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Monitoring the circulation of influenza viruses is essential to public health authorities around the world. The urgency of the problem is associated with the damage caused by annual epidemics and pandemics of influenza, accompanied by high morbidity and mortality. According to the World Health Organization (WHO), every year in the world 3 to 5 million people suffer from severe forms of influenza, mortality from influenza and its complications ranges from 250 000 to 500 000 cases, economic damage is estimated from 1 to 6 million dollars per 100 thousand people. In the Russian Federation, economic losses from influenza and SARS in 2004 amounted to 82.6 billion rubles or 86.0% of the total damage caused by infectious diseases [1]. The presence of a natural reservoir of type A influenza viruses maintained by aquatic and semiaquatic birds, as well as by some mammalian species, determines the risk of the formation of new antigenic variants, including those with pandemic potential, which was confirmed by the development of pandemics of the last century, which were caused by influenza A (H1N1) - "Spanish flu" in 1918-1919, A (H2N2) - "Asian flu" in 1957-1958, and A (H3N2) - "Hong Kong flu" in 1968-1969, as well as a new pandemic in 2009 caused by a reassortant of swine influenza A (H1N1) viruses of the American and Eurasian lines. By September 25, 2010 214 countries and territories have reported cases of infection with the new pandemic strain, with a total of 18,449 laboratory confirmed fatalities, incl. in Russia, about 4.09% of the population fell ill, the number of deaths amounted to 654.

Background. In 1947 on the initiative of the WHO, the International System for the Surveillance of the Circulation of Influenza Viruses was created, which over time admitted new participating countries and currently has 113 National Influenza Centers, actively working in 84 countries of the world, including two of them in Russia, which are operating at the Research Institute of Influenza, St. Petersburg and at the D.I. Ivanovsky Institute of Virology, Moscow.

D.I. Ivanovsky Institute of Virology has more than half a century of experience in monitoring the circulation of influenza viruses, a great contribution to the study of the properties of which was made by Professor, Honored Scientist of the RSFSR, M.I. Sokolov and Professor, MD A.S. Gorbunova. In 1959 at the Institute, the All-Union Center of the USSR for Influenza was created, collaborating with the WHO, headed by prominent scientists of the country – Academician of the USSR Academy of Medical Sciences, Professor V.M. Zhdanov, Honored Scientist of the RSFSR, Doctor of Medical Sciences, Professor L.Ya Zakstelskaya. Since 1987 Academician of the Russian Academy of Medical Sciences, Professor, Doctor of Medical Sciences, D.K. Lvov was elected director of the Institute. He headed the work of the Center and has been in charge of its activities for over 20 years.

In the interests of scientific support for the solution of tasks to improve epidemiological surveillance of influenza in the Russian Federation December 12, 1994 by order No. 160/85 of the State Committee for Sanitary and Epidemiological Surveillance of the Russian Federation, together with the Russian Academy of Medical Sciences (RAMS), the Scientific and Practical Center for Influenza Ecology and Epidemiology (CIEE) was established. Considering the likelihood of an influenza pandemic and the need to increase the efficiency of the system of current epidemiological surveillance of influenza and acute respiratory viral infections (ARVI) in the Russian Federation with the integration of laboratory diagnostic and virological data with morbidity indicators into it, by Order No. 373 of the Federal Service for Surveillance on Consumer Rights Protection and Human Well-Being (Rospotrebnadzor) dated March 31, 2005 "On improving the system of epidemiological surveillance and control of influenza and acute respiratory viral infections" the system of surveillance for influenza and SARS was updated. It was followed by the establishment of the Center for Influenza Ecology and Epidemiology of the DI. Ivanovsky Institute of Virology of RAMS" and its support bases: in Novgorod (Veliky Novgorod), Lipetsk (Lipetsk), Vladimir (Vladimir), Yaroslavl (Yaroslavl), Penza (Penza) regions, the Republic of Chuvashia (Cheboksary), Orenburg (Orenburg), Tomsk (Tomsk) regions, Primorsky Krai (Vladivostok) and Jewish Autonomous Okrug (Birobidzhan) [1]. 

In order to further implement the International Health Regulations (IHR 2005) on the territory of the Russian Federation, by Order of Rospotrebnadzor No. 88 dated March 17, 2008, Appendix No. 3 "On measures to improve monitoring of pathogens of infectious and parasitic diseases", the D.I. Ivanovsky Institute of Virology was assigned the functions of one of four Reference Centers for influenza monitoring in the Russian Federation.

