Antigenic Site Variation in the Hemagglutinin of Pandemic Influenza A(H1N1)pdm09 Viruses between 2009–2017 in Ukraine

The hemagglutinin (HA) is a major influenza virus antigen, which, once recognized by antibodies and substitutions in HA genes, helps virus in escaping the human immune response. It is therefore critical to perform genetic and phylogenetic analysis of HA in circulating influenza viruses. We performed phylogenetic and genetic analysis of isolates from Ukraine, the vaccine strain and reference strains were used to phylogenetically identify trends in mutation locations and substitutions. Ukrainian isolates were collected between 2009–2017 and clustered in the influenza genetic groups 2, 6, 7, and 8. Genetic changes were observed in each of the antigenic sites: Sa – S162T, K163Q, K163I; Sb – S185T, A186T, S190G, S190R; Ca1 – S203T, R205K, E235V, E235D, S236P; Ca2 – P137H, H138R, A141T, D222G, D222N; Cb – A73S, S74R, S74N. In spite of detected mutations in antigenic sites, Ukrainian isolates retained similarity to the vaccine strain A/California/07/09 circulated during 2009–2017. However, WHO recommended a new vaccine strain A/Michigan/45/2015 for the Southern Hemisphere after the emergence of the new genetic groups 6B.1 and 6B.2. Our study demonstrated genetic variability of HA protein of A(H1N1)pdm09 viruses isolated in 2009–2017 in Ukraine. Influenza surveillance is very important for understanding epidemiological situations.


Introduction
The pandemic influenza virus A(H1N1)pdm09 emerged in the human population in the spring of 2009 and caused a serious pandemic [1]. The emergence of a new highly virulent virus in the human population can cause pandemics, thus, it is important to conduct epidemiological, genetic and antigenic analyses [2]. The variability of influenza viruses is caused by changes in the sequences of their eight genes (PB2, PB1, PA, HA, NP, NA, M, NS). Mutations are the reason for genes oligonucleotide sequence changes of epidemic influenza viruses in the interpandemic period. In turn, the accumulation of amino acid substitutions leads to changes in the antigenic properties of a virus [2,3]. The antigenic properties of hemagglutinin and neuraminidase change rapidly in order to escape neutralization by human antibodies. Mainly because of an antigenic drift, WHO revises the vaccine composition on a yearly basis to include only actual circulating strains and to develop effective vaccines [3]. In addition, a number of cases of human infection with zoonotic influenza viruses (A(H5N1), A(H7N9) ect.)-which may cause severe disease and have potential risk to cause pandemic [4]-were reported. Viruses circulating in Ukraine throughout the research period belonged to genetic groups 2, 6 (6A, 6B, 6B.1, 6B.2, 6C), 7 and 8 as shown in Figure 1. Acquired amino acid substitutions, N31D and A186T, A/Dnipropetrovsk/445/2010 and A/Ukraine/220/2010 belonged to genetic group 2. These viruses were isolated in the 2009-2010 season, although they slightly differed from other isolates from the same season. Only two of the analyzed isolates obtained in the 2010-2011 season belonged to genetic group 8, namely, A/Ukraine/60/2011 and A/Ukraine/3/2011, and had an A186T specific substitution.  group 6C which became more widespread worldwide during that same season. Our isolates possessed the typical V234I substitution common to this group.
Starting in the 2013-2014 season, the genetic group 6B became widespread not only in Ukraine, but also throughout the world [14]. All viruses from genetic group 6B were characterized by acquired amino acid substitutions D97N, S185T, E47K and S124N. Since the 2015-2016 season, the vast majority of the viruses worldwide has become part of the 6B.1 subgroup according to Nextstrain data [18]. Group 6B.2 is somewhat less numerous, and acquired substitutes are unique to this group, including V152T and V173I in HA1, and E164G and D174E in HA2.
As showcased in Figure 2, in the early 2015-2016 season a division of genetic group 6B into two subgroups occurred. Approximately 63% of viruses belonged to genetic group 6B.1 and around 16%-to genetic group 6B.2. The composition changed later in the 2016-2017 season; the portion of 6B.2 viruses detected dropped to approximately 1% and genetic group 6B.1 viruses became widespread, accounting for 99% worldwide. All other viruses starting in the 2012-2013 season, to the 2016-2017 season belonged to genetic group 6 and were divided into separate subgroups. Genetic subgroup 6A has not been widely distributed in the world [14], but includes the 2010-2011 isolate A/Ukraine/104/2011 and the 2012-2013 isolates A/Ukraine/3/2013 and A/Ukraine/523/2013. These isolated viruses were characterized by the specific substitutions H138R and V249L. The majority of the 2012-2013 isolates fell within genetic group 6C which became more widespread worldwide during that same season. Our isolates possessed the typical V234I substitution common to this group.
Starting in the 2013-2014 season, the genetic group 6B became widespread not only in Ukraine, but also throughout the world [14]. All viruses from genetic group 6B were characterized by acquired amino acid substitutions D97N, S185T, E47K and S124N. Since the 2015-2016 season, the vast majority of the viruses worldwide has become part of the 6B.1 subgroup according to Nextstrain data [18]. Group 6B.2 is somewhat less numerous, and acquired substitutes are unique to this group, including V152T and V173I in HA1, and E164G and D174E in HA2.
As showcased in Figure 2, in the early 2015-2016 season a division of genetic group 6B into two subgroups occurred. Approximately 63% of viruses belonged to genetic group 6B.1 and around 16%to genetic group 6B.2. The composition changed later in the 2016-2017 season; the portion of 6B.2 viruses detected dropped to approximately 1% and genetic group 6B.1 viruses became widespread, accounting for 99% worldwide.

