Sigma Virocult) and Virocult (M40 Compliant, Virocult) with the transport medium as compared with the efficiency of influenza strains isolation from nasopharyngeal swabs taken in test tubes with the medium EMEM with respect to all estimated indicators: efficiency of isolation, a passage of isolation and the titer of isolates. The possibility of the long-term storage of a clinical material at room temperature and at 4 degrees C was confirmed, without resorting to freezing, which is significant in the absence of the necessary equipment.

[Efficacy of anti-neuraminidase drugs application during and after an influenza pandemic].

The emergent 2009 A(H1N1) pandemic brought into acute focus the problem of choosing the most effective anti-influenza drugs for successive influenza infection spreading control. Oseltamivir and zanamivir, influenza virus neuraminidase inhibitors (NAIs), were recommended by the WHO experts for the treatment and prevention of influenza, including that caused by pandemic strains. A major concern regarding the use of specific antiviral compounds is the emergence of the drug-resistant strains. Oseltamivir carboxylate and zanamivir IC50 values were equal to 0.3-5.2 microM for the most of A(H1N1)pdm09 pandemic strains and 1.6-8.6 microM for the strains of influenza B virus in cell-based ELISA assay (2009-2010 season). All the studied strains of influenza A(H1N1 ) pdm09 (151) and B (22) viruses were sensitive to NAIs (2009-2011 seasons). For the first time in Russia oseltamivir-resistant A(H1N1) pdm09 influenza virus was isolated from the patient on the 5th day of a treatment course of this drug.

[Development of the influenza epidemic in season 2011-2012 in some areas of Russia: results of activity of the Influenza Etiology and Epidemiology Center of the Ivanovsky Institute of Virology].

The results of analysis of the peculiarities of the epidemic 2011-2012 development in the areas of 10 cities of Russia obtained by basic laboratories of IEES on the base of D.I. Ivanovsky Research Institute of Virology, Ministry of Public Health and Social Development of Russia, are presented. The increasing ARD morbidity caused by the influenza viruses was detected rather late--in February-March 2012. The highest indices of the morbidity were detected during weeks 10-13 followed by decreasing to threshold levels by week 27. Children 0-2 and 3-6 years old were involved the most, meantime the high rate of hospitalization was found for 15-64 years old aged group (25%). Influenza A(H3N2) and B viruses were the cause of the epidemic. The results of studies of the antigenic and genetic properties of the influenza strains showed most of them to be close relatives to the vaccine strains. Some heterogeneity of circulating strains and their drift variants were found as well. All tested strains were sensitive to arbidol, oseltamivir and zanamivir, and saved resistance to rimantadine. The ratio of ARD viruses was comparable with the last epidemic seasons.

[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.

[Monoclonal antibodies with high virus-neutralizing activity against pandemic influenza virus A/llV-Moscow/01/2009 (H1N1)swl].

The authors have obtained a panel of 7 monoclonal antibodies (MAbs) against pandemic influenza virus A/IIV-Moscow/01/2009 (HIN1)swl isolated in Russia. One MAb is directed to a NP protein linear epitope and interacts with all the influenza A viruses under study. Six other MAbs are directed to H1 hemagglutinin conformation-dependent determinants and detect homologous virus in the hemagglutination-inhibition test, enzyme immunoassay, immunofluorescence and virus neutralization tests. MAbs differentiate pandemic influenza viruses A(H1N1)swl from seasonal influenza A(H1N1), A(H3N2), and B viruses. The high neutralizing activity of MAbs permits their use to study the fine antigen structure of influenza virus hemagglutinin and to differentiate the A(H1N1) pandemic influenza viruses and offers promise for obtaining humanized antibodies in order to make specific prevention and treatment of influenza.

[Contribution of D I Ivanovsky Research Institute of Virology to monitoring influenza viruses during epidemics and 2009 pandemic in Russia].

The data on monitoring influenza viruses in Russia are presented based on the research underway at Ivanovsky Research Institute of Virology since 1959. The Institute's priority in isolation and identification of influenza viruses during epidemics and 2009 pandemic is confirmed. Results of assessment of influenza vaccines and etiotropic preparations, development and introduction of new methods for diagnostics of influenza are discussed.

[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.

[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.

[The characteristics of epidemic influenza A and B virus strains circulating in Russia during the 2007-2008 season].

