Production of chemokines by MOPV- and LASV-infected DC cocultured with T cells and correlation with type I IFN synthesis.

<p>(A) Levels of CC chemokine and CXCL11 mRNA were quantified relative to GAPDH mRNA levels, after 2 days of coculture of mock- (white bars), MOPV- (gray bars), or LASV- (black bars) infected iDC and autologous T cells. Levels of CXCL10 mRNA were evaluated in iDC/T cell coculture 2 days after each stimulation of the T cells with infected or inactivated virus-stimulated iDC. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± standard error (SE) of 4 independent experiments. Significant differences are indicated as follows: * (<i>p</i><0.05) and ** (<i>p</i><0.01). (B) The levels of CC and CXC chemokines were quantified by ELISA in the supernatants after 2 days of coculture of mock- (white bars), MOPV- (gray bars), or LASV- (black bars) infected iDC and autologous T cells. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed in pg/ml, except for CXCL10 expressed in ng/ml. Significant differences are indicated as follows: * (<i>p</i><0.05). (C) Correlation between CCL4, CCL7, CXCL10, and CXCL11 mRNA levels and type I IFN synthesis by MOPV- or LASV-infected iDC cultured with naïve T cells 2 days after infection, represented by a linear regression with a correlation coefficient <i>r</i> and a probability of correlation <i>p</i>.</p

Release of chemokines into the supernatants of DC and MP infected with LASV and MOPV.

<p>The levels of CC and CXC chemokines were quantified by ELISA in the supernatants of MP (A) and DC (B) 24 and 72 h after mock (white bars), MOPV (gray bars), and LASV (black bars) infection. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed in pg/ml and ng/ml for CC and CXC chemokines, respectively. Significant differences are indicated as follows: * (<i>p</i><0.05), ** (<i>p</i><0.01), and *** (<i>p</i><0.001).</p

Role of type I IFN in the production of chemokines in MOPV- and LASV-infected iDC/T-cell cocultures.

<p>(A) The synthesis of mRNAs for CCL4, CCL7, CXCL10 and CXCL11 was evaluated in mock- (gray bars) or MOPV- (black bars) infected iDC cocultured with naïve T cells for 2 days in the presence of neutralizing Ab against CD118. (B) The levels of these mRNAs then were quantified 2 days after restimulation with mock- (gray bars) or inactivated MOPV- (black bars) pulsed iDC of T cells previously cocultured with mock- or MOPV-infected iDC in the presence of irrelevant IgG2a or CD118-neutralizing Ab. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± SE of 3 independent experiments.</p

Production of chemokines by APC infected with recombinant LASV.

<p>The production of CC and CXC chemokines by DC (A, B) and MP (C, D) was assessed after mock infection (white bars), or infection with recombinant wild-type LASV (gray bars) or rLASV NP-D389A/G392A (rNP LASV) (black bars). (A, C) The synthesis of mRNAs was analyzed by RT-qPCR 24 h after infection. (B, D) The protein levels released in the supernatants were quantified by ELISA 24 h (only for CXCL9, 10 and 11) and 48 h after infection. <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± SE of 4 (A, C) and 3–7 (B, D) independent experiments. Significant differences are indicated as follows: * (<i>p</i><0.05), ** (<i>p</i><0.01) and *** (p<0.001).</p

Production of type I IFN and chemokine mRNA in DC and MP infected with LASV and MOPV.

<p>We obtained mRNA from DC and MP 24(black bars), MOPV (gray bars), or after mock infection (white bars). (A) The amounts of type I IFN mRNA produced by DC and MP are shown as a gene mRNA/β-actin mRNA ratio. The synthesis of chemokine mRNA was evaluated with the GAPDH gene used as a housekeeping gene, in both DC (B) and MP (C). <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002637#s3" target="_blank">Results</a> are expressed as the mean ± standard error (SE) of 4 and 5 independent experiments, for LASV- and MOPV-infected cells, respectively. Significant differences between mock- and virus-infected cells are indicated as follows: * (<i>p</i><0.05), ** (<i>p</i><0.01), and *** (<i>p</i><0.001).</p

H1N1 influenza viruses varying widely in hemagglutinin stability transmit efficiently from swine to swine and to ferrets

<div><p>A pandemic-capable influenza virus requires a hemagglutinin (HA) surface glycoprotein that is immunologically unseen by most people and is capable of supporting replication and transmission in humans. HA stabilization has been linked to 2009 pH1N1 pandemic potential in humans and H5N1 airborne transmissibility in the ferret model. Swine have served as an intermediate host for zoonotic influenza viruses, yet the evolutionary pressure exerted by this host on HA stability was unknown. For over 70 contemporary swine H1 and H3 isolates, we measured HA activation pH to range from pH 5.1 to 5.9 for H1 viruses and pH 5.3 to 5.8 for H3 viruses. Thus, contemporary swine isolates vary widely in HA stability, having values favored by both avian (pH >5.5) and human and ferret (pH ≤5.5) species. Using an early 2009 pandemic H1N1 (pH1N1) virus backbone, we generated three viruses differing by one HA residue that only altered HA stability: WT (pH 5.5), HA1-Y17H (pH 6.0), and HA2-R106K (pH 5.3). All three replicated in pigs and transmitted from pig-to-pig and pig-to-ferret. WT and R106 viruses maintained HA genotype and phenotype after transmission. Y17H (pH 6.0) acquired HA mutations that stabilized the HA protein to pH 5.8 after transmission to pigs and 5.5 after transmission to ferrets. Overall, we found swine support a broad range of HA activation pH for contact transmission and many recent swine H1N1 and H3N2 isolates have stabilized (human-like) HA proteins. This constitutes a heightened pandemic risk and underscores the importance of ongoing surveillance and control efforts for swine viruses.</p></div

HA activation pH values for swine H1N1, H1N2, and H3N2 influenza viruses.

<p>Each data point represents the pH of activation of an individual virus, as measured by syncytium assay with 2 to 4 duplicates. Activation pH values of prepandemic (<2009) swine H1 viruses were previously reported [<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006276#ppat.1006276.ref039" target="_blank">39</a>] and are used here for comparison. A list of contemporary swine H1 and H3 viruses is given in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006276#ppat.1006276.s007" target="_blank">S1</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006276#ppat.1006276.s008" target="_blank">S2</a> Tables.</p

Detection of transmission in contact pigs and ferrets.

<p>Detection of transmission in contact pigs and ferrets.</p

HA activation pH values for recombinant mutant viruses.

<p>pH1N1 mutants possessing one or more of the mutations were generated by reverse genetics, and the pH of HA activation was determined using a syncytium assay. The mean (± SD) of the pH values is shown.</p

Viral growth in contact pigs and ferrets during transmission.

<p>Transmission experiments were performed as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006276#ppat.1006276.t002" target="_blank">Table 2</a>. Mean (± SD) viral titers in the nasal cavities of pigs (A) (n = 3) and ferrets (B) (n = 3) were determined by TCID50 titration. Two-way ANOVA with Bonferroni multiple comparisons was performed. Significant differences between the WT and Y17H groups are indicated as follows: **<i>P</i> < 0.01, ***<i>P</i> < 0.001, ****<i>P</i> < 0.0001. There was no significant difference between the WT and R106K groups.</p