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, 7 (7), e1002134

Illumination of Parainfluenza Virus Infection and Transmission in Living Animals Reveals a Tissue-Specific Dichotomy

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Illumination of Parainfluenza Virus Infection and Transmission in Living Animals Reveals a Tissue-Specific Dichotomy

Crystal W Burke et al. PLoS Pathog.

Abstract

The parainfluenza viruses (PIVs) are highly contagious respiratory paramyxoviruses and a leading cause of lower respiratory tract (LRT) disease. Since no vaccines or antivirals exist, non-pharmaceutical interventions are the only means of control for these pathogens. Here we used bioluminescence imaging to visualize the spatial and temporal progression of murine PIV1 (Sendai virus) infection in living mice after intranasal inoculation or exposure by contact. A non-attenuated luciferase reporter virus (rSeV-luc(M-F*)) that expressed high levels of luciferase yet was phenotypically similar to wild-type Sendai virus in vitro and in vivo was generated to allow visualization. After direct intranasal inoculation, we unexpectedly observed that the upper respiratory tract (URT) and trachea supported robust infection under conditions that result in little infection or pathology in the lungs including a low inoculum of virus, an attenuated virus, and strains of mice genetically resistant to lung infection. The high permissivity of the URT and trachea to infection resulted in 100% transmission to naïve contact recipients, even after low-dose (70 PFU) inoculation of genetically resistant BALB/c donor mice. The timing of transmission was consistent with the timing of high viral titers in the URT and trachea of donor animals but was independent of the levels of infection in the lungs of donors. The data therefore reveals a disconnect between transmissibility, which is associated with infection in the URT, and pathogenesis, which arises from infection in the lungs and the immune response. Natural infection after transmission was universally robust in the URT and trachea yet limited in the lungs, inducing protective immunity without weight loss even in genetically susceptible 129/SvJ mice. Overall, these results reveal a dichotomy between PIV infection in the URT and trachea versus the lungs and define a new model for studies of pathogenesis, development of live virus vaccines, and testing of antiviral therapies.

Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. In vitro and in vivo phenotypes of luciferase-expressing Sendai viruses.
(A) Recombinant Sendai viruses that contain the firefly luciferase gene (luc) inserted into the P-M, M-F, and F-HN gene junctions were generated. (B) Multiple-step replication kinetics of wild-type (WT) and luciferase-expressing Sendai viruses in LLC-MK2 cell cultures infected at a multiplicity of infection (MOI) of 0.01 PFU/cell and incubated at 33°C (closed symbols) and 37°C (open symbols). (C) Kinetics of luciferase reporter gene expression in LLC-MK2 cells infected with recombinant Sendai viruses at an MOI of 5 PFU/cell, as measured by luminescence. (D) Changes in mean body weight after intranasal inoculation of Sendai viruses. (E) Percent survival after intranasal inoculation of Sendai viruses. (F) Total lymphocyte counts in bronchoalveolar lavage fluid (BALF) 10 days after infection. (G) Sendai virus-specific binding antibody titers in sera collected 10 days after infection, as measured by reciprocal endpoint dilutions in ELISA assays. For panels DG, groups of five 8-week-old 129/SvJ-strain mice were intranasally inoculated with 7,000 PFU of recombinant Sendai virus or phosphate buffered saline (PBS) and the experiments were performed twice. Cumulative data are shown in panels D and E, and representative data are shown in panels F and G.
Figure 2
Figure 2. Non-invasive bioluminescence imaging of Sendai virus infection in the respiratory tracts of living mice.
Eight-week-old mice were intranasally inoculated with 7,000 PFU of rSeV-luc(P-M), rSeV-luc(F-HN), or rSeV-luc(M-F*). Every 24 hours the mice were intraperitoneally injected with luciferin substrate, anesthetized with isoflurane, imaged with a Xenogen IVIS device, and then allowed to recover. Bioluminescence in one experiment is shown on day 2 (A) and day 7 (B) post-infection (p.i.) in 129/SvJ mice infected with rSeV-luc(P-M), rSeV-luc(F-HN), or rSeV-luc(M-F*). Bioluminescence in a second experiment is shown on day 2 (C) or day 7 (D) for 129/SvJ, DBA/2, BALB/c, or C57BL/6 mice infected with rSeV-luc(M-F*). The data are displayed as radiance (bioluminescence intensity) on a rainbow log scale with a range of 1×106 (blue) to 1×109 (red) photons/s/cm2/steradian. Red circles indicate regions of interest (ROI) used to calculate the total flux (photons/s) in the nasopharynx, and red rectangles indicate the ROI areas for the trachea and lungs.
Figure 3
Figure 3. Kinetics of Sendai virus spread and clearance in the respiratory tracts of 129/SvJ mice.
(A) The extent of infection was determined by non-invasive bioluminescence imaging of living, anesthetized mice every 24 h. Each data point represents the mean bioluminescence of 6 mice. The total flux (photons/s) of bioluminescence intensity is calculated as the sum of radiance in the region of interest. (B–D) Viral titers in the nasal turbinates, trachea, and lungs were determined by sacrificing groups of 3 mice at the reported days and performing plaque titrations of tissue homogenates in LLC-MK2 cells. Both experiments were repeated, and representative data are shown.
Figure 4
Figure 4. Virus replication and pathogenesis as a function of virus dose and mouse strain.
After intranasal inoculation of 129/SvJ mice with 70 to 7,000 PFU of rSeV-luc(M-F*), the total flux of bioluminescence intensity (A) and viral titers (B) were measured as described in Figure 3. (C) Percent body weight change was measured in groups of ten 129/SvJ mice after inoculation with 70 to 7,000 PFU. The experiment was performed in duplicate; representative data are shown. (D) Sendai virus-specific binding antibody titers in sera of 129/SvJ mice collected 10 days after inoculation with 70 to 7,000 PFU of rSeV-luc(M-F*). Titers are reported as the reciprocal endpoint dilutions in ELISA assays. Five infected and two control mice were used in the experiment, which was performed in duplicate. Representative data are shown. (E) The total flux of bioluminescence intensity in the nasopharynx, trachea, and lungs after 7,000-PFU intranasal inoculation of 129/SvJ, DBA/2, BALB/c, or C57BL/6-strain mice with rSeV-luc(M-F*). Values are the mean from six animals. The experiment was performed in duplicate, and the results from a representative experiment are shown. (F) Mean percent weight change in groups of 10 mice after infection with 7,000 PFU of rSeV-luc(M-F*). The experiment was performed in duplicate; representative data are shown.
Figure 5
Figure 5. Sendai virus infection and immune response after contact transmission.
Donor mice were directly inoculated with 70 PFU (A,C,E) or 7,000 PFU (B,D,F) of rSeV-luc(M-F*) and then introduced into a cage with 3 naïve animals one day later. The total flux of bioluminescence intensity in the nasopharyngeal cavities of individual 129/SvJ (A–B) and BALB/c (C–D) mice are shown. Serum was collected on day 60 and contact mice were challenged with 7,000 PFU of rSeV-luc(M-F*) on day 63 to monitor for re-infection by bioluminescence. Sendai virus-specific binding antibody titers were measured as reciprocal endpoint dilutions of sera collected on day 60 from mice co-housed with animals inoculated with 70 PFU (E) or 7,000 PFU (F). Open bars represent mice inoculated on day 0 and solid bars represent the contact mice. The experiment was performed in triplicate for 129/SvJ mice (3 donor and 9 contact mice) and duplicate for BALB/c mice (2 donor and 6 contact mice).
Figure 6
Figure 6. Timing and tissue-tropic spread of Sendai virus infection after contact transmission.
The co-housing of contact mice with mice inoculated with rSeV-luc(M-F*) is described in Figure 5. The total flux of bioluminescence intensity in individual, representative 129/SvJ (A–B) and BALB/c (C–D) mice is shown for the nasopharynx (triangles), trachea (circles), and lungs (squares). Time to detection of bioluminescence (>6 log10 photons/s) in the nasopharynx after inoculation of donors with either 70 PFU (E) or 7,000 PFU (F) of virus. Mean percent weight change in BALB/c (G) and 129/SvJ (H) mice. The contact transmission experiment was performed in triplicate for 129/SvJ mice and in duplicate for BALB/c mice. Open symbols and bars represent directly inoculated mice and solid symbols and bars represent contact mice. In panels G and H, the symbol X indicates PBS-inoculated control mice.

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