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In silico analysis and molecular characterization of Influenza A (H1N1) pdm09 virus circulating and causing major outbreaks in central India, 2009–2019

Background and Objectives: Influenza A/H1N1pdm09 causes respiratory illness and remains a concern for public health. Since its first emergence in 2009, the virus has been continuously circulating in the form of its genetic variants. Influenza A/H1N1pdm09 surveillance is essential for uncovering emerging variants of epidemiologic and vaccine efficacy. The present study attempts in silico analysis and molecular characterization of Influenza A (H1N1) pdm09 virus circulating and causing major outbreaks in central India during 2009–2019. Materials and Methods: We have investigated the antigenic drift analysis of 96 isolates’ hemagglutinin (HA) gene sequences (59 central Indian and 37 local Indian and 28 global reference HA gene sequences) of Influenza A/H1N1pdm09 viruses from 2009 to 2019. The study includes mutational (Multiple sequence Alignment), phylogenetic (Maximum Likelihood Method), and statistical analysis (Covariance and correlation) of HA sequences submitted in NCBI, IRD and GISAID from central India. Results: Phylogenetic analysis indicated maximum clustering of central Indian HA gene sequences in genogroup 6B. Analysis of amino acid sequence alignment revealed changes in receptor binding site (RBS). The frequency of S220T amino acid substitution was found to be high followed by S202T, K300E A273T, K180Q. The Karl Pearson correlation coefficient (r) and covariance between the number of mutations and the death toll was found +0.246 and +100.3 respectively. Conclusion: The study identifies the continuous genetic variations in the HA gene sequences of circulating Influenza A/H1N1pdm09 in central India from the year 2009 to 2019. Further suggesting importance of monitoring the gradual evolution of the virus with regards to an increase in virulence, pathogenicity and vaccine efficacy timely.


INTRODUCTION
Influenza A (H1N1) pdm09 is one of a subtype of Influenza A viruses of Orthomyxoviridae family, like, influenza B and C which infects only humans, influenza A infects avian and mammalian hosts (5). Some aquatic avian species are the natural reservoir of all influenza A subtypes (6.) On the other hand, swine/pigs are susceptible to both avian and mammalian influenza viruses and act as an intermediate host and play an important role in the epidemiology and evolution of the virus (6).
In India, the first positive case of Influenza A (H1N1) pdm09 was reported in 2009 from Hyderabad and continued to spread across the country (12,13). Thereafter, the virus reappeared in the country during 2012-2013 with increased morbidity and mortality (14). The latest re-emergence in 2014-2015 caused the outbreak in the country with the highest human mortality (14). In central India, Madhya Pradesh has reported 5.74% positive cases in 2009 and 12.27% in the 2015 outbreak (15). So far, from 2010 to 2019 total of 4,790 positive cases and 905 deaths from central India have been recorded (15). Further continuous circulation of Influenza A (H1N1) pdm09 virus in the human population in the last decade (2009-2019) has resulted in its 8 genetic genogroups (4,16). These data indicate the increasing virulence and pathogenicity of the virus with the time, mainly attributed to the continuous antigenic shift and drift in HA and/or NA genes of the virus (17).
Vaccination is the principal strategy for the prevention of the disease caused by the influenza virus (18). The surface protein HA helps the virus to attach and evade the host cell, thereby have become the prime targets of the host's neutralizing antibodies (19). Hence, the Influenza vaccine comprises HA antigens from A (H3N2), A (H1N1) pdm09, and one lineage of B (trivalent vaccine) or both lineages (quadrivalent vaccine) (20). However, accumulation of amino acid mutations coupled with N-linked glycosylation at epitope sites diminishes the antibody recognition leading to reduced vaccine efficacy and intermittent seasonal epidemics (21). In 2010, the World Health Organization (WHO) recommended using A/California/7/2009 as the vaccine component for A (H1N1) pdm09 virus (22). Due to the emergence of antigenic drift variants, it was later replaced in 2017 with A/Michigan/45/2015-like virus (23).
Hence, the routine influenza surveillance and continuous monitoring of the genetic changes in the major antigenic sites of these viruses are needed and are important for improving the efficacy of the vaccine for the slowly altering variants of the influenza virus.
The present study aims to investigate the in-silico analysis for antigenic and phylogenetic aspects of influenza A (H1N1) pdm09 viruses circulating in central India during 2009-2019, when there was increased evolution of the virus, focusing on the hemagglutinin protein (HA).