Main Achievements.

The circulation of influenza viruses is supervised in several areas, including: isolation and identification of epidemic strains, study of their biological and molecular genetic properties, sensitivity to etiotropic drugs, assessment of the vaccine properties and effectiveness of influenza vaccines, participation in the development of diagnostic test systems, and development of new methods of antiviral protection using modern nanomaterials. CIEE works closely with the WHO International Influenza Centers in the United States and Great Britain, regularly submitting samples of epidemic strains for inclusion in international monitoring to develop recommendations for the composition of influenza vaccines. Since 2008 the Institute has been actively cooperating with the WHO European Office within the framework of the platform for monitoring the circulation of influenza and ARVI viruses in European countries.

During the period of its activity, the Research Institute of Virology had a number of priorities in the supervision of the influenza viruses circulation in our country. 

In 1963, 5 years before the "Hong Kong flu" pandemic, the Institute isolated the avian influenza virus A/Duck/Ukraine/1/63(H3N8), the whose hemagglutinin was similar in structure to the hemagglutinin of the reference strain A/HongKong/1/68(H3N2) that caused this pandemic. in 1968, which became a global priority [4]. 

In 1977 for the first time in the world, the Institute established the fact of the return of the A(H1N1) virus strains into circulation after their 20-year absence, in connection with which the journalists dubbed it the “Russian flu” [5].

In 1990, for the first time in the Russian Federation, a strain of a new evolutionary line of influenza virus B, B/Moscow/1/90, similar to B/Yamagata/16/88, was isolated. Viruses of this evolutionary line, since 1990 along with the strains of the evolutionary line B/Victoria/2/87 and the like, co-circulate as etiological agents during the last epidemics [6].

The next priority of the CIEE, which was of global importance, was the A/Moscow/10/99 (H3N2) strain, which was chosen by WHO experts as a standard for the production of influenza vaccines in the period 2000-2004. [7]. It should be noted that most of the reference strains of influenza A and B viruses selected by WHO experts in the composition of influenza vaccines usually originate from countries of the Southern Hemisphere and Southeast Asia.

Influenza viruses are known to be subject to frequent changes, the mechanisms of which fit into "antigenic drift" (point mutations of surface proteins, hemagglutinin and neuraminidase), which leads to the formation of a strain with epidemic potential, and "antigenic shift" (adaptation to a new host and reassortment genes of influenza A viruses of different hosts), which leads to the formation of a strain with pandemic potential.

With the recurrence of influenza A (H1N1) virus strains to active circulation in 1977 the etiology of epidemics was determined by three influenza viruses A (H1N1), A (H3N2) and B, moreover, their activity within one season and the rate of evolutionary variability were different. Studying the antigenic properties of the epidemic strains circulating in Russia since 1986, it was found that the evolutionary drift in the hemagglutinin molecule of the strains went in a direction similar to the reference strains of influenza A and B. It should be noted that the antigenic drift in the hemagglutinin molecule of influenza B viruses was less more intense than in influenza A strains.

The most active changes occurred in the protein structure of influenza A (H3N2) viruses, which drifted in the direction from A/Moscow/10/99 → A/Fujian/411/02 → A/California/7/04 → A/Wisconsin/67/05 → A/Brisbane/10/07.

The antigenic drift of hemagglutinins of influenza A(H1N1) virus strains went in the direction of A/New Caledonia/20/99 → A/Solomon Islands/3/06 → A/Brisbane/59/07, while the rate of variation was unequal: strains similar to A/Moscow/10/99New Caledonia/20/99 circulated in the Russian Federation from 2000 to 2006, and strains like A/Solomon Islands/3/06 did so for only two seasons (2006-2008), A/Brisbane/59/07-like ones occurred from 2008 to 2009 and, presumably, were pushed out of active circulation by a new pandemic strain A(H1N1)v.

Until 1990 in Russia, influenza B viruses belonging to the evolutionary branch B/Victoria/2/87-like were circulating. Since 1990, influenza B viruses belonging to an evolutionary branch the first representative of which was the B/Yamagata/16/88 strain [6] began to circulate in Russia. Since 1995 in the world, and since 2001 in Russia, there is a co-circulation of representatives of both branches. Moreover, depending on the country and season, viruses of one of them dominate in the population. In the B/Yamagata-like group in recent years, the hemagglutinin of epidemic strains drifted towards B/Sichuan/379/99 → B/Shanghai/361/02 → B/Florida/07/04. In group B/Victoria/2/87-like the drift went to B/Hong Kong/330/01 → B/Malaysia/2506/04 → B/Brisbane/60/2008.

