Reverse Genetics for Fusogenic Bat-Borne Orthoreovirus Associated with Acute Respiratory Tract Infections in Humans: Role of Outer Capsid Protein σC in Viral Replication and Pathogenesis

Abstract

Nelson Bay orthoreoviruses (NBVs) are members of the fusogenic orthoreoviruses and possess 10-segmented double-stranded RNA genomes. NBV was first isolated from a fruit bat in Australia more than 40 years ago, but it was not associated with any disease. However, several NBV strains have been recently identified as causative agents for respiratory tract infections in humans. Isolation of these pathogenic bat reoviruses from patients suggests that NBVs have evolved to propagate in humans in the form of zoonosis. To date, no strategy has been developed to rescue infectious viruses from cloned cDNA for any member of the fusogenic orthoreoviruses. In this study, we report the development of a plasmid-based reverse genetics system free of helper viruses and independent of any selection for NBV isolated from humans with acute respiratory infection. cDNAs corresponding to each of the 10 full-length RNA gene segments of NBV were cotransfected into culture cells expressing T7 RNA polymerase, and viable NBV was isolated using a plaque assay. The growth kinetics and cell-to-cell fusion activity of recombinant strains, rescued using the reverse genetics system, were indistinguishable from those of native strains. We used the reverse genetics system to generate viruses deficient in the cell attachment protein σC to define the biological function of this protein in the viral life cycle. Our results with σC-deficient viruses demonstrated that σC is dispensable for cell attachment in several cell lines, including murine fibroblast L929 cells but not in human lung epithelial A549 cells, and plays a critical role in viral pathogenesis. We also used the system to rescue a virus that expresses a yellow fluorescent protein. The reverse genetics system developed in this study can be applied to study the propagation and pathogenesis of pathogenic NBVs and in the generation of recombinant NBVs for future vaccines and therapeutics.

Establishment of different plasmid only-based reverse genetics systems for the recovery of African horse sickness virus

“…These uncapped transcripts can nevertheless functionally substitute for viral transcripts at all stages of the AHSV replication cycle, as evidenced by the recovery of viable AHSV-4. These results are in agreement with those reported for reoviruses (Kobayashi et al, 2007;Kawagishi et al, 2016) and BTV (Pretorius et al, 2015). It is plausible that the sustained synthesis of RNA transcripts in situ results in sufficient levels of expression to allow the assembly of core particles, which themselves are transcriptionally active (Vermaak et al, 2015), and thus lead to an amplification of gene transcription.…”

“…AHSV, BTV and EHDV can be recovered by the transfection of in vitro-transcribed and capped RNAs into permissive cell lines (Boyce et al, 2008;Vermaak et al, 2015) or into cells that had previously been transfected with helper expression plasmids that synthesize the core proteins and the two non-structural proteins NS1 and NS2 (Kaname et al, 2013;Matsuo and Roy, 2013;Yang et al, 2015). An entirely plasmid only-based reverse genetics system has been developed for mammalian orthoreovirus (Kobayashi et al, 2007) and, more recently, for BTV (Pretorius et al, 2015) and a fusogenic bat-borne orthoreovirus (Kawagishi et al, 2016). The development of reverse genetics has revolutionized the study of reoviruses and BTV (Roy, 2013) by providing a powerful tool for investigating virus replication and disease.…”

Lethal murine infection model for human respiratory disease-associated Pteropine orthoreovirus

“…PRV strain MB (Yamanaka et al, 2014) was amplified in L929 cells and approximately 10-fold concentrated virus stock was generated using a 10% polyethylene glycol concentration method as described elsewhere (Lewis and Metcalf, 1988). Infectious virus titer was determined by a plaque assay (Kawagishi et al, 2016). Avian reovirus strain 58-132 (Takase et al, 1985), simian rotavirus strain SA11 (Taniguchi et al, 1994), and mammalian orthoreovirus prototype strain T1L were amplified in QT6, MA104, and L929 cells, respectively.…”

“…After incubation, 50 μl neutralized viruses were inoculated onto a monolayer of A549 in a 96-well microplate. After incubation for 16 h, cells were fixed with absolute ethanol and cells expressing viral antigens were detected using murine anti-PRV strain MB σC antibody (Kawagishi et al, 2016) and goat antimouse IgG antibody Alexa488 conjugate. The NT titer was indicated by the reciprocal value of the maximum dilution at which the number of foci showed 50% reduction compared with the positive control.…”

Rethinking the Significance of Reovirus in Water and Wastewater

“…ReoVs have been occasionally found in fecal specimens associated with childhood diarrhea (Giordano et al 2005). More recently novel ReoVs from nonhuman animals have been identified with some infecting humans and thus raising concerns about their zoonotic potential (Chua et al 2007;Zhang et al 2011;Kohl et al 2012;Dermody et al 2013;Yamanaka et al 2014;Kawagishi et al 2016). …”