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REASSORTMENT EVENTS IN HA, NA AND NP GENES DETECTED BY PHYLOGENETIC ANALYSIS OF INFLUENZA A VIRUS STRAINS OF SUBTYPES A (H1N1) AND A (H7N9)
Buriachenko S.
Department ofpoultry Diseases NSC Institute of Experimental and Clinical Veterinary Medicine, NAAS
Ukraine, Kharkov, Ukraine Stegniy B.
Director of IECVM NSC, Doctor of Veterinary Sciences, honored Worker of Science and Technology of
Ukraine, professor, Academician of NAAS of Ukraine
ABSTRACT
The influenza virus is a serious pathogens of animals, humans and birds that regularly causes epidemics, as well as high-mortality pandemics; therefore, an analysis of the assessment of reassortment in the hemagglutinin (HA), neuraminidase (NA) and nucleoproteine (NP) genes is necessary. Reassortment causes the necessary genetic variability, which allows a virus with high efficiency to overcome the interspecies barrier. Determination of the reassortment events will allow assessing the degree of variability of the genes of the proteins responsible for the infection process in the infection of the cell.Reassortment events in HA, NA and NP genes of H1N1 and H7N9 strains of Influenza A virus were studied. To characterize the reassortant viruses that have obtained the genes for surface (HA, NA) and internal proteins (NP) from the low pathogenic avian influenza virus subtype H7, and the genes for the highly productive human influenza virus H1, the study of post-reassortment interaction of genes, optimization of the gene composition of highly productive reassortants. Materials and methods. The nucleotide sequences of the investigated genes of hemagglutinin, neuraminidase, and nucleoproteine proteins for determining the reassortment events were taken from the National Center for Biotechnological Information (NCBI) GenBank (http://www.ncbi.nlm.nih.gov/). The assessment of the reassortment was carried out by aligning nucleotide sequences using the ClustalW algorithm. Phylogenetic analysis was calculated by the method of maximum likelihood. The dendrogram was built using the MEGA6 program. The reliability of the resulting phylogenetic tree was obtained using bootstrap analysis. The level of reassortment was determined by the GiRaF program. Results. It has been shown that events of the reassortment of the HA gene on two clusters are present within and between subtypes H1N1 and H7N9. The reassortment of the NA genes of two subtlety subtypes shows that among them, each is produced by the genes of the reassortment and have sequences of genes of only one subtype of the influenza A virus. These events of the reassortment of the NA genes are present within the subtypes of H1N1 and H7N9, but not between them. It has been established that events of reassortment of NP genes are not present between subtypes H1N1 and H7N9. The absence of reassortment events in NA, NA and NP genes of the analyzed strains was shown. Conclusions. The assortment events of the HA, NA, and NA genes in subtypes of the influenza A H1N1 and H7N9 viruses show the presence of subtypes inside and not between them. This suggests that genetic polymorphism should be investigated in the subtypes by the definition of mutations, with further evaluation of the variability of the genetic markers of the genes of the proteins studied. Consequently, the absence of reassortment events in HA, NA, and NP genes of the Influenza A strain H1N1 and H7N9 was shown. The results of this research confirm previously conducted studies and explain the evolution of NA, NA and NP genes of the Influenza A virus.
Keywords: Influenza A virus, haemagglutinin, neuraminidase, nucleoprotein, reassortment events, phylogenetic analysis, H1N1, H7N9.
