Diminished Reovirus Capsid Stability Alters Disease Pathogenesis and Littermate Transmission

<div><p>Reovirus is a nonenveloped mammalian virus that provides a useful model system for studies of viral infections in the young. Following internalization into host cells, the outermost capsid of reovirus virions is removed by endosomal cathepsin proteases. Determinants of capsid disassembly kinetics reside in the viral σ3 protein. However, the contribution of capsid stability to reovirus-induced disease is unknown. In this study, we found that mice inoculated intramuscularly with a serotype 3 reovirus containing σ3-Y354H, a mutation that reduces viral capsid stability, succumbed at a higher rate than those infected with wild-type virus. At early times after inoculation, σ3-Y354H virus reached higher titers than wild-type virus at several sites within the host. Animals inoculated perorally with a serotype 1 reassortant reovirus containing σ3-Y354H developed exaggerated myocarditis accompanied by elaboration of pro-inflammatory cytokines. Surprisingly, unchallenged littermates of mice infected with σ3-Y354H virus displayed higher titers in the intestine, heart, and brain than littermates of mice inoculated with wild-type virus. Together, these findings suggest that diminished capsid stability enhances reovirus replication, dissemination, lethality, and host-to-host spread, establishing a new virulence determinant for nonenveloped viruses.</p></div

A reassortant reovirus strain containing σ3-Y354H displays enhanced virulence following peroral inoculation.

<p>Newborn C57BL/6J mice were inoculated perorally with 10⁴ PFU of either T1L/T3Dμ1σ3 or T1L/T3Dμ1σ3Y354H. Mice (n = 21 and 18 for T1L/T3Dμ1σ3 and T1L/T3Dμ1σ3Y354H, respectively) were monitored for survival for 21 days. *, <i>P</i> < 0.001 as determined by log-rank test in comparison to T1L/T3Dμ1σ3.</p

Construction of T1L × T3D reassortant reovirus strains.

<p>(A) Schematic of the genomes of the reassortant reovirus strains used in this study. Gene segments derived from T1L and T3D are shown in red and blue, respectively. Two reassortant strains were recovered by reverse genetics, incorporating either the wild-type T3D S4 gene segment encoding σ3 or T3D σ3-Y354H. (B) Electrophoretic analysis of the dsRNA genomes of recombinant reassortant viruses. Purified virions of T1L, T3D, T1L/T3Dμ1σ3, and T1L/T3Dμ1σ3Y354H were electrophoresed in an SDS-polyacrylamide gel, which was stained with ethidium bromide to visualize viral gene segments. Gene segments are labeled on the left. (C) Ammonium chloride sensitivity of reassortant viruses. Murine L929 cells were pretreated with the concentrations of ammonium chloride shown, adsorbed with T1L/T3Dμ1σ3 or T1L/T3Dμ1σ3Y354H at an MOI of 25 PFU/cell, and incubated for 18 h. Cells were fixed with methanol, and reovirus-infected cells were quantified for three independent experiments. Error bars indicate standard errors of the means. *, <i>P</i> < 0.05 as determined by Mann-Whitney test in comparison to T3D.</p

Elevated cytokine levels following infection of mice with T1L/T3Dμ1σ3 and T1L/T3Dμ1σ3Y354H.

<p>(A-D) Newborn C57BL/6J mice were inoculated perorally with 10³ PFU of either T1L/T3Dμ1σ3 or T1L/T3Dμ1σ3Y354H. At days 2, 4, and 8 post-inoculation, mice were euthanized, and hearts were excised, frozen at -80°C, thawed, and homogenized in PBS. Levels of IFNβ (A), IFNγ (B), IL-6 (C), and IL-10 (D) in heart homogenates were quantified by ELISA. Results are expressed as mean cytokine levels for 4–7 animals per time point. (E) Human embryonic kidney cells (293-T) were cotransfected with a plasmid encoding <i>Renilla</i> luciferase as a transfection control and either a PGL firefly luciferase reporter plasmid under the control of the IFN-sensitive reporter element (ISRE) or a PGL-basic vector as a general transcription control. Cells were incubated for 24 h and inoculated with the viruses shown at an MOI of 100 PFU/cell. Luciferase activity was quantified 24 h post-inoculation. Values for cells expressing the ISRE reporter were normalized to the corresponding values for the PGL-basic control vector as a transcription control. Data represent an experiment conducted twice in triplicate. Error bars indicate standard errors of the mean. *, <i>P</i> < 0.05, **, <i>P</i> < 0.01 as determined by Student’s <i>t</i> test.</p

Viral loads are comparable in mice infected with reassortant viruses.

<p>Newborn C57BL/6J mice were inoculated perorally with 10⁴ PFU of either T1L/T3Dμ1σ3 or T1L/T3Dμ1σ3Y354H. At days 2 (A), 4 (B), 8 (C) post-inoculation, animals were euthanized, intestine, spleen, liver, heart, and brain were excised, and viral titers in organ homogenates were determined by plaque assay. Results are expressed as mean viral titers for 6 to 9 animals for each time point. Error bars indicate standard errors of the mean.</p

The σ3-Y354H mutation is associated with higher viral loads after transmission of reovirus between littermates.

<p>Two newborn C57BL/6J mice from a litter of eight animals were inoculated perorally with 10⁴ PFU of either T1L/T3Dμ1σ3 (black) or T1L/T3Dμ1σ3Y354H (white). The inoculated mice (squares) were placed with their uninoculated littermates (circles) and housed together. Eight days later, inoculated mice and uninoculated littermates were euthanized, intestine, heart, and brain were excised, and viral titers were determined by plaque assay. Results are expressed as viral titers for each animal assayed. *, <i>P</i> < 0.05 as determined by Mann-Whitney test in comparison to T1L/T3Dμ1σ3.</p

Reovirus strain T1L/T3Dμ1σ3Y354H causes pronounced myocarditis.

<p>Newborn C57BL/6J mice were inoculated perorally with 10³ PFU of either T1L/T3Dμ1σ3 or T1L/T3Dμ1σ3Y354H. On day 8 post-inoculation, mice were euthanized, and hearts were excised and photographed (A). Cardiac tissue was fixed in formalin, embedded in paraffin, sectioned, and stained with H&E (B) or reovirus-specific polyclonal antiserum (C). Images are shown at 20X (left) and 400X (right) magnification.</p