Genetic Variability of Pandemic Influenza Viruses
We conducted an analysis of all the substitutions that took place in pandemic influenza viruses isolates in Ukraine from 2009 to 2017 and established their antigenic sites. All viruses were analyzed in comparison with A/California/07/2009 vaccine strain. The H1 molecule НА has five antigenic sites: Sa, Sb, Ca1, Ca2, and Cb; many isolates had mutations in this area that could impact pathogenicity. In this study, we analyzed all genetic changes that occurred in the five antigenic sites of pandemic influenza viruses during seven epidemic seasons. Interestingly, some of the site mutations were conserved and then identified through multiple years. Genetic changes were observed in each of the antigenic sites, but their greatest number was detected on the sites Ca1 and Ca2. Table 2 shows that the smallest number of amino acid substitutions was detected in the antigenic site Cb. In the antigenic site Sa, three substitutions were found. The first amino acid substitution in position 162, where serine substituted for threonine mutation, was initially detected in the 2014-2015 season, only among isolates from the Dnipro region. During the 2015-2016 season, when the 6B viruses were divided into the 6B.1 and 6B.2 genetic groups, all 6B.1 viruses acquired this substitution, which appears in the isolates now. The acquisition of a potential glycosylation site, which may increase the virulence of influenza viruses, determines significance of the S162T substitution [20]. Another replacement was detected in position 163 and had a polymorphism. In 2013-2014, the genetic clade 6B emerged with a featured amino acid substitution from lysine (K) to glutamine (Q) at position 163 (K163Q, H1