1. Introduction
Influenza viruses (from family Orthomyxoviridae) are among the most common causes of human respiratory and gastrointestinal infections. Influenza viruses are derived into three types — A, B and C [1]. Influenza B and C viruses infect humans predominantly, causing respiratory diseases [2, 3]. Avian influenza poses a significant risk of zoonotic infection, change of host and the appearance of pandemic viruses. It infects humans and farm animals (pigs, horses, marine mammals, cats, dogs and birds). The appearance of bird flu was recorded in Ancient Greece as early as 413 ad. [4], the first confirmed pandemic was the Russian flu, which occurred in 1889-1892. [5]. Three main characteristics predetermine the rapid evolution of viruses: large populations, short generation times and high mutation rates. Formed mutations help viruses escape the host's
immune system, and can be positively selected, passed on to the next generation, and distributed. During the last 100 years, four pandemics of influenza have occurred: the Spanish H1N1 flu of 1918 [6], which was described as the "greatest medical holocaust in history" [7], the Asian influenza H2N2 of 1957, the Hong Kong influenza H3N2 of 1968 and 2009 H1N1 swine flu [8,9]. Recently, the low pathogenic avian influenza virus (LPAI) (H7N9), first identified in the East China region, caused an outbreak of the disease among people with a mortality rate of up to 40% [10]. Membrane surface protein plays an important role in establishing the viral infection of cells. The rate of reassortment of influenza virus in natural reservoirs is extremely high [11]. Avian influenza viruses contain 16 types of hemagglutinin and 9 types of neuraminidase. In wild birds, at least 103 of 144 types of avian influenza virus,
which are possible with a free combination of different types of HA and NA segments, have been found [12]. The fact that most of the possible combinations of HA/NA subunits form in the body of wild birds proves a high frequency of reassortment. Domestic ducks are also natural reservoirs of the influenza virus, and reassortment of various strains occurs in them [13]. Phylo-genetic analysis of the genomes of the avian influenza virus showed that the virus in the body of wild birds exists in the form of a large pool of functionally equivalent and interchangeable gene segments that form the so-called "genomic constellations" [14]. When assembling virions from "genomic constellations," almost any combination of viral segments easily emerges. In fact, the body of wild waterfowl is constantly shuffling the genome of the influenza virus. Apparently, it is the increased survival of the virus in natural reservoirs that makes it possible to successfully replicate various combinations of RNA segments and contributes to the high efficiency of reassortment. For avian influenza viruses characterized by a constant change of owners. Viruses, overcoming the species barrier, infect poultry, pigs, horses, people. In all likelihood, it is the ongoing reassortment in the body of birds that creates the necessary genetic variability, which allows the virus to overcome interspecific barriers with high efficiency [15]. Thus, reassortment in natural reservoirs is similar to sexual reproduction and creates genetic polymorphism, which allows the virus to prepare for unpredictable changes in the external environment, including a change in the host organism. At the same time, reassortment takes place not only in natural reservoirs. From wild birds, the flu virus is easily transmitted to domestic birds, which in turn infect pigs. However, despite the fact that swine flu virus is widespread among poultry, avian flu virus is only sometimes found in pigs [16]. On the other hand, the human flu virus also infects pigs. A case has been described where pigs were infected with the flu virus after contact with farmers, and also when farmers were infected with the flu virus after contact with pigs [17]. Consequently, pigs can be coinfected by various strains of the avian and human influenza viruses, and in their bodies can be mixed avian, porcine viruses and human influenza virus [18, 19]. It is shown that viruses isolated from environmental and poultry samples in Guangdong from April to May 2013 were very similar to other H7N9 strains found in eastern China. The H7N9 virus, isolated from a clinical patient in Guangdong in August 2013, differed from the previously identified H7N9 viruses, with the NS and NP genes derived from the recent H9N2 viruses circulating in the province. This study provides direct evidence that reas-sortment has continued and led to the emergence of a new H7N9 flu virus in Guangdong Province, China. These results also shed light on the evolution of the H7N9 virus, which is critical for future monitoring and tracking of viral transmission. Phylogenetic analysis showed that the four internal A/Guangdong/1/2013 (H7N9) virus genes — the NS, NP, PB1, and PB2 genes — were in clusters other than the H7N9 genes previously identified in other provinces of China. [20]. Circulating viruses H7N9 2015-16. belong to different
