Mammalian orthoreoviruses exhibit rare genotype variability in genome constellations.

Mammalian orthoreoviruses (reoviruses) are currently classified based on properties of the attachment protein, σ1. Four reovirus serotypes have been identified, three of which are represented by well-studied prototype human reovirus strains. Reoviruses contain ten segments of double-stranded RNA that encode 12 proteins and can reassort during coinfection. To understand the breadth of reovirus genetic diversity and its potential influence on reassortment, the sequence of the entire genome should be considered. While much is known about the prototype strains, a thorough analysis of all ten reovirus genome segment sequences has not previously been conducted. We analyzed phylogenetic relationships and nucleotide sequence conservation for each of the ten segments of more than 60 complete or nearly complete reovirus genome sequences, including those of the prototype strains. Using these relationships, we defined genotypes for each segment, with minimum nucleotide identities of 77-88% for most genotypes that contain several representative sequences. We applied segment genotypes to determine reovirus genome constellations, and we propose implementation of an updated reovirus genome classification system that incorporates genotype information for each segment. For most sequenced reoviruses, segments other than S1, which encodes σ1, cluster into a small number of genotypes and a limited array of genome constellations that do not differ greatly over time or based on animal host. However, a small number of reoviruses, including prototype strain Jones, have constellations in which segment genotypes differ from those of most other sequenced reoviruses. For these reoviruses, there is little evidence of reassortment with the major genotype. Future basic research studies that focus on the most genetically divergent reoviruses may provide new insights into reovirus biology. Analysis of available partial sequences and additional complete reovirus genome sequencing may also reveal reassortment biases, host preferences, or infection outcomes that are based on reovirus genotype.

Reovirus Efficiently Reassorts Genome Segments during Coinfection and Superinfection.

Reassortment, or genome segment exchange, increases diversity among viruses with segmented genomes. Previous studies on the limitations of reassortment have largely focused on parental incompatibilities that restrict generation of viable progeny. However, less is known about whether factors intrinsic to virus replication influence reassortment. Mammalian orthoreovirus (reovirus) encapsidates a segmented, double-stranded RNA (dsRNA) genome, replicates within cytoplasmic factories, and is susceptible to host antiviral responses. We sought to elucidate the influence of infection multiplicity, timing, and compartmentalized replication on reovirus reassortment in the absence of parental incompatibilities. We used an established post-PCR genotyping method to quantify reassortment frequency between wild-type and genetically barcoded type 3 reoviruses. Consistent with published findings, we found that reassortment increased with infection multiplicity until reaching a peak of efficient genome segment exchange during simultaneous coinfection. However, reassortment frequency exhibited a substantial decease with increasing time to superinfection, which strongly correlated with viral transcript abundance. We hypothesized that physical sequestration of viral transcripts within distinct virus factories or superinfection exclusion also could influence reassortment frequency during superinfection. Imaging revealed that transcripts from both wild-type and barcoded viruses frequently co-occupied factories, with superinfection time delays up to 16 h. Additionally, primary infection progressively dampened superinfecting virus transcript levels with greater time delay to superinfection. Thus, in the absence of parental incompatibilities and with short times to superinfection, reovirus reassortment proceeds efficiently and is largely unaffected by compartmentalization of replication and superinfection exclusion. However, reassortment may be limited by superinfection exclusion with greater time delays to superinfection. IMPORTANCE Reassortment, or genome segment exchange between viruses, can generate novel virus genotypes and pandemic virus strains. For viruses to reassort their genome segments, they must replicate within the same physical space by coinfecting the same host cell. Even after entry into the host cell, many viruses with segmented genomes synthesize new virus transcripts and assemble and package their genomes within cytoplasmic replication compartments. Additionally, some viruses can interfere with subsequent infection of the same host or cell. However, spatial and temporal influences on reassortment are only beginning to be explored. We found that infection multiplicity and transcript abundance are important drivers of reassortment during coinfection and superinfection, respectively, for reovirus, which has a segmented, double-stranded RNA genome. We also provide evidence that compartmentalization of transcription and packaging is unlikely to influence reassortment, but the length of time between primary and subsequent reovirus infection can alter reassortment frequency.

Reovirus Low-Density Particles Package Cellular RNA.