MATERIALS AND METHODS
Sequence retrieval. A total of 96 influenza A (H1N1) pdm09 HA gene sequences from the period of 2009 to 2019 retrieved from the Gen Bank, IRD (Influenza Research Database), GISAID (Global Initiative on Sharing All Influenza Data databases) in FASTA format (Table 1). Out of 96, 59 were central Indian sequences, and rest was reference sequences (27 global and 10 local reference sequences). Both partial and complete HA sequences used in the study.
Phylogenetic analysis. All the 96 HA gene sequences used for phylogenetic analysis. Both partial and complete nucleotide sequences of all the isolates aligned using CLUSTAL-W program. After alignment, the deduced HA sequences trimmed from both sides in Bioedit software and consensus sequences of 688 base pair of HA sequences analyzed to infer the evolutionary history through phylogenetic analysis. The phylogenetic tree generated by using the Maximum likelihood method and Tamura-Nei method of nucleotide substitution implemented in the MEGA X particular year, we couldn't include it in calculation ( Table 2).

Formula for calculation of Karl Pearson Coefficient of Correlation
Formula for calculating Co-variance  Table 3). The intergenogroup antigenic divergence investigated with regards to genetic changes in HA gene of H1N1 viruses. Analysis of amino acid sequence alignment revealed changes at two positions (T151A, D239G) at RBS of HA between genogroup 2 and 3, three positions (A151T, S200P, S202T) between genogroup 3 and 4, two positions (N114D, E279G) between 4 and 6A, four positions (N101S, Q180K, G279E, E300K) between 6A and 6B, three positions (S101N, K180Q, I251V) between 6B and 6C genogroup, four positions (G101S, T214A, V251I, K300E) between 6C and 7 genogroup (Table 4).   I  I  I  I  I  I  I 172 T  T  T  T  T  T  T   221  S  S  S  S  S  S  S   233  I  I  I  I  I  I  I 239 T  T  T  T  T  T  T   266  T  T  T  T  T  T  T 273 Frequency of variation of amino acid. Alignment of 59 HA protein sequences during the period of 2009 to 2019 was investigated to know the frequency of variation of amino acid at different key positions (151, 155, 166, 172, 175, 177, 179, 180, 181, 200, 202,  214, 216, 220, 221, 233, 239, 245, 246, 251, 262, 266,  273, 279, 300) of HA protein. The study included both partial and complete sequences and to keep up homogeneity, amino acid from position 151 to 300 used in study which revealed S220T highly frequent followed by S202T, K300E, A273T, K180Q and so on (Fig. 2).
Correlation and co-variance. Correlation between annual death toll and number of amino acid substitutions was calculated. The Karl Pearson Coefficient of correlation (r) came out +0.246. The co-variance between number of amino acid substitution and death toll came out +100.3.