In 2007-2008 in Russia, the epidemic upsurge of influenza morbidity was caused by the active circulation of influenza A(H1N1, A(H3N2), and B viruses. The center for Ecology and Epidemiology of Influenza studied 334 epidemic strains. The results of a comparative study of the svirus specificity of commercial test systems (AmpliSens Influenza virus A/B and AmpliSens Influenza virus A/H5N1) for the polymerase chain reaction diagnosis and virological assays, including virus isolation, revealed their high correlation, which confirms that they may be expensively used to monitor the circulation of influenza viruses in the Russian Federation. All the strains were isolated in the MDCK cell culture. Influenza A(H1N1) viruses (n = 127) were antigenic variants of the reference strains A/Solomon Islands/3/06 and A/Brisbane/59107. Influenza A(H3N2) viruses (n = 49) were antigenic variants of the reference strains A/Wisconsin/67/05 and A/Brisbane/10/08. One hundred and fifty seven Influenza B strains were drift variants of the reference strains B/Florida/4/06 and B/Shanghai/361/02 of lineage B/Yamagata/16/88 and one strain, a variant of Malaysia/2506/04 related to lineage B/victoria/2/87. The isolates interacted actively with human 0(I) blood group erythrocytes and much more weakly with chicken ones. All study influenza A(H1N1) viruses (n = 74) preserved their sensitivity to rimantadine while 24 (77%) of the 31 study influenza A(H3N2) virus strains were resistant. A study of the time course of changes in the generation of antibodies in the donor sera obtained in Moscow and the Moscow Region in different periods of the epidemic process revealed an increase in antibodies to the reference influenza A and B virus strains circulating in this period.

[Monitoring of the sensitivity of epidemic influenza virus strains isolated in Russia to etiotropic chemical agents].

The paper presents the results of studying the spectrum of influenza A and B viruses to rimantadine, arbidol, and oseltamivir and describes the methods used for these purposes for epidemiological surveillance. Different sensitivities to rimantadine were found among influenza A viruses. During the 2007-2008 season, the vast majority of influenza A(H3N2) virus strains were resistant to rimantadine (77%) while all influenza A(H1N1) virus strains preserved their resistance to this drug. The fact that the epidemic influenza A(H1N1) virus strains that carry the mutation responsible for resistance to the neuraminidase inhibitor oseltamivir (Tamiflu) circulated in the Russian Federation was first established. At the same time all the study influenza A(H1N1) virus strains preserved their susceptibility to rimantadine. The sensitivity of the epidemic strains to arbidol has been confirmed.

[The spread and biological properties of epidemic influenza viruses A and B strains circulating in the 2006-2007 season in Russia].

The epidemic upsurge of influenza morbidity in the 2006-2007 season in Russia was caused by the active circulation of influenza A(H1N1), A(H3N2) and B viruses. The Center for Ecology and Epidemiology of Influenza studied 259 epidemic strains; All the strains were isolated on MDCK cell cultures. Influenza A(H1N1) viruses (n = 101) were antigenic variants of the references A/New Caledonia/20/99 and A/Solomon Islands/3/06. Influenza A(H3N2) viruses (n = 98) were antigenic variants of the references A/California/7/07 and A/Wisconsin/67/05. Twenty four influenza B virus strains were drift variants of the reference BI Shanghai/361/02 - lineage B/Yamagata/16188 and 36 were related to A/Malaysia/2506/05 - lineage B/Victoria/2/87). All the isolated strains actively interacted with human erythrocytes of the blood groups 0(I) and A(II) and very slightly with chicken ones. Twenty-two (48%) A(H1N1) strains and 19 (35%) A(H3N2) strains which were resistant to rimantafine were revealed. Studies of the donor sera collected in Moscow and the Moscow Region, the Jewish Autonomous Region, and Primorye revealed antibodies to today's strains; the level of antibodies in the preepidemic period depended on the region and strain. A study of changes in antibody formation in different periods of an epidemic process showed a rise in antibodies mainly to influenza A and B virus strains circulating in this period.

[Epidemic strains influenza viruses A and B in the 2005-2006 season in Russia].

Investigations indicated that the epidemic upsurge of influenza morbidity in the 2005-2006 season in Russia was caused by the active circulation of influenza viruses A and B. The Center for Ecology and Epidemiology of Influenza, D. I. Ivanovsky Institute of Virology, Russian Academy of Medical Sciences, studied 182 epidemic strains. A hundred and thirteen influenza viruses A(H3N2) were similar to the reference A/California/07/2004 or were its antigenic variants. Thirteen influenza virus A(H1N1) strains that were antigenic variants of the reference A/New Caledonia/20/99 were isolated in sporadic cases. Influenza viruses B were similar to B/Malaysia/2506/2004--lineage B/Victoria/2/87). All the strains were isolated in the MDCK cell culture. Comparative study of the sensitivity of the chicken embryo (CE) and MDCK isolation system to the 1999-2006 epidemic strains showed that CE tropism was least pronounced in influenza viruses A(H3N2). Analysis of the 2002-2006 strains demonstrated that influenza viruses A reacted actively with human erythrocytes of the blood groups 0(I) and A(II) and very slightly with chicken ones. Eighty-five influenza virus A(H3N2) strains from the 2005-2006 epidemic season were investigated for rimantadine susceptibility. The frequency of rimantadine-resistant influenza virus A(H3N2) strains was 38.0%. Studies of 79 paired sera from patients revealed a rise of antibodies to influenza viruses A(H3N2) and B in 25.9-33.3 and 20.7-23.8% of cases, respectively. There was an increase in antibodies to influenza viruses A and B in the sera collected from donors in Moscow and its region in September 2005 to June 2006.

