17 research outputs found
Mutations in NA That Induced Low pH-Stability and Enhanced the Replication of Pandemic (H1N1) 2009 Influenza A Virus at an Early Stage of the Pandemic
<div><p>An influenza A virus that originated in pigs caused a pandemic in 2009. The sialidase activity of the neuraminidase (NA) of previous pandemic influenza A viruses are stable at low pH (≤5). Here, we identified the amino acids responsible for this property. We found differences in low-pH stability at pH 5.0 among pandemic (H1N1) 2009 viruses, which enhanced the replication of these viruses. Low-pH-stable NA enhancement of virus replication may have contributed to the rapid worldwide spread and adaptation to humans of pandemic (H1N1) 2009 viruses during the early stages of the 2009 pandemic.</p></div
Effects of <i>Met</i> RNAi on DNA replication and vitellogenesis.
<p>(A) <i>Met</i> knockdown efficiency and <i>VgA</i> mRNA levels in the fat body of the dsGFP control (iGFP), <i>Met</i> RNAi (iMet), and iMet further treated with methoprene for 48 h (iMet+JHA). **, <i>P</i><0.01 and ***, <i>P</i><0.001; n.s, no significant difference; n = 4–6. (B) Fat body cell ploidy (a–f) and DNA synthesis (g–i) of iGFP, iMet and iMet+JHA. Blue, nuclei; green, F-actin; red, <i>de novo</i> DNA synthesis. Enlarged images (4×) of a–c are shown in d–f, respectively with the white bar, 20 µm and red bar, 5 µm. (C) Statistical analysis for the diameter of cell nuclei. **, <i>P</i><0.001; n = 80–100. (D) FACS analysis of DNA contents in fat body cells. (E) Representative phenotypes of ovaries and ovarioles after iMet, iMet+JHA <i>vs.</i> iGFP. (F) Statistical analysis for length*width index of primary oocytes. ***, <i>P</i><0.001; n = 30.</p
Effects of JH deprivation and JHA application on DNA replication and vitellogenesis.
<p>(A) Fat body cell ploidy (a–h) and DNA synthesis (i–l). PAE10, 10-day-old adult females; P10d, adult females treated with precocene for 10 d; PM24h and PM48h, precocene-treated adult females further treated with methoprene for 24 h and 48 h, respectively. Blue, nuclei; green, F-actin; red, <i>de novo</i> DNA synthesis. Enlarged images (4×) of a–d are shown in e–h, respectively. White bar, 20 µm; red bar, 5 µm. (B) Statistical analysis for the diameter of cell nuclei. **, <i>P</i><0.01; ***, <i>P</i><0.001; n = 80–100. (C) FACS analysis of DNA contents in fat body cells. (D) Relative mRNA levels of <i>VgA</i> in the fat body. ***, <i>P</i><0.001; n = 8. (E) Morphology of ovaries and ovarioles. Scale bars: ovary, 5 mm; primary oocyte, 0.5 mm. (F) Statistical analysis for length*width index of primary oocytes. **, <i>P</i><0.01; ***, <i>P</i><0.001; n = 30.</p
Differential gene expression profiles in JH-deprived and methoprene-exposed fat bodies.
<p>(A) Number of up-regulated (red) and down-regulated (blue) gene transcripts in JH-deprived fat bodies further treated with methoprene for 24 h. Fold change ≥2 and <i>P</i><0.05 were used as the cutoff criteria. (B) Significantly enriched gene ontology terms associated with up-regulated genes. (C) Significantly enriched KEGG pathways (top 10) of up-regulated genes. (D) Validation of RNA-seq data by qRT-PCR for genes associated with DNA replication. Fold change was calculated as: mRNA levels in methoprene-treated females/mRNA levels in precocene-treated females.</p
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Responsiveness of <i>Mcm4</i> and <i>Mcm7</i> to JH and <i>Met</i> RNAi.