Packaging of segmented, double-stranded RNA viral genomes requires coordination of viral proteins and RNA segments. For mammalian orthoreovirus (reovirus), evidence suggests either all ten or zero viral RNA segments are simultaneously packaged in a highly coordinated process hypothesized to exclude host RNA. Accordingly, reovirus generates genome-containing virions and "genomeless" top component particles. Whether reovirus virions or top component particles package host RNA is unknown. To gain insight into reovirus packaging potential and mechanisms, we employed next-generation RNA-sequencing to define the RNA content of enriched reovirus particles. Reovirus virions exclusively packaged viral double-stranded RNA. In contrast, reovirus top component particles contained similar proportions but reduced amounts of viral double-stranded RNA and were selectively enriched for numerous host RNA species, especially short, non-polyadenylated transcripts. Host RNA selection was not dependent on RNA abundance in the cell, and specifically enriched host RNAs varied for two reovirus strains and were not selected solely by the viral RNA polymerase. Collectively, these findings indicate that genome packaging into reovirus virions is exquisitely selective, while incorporation of host RNAs into top component particles is differentially selective and may contribute to or result from inefficient viral RNA packaging.

Reovirus RNA recombination is sequence directed and generates internally deleted defective genome segments during passage.

For viruses with segmented genomes, genetic diversity is generated by genetic drift, reassortment, and recombination. Recombination produces RNA populations distinct from full-length gene segments and can influence viral population dynamics, persistence, and host immune responses. Viruses in the Reoviridae family, including rotavirus and mammalian orthoreovirus (reovirus), have been reported to package segments containing rearrangements or internal deletions. Rotaviruses with RNA segments containing rearrangements have been isolated from immunocompromised and immunocompetent children and in vitro following serial passage at relatively high multiplicity. Reoviruses that package small, defective RNA segments have established chronic infections in cells and in mice. However, the mechanism and extent of Reoviridae RNA recombination are undefined. Towards filling this gap in knowledge, we determined the titers and RNA segment profiles for reovirus and rotavirus following serial passage in cultured cells. The viruses exhibited occasional titer reductions characteristic of interference. Reovirus strains frequently accumulated segments that retained 5' and 3' terminal sequences and featured large internal deletions, while similarly fragmented segments were rarely detected in rotavirus populations. Using next-generation RNA-sequencing to analyze RNA molecules packaged in purified reovirus particles, we identified distinct recombination sites within individual viral genome segments. Recombination junctions were frequently but not always characterized by short direct sequence repeats upstream and downstream that spanned junction sites. Taken together, these findings suggest that reovirus accumulates defective gene segments featuring internal deletions during passage and undergoes sequence-directed recombination at distinct sites.IMPORTANCE Viruses in the Reoviridae family include important pathogens of humans and other animals and have segmented RNA genomes. Recombination in RNA virus populations can facilitate novel host exploration and increased disease severity. The extent, patterns, and mechanisms of Reoviridae recombination and the functions and effects of recombined RNA products are poorly understood. Here, we provide evidence that mammalian orthoreovirus regularly synthesizes RNA recombination products that retain terminal sequences but contain internal deletions, while rotavirus rarely synthesizes such products. Recombination occurs more frequently at specific sites in the mammalian orthoreovirus genome, and short regions of identical sequence are often detected at junction sites. These findings suggest that mammalian orthoreovirus recombination events are directed in part by RNA sequences. An improved understanding of recombined viral RNA synthesis may enhance our capacity to engineer improved vaccines and virotherapies in the future.

Restoration of the type I IFN-IL-1 balance through targeted blockade of PTGER4 inhibits autoimmunity in NOD mice.

Type I IFN (IFN-I) dysregulation contributes to type 1 diabetes (T1D) development, and although increased IFN-I signals are pathogenic at the initiation of autoimmune diabetes, IFN-I dysregulation at later pathogenic stages more relevant for therapeutic intervention is not well understood. We discovered that 5 key antigen-presenting cell subsets from adult prediabetic NOD mice have reduced responsiveness to IFN-I that is dominated by a decrease in the tonic-sensitive subset of IFN-I response genes. Blockade of IFNAR1 in prediabetic NOD mice accelerated diabetes and increased Th1 responses. Therefore, IFN-I responses shift from pathogenic to protective as autoimmunity progresses, consistent with chronic IFN-I exposure. In contrast, IL-1-associated inflammatory pathways were elevated in prediabetic mice. These changes correlated with human T1D onset-associated gene expression. Prostaglandin E2 (PGE2) and prostaglandin receptor 4 (PTGER4), a receptor for PGE2 that mediates both inflammatory and regulatory eicosanoid signaling, were higher in NOD mice and drive innate immune dysregulation. Treating prediabetic NOD mice with a PTGER4 antagonist restored IFNAR signaling, decreased IL-1 signaling, and decreased infiltration of leukocytes into the islets. Therefore, innate cytokine alterations contribute to both T1D-associated inflammation and autoimmune pathogenesis. Modulating innate immune balance via signals such as PTGER4 may contribute to treatments for autoimmunity.