Development of an entirely plasmid-based reverse genetics system for 12-segmented double-stranded RNA viruses

The family Reoviridae is a nonenveloped virus group with a double-stranded (ds) RNA genome comprising 9 to 12 segments. In the family Reoviridae, the genera Cardoreovirus, Phytoreovirus, Seadornavirus, Mycoreovirus, and Coltivirus contain virus species having 12-segmented dsRNA genomes. Reverse genetics systems used to generate recombinant infectious viruses are powerful tools for investigating viral gene function and for developing vaccines and therapeutic interventions. Generally, this methodology has been utilized for Reoviridae viruses such as Orthoreovirus, Orbivirus, Cypovirus, and Rotavirus, which have genomes with 10 or 11 segments, respectively. However, no reverse genetics system has been developed for Reoviridae viruses with a genome harboring 12 segments. Herein, we describe development of an entire plasmid-based reverse genetics system for Tarumizu tick virus (TarTV) (genus Coltivirus, family Reoviridae), which has a genome of 12 segments. Recombinant TarTVs were generated by transfection of 12 cloned complementary DNAs encoding the TarTV genome into baby hamster kidney cells expressing T7 RNA polymerase. Using this technology, we generated VP12 mutant viruses and demonstrated that VP12 is an N-glycosylated protein. We also generated a reporter virus expressing the HiBiT-tagged VP8 protein. This reverse genetics system will increase our understanding of not only the biology of the genus Coltivirus but also the replication machinery of the family Reoviridae.

The family Reoviridae is a nonenveloped virus group classified into 15 genera. These viruses have double-stranded (ds) RNA genomes with 9 to 12 segments. This family includes several important pathogens in both humans and animals. Mammalian orthoreovirus (MRV) and Nelson Bay orthoreovirus (NBV), which belong to the genus Orthoreovirus, have genomes consisting of 10 segments of dsRNA. MRV is an experimental model for studies of Reoviridae virus replication and pathogenesis. NBV, classified into the fusogenic subgroup of this genus, is associated with acute respiratory tract infections in humans (1–3). The fusogenic orthoreoviruses encode a unique fusion-associated small transmembrane (FAST) protein associated with cell–cell fusion and viral pathogenesis (4–6). Bluetongue virus (BTV) and African horse sickness virus (AHSV) belong to the genus Orbivirus, have genomes with 10 segments of dsRNA, and cause severe diseases in domestic animals (7, 8). Rotavirus (RV, genus Rotavirus) has a genome with 11 segments of dsRNA and causes severe diarrhea in young children. RV infection is responsible for 128,500 deaths per year worldwide, predominately in developing countries (9). Colorado tick fever virus (CTFV), an arthropod-borne virus transmitted by ticks and belonging to the genus Coltivirus, has a dsRNA genome comprising 12 segments. CTFV causes a variety of symptoms in humans, including abrupt fever, chills, headache, myalgia, and abdominal pain (10, 11). Within the genus Coltivirus (in addition to CTFV), Eyach virus, Shelly Headland virus, Kundal virus, and Tarumizu tick virus (TarTV) have been isolated from, or detected in, ticks in Europe, Australia, India, and Japan, respectively (12–17). Taï Forest reovirus was isolated from free-tailed bats in Côte d’Ivoire (18), and Lishui pangolin virus was detected in pangolins in China (19). These reports suggest that coltiviruses are distributed in multiple species worldwide. However, the molecular mechanism underlying the propagation and pathogenesis of these viruses remains largely unknown.Reverse genetics systems are powerful tools used to study many aspects of viral biology and virus–host interactions and also provide an opportunity to generate recombinant viruses that can be used for vaccines or as viral vectors. This technology has been used for several viruses in the family Reoviridae. In the genus Orthoreovirus, an entirely plasmid-based reverse genetics system was developed for MRV in 2007 (20). This is the first example of engineering recombinant Reoviridae viruses entirely from cloned complementary DNAs (cDNAs). This system was established by cotransfection of cloned cDNAs representing 10 MRV gene segments, each flanked by the T7 promoter and hepatitis delta virus (HDV) ribozyme, into cells expressing T7 RNA polymerase. Subsequently, a reverse genetics system for NBV, belonging to a fusogenic reovirus group, was established based on the MRV rescue system (21). In the genus Orbivirus, RNA- and DNA-based reverse genetics systems were developed for BTV, AHSV, and Epizootic hemorrhagic disease virus (22–25). Although development of a reverse genetics system for the genus Rotavirus has lagged behind those for the genera Orthoreovirus and Orbivirus, our group recently developed the first plasmid-based reverse genetics system for RV by cotransfecting plasmids encoding unique heterogeneous viral proteins, NBV FAST, and vaccinia virus capping enzyme as rescue enhancers along with the 11 RV T7 promoter-based rescue plasmids (26). In addition, reverse genetics systems were also developed for Bombyx mori cypovirus and Dendrolimus punctatus cypovirus (genus Cypovirus), which are insect pathogens and have a genome consisting of 10 segments of dsRNA (27, 28). Since reverse genetics systems were developed for Reoviridae viruses with genomes containing 10 or 11 segments of dsRNA, studies of the family Reoviridae have advanced markedly. However, to date, no reverse genetics system has been established for Reoviridae viruses with 12 genome segments.In this study, we isolated a TarTV strain from a raccoon dog postmortem. Using this TarTV strain, we established an entire plasmid-based reverse genetics system and rescued VP12 mutant viruses and a HiBiT-tagged reporter virus. This system is a useful tool to generate recombinant coltiviruses with 12 dsRNA genome segments.