Pandemic influenza A(H1N1)pdm09 virus is usually a global health threat and between 2009-2011 it became the predominant influenza virus subtype circulating in the world. or the presence of small genetic variants the isolates were further analyzed. Bearing in mind the crucial part of hemagglutinin mutations for influenza computer virus virulence representative gene fragments encompassing nucleotides 125 to 302 were amplified from cDNAs. This region corresponds to a fragment of influenza computer virus HA1 polypeptide starting 25 amino acids (H1 numbering is used throughout this paper) after the N-terminal transmission peptide of hemagglutinin. A crucial part of the influenza A/H1N1 epitope reacting with neutralizing antibodies is located within this region. To check for the presence of small genetic variants of the A(H1N1)pdm09 pandemic strains within the acquired amplicones there was performed MSSCP (Multitemperature URB597 Solitary Strand Conformation Polymorphism) analysis. Table?1. Sample information and medical symptoms of flu illness URB597 among A(H1N1)pdm09 Taiwan individuals MSSCP is definitely URB597 a native electrophoretic separation performed under sequentially changed gel heat. This enhances the level of sensitivity of mutation detection and reduces time of analysis. The temperature changes increase the probability for the PCR products to adopt different ssDNA conformations during the electrophoretic run if they contain nucleotide substitutions All the amplified fragments including related fragments of the research seasonal (s) (A/Brisbane/59/2007) and pandemic (p) (A/Mexico/4486/09) strains were denatured and the producing ssDNA fragments were subjected to the native electrophoresis in ideal conditions for the MSSCP analysis (15-10-5 °C 450 Vxh/per phase 10 polyacrylamide gel). Results of this experiment (after visualization with metallic stain) are demonstrated in Number?1. According to the electrophoretic profiles (Fig.?1) none of the samples contains fragments corresponding to the predominant influenza A seasonal strain (s) which excludes the possibility of co-infection with seasonal and pandemic strains. Samples designated as 2009-02626 2009 2009 2010 2010 2011 2011 2009 2009 2009 2009 2011 2011 and 2011-04611 exhibited MSSCP profiles identical to the research pandemic strain while the electrophoretic profiles of five samples: 2010-03994 2011 2010 2010 and 2010-05347 URB597 were different from that of the pandemic research strain. For further analysis if profiles reflected unique DNA sequences ssDNA bands from your samples indicated by arrows in Number?1 were extracted from your gel re-amplified and the PCR products were Sanger sequenced. Additionally the research pandemic ssDNA bands were analyzed in the same manner. Number?1. New genetic variants among A(H1N1)pdm09 isolates collected at Taiwan between 2009-2011 recognized by MSSCP genotyping. RT-PCR products of hemagglutinin gene from pandemic Taiwan A(H1N1)pdm09 computer virus isolates as well as … Sanger sequencing of the ssDNA bands confirmed that 14 out Rabbit Polyclonal to LFNG. of the 19 analyzed samples were identical with the A(H1N1)pdm09 pandemic strain reference sequence (Table 2). For the five samples with electrophoretic profiles different from the research strain Sanger sequencing exposed the presence of many point mutations. Schematic representation of all recognized mutations and their localization within analyzed HA amplicone are offered in Number?2. Sample 2010-03994 URB597 contained two point mutations 2011 – eight 2010 – three 2010 – seven and 2010-05347 – five. Six mutations were present in more than one sample (Fig.?2) and nine were unique to solitary isolates. It seems unlikely that mutations arose during the short passages of the original computer virus from swabs in MDCK cells. Table?2. Genetic diversity of HA gene fragment in Taiwan A(H1N1)pdm09 isolates Number?2. Schematic representation of genetic diversity of hemagglutinin (HA) sequence in five Taiwan isolates of A(H1N1)v pan09 strain. Black arrows above and below A(H1N1)v pdm09 research sequence indicate altered DNA codons. Red letters show … DNA codons comprising detected point mutations were translated to amino acids and compared with the pandemic research sequence. Furthermore their physico-chemical properties and localization within HA.