Viral challenge and sample collections in pregnant and non-pregnant mice.

<p>Four groups of the mice (12-13 mice/group) were intranasally inoculated with wild type or mutant viruses. Four groups of mice were sampled at day 3 and 6 post-infection, respectively. The experiment was duplicated. This table showed the total numbers of mice in these two experiments.</p

Pathological and immunological changes in placenta and fetus.

<p>A. Placentas collected from pregnant mice infected with mutant (a & b) or wild type (c & d) virus at day 3 post-infection were examined after H&E staining. a, ** indicated large area of necrosis, c, * indicated two smaller foci of necrosis in the junctional zone of the placenta. Arrows in b &d indicated apoptotic bodies and nuclear debris in the trophoblast. e & f are placentas from uninfected control mice showing no significant pathological changes. Magnification of a, c, e & f 200x and b & d 400x. B. Representative sections from fetal lung (a), myocardium (c) and liver (e) tissues from mutant virus infected mice but not obvious in wild type virus infected mice (b, d & f). Original magnification: 200×. C. Levels of indicated cytokines and chemokines in amniotic fluids collected from mice infected with wild type (WT) or mutant (Mut) virus and uninfected mice (Ctl) were detected by ELISA. * indicates significant differences (<i>P</i><0.05) between pregnant mice infected with wild type and mutant viruses. IL-1β: interleukin-1β, IL-6: interleukin-6, IFN-γ: interferon-γ, MIP-1α: macrophage inflammatory protein 1α, MIP-2: macrophage inflammatory protein 2; TNF-α: tumor necrosis factor-α.</p

Effect of wild type and mutant pandemic virus infection on the development of mouse fetus.

<p>Effect of wild type and mutant pandemic virus infection on the development of mouse fetus.</p

Viral infection and replication in lungs.

<p>A. Virus titers in lung homogenates collected from mice infected with wild type (WT) or mutant (Mut) virus on indicated days post-infection were measured by plaque forming assay. ** indicates significant differences (<i>P</i><0.001) between pregnant and non-pregnant mice. B. Viral RNA copies in the same lung homogenates were also determined by real-time RT-PCR. * indicates significant differences (<i>P</i><0.001) between pregnant and non-pregnant mice. C. Immunohistochemical staining for influenza viral nucleoprotein (NP) protein in lung sections of infected mice. Representative images of lung sections from wild type virus infected bronchial epithelium of non-pregnant (a) and pregnant (b) mice, and mutant virus infected alveoli of non-pregnant (c) and pregnant (d) mice. Original magnification: 100×. The NP positive cells were morphologically identified to be type I alveolar cells (few) indicated by arrow heads and type II alveolar cells (majority) indicated by arrows (e). Original magnification: 200×. Dual-labeling of mouse lung type II alveolar cells with anti-NP antibody conjugated with FITC and anti-SP-C antibody conjugated with Texas red further confirmed that the virus mainly infected type II alveolar cells (f). Arrows indicates NP and SP-C double positive alveolar cells. Original magnification: 400×.</p

Proinflammatory cytokines and chemokines in lung homogenates.

<p>Proinflammatory cytokines and chemokines in lung homogenates collected from wild type (WT) or mutant (Mut) virus infected mice at day 3 post-infection and uninfected mice (Ctl) were detected by ELISA. ** indicates significant differences (<i>P</i><0.001) and * indicates significant differences (<i>P</i><0.05) between pregnant and non-pregnant mice. IL-1β: interleukin-1β, IL-6: interleukin-6, IFN-γ: interferon-γ, MIP-1α: macrophage inflammatory protein 1α, MIP-2: macrophage inflammatory protein 2; TNF-α: tumor necrosis factor-α.</p

Histopathological changes in lungs, livers and kidneys of infected mice.

<p>Representative H&E stained sections showing changes in lung, liver and kidney tissues of mice infected with wild type (a & b) or mutant virus (c & d). A. Various degrees of interstitial bronchitis, epithelial necrosis, alveolitis and alveolar edema were found in lung tissues. B. Various degrees of degeneration were shown in liver cells. C. Degeneration of renal tubular epithelial cells was found in kidney tissues. Original magnification: 200×.</p

Virus-turkey erythrocyte receptor binding avidity assay.

<p>Influenza virus cellular receptor binding affinity was tested with turkey erythrocytes pre-treated with <i>Vibrio cholerae</i> neuraminidase which mainly removes 2, 3-linked sialic acids as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013757#s2" target="_blank">Materials and Methods</a>. A) Illustration of assay for receptor binding avidity. B) Receptor binding avidity of mut and wt viruses. Data are representative of three independent experiments. Data was analyzed by one-way ANOVA using the mean square in the ANOVA to estimate of variability. P-value<0.001. PR8, A/Puerto Rico/8/34; Mut, mutant virus; WT, wild type virus.</p

Survival rates of infected mice.

<p>Pregnant and non-pregnant BALB/c mice infected with wild type (WT) and mutant (Mut) viruses and their survivals were recorded in indicated days. ** indicates significant difference (<i>P</i><0.001) between pregnant and non-pregnant mice.</p