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.

Author Summary

Nelson Bay orthoreoviruses (NBVs) are members of the fusogenic orthoreoviruses that have various host species, including reptiles, birds, and mammals. Recently, several NBV strains have been isolated from patients with acute respiratory tract infections. Isolation of these pathogenic reoviruses raises concerns about the potential emerging infections of bat-borne orthoreoviruses in humans. The development of an entirely plasmid-based reverse genetics system for double-stranded RNA viruses has trailed other systems of major animal RNA virus groups because of the technical complexities involved in the manipulation of genomes composed of 10 or more segments. In this study, we developed a plasmid-based reverse genetics system for a pathogenic NBV strain. We used this system to generate viruses incapable of expressing the cell attachment protein σC and to rescue a replication-competent virus that expresses a yellow fluorescent protein. Our studies using σC-deficient viruses suggest that NBVs may engage multiple independent viral ligands and cellular receptors for efficient cell attachment and viral pathogenesis, thus providing new insight into the biology of orthoreoviruses. The reverse genetics approach described in this study can be exploited for fusogenic orthoreovirus biology and used to develop vaccines, diagnostics, and therapeutics.