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

RESEARCH ARTICLE 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 Takahiro Kawagishi1,2, Yuta Kanai1, Hideki Tani3, Masayuki Shimojima3, Masayuki Saijo3, Yoshiharu Matsuura2, Takeshi Kobayashi1* 1 Laboratory of Viral Replication, International Research Center for Infectious Diseases, Osaka University, Suita, Osaka, Japan, 2 Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka, Japan, 3 Special Pathogens Laboratory, Department of Virology I, National Institute of Infectious Diseases, Musashimurayama, Tokyo, Japan * OPEN ACCESS Citation: Kawagishi T, Kanai Y, Tani H, Shimojima M, Saijo M, Matsuura Y, et al. (2016) 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. PLoS Pathog 12(2): e1005455. doi:10.1371/journal.ppat.1005455 Editor: Mark T. Heise, University of North Carolina at Chapel Hill, UNITED STATES Received: September 1, 2015 Accepted: January 24, 2016 Published: February 22, 2016 Copyright: © 2016 Kawagishi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported in part by grantsin-aid from the Ministry of Health, Labor, and Welfare of Japan (H25-Shinko-Ippan-004) and the Japan Society for the Promotion of Science (Grant Numbers 15J04209 and 26292149). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. 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 plasmidbased 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. PLOS Pathogens | DOI:10.1371/journal.ppat.1005455 February 22, 2016 1 / 30 Reverse Genetics for Fusogenic Bat-Borne Reovirus Competing Interests: The authors have declared that no competing interests exist. 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 batborne 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 plasmidbased 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 σCdeficient 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. Introduction Members of the genus Orthoreovirus belonging to the family Reoviridae are nonenveloped viruses. Their genomes contain 10-segmented double-stranded RNA (dsRNA) divided into three classes based on their sizes: large (L1–L3), medium (M1–M3), and small (S1–S4). The orthoreoviruses are classified into fusogenic and nonfusogenic subgroups based on their ability to induce cell-to-cell fusion during cell culture [1]. The fusogenic subgroup comprises the avian orthoreovirus (ARV), baboon orthoreovirus (BRV), reptilian orthoreovirus, Broome reovirus (BroV), and Nelson Bay orthoreovirus (NBV), whereas the nonfusogenic subgroup comprises the prototypical mammalian orthoreovirus (MRV) [1–3]. Nonfusogenic MRVs are quite common and generally asymptomatic in humans. Although natural infections involving fusogenic orthoreoviruses cause severe diseases in infected animals, infections involving these orthoreoviruses in humans have not been reported. However, in 2006, the Melaka (Mel) virus, a new fusogenic orthoreovirus, was isolated from a patient with acute respiratory tract infection in Malaysia [4]. This newly isolated virus is genetically related to the NBV strains Nelson Bay (NB) and Pulau, which were isolated from fruit bats in Australia and Malaysia, respectively [5, 6]. Subsequently, other related NBV strains have been isolated from patients with severe respiratory illness in Malaysia and Hong Kong [7–10]. Recently, we reported an imported case of a respiratory tract infection as (...truncated)