No influenza type B was identified in this study. Agarose gel electrophoresis of RT-PCR products are shown in figure 1 and ​and2.2. The sensitivity cut-off of RT-PCR was 0.1 ng of total template RNA genome as described previously.17 Figure 1 Agarose gel electrophoresis

of RT-PCR products for influenza typing. Lane1: Negative control, Lane 2-6 & 9-14: clinical samples, Lane 7: influenza type A, Lane 15: influenza type B, Lane 1 & 10: Gene Ruler 100bp (CinnaGen, Iran). Figure 2 Agarose gel electrophoresis of RT-PCR products for influenza A virus subtyping. Lane 1: Negative control, Inhibitors,research,lifescience,medical Lane 2-9: clinical samples, Lane 10: Gene Ruler 100bp (CinnaGen, Iran), Lane 11: A/H1N1, Lane 12: A/H3N2. Sequence and Amino Acid Analysis

All 17 influenza A positive samples were sequenced. The nucleotide and deduced amino acid sequences of the HA1 from 17 isolated samples were compared with other GenBank sequences as well as with current vaccine strains. Based on nucleotide alignments, the Tehran/2008/H1N1 Inhibitors,research,lifescience,medical GSK2118436 isolates had maximum similarity (98.5%) with New South Wales/18/99 isolates and 98% with those of Auckland/176/99, New Caledonia/20/99 and Tehran/7/2006. In the alignment generated based on the HA1 portion amino acid sequences, Tehran/2008/H1N1 isolates demonstrated 4-6 amino acid differences compared with vaccine candidate strain A/Brisbane/59/2007 (table 2). Inhibitors,research,lifescience,medical The Tehran/2008/ H3N2 Inhibitors,research,lifescience,medical isolates showed maximum similarity (100%) with the Nagasaki/N03/2005 strain and 99% with the Brisbane/10/2007. Alignment of the amino acids of the HA protein from these isolates demonstrated one amino acid change with the vaccine strain A/Brisbane/10/2007

(table 3). Table 2 Amino acid substitutions of hemagglutinin gene from Tehran/2008/H1N1 isolates compared with the vaccine strain (A/Brisbane/56/2007) Table 3 Amino acid substitutions of hemagglutinin gene from Tehran/2008/H3N2 Isolates compared with the vaccine strain (A/Brisbane/10/2007) Phylogenetic Analysis Nucleotide sequence Inhibitors,research,lifescience,medical of the HA1 region of the Tehran/2008/H1N1 and Tehran/2008/H3N2 isolates were compared with the vaccine strains and other influenza viruses, and their genetic relationships were considered by neighbor joining analysis with 1000 bootstrapped replicates. These analyses revealed that our H1N1 isolates were linked with A/Brisbane/59/2007 until vaccine strain and also with the Iranian isolates from previous years that all clustered in a distinct clade with 98% bootstrap value (figure 3a). Moreover, phylogenetic analysis showed that our H3N2 isolates and Nagasaki/N03/2005 strain branched in a unique cluster close to A/Brisbane-like vaccine virus, with a 99% bootstrap value (figure 3b). The phylogenetic tree is available at: http://ijms.sums.ac.ir/images/userfiles/Sep%202011/fig1a.jpg http://ijms.sums.ac.ir/images\userfiles\Sep 2011\fig1b.