Reverse Genetics with a Full-length Infectious cDNA Clone of Bovine Torovirus

Historically part of the coronavirus (CoV) family, torovirus (ToV) was recently classified into the new family Tobaniviridae . While reverse genetics systems have been established for various CoVs, none exist for ToVs. Herein, we developed a reverse genetics system using an infectious full-length cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV harboring genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the hemagglutinin-esterase (HE) gene was edited, as cell-adapted wtBToV generally loses full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued. These exhibited no significant differences in their effect on virus growth in HRT18 cells, suggesting that HE is not essential for viral replication in these cells. Thereafter, we generated recombinant virus (rEGFP), wherein HE was replaced by the enhanced green fluorescent protein (EGFP) gene. The rEGFP expressed EGFP in infected cells, but showed significantly lower viral growth compared to wtBToV. Moreover, the rEGFP readily deleted the EGFP gene after one passage. Interestingly, rEGFP variants with two mutations (C1442F and I3562T) in non-structural proteins (NSPs) that emerged during passages exhibited improved EGFP expression, EGFP gene retention, and viral replication. An rEGFP into which both mutations were introduced displayed a similar phenotype to these variants, suggesting that the mutations contributed to EGFP gene acceptance. The current findings provide new insights into BToV, and reverse genetics will help advance the current understanding of this neglected pathogen. Importance ToVs are diarrhea-causing pathogens detected in various species, including humans. Through the development of a BAC-based BToV, we introduced the first reverse genetics system for Tobaniviridae . Utilizing this system, recombinant BToVs with a full-length HE gene were generated. Remarkably, although clinical BToVs generally lose the HE gene after a few passages, some recombinant viruses generated in the current study retained the HE gene for up to 20 passages while accumulating mutations in NSPs, which suggested that these mutations may be involved in HE gene retention. The EGFP gene of recombinant viruses was unstable, but rEGFP into which two NSP mutations were introduced exhibited improved EGFP expression, gene retention, and viral replication. These data suggested the existence of an NSP-based acceptance or retention mechanism for exogenous RNA or HE genes. Recombinant BToVs and reverse genetics are powerful tools for understanding fundamental viral processes, infection pathogenesis, and BToV vaccine development.

A full-length infectious cDNA clone of a dsRNA totivirus-like virus

Totivirus-like viruses are icosahedral non-enveloped double-stranded (ds)RNA viruses belonging to a group recently discovered and provisionally assigned in the Totiviridae family. Unlike fungal and protozoan Totiviridae viruses, these totivirus-like viruses infect a diverse spectrum of metazoan hosts and currently have enormous impacts on fisheries and agriculture. We developed the first totivirus-like virus Omono River virus (OmRV) infectious full-length DNA clone and produce the infectious particles using an RNA-transcript-based method. Unlike the parent wild-type particles from nature, the reverse-genetically-generated OmRV particles had an indistinguishable cytopathic effect, infectivity, and morphology. The established system is one of the few systems that have been reported for generating a non-segmented dsRNA virus DNA clone.

Molecular Identification of Prune Dwarf Virus (PDV) Infecting Sweet Cherry in Canada and Development of a PDV Full-Length Infectious cDNA Clone

Prune dwarf virus (PDV) is a member of ilarviruses that infects stone fruit species such as cherry, plum and peach, and ornamentally grown trees worldwide. The virus lacks an RNA silencing suppressor. Infection by PDV either alone, or its mixed infection with other viruses causes deteriorated fruit marketability and reduced fruit yields. Here, we report the molecular identification of PDV from sweet cherry in the prominent fruit growing region of Ontario, Canada known as the Niagara fruit belt using next generation sequencing of small interfering RNAs (siRNAs). We assessed its incidence in an experimental farm and determined the full genome sequence of this PDV isolate. We further constructed an infectious cDNA clone. Inoculation of the natural host cherry with this clone induced a dwarfing phenotype. We also examined its infectivity on several common experimental hosts. We found that it was infectious on cucurbits (cucumber and squash) with clear symptoms and Nicotiana benthamiana without causing noticeable symptoms, and it was unable to infect Arabidopsis thaliana. As generating infectious clones for woody plants is very challenging with limited success, the PDV infectious clone developed from this study will be a useful tool to facilitate molecular studies on PDV and related Prunus-infecting viruses.