Evolutionary changes developed not only in the area of the antigenic sites of the hemagglutinin molecule, but also affected the receptor area of the molecule, which determines the specificity of viruses to cells of different origins (birds, mammals). As a result, since the beginning of the 1990s, the tropism of epidemic strains towards virus isolation systems has changed, and this, in turn, has led to the replacement of the traditional chicken embryo isolation system with MDCK tissue culture cells. Most of the strains were more successfully isolated only on this culture, especially the influenza A(H3N2) and B viruses [8]. 

Along with the evolutionary processes leading to the emergence of new epidemic strains, reassortment of genes of influenza A viruses belonging to different hosts is extremely rare. Striking examples of such reassortment are the strains that caused the pandemics in 1957-1958 – A(H2N2), 1968-1969 – A(H3N2) and 2009 – A(H1N1)v.

CIEE also has a number of priorities based on the results of studying the peculiarities of the development of the 2009 pandemic caused by the circulation of the influenza A(H1N1)v virus, similar to the swine flu [9,10,11,12,13,14]. On May 21, 2009 at the D.I. Ivanovsky Institute of Virology of RAMS real-time PCR (CDC&P, Atlanta, USA) detected the 1st case of the disease in the Russian Federation in a patient who returned from the USA. On May 24 the 1st strain of pandemic influenza was isolated on MDCK tissue culture cells, on May 25 it was isolated on chicken embryos. The priority of this fact was formalized in the form of an application for a patent of the Russian Federation for the strain A|IIV-Moscow/01/2009 (H1N1)swl, which was granted a patent in September 2010. CIEE held the studies to detect amino acid substitutions in the receptor-binding site of hemagglutinin (HA1) in strains of pandemic influenza A(H1N1)v. Analysis by sequencing of 80 samples (lung tissue, trachea, bronchi, spleen) from lethal cases, including from 10 pregnant women, in 65.0% revealed the presence of pandemic virus mutants in the lung tissue of the deceased patients. The results of a molecular genetic study of the 18 strains isolated from patients with moderate pneumonia (1 patient) and a fatal outcome after pneumonia (17 patients, including pregnant women) in November 2009 showed that nine of them contained HA1 aspartic acid (D) in site 222. Mutations were found in nine samples at this site: D222G (in 3 samples), D222N (3), and a mixture D222G/D222N (3); in one case from a patient with a moderate form of the disease, the D222G mutation was detected after the 2nd passage of the virus in developing chick embryos in the absence of it in the primary material. At the same time, among 82 strains of the pandemic influenza A(H1N1)v virus isolated in May-October 2009 from patients with a favorable outcome, no such mutations were found. The data obtained indicate the beginning of circulation among the population of mutant variants of the pandemic influenza virus with altered receptor specificity, ensuring its multiplication not only in the upper, but also in the lower parts of the respiratory tract– in the bronchioles and alveoli – which can cause the development of pneumonia, often with a fatal outcome especially in the absence of early etiological diagnosis and early etiotropic treatment. The International Genebank contains information on the complete genome sequence of 15 strains of pandemic influenza A(H1N1)v; 378 samples (nasopharyngeal swabs, sectional material, strains) were partially sequenced for the nucleotide sequence of individual genes. A high degree of homology of the isolated strains genome was established at the early stage of the development of the pandemic.

One of the areas of CIEE is to assess the effectiveness of specific agents for the prevention and treatment of influenza infection, including vaccines and etiotropic drugs.

As a result of large-scale epidemiological observations (1989-94) among school-age children, the advantages of live influenza vaccine (LIV) were revealed when compared with inactivated influenza vaccine (IIV) in terms of reactogenicity, the ability to restrict the circulation of influenza viruses among the unvaccinated (with vaccination coverage of more than 50% of the population) and protection from new drift variants [15 ]. The protective efficacy of LIV and IIV in elderly and senile persons was determined. Higher rates of vaccine properties were observed in those vaccinated with IIVs or IIVs with LIVs administered simultaneously or sequentially, which should be taken into account when organizing vaccine prophylaxis in these age groups [16]. The results obtained in the framework of the State tests of the domestic polymer-subunit vaccine Grippol (1999-2001) enabled to reveal its low reactogenicity, high immunogenicity and protective efficacy in risk groups and to expand the range of its use in all age groups of the population (children, the elderly, persons with allergic pathology, etc.), including those with various somatic diseases, which served as the basis for the compilation of two "Instructions for the use of influenza trivalent polymer-subunit liquid vaccine Grippol", approved by the Ministry of Health of the Russian Federation on 08.07.1999. and 22.06.2001. [17].

