Figure 1.
Schematic procedure of virus in-capsule-mucus penetration system.
(1) 150 µl of mucus were brought at the bottom of a gelatin capsule. (2) 8 µl of SIV suspension were added on top of the surface of the mucus. (3) Mucus together with virus was embedded and snap-frozen. (4) Cryosections were made vertically to the mucus surface. (5) Immunofluorescence staining was performed to visualize the Muc5AC (representing the mucus) and viral particles. (6) Penetration depth (shown by yellow arrows) was measured from the surface of mucus to the furthest point of the viral signal.
Figure 2.
Expression of α2,3- and α2,6-SA on porcine respiratory mucus determined by fluorescence lectin staining.
(A) Representative confocal microscopy images. Green color shows α2,3-SA staining and red color represents α2,6-SA staining. The scale bars indicate 50 µm. (B) Semi-quantification of the sialic acids. Three independent mucus samples were analyzed and error bars indicate the standard deviation. The asterisks (**) indicate statistical significance (P<0.01, Student’s t-test).
Figure 3.
Purity of SIV determined by Dio labeling and immunofluorescence staining.
(A) Confocal microscopy of the double staining of the virus preparations. Green represents Dio labeled particles; viral antigens are shown in red. Merged signals represent virus particles which are also labeled with Dio. (B) Bands form in the discontinuous iodixanol gradient separation. Three bands were identified, named Band 1, Band 2 and Band 3 from up downwards. (C) Ratio of double positive particles versus Dio positive particles for the particles from three different bands. Three independent experiments were performed and error bars indicate the standard deviation.
Table 1.
Characteristics of SIV and Dio-labeled SIV.
Figure 4.
Diffusion coefficient of SIV in mucus and comparison with PRV and 100 nm PEGylated beads.
(A) Distributions of the apparent diffusion coefficient of SIV, PRV and 100 nm PEGylated beads in porcine respiratory mucus. Trajectories of 8 steps were analyzed for each of the 1500 diffusion coefficients. Distributions were refined with MEM. Dashed line indicates the boundary of mobile and immobile diffusion. (B) Average diffusion coefficient of SIV, PRV and 100 nm PEGylated beads. Data were obtained from three independent experiments, and error bars indicate the standard deviation. The asterisks (**) indicate statistical significance (P<0.01, by Student’s t-test).
Figure 5.
Penetration of SIV through porcine respiratory mucus.
(A) Confocal microscopic analysis of the virus penetration. Representative confocal photomicrographs of the penetration of SIV at 2, 10 and 30 min post virus addition. Mucin 5AC and SIV antigens were visualized by red and green color, respectively. (B) Penetration depths of SIV with time. Hundred and twenty measurements were obtained from three independent mucus samples. (C) Average penetration depth of SIV at 2, 10 and 30 min post virus addition. Three independent experiments were performed. Error bars indicate the standard deviation. The asterisk (*) indicates statistical significance (P<0.05, by Student’s t-test).
Figure 6.
Effects of NAI and Arthrobacter ureafaciens neuraminidase (AUNA) on microscopic diffusion and macroscopic penetration of SIV in porcine respiratory mucus.
(A) Distribution of diffusion coefficient of NAI, AUNA and mock treated SIV in mucus. 1500 trajectories were analyzed. Distributions were refined with MEM. (B) Proportion of mobile fraction (Da>0.2 µm2/s) of NAI, AUNA and mock treated SIV in mucus. Error bars represent the standard deviation from three independent experiments. The asterisks (**) indicate statistical significance (P<0.01, by Student’s t-test) (C) Distribution of the penetration depth of NAI, AUNA and mock treated SIV through mucus at 30 min post virus addition. Hundred and twenty measurements were performed on three independent mucus samples. (D) Average penetration depth of NAI, AUNA and mock treated SIV through mucus at 30 min post virus addition. Three independent samples were performed, and error bars indicate the standard deviation. The asterisks (** and *) indicate statistical significance (P<0.01, and P<0.05, respectively, by Student’s t-test).
Figure 7.
Effects of NA on SIV binding to porcine respiratory mucus shown by confocal microscopy.
(A) Virions (green) bound to mucus sections in the presence or absence (Mock) of Zanamivir (NAI) or Arthrobacter ureafaciens neuraminidase (AUNA). (B) Quantification of viral particles bound to mucus (per 105 µm2). The error bars indicate the standard deviation from 3 independent experiments. The asterisks (** and *) show the significance difference (P<0.01, and P<0.05 by Student’s t-test).