The RT-PCR methods were optimized previously.3 Two sets of primers were used to distinguish type of viruses: the matrix protein gene of influenza A virus (M-A) and nucleoprotein gene of influenza B virus (NP-B). Primers designed for the hemagglutinin glycoprotein gene of influenza

A/H1N1 and A/H3N2 viruses (H1-A and H3-A) were used for subtyping. These primers were designed from conserved and consensus regions of about 30 different relevant isolates retrieved from GenBank database using multiple alignments.3 The reaction mixture contained 6 µl of the sample’s cDNA, 12.5 µl of master mix containing 1x PCR buffer, 1.5 U Taq polymerase enzyme (CinnaGen), 1.5 mM MgCl2, 0.2 mM dNTPs mix (Fermentas, Vilnius, Inhibitors,research,lifescience,medical Lithuania), and 0.5 µM Inhibitors,research,lifescience,medical of each appropriate primers (CinnaGen) shown in table 1. Sterile, distilled water was added to reach a final volume of 25 µl. The PCR conditions were 95°C for 5 min, followed by 35 AC220 cost cycles of 94°C for 40 sec, 63°C (for MA and NP-B primers annealing) or 58°C (for H1and H3 primers annealing) for 40 sec, 72°C for 40 sec and a final extension at 72°C for 5 min. Gel Inhibitors,research,lifescience,medical electrophoresis of the PCR products using 2% agarose gel and ethidium bromide staining was performed. Table 1 Primers used for the typing and subtyping of influenza viruses Sequencing and Phylogenetic Analysis

All 17 subtyped positive samples were assessed for molecular characterization of HA1 gene. Gene sequencing and phylogenetic analysis were carried out for H1 (543 bp) and H3 (292 bp) fragments from influenza A virus. The resulting amplicons of HA1 fragment from H1 and H3 genes of the isolates were cleaned up followed by sequencing in both directions Inhibitors,research,lifescience,medical which was performed on ABi 3730×1 genome analyser (Source BioScience, UK). Alignment of H1 and H3 gene sequences from Iranian isolates with about 60 H1 and H3 gene sequences as reference was performed by CLUSTALX software, version 1.81.12 Genetic distance was calculated using the Kimura two-parameter

matrix.13 The neighbor-joining Inhibitors,research,lifescience,medical method was used to construct phylogenetic trees.14 Bootstrap analysis (n=1,000) was performed to confirm the reliability of phylogenetic tree.15 Molecular Evolution Genetic Analysis (MEGA) computer software, version 4,16 was utilized in this study for phylogenetic and molecular evolutionary analysis and nucleotide differences see more within and between the isolate sequences. Nucleotid GenBank Accession Numbers The nucleotide sequences determined in this study have been submitted to GenBank under the following accession numbers: “type”:”entrez-nucleotide-range”,”attrs”:”text”:”HM346544 -HM346560″,”start_term”:”HM346544″,”end_term”:”HM346560″,”start_term_id”:”297185520″,”end_term_id”:”297185552″HM346544 -HM346560. Results The molecular typing and subtyping of the isolates revealed that 50 out of 142 samples were positive for human influenza A virus.