lines with different spatial distribution. Hemagglutina-tion inhibition analyzes performed on serum samples from patients infected with these viruses identified 3 antigenic clusters for 16 strains of different viral lines. Using the reconstruction of hereditary sequences to identify parallel amino acid changes on several separate lines, mutations in the hemagglutinin occur mainly at sites involved in receptor recognition or antigenicity [21]. Recent studies have shown that the internal genes of the H7N9 virus continue to undergo dynamic redistribution with bird H9N2 viruses. In accordance with the evolutionary distance and re-sorting style, H7N9 viruses were classified into 27 genotypes during the first three months after the outbreak and into 48 other genotypes by our and other groups, respectively [22]. Among genotypes, the G0 or W1 genotype (represented by A/Anhui/1/2013) acts as the dominant viral cluster in humans [22]. None of the G4, G5 and G6 viruses, which have 4, 5 and 6 phylogenetically distinct internal genes from G0, was observed in humans based on surveillance data from 109 isolates [22]. The significant hazard of zoonotic infection is caused by influenza A viruses. The most important factors of virulence of Influenza A viruses are cover-proteins hemagglutinin (HA, or H), neuraminidase (NA, or N), which provide attachment to the host cell, and replication factor nucle-oprotein (NP), which are encoded by HA, NA and NP genes, respectively. Different combinations of HA and NA originate different subtypes Influenza virus A. There are 18 HA (H) and 11 NA (N) subtypes. Epizootics usually are caused by high-virulence strains H1N1 and H7N9 [23]. Influenza A viruses contain negativestrand RNA with segmented genomes containing seven to eight gene segments which encode at least 10 proteins [24]. According to the results of phylogenetic study the possibility of Influenza A viruses to overcome the interspecific barrier was detected, but molecular processes which lead to a change in the host cell, has not been sufficiently examined. RNA mutations and re-assortments are general mechanisms of genome changing. Domination of reassortments processes was widely shown before [25]. There is a direct correlation between the presence of quantitative differences between the two niches of the influenza virus and the significance of the reassortment for changing the ecological niche. When there is a reassortment that leads to a change in the host cell, it remains unknown [26]. The aim of current research was to detect reassortment events in HA, NA and NP genes by phylogenetic analysis of Influenza A viruses strains of subtypes A (H1N1) and A (H7N9). The influenza virus is a serious pathogens of animals, humans and birds that regularly causes epidemics, as well as high-mortality pandemics; therefore, an analysis of the assessment of reassortment in the hemagglu-tinin (HA), neuraminidase (NA) and nucleoproteine (NP) genes is necessary. Reassortment causes the necessary genetic variability, which allows a virus with high efficiency to overcome the interspecies barrier. Determination of the reassortment events will allow assessing the degree of variability of the genes of the proteins responsible for the infection process in the infection of the cell. Reassortment events in HA, NA and NP genes of H1N1 and H7N9 strains of Influenza A virus
were studied. To characterize the reassortant viruses that have obtained the genes for surface (HA, NA) and internal proteins (NP) from the low pathogenic avian influenza virus subtype H7, and the genes for the highly productive human influenza virus H1, the study of post-reassortment interaction of genes, optimization of the gene composition of highly pathogenic reassortants. To achieve the goal, the following tasks were set: 1. analyze 8000 strains of influenza A type virus (H1N1) and (H7N9) isolated from humans and birds from the database. 2. determine the sample of nucleotide sequences of the studied strains. 3. to conduct phylogenetic analysis. 4. Obtain a set of reassortants of the low pathogenic avian influenza virus subtype H7 and the highly pathogenic human influenza virus H1 2. Material and methods Reassortment events were defined on nucleotide sequences of Influenza A virus HA, NA and NP genes from National Centre of Biotechnology Information (NCBI) GenBank (http://www.ncbi.nlm.nih.gov/). Each gene sequences were aligned by ClustalW algorithm. Phylogenetic analysis of data sequences was calculated by maximum likelihood method. The construction of kinship dendrograms (cluster algorithms) was performed using the DENDRO UPGMA computer program (http://genomes.urv.cat/UPGMA/). This program is based on the use of the UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method, which
allows you to create trees that graphically display the Similarity and Distance matrix. Dendrogram was built with the help of program MEGA6. The reliability of the inferred tree was detected by bootstrap test. Reliable result was considered at 70 and more. Reassortment events were analyzed on each obtained dendrogram. If reassortment events between two strains were found, reassortment level would be detected by GiRaF program.
3. Results
As a result of the studies, profiles of reassortment events of two strains H1N1 and H7N9 were obtained by genes HA, NA and NP. In particular, in the hemagglutinin (HA), neuraminidase (NA) and nucleoprotein (NP) genes, recombinations in the hemagglutinin, neuraminidase and nucleoprotein genes were found within the H1N1 and H7N9 strains. Between the strains, no combined and different replaced genes of the two strains of influenza A viruses, hemagglutinin (NA), neuraminidase (NA) and nucleoprotein (NP), were revealed. Results of phylogenetic analysis of Influenza A virus HA gene are shown on Figure 1. There was no exchange of genome segments between the strains of these viruses. The results obtained by us indicate the absence of recombinations among these strains among themselves, but a rather high level of intrastate reassortment events remains.
