Real-time cell analysis of recombinant HMPV strains.

<p>LLC-MK2 monolayers in 96 well-plates were infected with rHMPV at an MOI of 0.01 (a) Output of one real-time cell analysis (RTCA) experiment; data was normalized using mock-infected wells and normalized cell index is plotted. (b) Mean time until cell index is reduced by 50% from 4 independent experiments is plotted. *, p < 0.05 using unpaired, two-tailed Student t-test.</p

Pulmonary cytokine levels of BALB/c mice infected with recombinant HMPV strains.

<p>BALB/c mice were infected with 6x10⁵ TCID₅₀ of rHMPV (as determined by back-titration). On days 3 through 6, four mice per group were euthanized to determine pro-inflammatory cytokine/chemokine levels in the lungs of infected mice. Mock-infected mice are representad as day 0. ***, p < 0.001; **, p < 0.01; * p < 0.05 comparing all other strains to rC-85473.°°°, p < 0.001; °°, p < 0.01; °, p < 0.05 comparing all other strains to rCAN98–75 using Repeated Measures Two-way ANOVA.</p

Cytopathic effects of HMPV strains and recombinant HMPV viruses.

<p>(a) microscopic images of cytopathic effects induced by HMPV infection of LLC-MK2 monolayers. CAN98–75 (B2) induces focal cell-rounding (left) whereas C-85473 (A1) induces multinucleated syncytia (right). Magnification = 10x. (b) Representation of the genomes of the 4 recombinant viruses used in this study; rC-85473 and rCAN98–75 represent the wild-type strains, rC-85473_F represents the chimeric rC-85473 strain in which the F gene has been replaced with that of CAN98–75 and rCAN98–75_F represent the chimeric rCAN98–75 in which the F gene has been replaced with that of C-85473.</p

Lung viral titers and weight loss of BALB/c mice infected with recombinant HMPV strains.

<p>BALB/c mice were infected with 6x10⁵ TCID₅₀ of rHMPV (as determined by backtitration). (a) On days 3 through 6, four mice per group were euthanized to determine pulmonary viral titers. (b) Six mice per group were monitored for weight loss on a daily basis for 14 days. ***, p < 0.001; * p < 0.05 comparing all other strains to rC-85473.°°°, p < 0.001; °, p < 0.05 comparing all other strains to rCAN98–75 using Repeated Measures Two-way ANOVA.</p

Syncytium formation induced by recombinant HMPV strains.

<p>(a) LLC-MK2 monolayers in 24 well-plates were infected with rHMPV at an MOI of 0.01 in quadruplicate. On days 2 through 4 pi, pictures were taken using fluorescent microscopy in 3 random fields (20x magnification) per well and the number of nuclei per GFP-expressing cell was calculated. ***, p < 0.001 comparing all other strains to rC-85473 and °°°, p < 0.001 comparing all other strains to rCAN98–75 using Repeated Measures Two-way ANOVA. (b) An example of the observed difference in syncytium formation between the 4 recombinant strains on day 3 pi.</p

PAR1 agonist or antagonist dose-dependent effect on hMPV infection during a 3-day treatment in mice.

<p>Groups of 12 mice were infected intranasally with hMPV (4-6 x10⁵ TCID₅₀) or mock infected and simultaneously treated for 3 days with a single daily dose of 50 or 500 µM of PAR1 agonist (TFLLR-NH₂), PAR1 antagonist (SCH79797) or their respective vehicles. A) and B) Weight loss and mortality were monitored daily for 14 days for mice treated with the PAR1 agonist and antagonist, respectively. The horizontal bar underneath the graphic indicates the timing and duration of treatment. Arrows and numbers indicate the mice that reached the endpoint and were sacrificed (full line: mice treated with 50 µM of PAR1 agonist, dotted line: mice treated with 500 µM of PAR1 agonist). Significant differences in weight loss were observed between mice treated with 50 µM (†) or 500 µM (*) of compound compared to untreated mice based on a two-way ANOVA (* p<0.05, ** p<0.01, *** p<0.001, † p<0.05, † † p<0.01). C) Viral titers were determined by TCID₅₀ in lung homogenates at day 5 pi. Significant differences were observed between treated or untreated mice as determined by one-way ANOVA. (* p<0.05, ** p<0.01) The bar indicates the lower limit of detection.</p

Effect of PAR1 agonist or antagonist treatment of hMPV-infected mice on furin expression.