Complete genome sequence and construction of an infectious full-length cDNA clone of a cucumber vein yellowing virus (CVYV) isolate from Portugal

Cucumber vein yellowing virus (CVYV) is a member of the genus Ipomovirus in the family Potyviridae. In the National Center for Biotechnology Information (NCBI) database, three complete genome sequences of CVYV isolates from Spain (NC_006941), Israel (KT276369), and Jordan (JF460793) are available. In this study, we report the complete sequence of an isolate of CVYV from Portugal (DSMZ PV-0776) along with the construction of an infectious full-length cDNA clone via Gibson assembly. The sequence of CVYV Portugal shows the closest relationship to a CVYV isolate from Spain (genome, 99.7% identity; polyprotein, 99.7% identity). The CVYV full-length cDNA clone was introduced by electroporation into Rhizobium radiobacter and infiltrated into the cotyledons of Cucumis sativus plantlets, resulting in symptoms resembling those of the wild-type virus. Transmission of the infectious CVYV full-length clone by the whitefly Bemisia tabaci was confirmed. This first report confirming the infectivity of a CVYV cDNA clone provides the opportunity to study gene functions in a consistent genomic background.

Molecular characterization and pathogenicity of an infectious cDNA clone of tomato brown rugose fruit virus

Tomato brown rugose fruit virus (ToBRFV) is a new member of the genus Tobamovirus, and has the potential to affect the production and marketability of tomatoes and peppers. In this study, we sequenced and analyzed the complete genome of ToBRFV isolates from tomato plants showing mosaic and mottling symptoms in Yunnan Province of China. We constructed a full-length infectious cDNA clone of ToBRFV, which could induce systemic infection with typical symptoms in tomato, Nicotiana benthamiana, and N. tabacum cv. Samsun nn plants through Agrobacterium-mediated inoculation. Further experimental evidence demonstrated that the rod-shaped virions accumulating in agroinfiltrated plants are sap-transmissible. This is the first report on the construction of a biologically active, full-length infectious cDNA clone of ToBRFV. The system developed herein will facilitate further research on functions of ToBRFV-encoded proteins and plant-ToBRFV interactions through reverse genetic approaches.

Synthesis and Characterization of a Full-Length Infectious cDNA Clone of Tomato Mottle Mosaic Virus

Tomato mottle mosaic virus (ToMMV) is a noteworthy virus which belongs to the Virgaviridae family and causes serious economic losses in tomato. Here, we isolated and cloned the full-length genome of a ToMMV Chinese isolate (ToMMV-LN) from a naturally infected tomato (Solanum lycopersicum L.). Sequence analysis showed that ToMMV-LN contains 6399 nucleotides (nts) and is most closely related to a ToMMV Mexican isolate with a sequence identity of 99.48%. Next, an infectious cDNA clone of ToMMV was constructed by a homologous recombination approach. Both the model host N. benthamiana and the natural hosts tomato and pepper developed severe symptoms upon agroinfiltration with pToMMV, which had a strong infectivity. Electron micrographs indicated that a large number of rigid rod-shaped ToMMV virions were observed from the agroinfiltrated N. benthamiana leaves. Finally, our results also confirmed that tomato plants inoculated with pToMMV led to a high infection rate of 100% in 4–5 weeks post-infiltration (wpi), while pepper plants inoculated with pToMMV led to an infection rate of 40–47% in 4–5 wpi. This is the first report of the development of a full-length infectious cDNA clone of ToMMV. We believe that this infectious clone will enable further studies of ToMMV genes function, pathogenicity and virus–host interaction.

Rescue of an infection clone of barley yellow dwarf virus -GAV

Barley yellow dwarf virus-GAV (BYDV-GAV) is one of the most prevalent viruses causing yellow dwarf disease in wheat in China. The biology and pathology of BYDV-GAV are well studied; however, gene functions and molecular mechanisms of BYDV-GAV disease development are unclear due to the lack of a reverse genetics system. In this study, a full-length cDNA clone of BYDV-GAV was constructed and expressed using Agrobacterium-mediated inoculation of Nicotiana benthamiana. Virions produced by BYDV-GAV in N. benthamiana were transmitted to wheat by an aphid vector after acquisition via a sandwich feeding method. Infectivity of the cDNA clone in wheat was verified through RT-PCR and Western blot assays, and the recombinant virus elicited typical reddening symptoms in oats and was transmitted between wheat plants. These results confirm the production of biologically active transmissible virions. Using the BYDV-GAV infectious clone, we demonstrate that the viral protein P4 was involved in cell-to-cell movement and stunting symptoms in wheat. This is the first report describing the development of an infectious full-length cDNA clone of BYDV-GAV and provides a useful tool for virus-host-vector interaction studies.