Preparation of SmartAmp primers to detect the HA segment of the 2009 pdm influenza A(H1N1) virus.

<p>A. Mutation rate and difference score in the consensus sequence of the HA segment. Nucleotide sequences of the HA segment of 2009 pdm influenza A(H1N1) viruses were obtained from the NCBI Influenza Virus Resource database and aligned by using the MUSCLE program to gain the consensus sequence of the HA segment. The mutation rate at each base position was calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030236#s4" target="_blank">Materials and Methods</a>. The difference between 2009 pdm and seasonal A(H1N1) viruses was calculated at each position in the nucleotide sequence of the HA segments to gain the difference score. B: Comparison of data acquired in 2009 and 2011 as to the mutation rates in the HA segment of the 2009 pdm influenza A(H1N1) viruses.</p

Time after the onset of fever and the number of patients who were diagnosed by the RT-SmartAmp assay as positive for infection with the 2009 pdm influenza A(H1N1) virus.

<p>Time after the onset of fever and the number of patients who were diagnosed by the RT-SmartAmp assay as positive for infection with the 2009 pdm influenza A(H1N1) virus.</p

Detection of the 2009 pdm influenza A(H1N1) virus by RT-SmartAmp assay in the fatal case.

<p><b>A:</b> Nasopharyngeal swab samples were collected at 11, 28, and 52 hours after the onset of fever from the patient who was transferred by ambulance to the National Center for Global Health and Medicine. The 2009 pdm influenza A(H1N1) virus was immediately detected by the RT-SmartAmp assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030236#s4" target="_blank">Materials and Methods</a>. <b>B:</b> Chest radiography of the patient was taken at 11 and 28 hours after the onset of fever. <b>C:</b> Partial sequence of the HA segment of the 2009 pdm influenza A(H1N1) virus was analyzed after extraction of viral genome RNA from the swab samples. An arrow indicates the mutation that caused an amino acid substitution at 185 from aspartate to asparagine (N) in the HA protein.</p

Comparison of results detected by the rapid diagnosis kits and the RT-SmartAmp assay.

<p>As depicted in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030236#pone.0030236.s003" target="_blank">Figure S3</a>, two swab samples were collected as one pair from each patient. Swab samples were separately used for the rapid influenza diagnostic tests and RT-SmartAmp assay. All the samples were transferred to RIKEN Yokohama Institute for sequence analysis and RT-Q-PCR detection.</p

Detection of the 2009 pdm influenza A(H1N1) virus by the RT-SmartAmp assay in the oseltamivir-resistance case.

<p><b>A:</b> Images of the head CT scanning taken on hospital day 1 (left) and day 13 (right). <b>B:</b> Chest radiography of the patient taken on hospital day 1 (left) and day 5 (right). <b>C:</b> The RT-SmartAmp assay with tracheal fluid (○) and nasopharyngeal swab (•) samples collected on hospital day 9. This figure depicts the time courses of the RT-SmartAmp assay reactions with those samples as well as with positive (▴) and negative (▵) controls. <b>D:</b> Partial sequence of the NA segment of the 2009 pdm influenza A(H1N1) virus was analyzed after extraction of viral genome RNA from the swab samples. An arrow indicates the mutation that caused an amino acid substitution at 275 from histidine (N) to tyrosine (Y) in the NA protein.</p

The sequence of nucleotides amplified by the SmartAmp reaction and primer annealing sites.

<p><b>A:</b> cDNA encoding the partial sequence of the HA segment of the 2009 pdm influenza virus and primer annealing sites. <b>B:</b> Schematic illustration of annealing sites of the TP, FP, OP1, OP2, and BP primers. The size of each annealing site is numerically indicated as the number of base units (b).</p

SmartAmp primers designed for detection of the 2009 pdm influenza A(H1N1) virus.

<p>TP: The turn back region is underlined.</p><p>FP: The folding region is underlined.</p><p>BP-Ex: The Exciton dye is covalently bound with thymidine that is marked with a bold “Z”.</p

RT-SmartAmp detection of the 2009 pdm influenza A(H1N1) virus with different dilutions as well as the cross activity with seasonal A(H1N1), seasonal A(H3N2), and B(Victoria) viruses.

<p>Isolated and cultured influenza viruses, <i>i.e.</i>, 2009 pdm A(H1N1), seasonal A(H1N1), seasonal A(H3/N2), and seasonal B/Victoria, were prepared at a viral titer of 10⁷ pfu/ml. Each viral sample (10 µl), except for the 2009 pdm influenza A(H1N1) virus, was mixed with 90 µl of the pretreatment medium (5% SDS) to dissolve the viral membrane and to facilitate viral RNA extraction. A sample (15 µl) of the resulting medium was subjected to spin column chromatography, and the eluted solution (5 µl) was applied to the RT-SmartAmp reaction mixture. In the case of the 2009 pdm influenza A(H1N1) virus, the viral sample was diluted by 10³-, 10⁴-, or 10⁵-fold as indicated in the figure, and then processed in the same manner as described above. The RT-SmartAmp assay was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030236#s4" target="_blank">Materials and Methods</a>.</p