Genetic Variability of Pandemic Influenza Viruses
We conducted an analysis of all the substitutions that took place in pandemic influenza viruses isolates in Ukraine from 2009 to 2017 and established their antigenic sites. All viruses were analyzed in comparison with A/California/07/2009 vaccine strain. The H1 molecule HA has five antigenic sites: Sa, Sb, Ca1, Ca2, and Cb; many isolates had mutations in this area that could impact pathogenicity. In this study, we analyzed all genetic changes that occurred in the five antigenic sites of pandemic influenza viruses during seven epidemic seasons. Interestingly, some of the site mutations were conserved and then identified through multiple years. Genetic changes were observed in each of the antigenic sites, but their greatest number was detected on the sites Ca1 and Ca2. Table 2 shows that the smallest number of amino acid substitutions was detected in the antigenic site Cb. In the antigenic site Sa, three substitutions were found. The first amino acid substitution in position 162, where serine substituted for threonine mutation, was initially detected in the 2014-2015 season, only among isolates from the Dnipro region. During the 2015-2016 season, when the 6B viruses were divided into the 6B.1 and 6B.2 genetic groups, all 6B.1 viruses acquired this substitution, which appears in the isolates now. The acquisition of a potential glycosylation site, which may increase the virulence of influenza viruses, determines significance of the S162T substitution [20]. Another replacement was detected in position 163 and had a polymorphism. In 2013-2014, the genetic clade 6B emerged with a featured amino acid substitution from lysine (K) to glutamine (Q) at position 163 (K163Q, H1 numbering) in the hemagglutinin (HA), located at the Sa antigenic site. Only isolate A/Zaporizza/417/2013 was somewhat different as lysine was replaced with isoleucine in position 163 instead of glutamine, as with all other viruses. In antigenic site Sb, four amino acid substitutions were noticed in three positions. The first substitution S185T was detected among isolates from the 2010-2011 season and appeared consolidated in the virus population at the antigenic level. The adjacent A186T substitution was discovered in two isolates: showed that substitution D222G in Ca HA causes the transition to a double a-2,3/a-2,6-mediated affinity for the upper and lower respiratory tract epithelium [21]. This mutation was associated with higher replication rates and more severe disease [21]. It has been shown that viruses with such substitutions are found only in fatal and severe cases and are not found in milder forms of infection [22]. Substitution D222N also causes changes in the specificity of the receptors resulting in viral infection of the lower respiratory tract. Supposedly, this substitution is to a lesser extent pathogenic compared with D222G [23]. This substitution was detected in isolate

Discussion
Viruses belonged to 8 genetic groups that circulated worldwide during 2009-2017. Ukrainian isolates within the indicated period belonged to genetic groups 2, 6, 7, and 8. All viruses acquired a number of mutations, which were located in antigenic sites of A(H1N1)pdm09 influenza viruses.
The emergence of S162T and D127E substitutions has led to the acquisition of a potential site for glycosylation, which may increase the virulence of influenza viruses. This has been maintained in the viral population and, in turn, elicits the assumption that the mutation is conducive for infection [10].
By gaining glycosylation sites, virus may escape human immune response. Glycosylation of antigenic sites has been proved to change surface topology of HA glycoprotein. Whereas, virus antigenic properties also change resulting in antibodies failing to recognize the virus [10]. Information about the changes in antigenic sites is very important for prediction of the next dominant strains. The acquisition of carbohydrate side chains by the HA protein is known for the ability to sometimes affect the antigenic properties of the influenza viruses. The carbohydrate chains in or near the antigenic sites may hide them from recognition by antibodies. The substitutions, leading to an acquisition of carbohydrate chains, can create new antigenic variants of influenza viruses [11].
The S185T substitution in antigenic site Sb falls within a domain defining the receptor-binding site (RBS). It is well documented that amino acid changes near or in the RBS may have influence on the antigenic properties of pandemic influenza viruses A(H1N1)pdm09. The substitution in antigenic site Sb A186T was also in the RBS [27].
Importantly, substitution D222G is located in the receptor-binding pocket in the antigenic site Ca HA and may alter the properties of the RBS [21]. In addition, the K163I substitution observed in A/Zaporizza/417/2013 may impart altered antibody-binding properties [28,29].

