Whole genomic analysis of two influenza H3N2 virus strains isolated from Qinghai, China

Background Influenza H3N2 virus has a faster evolution rate than other types of influenza viruses. This study was performed to better understand the molecular evolution of influenza H3N2 in Qinghai Province, China in 2017. Methods Complete sequences of eight gene segments of two influenza H3N2 isolates in 2017 in Qinghai Province were sequenced and analyzed by MEGA 6.06 software. Results Phylogenetic analysis showed that two Qinghai H3N2 isolates were relatively close to the 2016–2017 vaccine strain, 3C.2a-A/Hong Kong/4801/2014. In HA protein, compared with the 2015-2016 WHO recommended vaccine strain A/Switzerland/971 5293/2013, six amino acid substitutions were observed in epitopes A and B in Qinghai isolates in 2017, however, only two amino acid substitutions were observed in epitopes A and B in Qinghai isolates compared with the A/Hong Kong/4801/2014, which indicated 2016-2017 vaccine strain might have a better protection against the strains circulating in Qinghai Province in 2017 . Besides, amino acid substitution of K160T at the glycosylation site of HA and H75P in PB1-F2 in the two Qinghai isolates might affect the antibodies binding ability and the virulence of influenza virus. And there was no key amino acid substitution in the key sites of segment NA, M, NP, NS, PA and PB2. Conclusions The presence of several antigenic site mutations in Qinghai H3N2 isolates confirms the evolution of circulating H3N2 strains. Enhancing the surveillance of influenza epidemic by whole genome sequencing is important to monitor whether the selected vaccine strains are protective against the circulating strains in Qinghai Province. has which a long-term threat to human health, and vaccination is the most effective way to prevent the infection. Exploring the characterization of genetic and antigenic evolution of influenza H3N2 virus is essential to formulating effective vaccine strategies. Periodic assessment and replacement of vaccine strains are important for influenza pandemic prevention and control.


Introduction
Influenza virus is an enveloped, single-stranded RNA virus with negative strand, containing eight fragments, haemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), non-structural (NS), matrix (M), polymerase acidic (PA), polymerase basic 1 (PB1) and polymerase basic 2 (PB2). By constantly changing the antigenicity, influenza virus can evade the identification and elimination of host specific immunity, and thus continuously causes epidemics which is one of the important public health issues worldwide [1]. H3N2 subtype of influenza virus was first identified in 1968 and caused the third flu pandemics of 20 th century [2]. Owing to the rapid evolution, influenza H3N2 virus have always been active in the crowds. Therefore, it is important to monitor and explore the variation rules of influenza virus for the prediction of influenza outbreak and the screening of influenza vaccine.
Influenza vaccines have been developed to protect the general population from developing severe disease [3]. Considering the rapid viral antigenic evolution of influenza virus, vaccines need to be reformulated periodically [4]. Twice a year, the World Health Organization (WHO) provides recommendations on influenza vaccines strains for the upcoming influenza season, for southern and northern hemispheres respectively. The application of complete influenza virus genomes is deepening our understanding of influenza evolutionary characteristics and promoting the selection of vaccine strains.
In this study, we firstly performed the whole genome sequencing of influenza H3N2 virus in Qinghai Province, located in the northwest of China. We analyzed eight gene segments of two influenza A/H3N2 virus strains circulating in 2017 and compared them with those of the WHO recommended vaccine strains and with some other H3N2 strains circulating in other areas at the same time from Gen Bank and GISAID, which benefit us for understanding the molecular epidemic characteristics of influenza H3N2 virus in Qinghai Province in 2017 and assessing the protective effect of WHO recommended vaccine strains in this area.  [7] and synthesized in Sangon Biotech (Shanghai, China). Then the mixture was run using the following program: 60℃1min, 42℃10min, 50℃30min, 95℃15min, (94℃30s, 50℃30s, 72℃1min)×35 cycles, 72℃10min. The PCR products were identified using 1.5% agarose gel electrophoresis and then sent to Sangon Biotech (Shanghai, China) for Sanger sequencing. Genbank accession numbers range from MN533932 to MN533947 for virus isolate sequences.

Sequence analysis
The nucleotide sequences were spliced by using SeqMan, analyzed by using BioEdit   (Table 1).
HA protein was spliced into two subunits HA1 and HA2 at site 329, showing characteristic differences in splicing sites between highly pathogenic influenza viruses and low-pathogenic influenza viruses. In our study, the splicing site of the two Qinghai isolates was PEKQTR↓G, which were typical low-pathogenic influenza viruses. HA protein have 5 epitopes, all of which are located in the HA1 subunit, including 129 sites of A, B, C, D and E. Among them, epitopes A and B are the most important, which can induce the protective antibodies [9].   Amino acid substitution  3  96  128  131  138  14  0  14  2  14  4  15  9  16  0  18  6  19  4  23  9  A/Switzerland/9715293/2013  L  N  A  T  S  R  G  N  S  K  V  P  P  A/Hong Kong/4801/2014  I  S  T  T  A  I  R  S  Y  K  G  L  P  A/QH/219/2017  I  N  T  K  A  I  K  S  Y  T  G  L  S  A/QH/245/2017  I  N  T  K  S  I  K  S  Y  T  G  L [18]. PB1-F2 protein is an important virulence factor of influenza virus, and its mitochondrial target sequence is located at sites 69-82, while sites 63-75 is related to mitochondrial localization [19]. In this study, amino acid substitution of H75P were observed in Qinghai isolates, which might have some influence on the virulence of influenza virus.

Discussion
Influenza H3N2 virus has a faster evolution rate than other types of influenza viruses, and

Conclusions
Influenza H3N2 virus has evolved rapidly, which epidemic is a long-term threat to human health, and vaccination is the most effective way to prevent the infection. nostril, posterior pharyngeal wall and bilateral tonsils by using sterile swabs gently, which was safety and non-invasive. Before the sample collection, all the subjects or their guardian were informed the purpose of this study, and signed the informed consent.

Consent for publication
All authors of this manuscript declare that we have approved the submission and publication of this manuscript.

Availability of supporting data
Not applicable.

Competing interests
The authors declare no competing interests. Authors' contributions HXR designed the study, performed sequence alignments and drafted the manuscript.
Viral nucleic acids were detected by HL and NNL, and viral isolation was performed by HXR, YJL and SCZ. JY designed the study and revised the manuscript. Figure 1 Phylogenetic tree of eight segments of influenza virus H3N2. Black dot represent Qinghai isolates, black circle represent strains from other areas of the same period, black triangle represent the northern hemisphere vaccine strains recommended by WHO.