Abstract
The pandemic influenza A(H1N1)pdm09 virus was first detected in India in May 2009 and continued to circulate in the postpandemic period. Whole-genome sequence analysis of postpandemic A(H1N1)pdm09 viruses showed the circulation of clade 6 and clade 7 viruses. The hemagglutinin (HA) gene showed increased diversity compared with that in the pandemic phase.
GENOME ANNOUNCEMENT
After the first reported case of infection with the influenza A(H1N1)pdm09 virus in India (1), the virus was found to be in circulation in many parts of the country. A previous report (2) of 31 whole-genome sequences of the A(H1N1)pdm09 virus during a pandemic revealed higher substitution rates than those observed globally. In the postpandemic period, the A(H1N1)pdm09 virus remained in circulation and showed unusually increased activity and severity in March and April, especially in the northern and western parts of the country, for three consecutive years, which is unseasonal for India. The National Institute of Virology in Pune carried out diagnostic and genetic analyses as part of postpandemic surveillance (institutional review board [IRB] approved). Thirteen whole-genome sequences from isolates obtained from April 2011 to April 2013 were studied and compared with the 31 whole-genome sequences from the pandemic period (2) to identify the genomic changes responsible for pathogenesis and drug susceptibility of A(H1N1)pdm09.
During the study period, 6,836 samples were analyzed, and of the 1,675 real-time PCR positives, 1,036 (61.8%) were A(H1N1)pdm09, 242 (14%) were A(H3N2), and 397 (23%) were type B positive. Isolation was carried out with MDCK cells. Thirteen A(H1N1)pdm09 isolates from a period of unusual activity (2 isolates from 2011, 8 isolates from 2012, and 3 isolates from 2013) were selected for whole-genome sequencing. All the eight influenza gene segments were amplified in an overlapping manner by one-step conventional reverse transcriptase PCR (RT-PCR) using the whole-genome primers recommended by the WHO and the CDC (3). Sequencing was carried out by using the BigDye Terminator version 3.1 cycle sequencing ready reaction kit (ABI) and processing for capillary electrophoresis on an ABI 3730 DNA analyzer; sequences were curated using Sequencing Analysis version 5.3, and MEGA version 5.2 was used for sequence alignments.
All gene segments were compared with those of the vaccine component strain A/California/07/2009, along with those of the globally circulating strains. The result showed that A(H1N1)pdm09 viruses of clade 7 (D97N mutation) and clade 6 (A197T mutation) were in circulation. A single isolate from 2013 possessed D222N (D239N) in the receptor binding region of the hemagglutinin (HA) molecule (4). The HA gene mutations P83S, S84G, S183P, S185T, S203T, R223Q, E374K S451N, and I321V observed during the pandemic were also noted in the postpandemic period (1, 2). Newly identified mutations in HA were S143G, K283E, and E499K. Increased diversity in the HA gene was observed from 2011 to 2013. All isolates remained sensitive to neuraminidase inhibitor (NAI) drugs and showed N369K and V241I amino acid changes in the neuraminidase (NA) gene, which may facilitate the stability of resistant viruses (5). Similarly, mutations noticed for the polymerase basic 2 (PB2) gene were D195N, V731I, and N456S; for PB1, the mutations were G154D, I397M, and I435T; for the polymerase acidic (PA) gene, they were V100I and I330V; for M1, K239R; for M2, D21G; for the nonstructural 1 (NS1) gene, L90I, N205S, and I111M/T; and for NS2, T48A and V49M.
Continued molecular surveillance and whole-genome sequencing are important for understanding significant evolutionary changes in this pandemic virus.
Nucleotide sequence accession numbers.
The whole-genome sequences of 13 Indian A(H1N1) pdm09 isolates from the period 2011 to 2013 have been deposited in GenBank under accession no. KF280652 to KF280755; accession numbers are listed in Table 1.
Table 1 .
Accession numbers of whole-genome sequences of A(H1N1) pdm09 isolates
PB2, polymerase basic 2; PA, polymerase acidic; HA, hemagglutinin; M, matrix; NP, nucleoprotein; NA, neuraminidase; NS, nonstructural.
ACKNOWLEDGMENTS
We acknowledge technical support from the NIV, Pune, India.
Financial support was received from the Indian Council of Medical Research, New Delhi.
Footnotes
Citation Dakhave M, Khirwale A, Patil K, Kadam A, Potdar V. 2013. Whole-genome sequence analysis of postpandemic influenza A(H1N1)pdm09 virus isolates from India. Genome Announc. 1(5):e00727-13. doi:10.1128/genomeA.00727-13.
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