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Figure 1. UPGM - dendrogram built by results of phylogenetic analysis of Influenza A virus HA gene
Dendrogram consists of two clades. Each of them is produced by reassortment genes and contains HA gene sequences of only one strain of Influenza A virus.
Thus, reassortment events of HA gene are present inside strains H1N1 and H7N9, but not between them.
Results of phylogenetic analysis of Influenza A virus NA gene is shown on Figure 2.
51, AH5397H.1 Influenza A virus H1N1 JM631D4fl. 1 Influenza A virus H INI JMD365S1.1 Influenza A virusHINI KP019726.1 InfluenzaA virusHINI JN207833.1 Influenza A virus H1H1 JH00648tt.1 InlluenzaA virusHINI » JN006487.1 InfluenzaAvirusHINI JN0D6471.1 Influenza A virusH INI 6U59C010. ■ Influenza A virus H INI CY123246.1 I nfluenza A virus H1N1 30 CYW59B7.1 llflueiuA VirusHINI JN006483.1 Influenza A virusHINI KP019721.1 I nfluenzaA virusHINI MS632S21.1 Influenza A virusHINI 37, JIJ036594.1 InfluenzaA virusHINI JJB2E034.1 Influenza A virusHINI 35 KC782242.1 Influenza A virusHINI KC78176C.' Influenza A virusHINI CY 124014.1 Influenza A virusHINI JNDG8436.1 Influenza A virusHINI KF50D629.1 Influenza AvirusH7N6 -KCS532ai.1 Influenza AvirusH7NB KJ033S1 Dr. 1 Influenza A vims H7M9 KF918e31.1 Influenza AvintsH7MB CY187452.1 Influenza AvirusH7M6 CY18762D.1 Influenza AvirusH7MG
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Figure 2. UPGM - dendrogram built by results of phylogenetic analysis of Influenza A virus NA gene
The obtained dendrogram includes two clades. Among them each is produced by reassortment genes and contains NA gene sequences of only one strain of Influenza A virus. Considering this fact we conclude
that reassortment events of NA gene are present inside strains H1N1 and H7N9, but not between them.
Results of phylogenetic analysis of Influenza A virus NP gene is shown on Figure 3.
Figure 3. UPGM - dendrogram built by results of phylogenetic analysis of Influenza A virus NP gene
The received dendrogram is reliable and consists of two clades as well. Each of them is found to be produced by reassortment genes and contains NP gene sequences of only one strain of Influenza A virus. Thus detained that reassortment events of NP gene are present not between but inside of strains H1N1 and H7N9.
According to the obtained results of the conducted phylogenetic analysis of НА, NA and NP genes there was no further purpose to calculate reassortment level by GiRaF program.
4. Discussion
The work analyzed 40 natural isolates of influenza A virus subtypes H1N1, H7N9.
As a result, it was revealed that reassortment events occur in the analyzed clades not between the subtype genes but within each. Genetics traits human influenza viruses reassorted with genes of non-human (probably avian) origin [27]. The subtypes of the virus A (H7N9), resulted from the recombination of three viral strains that infect only birds. This is consistent with our research, since no reassortment between the HA, NA and NP genes of all the H1N1 and H7N9 subtypes obtained in the treasures occurs. In late March 2013, a new H7N9 flu virus appeared in China, which infected 137 people, 45 of whom died. The virus causes almost no symptoms in poultry. However, in humans, the virus
causes severe pneumonia. The H7N9 virus was caused by the reassortment of the H7, N9 and H9N2 avian influenza viruses but does not contain the H1N1 subtype genes [28]. The H1N1 influenza virus subtypes acquired the NA gene, from the Eurasian swine influenza virus as a result of reassorting the human influenza virus, the avian influenza virus and the classic swine influenza virus [29]. The absence of reassorting events in the NA, NA, and NP genes of the influenza A strain H1N1 and H7N9 of the analyzed subtypes confirms previous studies and explains the evolution of the NA, NA, and NP genes of influenza A virus [30]. A comparative phylogenetic analysis of four strains of the H7N9 influenza virus with other well-known representatives of the A/H7 subtype for all eight genes showed reassortment events involving the H7N3 virus genes [31]. The 2013 H7N9 virus arose as a result of at least three reassortment events that combined the HA7 and NA9 genes from previously known lines. Thus, the H7 segment was closest to the NA gene of the H7N3 virus isolated from a duck in Zhejiang Province in eastern China, while the NA gene resembled that of the H7N9 isolates isolated in Korea from ducks and wild birds [32]. Beijing A/Beijing/01/2013 (H7N9) isolate suggests that the H7N9 virus did not acquire the ability to pass from person to person, the virus develops only in
