Full genomic analysis of an influenza A (H1N2) virus identified during 2009 pandemic in Eastern India: evidence of reassortment event between co-circulating A(H1N1)pdm09 and A/Brisbane/10/2007-like H3N2 strains

Background During the pandemic [Influenza A(H1N1)pdm09] period in 2009-2010, an influenza A (Inf-A) virus with H1N2 subtype (designated as A/Eastern India/N-1289/2009) was detected from a 25 years old male from Mizoram (North-eastern India). Objective To characterize full genome of the H1N2 influenza virus. Methods For initial detection of Influenza viruses, amplification of matrix protein (M) gene of Inf-A and B viruses was carried out by real time RT-PCR. Influenza A positive viruses are then further subtyped with HA and NA gene specific primers. Sequencing and the phylogenetic analysis was performed for the H1N2 strain to understand its origin. Results The outcome of this full genome study revealed a unique reassortment event where the N-1289 virus acquired it’s HA gene from a 2009 pandemic H1N1 virus with swine origin and the other genes from H3N2-like viruses of human origin. Conclusions This study provides information on possibility of occurrence of reassortment events during influenza season when infectivity is high and two different subtypes of Inf-A viruses co-circulate in same geographical location.

sequences of the HA1 gene for H1N1 and H3N2 strains showed that a subset of the circulating Kolkata strains precede the WHO recommended vaccine strains by 1-2 years [5]. Prior to pandemic of 2009, neuraminidase inhibitors were rarely used for influenza prophylaxis or treatment in India which could be the probable reason for drug sensitive strains; no genetic indication of mutations conferring neuraminidase drug resistance in the H3N2 (aa119, 152, 274, 292 or/and294) or in the H1N1 (aa275) viruses was observed during 2005-08 in eastern region of India [5]. However in 2009, oseltamivir resistant seasonal H1N1 viruses emerged with H275Y substitution [6][7][8]. Interestingly, inspite of oseltamivir   [11].
In the present study, we are reporting detection and molecular characterization of a H1N2 strain (designated as A/Eastern India/N-1289/2009); detected from a 25    years old male in North-eastern India who was suffering from fever, and acute respiratory tract infections (ARTIs) and had histories of close contact with a person suffering from influenza A(H1N1)pdm09. Since H1N2 is not common in this region, all gene segments were sequenced to characterize the virus.

Sample collection and screening
Nasal and/or throat swabs of hospitalized cases were referred from different hospitals in eastern and northeastern India during the pandemic period (June 2009-Dec 2010) [12,13]. The informed consent forms and detailed case histories were recorded before collection of sample. The study was approved by the Institutional Ethical Committees, NICED (Ethical Clearance No. A-1/ 2009-IEC). Initial detection of influenza viruses, was carried out as described previously [14]. The H1N2 virus characterized in this study was identified from a referred sample of a patient with severe respiratory infection in Mizoram (North-eastern India).

Full length amplification and sequencing of viral genes
For sequencing, viral genes were amplified as described earlier [14,15]. Nucleotide (nt) sequencing was carried out in an ABI Prism 3100 Genetic Analyzer (PE Applied Biosystems, Foster City, CA, USA) using gene specific primers. Nucleotide and protein sequence BLAST search was performed using the National Centre for Biotechnology Information (NCBI, National Institutes of Health, Bethesda, MD) Basic Local Alignment Search Tool (BLAST) server on GenBank database release 143.0. [16].

Phylogenetic analysis
Pairwise sequence alignments were performed using LALIGN software. Multiple alignments were done with Clustal W program which is available at DDBJ software. Phylogenetic tree was constructed by the neighborjoining method [17] using the MEGA program, version 4.1.

