Respiratory infection of mice with mammalian reoviruses causes systemic infection with age and strain dependent pneumonia and encephalitis

Abstract

Background: Because mammalian reoviruses are isolated from the respiratory tract we modeled the natural history of respiratory infection of adult and suckling mice with T1 Lang (T1L) and T3 Dearing (T3D) reoviruses.

Methods: Adult and suckling Balb/c mice were infected by the intranasal route and were assessed for dose response of disease as well as viral replication in the lung and other organs. Viral antigen was assessed by immunofluorescence and HRP staining of tissue sections and histopathology was assessed on formalin fixed, H + E stained tissue sections.

Results: Intranasal infection of adult mice resulted in fatal respiratory distress for high doses (10(7) pfu) of T1L but not T3D. In contrast both T1L and T3D killed suckling mice at moderate viral dosages (10(5) pfu) but differed in clinical symptoms where T1L induced respiratory failure and T3D caused encephalitis. Infections caused transient viremia that resulted in spread to peripheral tissues where disease correlated with virus replication, and pathology. Immunofluorescent staining of viral antigens in the lung showed reovirus infection was primarily associated with alveoli with lesser involvement of bronchiolar epithelium. Immunofluorescent and HRP staining of viral antigens in brain showed infection of neurons by T3D and glial cells by T1L.

Conclusions: These mouse models of reovirus respiratory infection demonstrated age and strain dependent disease that are expected to be relevant to understanding and modulating natural and therapeutic reovirus infections in humans.

Figure 1

Age and virus dependent fatal dose response of reovirus T1L and T3D infection in adult and suckling Balb-c mice. The number of animals monitored in each group is shown as values of n. Top panel. Adult mice were intranasaly infected with 10⁷ pfu of either T1L or T3D and monitored for 30 days. Lower dosages did not induce fatality (not shown). Bottom panel. Suckling mice at 2 days of age were intranasaly infected with graded dosages of either T1L or T3D and monitored for 30 days. The times to death were significantly different using the students t test for the comparisons indicated in brackets.

Figure 2

Reovirus T1L and T3D replicates in lung and peripheral tissues following respiratory inoculation. Groups of 2 adult mice were infected intranasaly with 10⁵ pfu of either T1L (white bars) or T3D (black bars) before collecting various organs 2 days pi. The yield of infectious virus was determined for each mouse and is shown + 1 SE for each virus and tissue type. The bars for T1L infected animals are open and for T3D are closed.

Figure 3

Yield of infectious virus in lung, brain and liver of adult and suckling mice. Groups of 2 adult or 2 day old suckling mice were infected intranasaly with 10⁵ pfu of either T1L or T3D before assaying infectious virus in lung, brain and liver. The titres represent the mean of 2 animals which where titrated in duplicate for all time points except for the later 15 and 21 day time points for the T1L infections that represent single animals with duplicate titrations. The yield of infectious virus is shown + 1 SE for each time point. The yield values are closed for T1L and open for T3D. The p statistics comparing yields of virus for T1L versus T3D are indicated for groups of values in brackets (n = 6–8) that were determined by the paired students t test with significant differences considered as p ≤ 0.05 for adult and suckling lung as well as sucking liver.

Figure 4

Respiratory infection of adult mice results in transient viremia. Groups of 2 adult mice were infected intranasaly with 10⁵ pfu of either T1L or T3D before assaying infectious virus in the blood at various times pi. The yield of infectious virus is shown + 1 SE for each time point. The yield values are closed for T1L and open for T3D.

Figure 5

Time course of immunofluorescent staining of virus infected adult lungs. Groups of 2 adult mice were infected intranasaly with 10⁷ pfu of either T1L or T3D before fluorescent antibody staining of frozen lung sections at 1, 2, or 3, days pi. T1L and T3D antigens were stained with virus specific antiserum as the primary antibody followed by CY3 labeled donkey anti-rabbit IgG and are colored in red. Cell nuclei were stained with DAPI and are shown in blue, either alone or merged with the CY3 stained images. Sagital sections show extensive staining in alveolar regions as well as sparse staining of epithelium in the bronchiolar airways (arrow) restricted primarily to T3D infection. Uninfected control lungs are shown as negative staining controls. Size bars are 100 μM.

Figure 6

High magnification images of T1L and T3D infected adult lungs. The 2 day post infection images shown in Figure 5 are enlarged here to demonstrate more clearly the presence of reovirus antigens in alveolar tissues. Size bars are 100 μM.

Figure 7

Time course of immunofluorescent staining of virus infected suckling lung. Two day old suckling mice were infected intranasaly with 10⁵ pfu of either T1L or T3D before fluorescent antibody staining of frozen lung sections after 1, 2, or 3 days of infection. T1L and T3D antigens were stained with virus specific antiserum as described in Figure 5. Cell nuclei were stained with DAPI, shown in blue. Size bars are 100 μM. Sagital sections show viral infection patterns similar to those of adults ( Figures 5 and 6) that again show sparse staining of epithelium in the bronchiolar airways (arrow) being restricted primarily to T3D infection.

Figure 8

Immunofluoresent staining of virus infected suckling mouse brain sections. a) Two day old suckling mice were infected intranasaly with 10⁵ pfu of either T1L or T3D before fluorescent antibody staining of frozen coronal brain sections at 7 days pi. T1L and T3D antigens were stained with virus specific antiserum as described in Figure 5. Cell nuclei were stained with DAPI, shown in blue. Size bars are 100 μM. b) Horse Radish Peroxidase staining (in brown) of formalin fixed paraffin embedded brains of T1L and T3D infected suckling mouse brains at 7 days pi. Staining of T3D infected brains showed the presence of neurons and neuronal processes whereas the cell type infected by T1L were non-neuronal and thus lacked processes. Staining of endothelial cells in blood vessels was also observed for both T3D (not shown ) as well as T1L infected brain that is shown in insert. Images were collected with the 20X objective lens and size bars are indicated.

Figure 9

Pathology of virus infected lung, liver and brain tissues. Adult or 2 day old suckling mice were infected intranasaly with 10⁷ or 10⁵ pfu respectively with either T1L or T3D before sectioning formalin fixed, paraffin embedded sections for histopathological assessment by H and E. staining. Panels a, d, g, and j show uninfected control images for adult or suckling lung, liver and brain sections of mice as labeled. Panels b, e, h, and k show the T1L infected lung, liver and brain of the indicated types (suckling or adult) of infected mouse organs. Panels c, f, i, and l show the T3D infected lung, liver and brain for the indicated types (suckling or adult) of mouse organs. Arrows show local areas of cellular damage in liver and suckling brain sections that show the dentate gyrus of the hippocampus. Lungs and liver were imaged 7 days pi and brains at 13 days pi. Images were captured with the 20X objective.