Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease

Reovirus-Induced Apoptosis in the Intestine Limits Establishment of Enteric Infection

Several viruses induce intestinal epithelial cell death during enteric infection. However, it is unclear whether proapoptotic capacity promotes or inhibits replication in this tissue. We infected mice with two reovirus strains that infect the intestine but differ in the capacity to alter immunological tolerance to new food antigen. Infection with reovirus strain T1L, which induces an inflammatory immune response to fed antigen, is prolonged in the intestine, whereas T3D-RV, which does not induce this response, is rapidly cleared from the intestine. Compared with T1L, T3D-RV infection triggered apoptosis of intestinal epithelial cells and subsequent sloughing of dead cells into the intestinal lumen. We conclude that the infection advantage of T1L derives from its capacity to subvert host restriction by epithelial cell apoptosis, providing a possible mechanism by which T1L enhances inflammatory signals during antigen feeding. Using a panel of T1L × T3D-RV reassortant viruses, we identified the viral M1 and M2 gene segments as determinants of reovirus-induced apoptosis in the intestine. Expression of the T1L M1 and M2 genes in a T3D-RV background was sufficient to limit epithelial cell apoptosis and enhance viral infection to levels displayed by T1L. These findings define additional reovirus gene segments required for enteric infection of mice and illuminate the antiviral effect of intestinal epithelial cell apoptosis in limiting enteric viral infection. Viral strain-specific differences in the capacity to infect the intestine may be useful in identifying viruses capable of ameliorating tolerance to fed antigen in autoimmune conditions like celiac disease

Congenital Zika virus infection as a silent pathology with loss of neurogenic output in the fetal brain

Zika virus (ZIKV) is a flavivirus with teratogenic effects on fetal brain, but the spectrum of ZIKV-induced brain injury is unknown, particularly when ultrasound imaging is normal. In a pregnant pigtail macaque (Macaca nemestrina) model of ZIKV infection, we demonstrate that ZIKV-induced injury to fetal brain is substantial, even in the absence of microcephaly, and may be challenging to detect in a clinical setting. A common and subtle injury pattern was identified, including (i) periventricular T2-hyperintense foci and loss of fetal noncortical brain volume, (ii) injury to the ependymal epithelium with underlying gliosis and (iii) loss of late fetal neuronal progenitor cells in the subventricular zone (temporal cortex) and subgranular zone (dentate gyrus, hippocampus) with dysmorphic granule neuron patterning. Attenuation of fetal neurogenic output demonstrates potentially considerable teratogenic effects of congenital ZIKV infection even without microcephaly. Our findings suggest that all children exposed to ZIKV in utero should receive long-term monitoring for neurocognitive deficits, regardless of head size at birth

Flavivirus Nonstructural Protein NS5 Dysregulates HSP90 to Broadly Inhibit JAK/STAT Signaling

Pathogenic flaviviruses antagonize host cell Janus kinase/signal transducer and activator of transcription (JAK/STAT) signaling downstream of interferons α/β. Here, we show that flaviviruses inhibit JAK/STAT signaling induced by a wide range of cytokines beyond interferon, including interleukins. This broad inhibition was mapped to viral nonstructural protein 5 (NS5) binding to cellular heat shock protein 90 (HSP90), resulting in reduced Janus kinase–HSP90 interaction and thus destabilization of unchaperoned JAKs (and other kinase clients) of HSP90 during infection by Zika virus, West Nile virus, and Japanese encephalitis virus. Our studies implicate viral dysregulation of HSP90 and the JAK/STAT pathway as a critical determinant of cytokine signaling control during flavivirus infection

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