poultry, and then infects the person through direct contact [33]. This assumption is confirmed with our data on the absence of reassortment between the HA, NA and NP genes of the H1N1 and H7N9 subtypes. It is important to note that avian influenza viruses "participate" in the emergence of new "human" influenza viruses, which are characterized by high pathogenicity and the ability to cause pandemics. The H1N1 virus has a set of internal genes, the origin of which indicates their phy-logenetic relationship with viruses of birds and pigs [34]. A permanent source of genes for pandemic influenza viruses exists (in a phenotypically unchanged state) in the natural reservoir of waterfowl and migratory avian [35]. Modern studies have shown that the gene structure of the new A/H1N1 virus is complex and includes the genes of swine flu affecting pigs in North America; swine flu genes affecting pigs in Europe and Asia; avian flu genes; human flu genes. In fact, the genes for the new virus come from four different sources. Influenza A viruses are characterized by a high incidence of reassortants as a result of mixed infection, due to the segmentation of the viral genome. Strain-specific properties of genomic segments can have a strong influence on the gene composition of reassortants in non-selective conditions. In other words, a distinctive feature of influenza viruses is the fact that in eight of the gene segments, especially in the NA gene, frequent and unpredictable mutations occur [36]. This is confirmed by phylogenetic analysis of nucleic acid sequences of different subtypes of influenza A viruses from different hosts and from different geographic regions conducted by us.
5. Conclusions
A sample of nucleotide sequences of the HA, NA and NP gene of the avian influenza virus A subtypes H1N1 and H7N9, causing an infectious process in mammals and poultry, was identified. It has been shown that the events of the reassortment of the HA gene in the two clusters are present inside the subtypes H1N1 and H7N9, but not between them. Reassortment of the NA genes of two subtle types of subtlety indicates that among them each is produced by the genes of the reassortment and have sequences of genes of only one subtype of the influenza A virus. These events of the reassortment of the NA genes are present within the subtypes of H1N1 and H7N9 but not between them. It has been established that events of reassortment of NP genes are present not between subtypes H1N1 and H7N9. The absence of reassortment events in NA, NA and NP genes of the analyzed strains was shown. Consequently, the absence of reassortment events in NA, NA and NP genes of the Influenza A strain H1N1 and H7N9 was shown. The results of this research confirm previously conducted studies and explain the evolution of NA, NA and NP genes of the Influenza A virus. Thus, as a result of constantly ongoing reassortment processes in natural reservoirs, new strains of influenza viruses are emerging that can overcome the interspecific barrier. For the transition of viruses to the third and fourth stages of emergence, additional reassortment events and mutations are needed, which, in all likelihood, occur in the human body, as well as in the pigs
that serve as "mixers" for genetic mixing of influenza viruses.
Acknowlegements
The author sincere thanks gratitude to Academician of the National Academy of Sciences of Ukraine professor B.T. Stegniy (NSC IECVM NAAS of Ukraine) and the PhD of biological sciences O. V. Bilynska (V. Ya. Yuriev Research Institute of NAAS of Ukraine)
References
1. A. B. Allison J. R. Ballard, R. B. Tesh, J. D. Brown, M. G. Ruder, M. K. Keel, B. A. Munk, R.l M. Mickley, S. E. J. Gibbs, A. P. A. Travassos da Rosa, J. C. Ellis, H. S. Ip, V. I. Shearn-Bochsler, M. B. Rogers, E. Ghedin, E. C. Holmes, C. R. Parrish, C. Dwyerj Cyclic Avian Mass Mortality in the Northeastern United States Is Associated with a Novel Orthomyxovirus.// //J Virol. - 2015. - № 89 (2). - P. 1389-1403.
2. B. Barrett Chapter 18—Viral Upper Respiratory Infection. In: Rakel D., editor. Integrative Medicine (Fourth Edition) Elsevier; Amsterdam, The Netherlands: 2018. Pp. 170-179.e7.
3. X. Cui, A. Das, A. N. Dhawane, J. Sweeney, X. Zhang, V. Chivukula, S. S. Iyer. Highly specific and rapid glycan based amperometric detection of influenza viruses. Chem. Sci. 2017;8:3628-3634. Doi: 10.1039/C6SC03720H.
