used in both the northern and southern hemispheres, indicating that these viruses may show less cross-reaction to the current vaccine strain (Winter et al., 2009; Canton et al., 1982).
Mutations Glu627Lys and Asp701Asn of the PB2 gene that are thought to be associated with adaptation to mammals and increased virulence of influenza viruses in mice were not present (Hatta et al., 2001; Gabriel et al., 2008; Le et al., 2009; Steel et al., 2009; Obenauer et al., 2006; Jackson et al., 2008). The C terminal of the NS1 gene is truncated in the A/California/04/2009-like viruses and four residues of the PDZ ligand domain are not present (Obenauer et al., 2006).
Viral RNA was directly extracted from infected allantoic fluid or cell culture using QIAamp viral RNA minikit (Qiagen, Inc., Valencia, Calif.). cDNA were synthesized by reverse transcription reaction and gene amplification by PCR were performed using specific primers for each gene segments. PCR products were purified with the QIAquick PCR purification kit (Qiagen Inc.) and sequenced by synthetic oligonucleotides. Reactions were performed using Big Dye- Terminator v3.1 Cycle Sequencing Reaction Kit on an ABI PRISM 3700 DNA Analyzer (Applied Biosystems) following the manufacturer’s instructions. All sequences were assembled and edited with Lasergene version 6.1 (DNASTAR, Madison, WI). Full genome sequences of these viruses are available for download at GISAID under the accession numbers (EPI177540 to EPI77658 and EPI177947).
Barr, I. G., Deng, Y. M., Iannello, P., Hurt, A. C. & Komadina, N. Adamantane resistance in influenza A (H1) viruses increased in 2007 in South East Asia but decreased in Australia and some other countries. Antiviral Research 80, 200–205 (2008).
Canton, A., Brownlee, G. G., Yewdell, J. W. & Gerhard, W. The antigenic structure of the influenza virus A/PR/*/34 hemagglutinin (H1 subtype). Cell 31, 417–27 (1982).
Gabriel, G. I., Herwig, A. & Klenk, H.–D. Interaction of polymerase subunit PB2 and NP with importin a1 is a determinant of host range of influenza A virus. PLoS Pathog. 4, e11 (2008).
Hatta, M., Gai, P., Halfmann, P. & Kawaoka, Y. Molecular basis of high virulence of Hong Kong H5N1 influenza A viruses. Science 7, 1840–1842 (2001)
Le, Q. M., Sakai-Tagawa, Y,, Ozawa, M., Ito, M. & Kawaoka, Y. Selection of H5N1 Influenza Virus PB2 during Replication in Humans. J. Virol. 83, 5278–5281 (2009).
Jackson D, Hossain MJ, Hickman D, Perez DR, Lamb RA (2008) A new influenza virus virulence determinant: The NS1 protein four C-terminal residues modulate pathogenicity. Proc. Natl. Acad. Sci. USA 105, 4381–4386 (2008).
Obenauer, J. C. et al. Large-Scale Sequence Analysis of Avian Influenza Isolates. Science 311, 1576–1580 (2006).
Rogers, G. N. et al. Single amino acid substitutions in influenza haemagglutinin change receptor binding specificity. Nature 304, 76–78 (1983).