[Rimantadine and arbidol sensitivity of influenza viruses that caused epidemic morbidity rise in Russia in the 2004-2005 season].

The study of the activity of arbidol against epidemic influenza A and B virus strains (2002-2005) in the cultured MDCK cells showed the higher sensitivity of enzyme immunoassay than that of hemagglutination test. The influenza A virus strains tested, including those resistant to rimantadine (5 microg/ml), were sensitive to arbidol (10 microg/ml). The population of influenza B virus strains was heterogeneous in this indicator, 43% of the strains being less sensitive to arbidol. There was an increase in the number of rimantadine-resistant influenza A(H3N2) virus strains (10-18%) in our country during 3 epidemic seasons. The sequencing analysis of protein M2-endoding gene revealed the amino acid replacement of serine by asparagine in position 31, which is characteristic of rimantadine-resistant strains. Arbidol in combination with rimantadine potentiated the effect of viral reproduction in the cultured cells, as compared with the effect produced by the same concentrations of the drugs used alone.

[Characterization of epidemic influenza virus A(H3N2) strains circulating in Russia in the 2003-2004 epidemic season].

Studies indicated that the epidemic rise in the incidence of influenza was caused by its virus A (H3N2) circulation in Russia in the 2003-2004 season. The Center of Influenza Ecology and Epidemiology investigated 101 epidemic strains isolated the MDCK culture. Antigenic analysis showed that all viruses A(H3N2) were similar to the reference virus A/Fujian/411/02(H3N2) and only 5 strains slightly differed from the latter. Twelve (14%) strains resistant to rimantadine at a concentration of 0.5 mg/ml were identified. Investigation of paired sera from the patients demonstrated a rise of antibodies to the references of influenza virus A(H3N2) in 68.7% of cases and a less increase in those to influenza viruses A(H1N1) and B. The active circulation of A(H3N2) viruses was due not only to changes in their antigenic structure, but also to the low level of antibodies to these viruses, as shown by the analysis of donor sera.

[The properties of the epidemic influenza viruses A and B strains circulating in Russia in the 2004-2005 epidemic season].

The epidemic upsurge of influenza morbidity in Russia in 2004-2005 was caused by the active circulation of influenza A(H3N2) and B viruses. A hundred and sixty-six epidemic strains were studied. All the strains were isolated in the MCK cell culture. Influenza A(H3N2) viruses (n=77) were antigenic variants of the reference A/Fujian/411/ 2002 and A/California/7/2004 strains. Three influenza A(H1N1) viral strains that were antigenic variants of A/New Caledonia/20/99 strains were isolated in sporadic cases. Influenza B virus strains (n=83) were antigenic variants of the reference B/Shanghai/361/02--lineage B/Yamagata/l6/88. In addition, 3 antigenic variants of B/Hong Kong/ 330/2002 (lineage B/Victoria/2/87) strains were isolated. Nine (20%) strains resistant to rimantadine at a concentration of 5 microg/ml were identified. Chromatographic analysis of B/Shanghai/361/02 and BIHong Kong/330/01 viral protein M1 trypsin hydrolysates revealed differences in the profiles of chromatograms of influenza A virus proteins M1. Examination of 121 pair sera from patients revealed an increase in antibodies to influenza A(H3N2) viruses in 10-21% of cases and to influenza B viruses in 20-36% of cases.

[The rimantadine sensitivity spectrum in influenza A viruses circulating in the 2002-2004 epidemic seasons].

A total of 200 influenza A virus strains were studied. Among them there 32 strains of A(H1N1) and 84 strains of A(H3N2) from the 2002-2003 epidemic season and 84 strains of A(H3N2) from the 2003-2004 epidemic season. Most rimantadine-sensitive strains whose infectivity and hemagglutinating activity were found to decrease by the drug given at a concentration of 0.5 microg/ml. In terms of infectivity and hemagglutinating activity, the number of rimantadine-resistant strains of A (H1N1) was equal to 9.0% for A(H1N1) influenza viruses and to 10.0 and 14.0% for A (H3N2) influenza viruses in different seasons, respectively.