Gene bank accession number
The nucleotide sequence data were submitted to the GenBank under the accession numbers JQ340003, JQ340004, JQ340005, JQ340006, JQ340007, JQ340008, JQ340009, and JQ340010 for HA, NA, NP, PA, PB1, PB2, Matrix and Non structural genes respectively. The sample was initially classified as influenza A(H1) pdm09, though NA gene could not be amplified using N1 specific primers. To rule out mix infection in patient, primers specific for H3 and N2 were also used, H3 did not amplify, however NA gene was amplified using N2 specific primers. Full length sequencing of HA and NA gene segments confirmed the virus to be of H1N2 subtype. For further genetic analysis, the complete coding sequences of all 8 segments were determined.  (Table 2). Among them two substitution were Figure 6 Phylogenetic comparison based on nucleotide sequence of NP gene with that of vaccine strains and the strains from different countries. Our strain is marked as '•'. The tree was rooted with cognate stretch of strain B/Florida/4/2006 for NP. The tree was created by using neighbor-joining method with 1000 bootstrap replicates. very important. One was located at the antigenic site B (E221K) and the second one was E432Q substitution. No mutation regarding resistance against neuraminidase inhibitor was observed.  Table S1).  Table S1). No mutation regarding amantadine resistance in the matrix protein gene was observed.

Discussion
Influenza A viruses of the H1N2 subtype were isolated previously from humans in India [18] and Japan [19] during the 2001-2002 influenza season. Genetic analysis revealed that they were reassortants of the human H1N1 and H3N2 viruses and that they were apparently distinct from the H1N2 swine influenza viruses. However, inspite of repeated attempts to isolate the recombinant virus (designated as A/Eastern India/N-1289/2009) by inoculation of susceptible cells as well as by egg inoculation, virus could not be isolated. The reason for failure could be loss of viability as the sample came from Mizoram (~1219 kms from Kolkata) by courier which took >48 hrs to deliver it in lab.
Co-circulation of both influenza A(H1N1)pdm09 and seasonal (A/H1N1, A/H3N2) influenza during 2009 and complete disappearance of seasonal A/H3N2 and A/ H1N1 strains in 2010 was observed in eastern India [13]. Such co-circulation is the prime cause of the generation of genetically reassortant viruses. Our study revealed that the H1N2 virus identified during 2009 was reassortant of influenza A(H1N1)pdm09 and H3N2-like viruses of human origin. Phylogenetic analysis of the HA gene revealed that the virus has got it's HA gene from Influenza A(H1N1)pdm09 viruses whereas the virus derived the NA gene and all other gene segments from the H3N2-like viruses (Additional file 2: Table S2). Comparison of antigenic sites of HA1 region of our strain with A/Brisbane/59/2007 (the vaccine strain; which was used to clarify whether the vaccination could provide protection against this virus), A/Eastern India/2499/2009 (one of the influenza A(H1N1)pdm09 strain) and A/India/ 77251/2001(an H1N2 strain isolated in 2001-02 sessions) revealed that there is an insertion of a strong basic amino acid Lysine (K) at position 147. Such insertion might affect electrostatic interactions of HA with its antibodies [20]. The substitution of one nonpolar neutral amino acid with another nonpolar neutral one will not make any difference in making the hydrophobic interaction and proper conformation into the binding pocket Figure 8 Phylogenetic comparison based on nucleotide sequence of matrix protein gene with that of vaccine strains and the strains from different countries. Our strain is marked as '•'. The tree was rooted with cognate stretch of strain B/Florida/4/2006 for HA. The tree was created by using neighbor-joining method with 1000 bootstrap replicates.
[21] but the S152A substitution is polar to nonpolar whereas L174S and A207S show substitution of a nonpolar aa with a polar one. It could be predicted that such shift may lead to the improper hydrophobic interaction and improper conformation into the binding pocket which may affect receptor binding. The aa sequence analysis of the NA gene show more substitutions than the HA1 region, but the important one is located at position 221 where a polar negative aa has been substituted with polar positive aa. Such positivity results in more hydrophobicity of the amino acids located in that particular region of the protein which attracts specific protein regions towards the hydrophobic region. Such type of substitution also occurred at position 18, 42, 50, 267, 370 and 372. However the effects of amino acid substitution in the internal genes are not known.

Conclusion
The strain A/Eastern India/N-1289/2009 is a classical case of sporadic reassortment event occurring during the 2009-2010 influenza season when infectivity is high and different subtypes co-circulate.