4. I. Dukhovlinov, R. Al-Shekhadat,E. Fedorova, L. Stepanova, M. Potapchuk, I. Repko, O. Rusova, A. Orlov, L. Tsybalova, O. Kiselev Study of immunogen-icity of recombinant proteins based on hemagglutinin and neuraminidase conservative epitopes of Influenza A virus. //Med Sci Monit Basic Res. - 2013. - № 19. -P. 221-227.
5. E. G. Deeva Influenza. On the verge of a pandemic. Rukovod-stvo dlja vrachey. Moskow: Geotar-Media; 2008 (in Russian)
6. B. Efron Bootstrap Methods: Another Look at the Jackknife.//B. Efron.//Ann. Statist. - 1979. - № 7 (1). - P.1 -26.
7. S. Faccini , A. De Mattia, C. Chiapponi, I. Barbieri, M. B. Boniotti, C. Rosignoli, G. Franzini, A. Moreno, E. Foni, A. D. Nigrelli. Development and evaluation of a new Real-Time RT-PCR assay for detection of proposed influenza D virus. J. Virol. Methods. 2017;243:31-34. doi: 10.1016/j.jviromet.2017.01.019.
8. A. Hushegyi, T. Bertok, P. Damborsky, J. Kat-rlik, J. Tkac. An ultrasensitive impedimetric glycan biosensor with controlled glycan density for detection of lectins and influenza hemagglutinins. Chem. Commun. 2015;51:7474-7477. doi: 10.1039/C5CC00922G.
9. U. Jarocka, R. Sawicka, A. Gora-Sochacka, A. Sirko, W. Dehaen, J. Radecki, H. Radecka. An electrochemical immunosensor based on a 4,4'-thiobisben-zenethiol self-assembled monolayer for the detection of hemagglutinin from avian influenza virus H5N1. Sens. Actuators B Chem. 2016;228:25-30. doi: 10.1016/j.snb.2016.01.001.
10. J. Lui, J. Wu, X. Zeng, D. Guad, L. Zou, L. Yi, L. Liang, H. Ni, M. Kang, X. Zheng, H. Zhong, X. He,
C. Monagin, J. Lin, C. Ke. Continuing reassortment leads to the genetic diversity of influenza virus H7N9 in Guangdong, China. J Virol. 2014 Aug;88(15):8297-306. doi: 10.1128/JVI.00630-14. Epub 2014 May 14.
11. J. Lui, J. Raghwani J, R. Pryce, T. A. Bowden, J. Theze, S. Huang, Y. Song, L. Zou, L. Liang, R. Bai, Y. Jing, P. Zhou, M. Kang, L. Yi, J. Wu, O. G. Pybus, C. Ke. Molecular Evolution, Diversity, and Adaptation of Influenza A(H7N9) Viruses in China. Emerg Infect Dis. 2018 0ct;24(10):1795-1805. doi: 10.3201/eid2410.171063.
12. T. T. Lam, et al. Systematic phylogenetic analysis of influenza A virus reveals many novel mosaic genome segments. Infect. Genet. Evol. 2013;18:367-378.
13. D. Liu, et al. Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses. Lancet. 2013;381:1926-1932.
14. R. Manzoor, M. Igarashi, A. Takada. Influenza A Virus M2 Protein: Roles from Ingress to Egress. Int. J. Mol. Sci. 2017;18 doi: 10.3390/ijms18122649.
15. www.ncbi.nlm.nih.gov
16. N. Nagarajan, C. Kingsford. GiRaF: robust, computational identification of influenza reassortments via graph mining./ //Nucleic Acids Research. - 2010. -P. 1-10.
17. Orthomyxoviridae—Negative Sense RNA Viruses—Negative Sense RNA Viruses. [(accessed on 6 September 2018)];2011 Available online: https://talk.ictvonline.org/ictv-reports/ictv_9th_re-port/negative-sense-rna-viruses-2011/w/negrna_vi-ruses/209/orthomyxoviridae.
18. M. Ozawa, Y. Kawaoka. Crosstalk between animal and human influenza viruses./M. Ozawa, Y. Ka-waoka//Annu Rev Anim Biosci. - 2013. - № 1. - P. 21-42.
