Transmission of pandemic Influenza virus is persisting in many continents but current activity levels are low in Asia. Recent peaks in the activity were noted during early 2010 in northern India, Nepal and Sri Lanka. Influenza activity remained stable but elevated in western India, continued to decline substantially in Northern India, and remained low overall in Southern and Eastern India 
. This virus was generated by multiple reassortment events, and each of its precursor gene segments has circulated in swine for more than 10 years. Infection of swine with H1N1/2009 virus has been observed in multiple countries. But, because of a paucity of systematic surveillance of swine influenza worldwide the question remains whether H1N1/2009 will become established in swine and become a reservoir of reassortment that may produce novel viruses of potential threat to public health 
. The H1N1/2009 virus has remained antigenically and genetically stable and are relatively low virulence in humans since its detection in April 2009. Most genetic changes in H1N1pdm to date have not been clearly linked to changes in antigenicity, disease severity, antiviral drug resistance, or transmission efficiency. However, rapid evolution rate characteristic of influenza viruses suggest that changes in antigenicity are inevitable in future 
. With the number of reported pandemic cases of H1N1 virus in many parts of the world and continued viral persistence in India and nearby countries (Nepal, Sri Lanka, Bangladesh), elevated activity has given an urgent need to track the global dispersion of this virus in humans.
In this particular study, the main focus was complete genome characterization of the circulating isolates of northern India (Gwalior region) and to decipher conservative and non conservative substitutions, its comparative analysis with respect to other Indian and global circulating H1N1pdm isolates. The continued circulation of virus in particular region from 2009-till date is also a serious concern and required in depth investigation. With the determined objective of molecular investigation of circulating H1N1pdm virus, Influenza like illness (ILI) in suspected clinical samples from Gwalior, India during 2010–2011 were investigated. The clinical picture of the patients revealed the same pattern as was reported in 2009 
but there was an increase in number of H1N1pdm cases in 2010. It was revealed during the study that the virus has affected all the age groups with the highest in young age group. The numbers of females were affected more than males during the period under observation. Fatality ratio (5.83%) was found prominently high in young persons. Young groups have least experience of influenza A (H1N1pdm) virus and are recognized as potential source in the transmission of influenza. It is also possible that propensity to consult doctor is greatest in younger age groups. However, in 2011 the numbers of positive cases were higher in young age group of 18–28 Yr. The possible reason of higher cases in 2011 may be increase in viral virulence and its better adaptation in the region, which may become severe in the coming years.
In this study four Indian isolates that are confirmed by virus specific CPE, HA, IFT as well as CDC Real time RT-PCR were selected for complete genome characterization. The nucleotide sequence analysis revealed that there is no significant difference among viruses recovered from two different places and of different years from India. Diversity of the Indian isolates at the amino acid level with respect to the prototype strain and within the Indian isolates was found to be maximum in the HA and NP gene. Substitution S220T (HA) specific to clade VII isolates was adequate to lineate the isolates in HA-based phylogeny. Most of the amino acid changes were conservative, involving interchanges of amino acids having same physicochemical properties. However, few major non-conservative changes between Indian isolates were also observed. Compared to the prototype strain, glutamic acid was replaced by a strongly basic amino acid lysine at position 391 (HA) among the four Indian H1N1pdm virus and at the position 71 (NS1) in one Indian H1N1pdm virus sequenced in this study. Two important non conservative substitutions involving acidic aspartic acid to basic histidine at position 441(PB2) in two Indian H1N1pdm virus and cyclic proline to acyclic serine at position 100(HA) among the four Indian H1N1pdm virus were also recorded. Similar non conservative substitutions involving shift in amino acids were also recorded in other gene segments. However, the significance of these substitutions need to be addressed.
To identify genetic lineage of H1N1pdm virus, phylogenetic analysis was conducted for concatenated whole genome sequences retrieved from GenBank from 2009–2012 including all the available H1N1pdm whole genome from India sequenced till date. Whole genome and full HA based phylogenetic analysis revealed existing seven discrete clades of H1N1pdm virus circulating globally. Both the trees based on genome information comprised of all representative H1N1pdm clades from diverse geographical origin which included maximum number of representative H1N1pdm from all the affected areas. Both the trees yielded similar topologies, with characteristic distribution of H1N1pdm isolates into seven distinct clades. Maximum numbers of isolates were grouped into clade VII. The clade I included prototype California/04 and California/07 virus isolated first during H1N1pdm 
. All Indian isolates (2009–2011) were grouped in clade VII except Hyd/NIV51/2009 and Pune/NIV6196/2009, Pune/NIV10604/2009 (HA gene phylogeny) virus isolated during initial pandemic phase grouped into clade V and VI respectively 
. Clade VII is identified as predominant circulating clade in India, Asia as well as globally 
. Phylogenetic analysis of all Indian H1N1pdm complete genome sequenced so far demonstrated that earliest isolate from Hyderabad (A/India/Hyd/NIV51/2009) during initial pandemic phase was a clade V isolate. Two other isolates from Pune during later pandemic phase (A/India/pune/NIV6196/2009, A/India/pune/NIV10604/2009) belonged to clade VI. Both the cases were not directly associated with any foreign travel history that is why it is not clear whether the clade evolved within the country or were imparted into the country. All other Indian isolates from last pandemic phase to post pandemic phase belonged to clade VII. Two initial Indian isolates belonging to clade VII had a foreign travel history and thus may be indicative of the fact that clade VII was introduced from an external source 
. Therefore it may be possible clade VII is favourably selected as dominant H1N1pdm lineage in India.
Influenza viruses comprise of segmented viral genome, and are more prone to genetic reassortment during mixed infections. Hence the circulating H1N1pdm strains also evolve and may favourably be selected with higher fitness at a particular time point. It is most likely that the H1N1pdm strains were also undergone similar evolutionary process and the viruses of higher fitness were favourably selected over time. The selection pressure analysis revealed 18 positively selected sites in major surface proteins of Influenza A (H1N1pdm) virus i.e. HA, NA and matrix proteins. Since these proteins plays crucial role in the attachment, assembly, release of the virus, these substitutions might have played important role in making these isolates more transmissible. Differential selection analysis also supported the pandemic 2009 strains being subject to distinctive selection compared to their progenitors 
. The results indicated HA gene may experience stronger positive selection compared to NA and matrix gene in process of adaptation to the human population globally. Out of 18 positive selected sites, the S220T (HA; found in Indian isolate) and I30V (NA; found in global isolate) were also reported in previous studies as clade VII specific markers 
. Position A151T/V and R222K, are situated within A and D epitopic regions of HA and is also associated with receptor binding 
. Since HA plays a crucial role in virus attachment, these substitution might have played an important role in virus transmission.
The present study is the first systematic study carried out to characterize the true genetic nature of recently circulating Indian H1N1pdm virus in post-pandemic phase. This study clearly indicates that the cosmopolitan clade VII is predominant in India. Few reported Clade VII markers revealed in this study indicates that the clade is undergone positive selection during virus evolution since last 3 years and a shift to clade VII in Indian isolates was observed from other circulating clades during 2009–2012. The complete genome information of recent H1N1pdm Indian virus isolate elucidated for the first time in this study will serve in future epidemiological surveillance in Indian subcontinent and abroad.