Discussion
Viruses belonged to 8 genetic groups that circulated worldwide during 2009-2017. Ukrainian isolates within the indicated period belonged to genetic groups 2, 6, 7, and 8. All viruses acquired a number of mutations, which were located in antigenic sites of A(H1N1)pdm09 influenza viruses.
The emergence of S162T and D127E substitutions has led to the acquisition of a potential site for glycosylation, which may increase the virulence of influenza viruses. This has been maintained in the viral population and, in turn, elicits the assumption that the mutation is conducive for infection [10].
By gaining glycosylation sites, virus may escape human immune response. Glycosylation of antigenic sites has been proved to change surface topology of HA glycoprotein. Whereas, virus antigenic properties also change resulting in antibodies failing to recognize the virus [10]. Information about the changes in antigenic sites is very important for prediction of the next dominant strains. The acquisition of carbohydrate side chains by the HA protein is known for the ability to sometimes affect the antigenic properties of the influenza viruses. The carbohydrate chains in or near the antigenic sites may hide them from recognition by antibodies. The substitutions, leading to an acquisition of carbohydrate chains, can create new antigenic variants of influenza viruses [11].
The S185T substitution in antigenic site Sb falls within a domain defining the receptor-binding site (RBS). It is well documented that amino acid changes near or in the RBS may have influence on the antigenic properties of pandemic influenza viruses A(H1N1)pdm09. The substitution in antigenic site Sb A186T was also in the RBS [27].
Importantly, substitution D222G is located in the receptor-binding pocket in the antigenic site Ca HA and may alter the properties of the RBS [21]. In addition, the K163I substitution observed in A/Zaporizza/417/2013 may impart altered antibody-binding properties [28,29].
Ukrainian isolates retained similarity to the vaccine strain A/California/07/09 during 2009-2017, despite the observed mutations present in the antigenic sites. However, after the emergence of the new genetic groups 6B.1 and 6B.2, WHO recommended a new vaccine strain with changed antigenic properties A/Michigan/45/2015 [30].
In this paper, we identified amino acid substitutions that occurred in isolates of pandemic influenza viruses isolated in Ukraine from 2009 to 2017, and analyzed their possible impact on virus virulence. The genetic characterization and surveillance of circulating influenza strains are extremely important for the evaluation of vaccine effectiveness, and compositing annual influenza vaccine development.

Sample Collection
This study used clinical samples of nasopharyngeal, pharyngeal, nasopharynx, nose swabs, and autopsy material collected in the first three days and no later than the fifth day of illness from patients with suspected influenza and SARI (severe acute respiratory infections). Autopsy material from dead bodies was obtained from the Regional Department of Morbid Anatomy and Office Forensics.
Samples were collected during routine diagnosis of influenza-like illness (ILI) in patients from sentinel clinics in four cities in Ukraine (Kyiv, Dnipro, Odesa, and Khmelnytsky). All the clinical samples were collected under bioethical guidelines research among human beings for human subjects research under Institutional Review Board approval #281 (11/01/2002); approving authority: Ministry of Health of Ukraine.

Extraction of Nucleic Acids and RT-PCR
Virological and molecular genetic methods were used to confirm influenza cases and analyze genetic sequences of isolates. Laboratory diagnosis was conducted by real-time PCR (polymerase chain reaction). To detect influenza virus types A and B, the following extraction kits were used: InnuPREP Virus DNA/RNA, Analytik Jena, Reinach, Switzerland, and primers and probes for respective markers: Univ inf A, sw A, sw H1, H1, H3, Univ inf B, RNase P (Biosearch Technologies, Petaluma, CA, USA). Molecular methods were carried out in accordance with the protocol provided by the WHO Collaborating Centre for Influenza at Atlanta's Centers for Disease Control and Prevention (CDC) (version 30 April 2009) [31]. The Thermo Scientific Verso 1-Step qRT-PCR ROX Kit (Waltham, Massachusetts, USA) was used for RT-PCR [31].

Virus Isolation, Sequencing and Genetic Analysis
Viral isolation and cultivation included passages in the MDCK cell line (epithelial kidneys cells from a Cocker Spaniel) obtained from CDC [32]. Isolates were later used for strain identification and sequencing. The sequencing of influenza virus isolates was performed by the World Influenza Center in London using RNA-SEQ technology. The influenza virus isolates in a cell culture medium were frozen in liquid nitrogen (-80 • C) and shipped following the cold chain principle. The sequences of influenza viruses from other countries were obtained from the GISAID (Global Initiative on Sharing All Influenza Data; http://platform.gisaid.org) website using BLAST analysis. Sequences were aligned using the ClustalW algorithm. The neighbor joining method applying Kimura 2-parameter model built phylogenetic trees. Nucleotide sequences were translated into amino acid sequences using MEGA 7 software [33]. The 3D structures were constructed in Chimera 1.11.2rc [34] software using crystallographic structures of HA (PDB files) from the Protein Data Bank [35]. Spread of A(H1N1)pdm09 influenza virus of genetic groups 6B.1 and 6B.2 was taken from Nextstrain https://nextstrain.org [18,19]. Funding: This research was funded via a cooperative agreement with CDC entitled: "Maintenance of Influenza Surveillance Capacity by National Health Authorities in Ukraine" -6NU51IP000855.