<p>(A) Relative mRNA levels of <i>Mcm4</i> and <i>Mcm7</i> in the fat body of adult females treated with precocene (P10d) and those further treated with methoprene for 6, 12, 24 and 48 h (PM6h, PM12h, PM24h and PM48h, respectively). mRNA levels in precocene-treated fat bodies were used as the calibrator. *, <i>P</i><0.05, **, <i>P</i><0.01 and ***, <i>P</i><0.001 compared to P10d; n = 4–6. (B) <i>Mcm4</i> and <i>Mcm7</i> mRNA abundance in fat bodies of female locusts from 0 to 10 days post adult eclosion (PAE). *, <i>P</i><0.05 and **, <i>P</i><0.01 compared to PAE0; n = 4–6. (C) Relative levels of <i>Mcm4</i> and <i>Mcm7</i> transcripts in fat bodies of dsGFP control (iGFP), <i>Met</i>-RNAi (iMet), and iMet further treated with methoprene for 48 h (iMet+JHA). *, <i>P</i><0.05; n = 10–12. (D) Alignment of DNA element sequences containing E-box and E-box-like motifs in the upstream promoter regions of locust <i>Mcm7</i> and <i>Mcm4</i> with experimentally tested Met-binding consensus sequences except that of <i>DmKr-h1</i><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Kayukawa1" target="_blank">[7]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Li1" target="_blank">[9]</a>, <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Zou1" target="_blank">[34]</a>–<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702-Cui1" target="_blank">[36]</a>. (E) EMSA using the Mcm4 probe listed in (D) with fat body nuclear extracts of iGFP and iMet (shown is a representative short exposure; longer exposures in some experiments showed additional two bands of less interest; see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#s2" target="_blank">Results</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004702#pgen.1004702.s007" target="_blank">Fig. S7</a>). (F) EMSA using Mcm7 probe listed in (D) and with fat body nuclear extracts of iGFP and iMet. For both (E) and (F), arrows indicate the most likely specific bands. FP, free probe.</p
<i>Mcm4</i> and <i>Mcm7</i> transcription and the JH-receptor complex.
<p>(A) Upper panels: western blot (WB) showing the expression of Flag-Met<sup>1-3108</sup> and V5-SRC in S2 cells; lower panel: immunoprecipitation (IP) showing the interaction of Flag-Met<sup>1-3108</sup> and V5-SRC in the presence of 10 µM JH III or methoprene. α-Flag, Flag antibody; α-V5, V5 antibody. (B) Luciferase assays after co-transfection of pGL4.10/<i>Mcm4</i><sup>−933 to −37</sup> or pGL4.10/<i>Mcm7</i><sup>−933 to −11</sup> into S2 cells compared with the pAc5.1 empty vector, pAc5.1/Flag-Met<sup>1-3108</sup> (Met) or/and pAc5.1/V5-SRC (SRC), with or without 10 µM JH III or methoprene (JHA) treatment. (C) EMSA using the Mcm4 probe and S2 cell nuclear extracts with expressed Flag-Met and V5-SRC and treated with JH III (10 µM). The arrow indicates the specific complex. FP, free probe. (D) EMSA using the Mcm7 probe and S2 cell nuclear extracts with expressed Flag-Met and V5-SRC and treated with JH III (10 µM). The arrow indicates the specific complex. FP, free probe.</p
Body temperature of nonhuman primates after challenge with AH/05 virus.
<p>Change in body temperature in nonhuman primates after challenge with AH/05 virus (A) or BHG/05 virus (B). Changes were calculated by subtracting the mean temperature 3 days before challenge from the temperature recorded on the indicated day.</p
Replication of the H5N1 <i>ca</i> reassortants and the wild-type H5N1 viruses in mice.
a<p>Six-week-old BALB/c mice (3 per group), inoculated intranasally with 10<sup>6</sup> EID<sub>50</sub> of the indicated virus in a 50-µl volume, were killed on day 3 postinoculation and their organs were collected for virus titration in eggs. <, no virus was isolated from that sample.</p>b<p>P value was<0.01 compared with the titers in the corresponding organs of the AH/05-inoculated mice.</p
Pathological lesions and antigen distribution in tissues of nonhuman primates infected with H5N1 viruses.
<p>Animals were vaccinated twice (4-week interval) with AH/AA<i>ca</i> and challenged with AH/05 or BHG/05 virus. Tissues for pathological examination were collected 3 days after viral challenge. Tra, trachea; Bro, Bronchus; TBLN, tracheobronchial lymph node.</p>b<p>Pathological lesions/viral antigens. −, no pathological change/antigen. +, limited pathological change/antigen. ++, moderate pathological change/antigen. +++, severe pathological change/abundant antigen.</p