Estimate of odds of a 4-fold conversion relative to conversions against the H3N2 virus.

<p>Estimate of odds of a 4-fold conversion relative to conversions against the H3N2 virus.</p

Affinity measurements isotyping and total anti-HA1 binding antibody in human sera following immunization.

<p><b>(A–B) Correlation between in vitro MN titers and rHA1 binding in human sera following immunization with H1N1 vaccine in TIV and LAIV groups.</b> Steady-state equilibrium analysis of the binding of vaccine serum IgG to properly folded functional HA1 oligomers was measured using surface plasmon resonance (SPR). Ten-fold diluted post-H1N1 vaccination sera from vaccine groups (LAIV in A, TIV in B) were injected simultaneously onto HA1 immobilized on a sensor chip, free of peptide. Binding was recorded in resonance units (RU) values. The maximum RU values for HA1 binding by serum antibodies obtained from vaccinated individuals with either LIAV (A) or TIV (B) vaccination is shown on the Y-axis. The MN titer is expressed as end-point neutralizing antibody titer of post-H1N1 vaccine sera and is depicted on the X-axis. <b>(C) The isotype of serum antibodies bound to rHA1 for the two vaccine groups.</b> Data shown are the means for serum from two independent experiments. <b>(D) Antibody avidity measurements in polyclonal serum by off-rate constants using SPR.</b> Antibody off-rate constants, which describe the stability of the complex were determined directly from the serum/plasma sample interaction with rHA1 protein using SPR in the dissociation phase. For accurate measurements, parallel lines in the dissociation phase for the 10-fold and 100-fold dilution for each post-vaccination human sera were required. The off-rate constants were determined from two independent SPR runs. SPR analysis of post-vaccinated human sera with LAIV (left) or TIV (right) from the vaccine trial was performed with properly folded H1N1pdm09 HA1 (A/CA/7/2009) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034581#pone.0034581-Khurana7" target="_blank">[60]</a>. Serum antibody off-rate constants for vaccinees (each symbol is one individual) were plotted. Correlation statistics of affinity measurement and off-rate constants of sera binding to rHA1 between LAIV and TIV vaccinees were statistically significant with <i>p</i><0.05 (T-test).</p

Serologic responses in vaccinated recruits as measured by microneutralization assay, LAIV-vaccinated recruits (n = 78).

<p>CI, confidence interval; GMT, geometric mean titer; LAIV, live attenuated influenza vaccine.</p>1<p>Seroconversion is defined as a 4-fold increase in titer from pre-vaccine to post-vaccine titer.</p>2<p>Fold change adjusted for pre-vaccine seroprotection levels.</p

Post-vaccination microneutralization titer distribution responses in vaccinated recruits based upon vaccine type.

<p>Post-vaccination microneutralization titer distribution responses in vaccinated recruits based upon vaccine type.</p

Confirmatory sequencing of full-genome amplicons generated by PCR.

<p>A. For each amplicons, the sequencing was carried out with primers other than those used for full-genome amplification. The linear sequencing template (black line), the PCR primers are shown at both ends of the amplicons. Bleu lines (amplicons 671F-892R), green lines (amplicons 6838F-6972R), red lines (amplicons 6838F-7076R) represent the sequenced regions attached to the corresponding sequencing primers. Numbers included in primer IDs represent the position relative to HPV_SD2 genome. B. Alignment of sequences generated by Sanger method along the HPV_SD2 generated by 454 sequencing. Bars in blue, green and red correspond to sequences shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058404#pone-0058404-g006" target="_blank">Figure 6A</a>. The newly generated sequences covered 4,622 nucleotides representing 63.3% of the total HPV_SD2 genome. C. Comparison of the sequences generated by Sanger method and HPV_SD2 contig generated by 454 sequencing. Size of each Sanger sequence and percentage identity are shown.</p

Maximum likelihood tree showing the clustering of HPV_SD2 with previously documented full-length genomes of human papillomaviruses.

<p>The tree was visualized with the Interactive Tree of Life (iTOL) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0058404#pone.0058404-Letunic1" target="_blank">[32]</a>. Bootstrap values with at least 50%, 75% or 100% (100 re-samplings) are indicated. Note, another HPV_SD1 from this study was added in this analysis clustered with reference HPV_49.</p

Genomic organization of the HPV_SD2 virus.

<p>Open reading frames (L2, L1, E6, E7, E1, E2, E4) and a 530 bp non-coding long control region (LCR) are shown. B. Details of the LCR region showing the TATA box (TATAAA, positions 3735–3740), a polyadenylated site (AATAAA, positions 3336–3341), 3 palindrome sites (ACCG-N₄-CGGT; positions 3483–3494, 3650–3661, 3720–3731) and 1 degenerate palindrome (ACC-N₆-GGT, positions 3524–3535). C. Metal-binding domains in deduced E6 and E7 proteins.</p

Coverage plot of the circular structure of HPV-SD2.

<p>A. Screenshot of the assembly of sequences using the <i>De Novo Assembler</i> set for 98% similarity and 45 bp overlap. The coverage plot is shown in green. Overhanging sequences at the 5′ and 3′ ends are partially assembled. B. Box at 5′ end and box at 3′ end are highlighted to show the similarity between unassembled overhang reads at 5′ and 3′ ends and corresponding ends of the consensus sequence.</p

Taxonomical assignment of sequence reads convenient respiratory waste samples.

<p>A. Overall proportions of sequences with homolog in Genbank (the known, 7.8%) compared to sequences with no homolog in Genbank (Unknown, 92.2%). Unknown/Divergent indicates the proportion of highly divergent and/or novel sequences with no homology to NCBI. B. Proportion of sequences classified as eukaryotes, bacteria and viruses. Sequences were classified using BLASTn search against all non-redundant nucleotide sequences in the NCBI nt database with an E-value cutoff of 10⁻⁵.</p

Confirmatory PCRs to verify HPV_SD2 is a circular double-stranded genome.

<p>A. Graphical representation of the binding sites of primers 671F-892R on the putative circular structure of HPV_SD2, and the predicted PCR product sizes (I: 222 bp, II: 7,591 bp, III: 14,890 bp). The predicted short band (I) indicates the amplification of the proximal region between primers 671F and 892R. The large band (II) indicates that <i>Taq</i> DNA polymerase would amplify the region between the primers 671F and 892R by making the full circle of the HPV genome. The large band (III) indicates the <i>Taq</i> DNA polymerase would make 2 full circle around the HPV genome. PCRs were also performed using primer sets 6838F-6972R, 6838F-7076R. B. Agarose gel (0.5%) showing the amplified HPV_SD2. Primer sets used are shown: primer sets 6838F-6972R, 6838F-7076R. For each PCR, the same sample pool was tested at different concentrations [1∶1 (lanes 1, 4, 7), 1∶10 (lanes 2, 5, 8) and 1∶50 (lanes (3, 6, 9)] using 1 µl per reaction. L: DNA ladder. Amplicons can be seen at the expected sizes.</p