<p>A) Groups of 6 mice were infected intranasally with hMPV (7x10⁵ TCID₅₀) or mock infected and simultaneously treated for 5 days with a single daily dose of 500 µM of PAR1 agonist (TFLLR-NH₂) or PAR1 antagonist (SCH79797). Mice were sacrificed on day 5 pi, lungs were removed and snap frozen. RNA was extracted and furin transcript levels were determined using RT-PCR. Significant differences in furin transcript levels were observed between treated and untreated mice based on a Student T-test (* p=0.05, ** p<0.01) B) COS-1 and HEK293 cells were co-transfected with a plasmid encoding the hMPV F protein containing a V5-tag and a plasmid encoding one of the proprotein convertases. Cell lysates were analyzed by western blot using a V5 mAb. Furin is the only convertase capable of cleaving the full length precursor protein (F0) into its activated form, resulting in the shorter C-terminal subunit (F1). C) Recombinant human PAR1 and furin are co-tranfected with the hMPV F protein containing a V5-tag in HEK293 cells with/without hPAR1 agonist, (100 μM) or antagonist (0.1 μM). Western blot analysis of cell lysates using an anti-V5 mAb is shown.</p

Cell recruitment in the lungs of hMPV-infected mice treated with the PAR1 antagonist.

<p>Groups of 6 mice were infected intranasally with hMPV (7 x10⁵ TCID₅₀) or mock infected and simultaneously treated for 5 days with a single daily dose of 500 µM of PAR1 antagonist (SCH79797). Mice were sacrificed on day 5 pi, lungs were removed, homogenized in HBSS and analyzed by flow cytometry for the presence of (A) dendritic cells expressing the MHC II molecules I-A/I-E (CD11c⁺CD11b^loLy6G ^loI-A/I-E⁺), (B) macrophages (CD11c⁺CD11b⁺F4/80^hi), (C) T lymphocytes (CD3⁺CD4⁺ or CD3⁺CD4^-) and (D) neutrophils (CD11c^-CD11b^hiLy6G⁺). Significant differences in recruited cells were observed between treated and untreated mice based on a one-way ANOVA. (* p<0.05, *** p<0.001).</p

Clinical outcomes including weight loss, nasal wash and lung virus titers by study group and day.

<p>Clinical outcomes are displayed including: (A) Mean percentage weight relative to baseline by study group and day, with standard errors. (B) Nasal wash virus titers by study group and day. (C) Lung homogenate virus titers at day 5 post-challenge. Box plots (B and C) display mean (dot) and median (line) virus titres as log pfu/mL. Per usual, the box extends to the 25^th/75^th percentiles and whiskers extend to minimum/maximum values. Ch refers to challenge day and Ch+1, Ch+2 etc indicate day post-challenge (i.e. day one post-challenge, day two post-challenge etc). Ch+5 indicates day five post-challenge on which four animals per group were randomly selected for sacrifice. Statistically significant between-group differences are as specified.</p

HA1 microarray serological values by study antigens, group and day.

<p>Box plots display median (dash) and mean (dot) of log₁₀-transformed HA1 protein microarray signal values. The box extends to the 25^th/75^th percentiles and whiskers extend to minimum/maximum values. H1-07 indicates A/Brisbane/59/2007 (H1N1)-like; H3-07 indicates A/Brisbane/10/2007 (H3N2)-like; H1-09 indicates A/California/7/2009 (H1N1)pdm09-like (<b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0086555#pone.0086555.s004" target="_blank">Table S3</a>;</b> grey-shaded). Sample size as follows: Pre-immunization Vaccine = 15, Placebo = 16 (3 ferrets each per group pre-shipment serum was substituted owing to insufficient day 0 available); Day 28 Vaccine = 14, Placebo = 15; Day 49 Vaccine = 12, Placebo = 11; Day 54 Vaccine = 2, Placebo = 4; Day 63 Vaccine = 9, Placebo = 8. **indicates statistical significance at p<0.01 and *indicates statistical significance at p<0.05 in comparing vaccine to placebo group at the designated time point. ΔΔ indicates statistical significance at p<0.01 and Δ indicates statistical significance at p<0.05 in comparing values within study groups at days 28, 49, 54 and 63 relative to pre-immunization, colour coded by vaccine (red) or placebo (blue). □□ indicates statistical significance at p<0.01 and □ indicates statistical significance at p<0.05 in comparing day 63 to day 49 within groups, colour coded per above by study group.</p