Development of a Mini-Replicon-Based Reverse-Genetics System for Rice Stripe Tenuivirus

Abstract

Negative-stranded RNA (NSR) viruses include both animal- and plant-infecting viruses that often cause serious diseases in human and livestock, and in agronomic crops. Rice stripe tenuivirus (RSV), a plant NSR virus with four negative-stranded/ambisense RNA segments, is one of the most destructive rice pathogens in many Asian countries. Due to the lack of a reliable reverse-genetics technology, molecular studies of RSV gene functions and its interaction with host plants are severely hampered. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV gene functional analysis in Nicotiana benthamiana . We first developed a mini-replicon system expressing RSV genomic RNA3 eGFP reporter (MR3 (-)eGFP ), a nucleocapsid (NP), and a codon usage optimized RNA-dependent RNA polymerase (RdRp opt ), respectively. Using this mini-replicon system we determined that RSV NP and RdRp opt are indispensable for the eGFP expression from MR3 (-)eGFP . The expression of eGFP from MR3 (-)eGFP can be significantly enhanced in the presence of NSs and P19-HcPro-γb. In addition, NSvc4, the movement protein of RSV, facilitated eGFP trafficking between cells. We also developed an antigenomic RNA3-based replicon in N. benthamiana . However, we found that the RSV NS3 coding sequence acts as a cis -element to regulate viral RNA expression. Finally, we made mini-replicons representing all four RSV genomic RNAs. This is the first mini-replicon-based reverse-genetics system for monocot-infecting tenuivirus. We believe that this mini-replicon system described here will allow the studies of RSV replication, transcription, cell-to-cell movement and host machinery underpinning RSV infection in plants. IMPORTANCE Plant-infecting segmented negative-stranded RNA (NSR) viruses are grouped into 3 genera: Orthotospovirus, Tenuivirus and Emaravirus . The reverse-genetics systems have been established for members in the genera Orthotospovirus and Emaravirus , respectively. However, there is still no reverse-genetics system available for Tenuivirus . Rice stripe virus (RSV) is a monocot-infecting tenuivirus with four negative-stranded/ambisense RNA segments. It is one of the most destructive rice pathogens and causes significant damages to rice industry in Asian countries. Due to the lack of a reliable reverse-genetics system, molecular characterizations of RSV gene functions and the host machinery underpinning RSV infection in plants are extremely difficult. To overcome this obstacle, we developed a mini-replicon-based reverse-genetics system for RSV in Nicotiana benthamiana . This is the first mini-replicon-based reverse-genetics system for tenuivirus. We consider that this system will provide researchers a new working platform to elucidate the molecular mechanisms dictating segmented tenuivirus infections in plant.

Monitoring SARS-CoV-2 Infection Using a Double Reporter-Expressing Virus

“…Replication-competent, reporter-expressing, recombinant viruses have been previously shown to represent an excellent approach to study, among others, viral infection, replication, pathogenesis, and transmission ( 21 – 31 ). We and others have described the feasibility of generating rSARS-CoV-2 expressing reporter genes encoding either fluorescent or luciferase proteins ( 24 , 38 , 45 , 46 , 48 ).…”

“…Reverse genetics systems have permitted the generation of recombinant RNA viruses entirely from cloned cDNA, facilitating studies to better understand multiple aspects of the biology of viruses, including, among others, mechanisms of viral infection, pathogenesis, transmission, and disease ( 21 – 31 ). Another application of reverse genetics is the generation of recombinant viruses containing gene mutations and/or deletions that result in viral attenuation for their implementation as safe, immunogenic, and protective live-attenuated vaccines ( 22 , 32 – 37 ).…”

Monitoring SARS-CoV-2 infection using a double reporter-expressing virus

“…Replication-competent, reporter-expressing, recombinant viruses have been previously shown to represent an excellent approach to study, among others, viral infection, replication, pathogenesis, and transmission (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). We and others have described the feasibility of generating rSARS-CoV-2 expressing reporter genes encoding either fluorescent or luciferase proteins (36,43,44,46,47).…”

“…The copyright holder for this preprint this version posted June 24, 2022. ; https://doi.org/10.1101/2022.06. 23.497376 doi: bioRxiv preprint Reverse genetics systems have permitted the generation of recombinant RNA viruses entirely from cloned cDNA, facilitating studies to better understand multiple aspects of the biology of viruses, including, among others, mechanisms of viral infection, pathogenesis, transmission, and disease (19)(20)(21)(22)(23)(24)(25)(26)(27)(28)(29). Another application of reverse genetics is the generation of recombinant viruses containing gene mutations and/or deletions that result in viral attenuation for their implementation as safe, immunogenic, and protective liveattenuated vaccines (LAV) (20,(30)(31)(32)(33)(34)(35).…”

Navigating migraine care through the COVID-19 pandemic: an update

“…Ibuprofen increases angiotensin-converting enzyme-2 (ACE-2) bioavailability, a binding site for COVID-19, and could potentially enhance initial infectivity, based on extrapolations from animal studies in cardiac muscle using sevenfold the weight-adjusted doses for humans [ 41 ]. However, NSAIDS also attenuate inflammation through reduced inflammatory cytokine activation [ 42 ]. Another proposed mechanism of harm of NSAIDs is the reduction of fever and host defence [ 40 ] implying that paracetamol and aspirin are also unsafe [ 40 ].…”