DISCUSSION
Since its emergence in 2009, Influenza A/H1N-1pdm09 is causing menace continuously (2). During 2009-2019, India saw a varied number of positive cases and deaths due to many factors (15). One of them is the mutations accumulated in the HA protein of Influenza A/H1N1pdm09 virus (21). HA protein is present as H0 which cleaved into HA1 and HA2 by the host cell (30). Influenza A/H1N1pdm09 HA1 subunit contains five antigenic sites Sb, Ca1, Ca2 and Cb (30). In addition to N-glycosylation, mutations in these sites change the antigenicity of influenza A/ H1N1pdm09 HA1 and generate different variants that escape the neutralizing antibodies (21). In silico analysis (Phylogenetic analysis, mutational analysis) of HA amino acid sequences is a convenient method to study genetic variations which leads to evolution (10,16).
We performed intergenogroup antigenic divergence investigation (by using Multiple sequence alignment and comparing the amino acid substitutions in HA gene sequences between different genogroups found in central India at different positions described in results) during the period of 2009-2019. It revealed many changes which showed that virus is mutating continuously comparable to other studies (4,35). As such, intergenogroup antigenic divergence investigation is not reported from studies on H1N1 influenza virus, but can observed in H5N1 influenza virus studies (26). Amino acid substitutions (S200, S202, D202, A214, I233) in receptor binding site envisaged to vary during adaptation process to α2-6-linked sialic acids receptors of human (42). Out of these, 4 sites were found mutated (S200, S202, A214, I233) in our study as similar to study conducted elsewhere (33). I223T amino acid substitution linked to increased binding affinity to human α2-6-linked sialic acids receptors (33). S200P and S202 T substitutions are responsible for enhancement of receptor-binding avidity whereas A214T substitution linked to decrease binding avidity (43).
Frequency distribution of amino acid substitution from 2009 to 2019 at different key positions of HA protein among 59 studied H1N1 viruses isolated in Central India resulted that S220T (100%) amino acid substitution was highly frequent similar to study conducted in MEENA (33) (Fig. 2). These substitutions have significant implications as they appeared in receptor binding site. K180Q substitution triggers conformational variation to ligand binding which might important for virulence (38). S179 N associated with glycosylation is responsible for enhanced pathogenicity of virus by prevention of antigenic sites for immune recognition (33). D239 amino acid substitution has deleterious effect on HA (33). S200P alter receptor binding affinity. S181 leads to changed glycan specificity (38). The amino acid substitutions such as P100S, S101N, D114N, K180Q, S181T, S202T, S220T, I233T, A273T, K300E present in the isolates from Central India in the HA gene are also reflected in the recent studies from other parts of India and world (38,39,44). Number of positive cases in the summer in central India, also shows that, the virus is getting more heat -resistant which might be due to the antigenic drift in H1N1 virus (45).
In our study, we reported a conceivable description for correlation between number of deaths and number of amino acid substitutions in HA gene of Influenza A/H1N1pdm09 virus in central India which may direct towards mortality. However in-depth lab study required for making these results operative. This type of study is yet to be enunciated in the literature. We calculated correlation and co-variance (Karl Pearson coefficient of correlation and Co-variance) of number of amino acid substitutions and number of deaths in central India. Positive correlation and co-variance indicated that there is a correlation between number of deaths and number of mutations in HA protein. Very less researches are in line with the possible cause of mortality. However, Wu et al. recognized quantitative relationship between amino acid substitution of human influenza virus and mortality. They reported positive correlation between mortality and antigenic distance to the first antigenic strain (46). Adam et al. (2019) reported the qualitative relationship of amino acid substitution and mortality. According to him, S202T and D239G are responsible for increased mortality and morbidity (47). However, the factor affecting mortality also includes age, gender, severity of infection, mutations in other genes of influenza like NA and PB2 (47)(48)(49).
There are limitations in this study. Due to unavailability of HA gene sequences submitted to the Gen-Bank from 2018, 2016 and 2014, the original evolution of Influenza A/H1N1pdm09 in central India is not completely explicit in this in-silico analysis. Moreover, unavailability of complete sequences of all HA gene is also a limitation for extensive molecular analysis. In addition to number of mutations in HA gene, mortality also depends on other factors described above. Therefore, consideration of other factors and in-depth wet lab study is required for making correlation perspicuous between mutations and mortality. Less data on HA gene sequences prevent investigating original genetic diversity of Influenza A/H1N1pdm09 in the region. This study has provided some direction towards changes that are occurring in the HA gene sequences of circulating Influenza A/H1N1pdm09 in the region and surely help scientific community to compare and further analyze their generated results to correlate findings about its outbreak.

CONCLUSION
In conclusion, these in silico findings direct a quickly changing Influenza A (H1N1) pdm09 virus during 2009 to 2019 in central India emphasizing the prerequisite for continuous surveillance together with molecular and antigenic analyses, to recommend suitable and proper influenza vaccine update.
Additionally, detailed laboratory studies is needed for making correlation clearer between mutations and mortality.