Virulence, pathology, and pathogenesis of Pteropine orthoreovirus (PRV) in BALB/c mice: Development of an animal infection model for PRV

<div><p>Background</p><p>Cases of acute respiratory tract infection caused by Pteropine orthoreovirus (PRV) of the genus <i>Orthoreovirus</i> (family: <i>Reoviridae</i>) have been reported in Southeast Asia, where it was isolated from humans and bats. It is possible that PRV-associated respiratory infections might be prevalent in Southeast Asia. The clinical course of PRV is not fully elucidated.</p><p>Methods</p><p>The virulence, pathology, and pathogenesis of two PRV strains, a human-borne PRV strain (isolated from a patient, who returned to Japan from Bali, Indonesia in 2007) and a bat-borne PRV (isolated from a bat [<i>Eonycteris spelaea</i>] in the Philippines in 2013) were investigated in BALB/c mice using virological, pathological, and immunological study methods.</p><p>Results</p><p>The intranasal inoculation of BALB/c mice with human-borne PRV caused respiratory infection. In addition, all mice with immunity induced by pre-inoculation with a non-lethal dose of PRV were completely protected against lethal PRV infection. Mice treated with antiserum with neutralizing antibody activity after inoculation with a lethal dose of PRV showed a reduced fatality rate. In this mouse model, bat-borne PRV caused respiratory infection similar to human-borne PRV. PRV caused lethal respiratory disease in an animal model of PRV infection, in which BALB/c mice were used.</p><p>Conclusions</p><p>The BALB/c mouse model might help to accelerate research on the virulence of PRV and be useful for evaluating the efficacy of therapeutic agents and vaccines for the treatment and prevention of PRV infection. PRV was shown for the first time to be a causative virus of respiratory disease on the basis of Koch’s postulations by the additional demonstration that PRV caused respiratory disease in mice through their intranasal inoculation with PRV.</p></div

Histopathological findings of the lungs of BALB/c mice infected with PRV-MB.

<p>The lungs were obtained from PRV-MB-1.0×10⁶ PFU mice on the 4th DPI. H&E staining (A, left panels, and B) and IHC analysis with an OCP antibody (A, middle panels) or with NRS (A, right panels) were performed. The H&E staining and IHC analysis with an OCP antibody or with NRS of the lung at 200× magnification (A, upper panels), of a bronchiole at 1000× magnification (A, middle panels), and of an alveolus at 1000× magnification (A, lower panels) are shown. The black boxes in the upper panels indicate the necrotic lesion, which was positive for PRV-MB antigen (A, upper-middle panel). These areas are shown at higher magnification in the middle panels. The black arrows in the middle panels indicate the area of bronchiolar epithelial cell necrosis. The black dotted boxes in the upper panels indicate the PRV-MB antigen-positive lesion in the alveolar area and the images are enlarged in the lower panels (A, lower-middle panel). The PRV-MB antigen-positive lesion did not show a positive reaction in the IHC analysis, in which NRS was used instead of the OCP antibody (A, right panels). The scale bars in A (upper panels) indicate 200 μm, whereas those in A (middle panels), and A (lower panels) indicate 20 μm. H&E staining images of the alveoli at 1000× magnification (B) are shown. The black arrows in the upper and lower panels indicate neutrophils and type II pneumocytes, respectively. The scale bars in (B) indicate 20 μm.</p

Changes in the survival rate (upper panels) and body weight (lower panels) in BALB/c mice treated with antiserum after lethal PRV-MB infection.

<p>The PRV-MB-1.0×10⁵ PFU mice were treated with the intraperitoneal administration of antiserum once daily during periods surrounded by the gray boxes (5 mice per group). Antiserum with a PRV-MB-specific serum neutralizing antibody titer of 1:10,240 was diluted 4-fold with PBS. The volume of the antiserum administered daily was 100 μL. The treatment was initiated at just after 1 h after the virus challenge (B) or from the 1st (C), 2nd (D), 3rd (E), and 4th (F) DPI. The control mice were administered the same volume of control serum, PRV antibody negative mouse serum diluted with PBS (A). Error bars indicate the standard error.</p

Temporal changes in the viral RNA loads in the lungs of the BALB/c mice infected with PRV-MB.

<p>The lungs were obtained from PRV-MB-1.0×10³ mice or PRV-MB-1.0×10⁵ PFU mice on the 1st, 3rd, and 5th DPI (5 mice per group for each day). Error bars indicate the standard error. N.S., not significant. ***, statistically significant (p < 0.0001).</p

Histopathological findings in the lungs of BALB/c mice infected with PRV-MB on the 1st, 3rd, and 5th DPI.

<p>The lungs were obtained from the PRV-MB-1.0×10³ PFU mice (A) and the PRV-MB-1.0×10⁵ PFU mice (B) on the 1st, 3rd, and 5th DPI. IHC analysis of the lungs was performed using the OCP antibody for detection of PRV antigen. The right panels show enlarged views of the areas of interesting lesions (black squares in the left panels). The magnification levels of the left and right panels are 200× and 1000×, respectively. Black arrows indicate the pneumocytes that were positive for PRV-MB antigen. The scale bars in the left and right panels indicate 200 μm and 20 μm, respectively.</p

GenBank accession numbers for the nucleotide sequences of the 10 RNA genome segments of the PRV-MB and PRV-Samal-24 strains used in this study.

<p>GenBank accession numbers for the nucleotide sequences of the 10 RNA genome segments of the PRV-MB and PRV-Samal-24 strains used in this study.</p