High vaccine properties of IIV of the split (Begrivaktm, Vaxigriptm, Fluorikstm) and subunit (Influvaktm) types of foreign manufacturers have been confirmed in risk groups for diseases and complications (2001-2004) [18]. The research of vaccines contributed to their active use for the population of the Russian Federation as preventive measures against epidemics caused by A(H1N1), A (H3N2) and B strains of influenza.

D.I. Ivanovsky Institute of Virology of RAMS participated in the State tests of the domestic drug Arbidol for the prevention of influenza in adults and children (1988-1995) [19, 20]. The specific activity of Arbidol against epidemic strains of influenza A and B viruses was revealed in in vitro experiments and clinical and epidemiological tests. The proposed optimal schemes and dosages for the use of the drug in children enabled to expand the recommendations and widely introduce it into practice for the prevention of influenza and ARVI. The data were taken into account when compiling three "Instructions for the use of the drug Arbidol", approved by the Pharmacological Committee of the Ministry of Health of the Russian Federation on May 11, 1995, December 11б 1997 and April 11, 2002. 

The CIEE monitors the sensitivity of epidemic strains of influenza A and B viruses to etiotropic drugs such as Remantadine, Arbidol, neuraminidase inhibitors (Oseltamivir and Zanamivir). A complex of virological and molecular genetic methods is widely used in this respect. For the 2008-2009 season, the heterogeneity of the population of epidemic strains in terms of sensitivity to etiotropic drugs became obvious: strains of the influenza A(H1N1) virus were sensitive to Rimantadine, Arbidol and Zanamivir; A(H3N2) and B viruses were sensitive to Arbidol, Oseltamivir and Zanamivir. Strains of the new pandemic influenza A(H1N1)v virus were found to be resistant to Rimantadine and sensitive to Arbidol, Oseltamivir and Zanamivir. The results obtained scientifically substantiate recommendations for the effective use of drugs for the treatment and prevention of influenza infection [21, 22].

One of the important activities of the CIEE is the development and implementation of new methods for the influenza diagnosis. The introduction of two modifications of the polymerase chain reaction (PCR) method with the identification of reaction products in agarose gel (EP-PCR), as well as real-time PCR (RT-PCR) with fluorescent labels made it possible to speed up the process of monitoring the circulation of influenza virus strains and to increase the number of investigated objects (nasopharyngeal swabs and sectional material). To assess the sensitivity of strains to etiotropic drugs, methods of cellular enzyme-linked immunosorbent assay (ELISA), fluorometric, RT-PCR and partial sequencing of the M2 protein gene and neuraminidases of influenza A and B viruses were used.

In connection with preparations for a pandemic associated with the emergence of a highly pathogenic strain of avian influenza A (H5N1) virus and its introduction into the human population, the problem of pathogen verification arose. This could not be done with the help of the traditional method, by hemagglutination inhibition (HI), therefore, the immunoblot and microneutralization methods were developed in the laboratory, which enabled to determine specific antibodies to influenza viruses. Differences in the spectrum and rates of production of antibodies to the internal proteins of influenza viruses (M and NP) in those who had been ill and those vaccinated with different types of inactivated vaccines were determined by the immunoblot.

The work of the Center's employees was presented at numerous international congresses and conferences held in the Russian Federation and abroad (in Europe, North America, Asia and Australia) on the surveillance of the spread and prevention of influenza in the Russian Federation. The research was supported by grants from the Russian Foundation for Basic Research and the International Science and Technology Center (ISTC grant No. 3070).

All of the above testifies to the many years of fruitful work on the problems of influenza by Academician of the Russian Academy of Medical Sciences, Professor D.K. Lvov as director of the Center for Influenza Ecology and Epidemiology, Director of the Reference Center for Influenza Monitoring, Director of the National Influenza Center, collaborating with WHO.