Over the last two decades, since the outbreak of Severe Acute Respiratory Syndrome coronaviruses (SARS-CoV) in 2003, several surveillance studies of viruses prevalent in wildlife and especially in bats have been conducted, revealing a number of novel viruses associated with newly emerging diseases. A few of these viruses originating from bats are thought to be associated with severe human diseases, including Ebola virus, Middle East respiratory syndrome coronavirus (MERS-CoV), Nipah and Hendra viruses, and the current pandemic viruses - Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) [3, 6, 36, 37]. Reoviruses were not known to be associated with severe human diseases and were studied as oncolytic viruses. MRVs not only efficiently replicate in many mammalian species but also can change drastically via gene reassortment events. Almost all novel reovirus isolates are hypothesized to have resulted from a reassortment event [9, 19, 20, 23, 24, 30, 35]. An isolate can contain genome segments from different serotypes and/or different hosts. It could increase the zoonotic potential of these viruses in the future.
Based on sequence comparison and phylogenetic analysis, we hypothesize that the novel type 1 BatMRV/B19-02 isolate may have resulted from a reassortment of the bat, human, rodent and/or swine MRV strains. Five of ten segments (L1, L2, M1, M3 and S2) were highly similar to those segments of MRVs isolated from human, including two classical MRV type 1 strain and a novel MRV type 2 strain caused acute necrotizing encephalopathy. Only segment S4 was closer to that of the bat MRV WIV5 isolate from Hipposideros sp. in China . However, due to limited number of references, we could not exclude the possibility of genetic drift from the MRV that had not found before in bats or other species.
In the previous reports, all three MRV serotypes are circulating in bat species in Europe and China. However, MRV serotypes 2 and 3 are more prevalent in bat populations [12, 17, 20, 35]. In South Korea, MRVs have been detected in swine, human and mouse belonging to serotype 2 and 3 [13, 16]. This study is the first description of the bat MRV serotype 1 strain found in South Korea, and almost all of the genome segments were more related to those of human MRVs than those of bat MRVs identified. Since Melaka virus was identified in 2006, several novel reovirus strains have been discovered which cause severe respiratory and enteric and encephalitis diseases in both animals and humans . Thus, the pathogenesis of the novel reovirus infections is needed to be further studied.
It is important to distinguish between genomic RNA and synthesized viral mRNA in infected cells. Unfortunately, reovirus mRNAs lack poly(A) tails . However, the viral genome comprises ten dsRNA segments, with a single copy of each viral gene segment incorporated per virion, encoding for eight structural proteins and four nonstructural proteins . In addition, the minus strands were synthesized using mRNA as templates, resulting in formation of nascent genomic dsRNA, concomitantly . The relative proportions of the 10 nascent genomic dsRNA segments are equimolar . Thus, the relative abundances of total RNA could reflect the expression level.
The level of viral mRNA synthesis could be divided into two stages: the early viral mRNA is synthesized before the newly viral genome is replicated, and the late mRNA. The previous data indicated that the S4 mRNA, which encodes the σ3 protein, is the most efficiently translated in both in vitro translation reactions and reovirus-infected cells . In this study, both the M2 gene and S4 gene, encoding for the two most abundant proteins in the reovirus virion (µ1 and σ3 protein, respectively), were related to the highest expression levels of mRNA during the late stage. In contrast, the genes encoding for enzymes related to the lower gene expression levels. The dsRNA activates innate immune response in host cells, which may be one disadvantage for MRVs replicating in the infected cells. As the M2 and S4 were related to apoptosis and inhibition of host RNA and protein synthesis , the high expression-associated level of M2 and S4 RNA might be associated with evading host cell innate immune responses. However, there should be additional studies to demonstrate this hypothesis and on whether those differences of RNA levels were due to either induced RNA synthesis or delayed RNA decay. In addition, further studies based on dsRNA-seq and/or strand-specific sequencing for preparing libraries could be useful for not only the role of junctions in RNA virus recombination and rearrangements but also the level gene expression in the virus life cycle.
Both recombination and reassortment are an evolutionary mechanism of segmented RNA viruses. The previous study demonstrated that recombination junctions affected non-canonical RNA synthesis. They used RNA-seq to analyze viral RNA packaged in reovirus particles, which reovirus strains rsT1L and rsT3D were engineered using plasmid-based reverse genetics . We also utilized RNA-seq to analyze viral RNA genomic reovirus type 1 isolated from bats using the Geneious software platform to analyze and visualize the RNA-seq data, an easy-to-use and flexible desktop software application framework . We identified massive junctions within individual viral genome segments in the infected cell. The percentage of deletion and rearrangement junctions based on size and mapped read was high in both M3 and S3 genes. In contrast, the junctions per size were the highest, while the junctions per mapped read were low in the S4 segment. The rates of junctions per size and mapped read were low (2.4% and 0.06%, respectively) in the S1 gene that is the most variant. The relative abundance of deletions and rearrangement varies seems not only increase along with the read coverage. Most studies on RNA viruses suggested that a large diverse population of defective viral genomes are generated by high MOI. In the case of Zika virus, the large deletions were more abundant under high MOI, while the small deletions were common in both low and high MOI passaging conditions . At a high MOI condition, our results also suggested that the relative abundance of the deletion or rearrangement junctions varies lead to the distribution of defective viral genomes (DVGs) diversity during infections. The defective gene segments featuring internal deletions undergoes sequence-directed recombination at distinct site . The deletions could lead to the mechanism of recombination. The role of deletion and rearrangement junctions in the recombination pathways may be explained based on the studies on the interaction between viral RNA and host-cell proteins or viral structure/non-structure proteins in the replication cycles of viruses. Moreover, deletions in different lengths could be donors for the recombination mechanisms within species or even cross family. A novel gene likely originated from the segment S1 gene of a bat orthoreovirus in a novel coronavirus isolated from Rousettus leschennaulti bat in China, which suggested the heterologous inter-family recombination between a single positive-strand RNA virus and a double-stranded segmented RNA virus . In the long-term, surveillance of prevalent viruses from bats, virus co-infections can be observed in a few cases. For instance, coronavirus frequently co-infect with paramyxovirus or reovirus (Data not shown). Thus, the cross-family recombination events could have probably occurred during co-infection in the reservoir host – bat.
In conclusion, the BatMRV/B19-02 strain, which belonged to the MRV type 1 isolated from Miniopterus schreibersii, may have resulted from a reassortment of the bat, human, rodent and swine MRV strains. The ratios of relative abundances in RNA levels of the ten reovirus segments is constant during the late stage, with higher-levels of abundance of M2 and S4 gene segments. Moreover, the massive junctions, including deletion and rearrangement junctions, were identified within all viral genome segments in the infected cell that supported the explanation of the recombination and reassortment mechanism of segmented RNA viruses. Considering that mammalian orthoreovirus isolated from bat was highly similar to human MRV strains, it could increase the zoonotic potential. The pathogenesis of the novel reovirus infections is needed for further study.