19. C. Paules, K. Subbarao. Influenza. Lancet. 2017;390:697-708.doi: 10.1016/S0140-6736(17)30129-0.
20. Past Pandemics|Pandemic Influenza (Flu)|CDC.[(accessed on 6 September 2018)]; Available online: https://www. cdc.gov/flu/pandemic -resources/basics/past-pandemics.html
21. S. Smith, M. Waterman Identification of Common Molecular Subsequences.//Journal of Molecular Biology. - 1981. - № 147. - P. 195-197.
22. C. B. Staats, R. G. Webster, R. J. Webby . Diversity of influenza viruses in swine and the emergence of a novel human pandemic influenza A (H1N1)./ // Influenza Other Respir Viruses. - 2009. - № 3 (5). - P. 207-213.
23. T. Shelby, T. Banerjee, J. Kallu, S. Sulthana, I. Zegar, S. Santra. Novel magnetic relaxation nanosen-sors: An unparalleled "spin" on influenza diagnosis. Nanoscale. 2016;8:19605-19613.doi: 10.1039/C6NR05889B.
24. Y. -T. Tseng, C.-H. Wang, C. - P. Chang, G. - B. Lee. Integrated microfluidic system for rapid detection of influenza H1N1 virus using a sandwich-based aptamer assay. Biosens. Bioelectron. 2016;82:105-111. doi: 10.1016/j.bios.2016.03.073.
25. N. J. Maclachlan; C. E. Mayo; P. W. Daniels; G. Savini; S. Zientara; E. P. Gibbs. Bluetongue. Rev. Sci. Tech.2015, 34, 329-340.
26. T. T. Lam, et al. Systematic phylogenetic analysis of influenza A virus reveals many novel mosaic genome segments. Infect. Genet. Evol. 2013;18:367-378.
27. R. G. Webster, W. J. Bean, O. T. Gorman, T. M. Chambers, Y. Kawaoka. Evolution and ecology of influenza A viruses.//Microbiol Rev. - 1992. - № 56 (1). - P. 152-179.
28. C. B. Staats, R. G. Webster, R. J. Webby . Diversity of influenza viruses in swine and the emergence of a novel human pandemic influenza A (H1N1)./ // Influenza Other Respir Viruses. - 2009. - № 3 (5). - P. 207-213.
29. D. Liu, et al. Origin and diversity of novel avian influenza A H7N9 viruses causing human infection: phylogenetic, structural, and coalescent analyses. Lancet. 2013;381:1926-1932.
30. K. H. Zhumatov, M. Kh. Sayatov, A. I. Kydyr-manov, A/H7 Flu viruses: disrtibution, genetic variability, pathogeneticy for avian and human.Eurasian Journal of Applied Biotechnology .2013;2:12-16
31. Bi Yuhai, Liu Jingyuan, Xiong Haofeng, Zhang Yue, Liu Di, Liu Yingxia, F. George. Gao, and Beibei Wang. A new reassortment of influenza A (H7N9) virus causing human infection in Beijing, 2014. Sci Rep. 2016; 6: 26624.
32. E. G. Deeva Influenza. On the verge of a pandemic. Rukovod-stvo dlja vrachey. Moskow: Geotar-Media; 2008 (in Russian)
33. R. G. Webster, T. Ito, J. N.S.S. Couceiro, S. Kelm, L. G. Baum, S. Krauss, M. R. Castrucci, I. Don-atelli, H. Kida, J. C. Paulson and Y. Kaawaoka.1998. Molecular basis for the generation in pigs of influenza A viruses with pandemic potential. J.Virol.72:7367-7373
34. N. L. Varich, A. K. Gitel'man, A. A. Shilov, Iu. A. Smirnov, N. V. Kaverin. Differential incorporation of genomic segments into the influenza A virus reassortants in mixed infection]. Vopr Virusol. 2009 Jan-Feb;54(1):7-11. Russian.
35. T. Tada, K. Suzuki, Y. Sakurai, M. Kubo, H. Okada, T. Itoh, K. Tsukamoto. Emergence of Avian Influenza Viruses with Enhanced Transcription Activity by a Single Amino Acid Substitution in the Nucleopro-tein during Replication in Chicken Brains.//J Virol. -2011. - № 85 (19). - P.10354-10363.
36. K. Tamura, D. Peterson, N. Peterson. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods//Mol. Biol. Evol. -2011. - № 28 (10). - P. 2731-2739.
