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.

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.

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

Torovirus (ToV) has recently been classified in the new family Tobaniviridae, although it belonged to the Coronavirus (CoV) family historically. Reverse genetics systems for many CoVs have been established, but none exist for ToVs. Here, we describe a reverse genetics system using a full-length infectious cDNA clone of bovine ToV (BToV) in a bacterial artificial chromosome (BAC). Recombinant BToV containing genetic markers had the same phenotype as wild-type (wt) BToV. To generate two types of recombinant virus, the Hemagglutinin-esterase (HE) gene was manipulated, since cell-adapted wtBToV generally loses the full-length HE (HEf), resulting in soluble HE (HEs). First, recombinant viruses with HEf and HA-tagged HEf or HEs genes were rescued; these showed no significant differences in cell growth, suggesting that HE is not essential for viral growth in cells. Then, recombinant virus in which HE was replaced by the Enhanced Green Fluorescent Protein (EGFP) gene expressed EGFP in infected cells, but showed significantly reduced viral growth compared to wtBToV. Moreover, the recombinant virus readily deleted the EGFP gene after one passage. Interestingly, one variant with mutations in non-structural proteins (NSPs) showed improved EGFP expression and viral growth during serial passages, although it eventually deleted the EGFP gene, suggesting that these mutations contributed to EGFP gene acceptance. These recombinant viruses provide new insights regarding BToV and its reverse genetics will help advance understanding of this neglected pathogen.ImportanceToVs are diarrhea-causing pathogens that have been detected in many species, including humans. BToV has spread worldwide, leading to economic losses. We developed the first reverse genetics system for Tobaniviridae using a BAC-based BToV. Using this system, we showed that recombinant BToVs with HEf and HEs showed no significant differences in cell growth. In contrast, clinical BToVs generally lose the HE gene after a few passages but some recombinant viruses retained the HE gene for up to 20 passages, suggesting some benefits of HE retention. The EGFP gene of the recombinant viruses was unstable and was rapidly deleted, likely via negative selection. Interestingly, one virus variant with mutations in NSPs was more stable, resulting in improved EGFP-expression and viral growth, suggesting that the mutations contributed to some acceptance of the exogenous EGFP gene without clear positive selection. The recombinant BToVs and reverse genetics developed here are powerful tools for understanding fundamental viral processes and their pathogenesis and for developing BToV vaccines.

Development of a Full-Length Infectious cDNA Clone of the Grapevine Berry Inner Necrosis Virus

Grapevine berry inner necrosis virus (GINV) belongs to the genus Trichovirus in the family Betaflexiviridae. The GINV isolate LN_BETA_RS was obtained from a “Beta” grapevine (Vitis riparia × Vitis labrusca) exhibiting chlorotic mottling and ring spot in Xingcheng, Liaoning Province, China. To verify the correlation between GINV and grapevine chlorotic mottling and ring spot disease, we constructed an infectious cDNA clone of GINV isolate LN_BETA_RS using the seamless assembly approach. Applied treatments of agroinfiltration infectious cDNA confirmed systemic GINV infection of the Nicotianaoccidentalis 37B by reverse transcription polymerase chain reaction (RT-PCR) and transmission electron microscopy, exhibiting chlorotic mottling symptoms on leaves. Infectious cDNA was also transmitted to new healthy N. occidentalis plants through rub-inoculation. Moreover, the cDNA clone was agroinfiltrated into “Beta” and “Thompson Seedless” grapevine plantlets, and the inoculated grapevines exhibited leaf chlorotic mottling and ringspot during the two years of observation. GINV-inoculated “Beta” grapevines had serious leaf chlorotic mottling and ringspot symptoms on the whole plant, while relatively few symptoms were observed on the leaves of agroinoculated “Thompson Seedless” grapevines in early spring and only weak ring spot gradually appeared later in the top young leaves. Our experiments fulfilled Koch’s postulates and revealed the causative role of GINV in grapevine chlorotic mottling and ring spot disease.

Rolling Circle Amplification is a high fidelity and efficient alternative to plasmid preparation for the rescue of infectious clones

Alphaviruses (genus Alphavirus; family Togaviridae) are a medically relevant family of viruses that include chikungunya virus, Eastern equine encephalitis virus, and the emerging Mayaro virus. Infectious cDNA clones of these viruses are necessary molecular tools to understand viral biology and to create effective vaccines. The traditional approach to rescuing virus from an infectious cDNA clone requires propagating large amounts of plasmids in bacteria, which can result in unwanted mutations in the viral genome due to bacterial toxicity or recombination and requires specialized equipment and knowledge to propagate the bacteria. Here, we present an alternative to the bacterial-based plasmid platform that uses rolling circle amplification (RCA), an in vitro technology that amplifies plasmid DNA using only basic equipment. We demonstrate that the use of RCA to amplify plasmid DNA is comparable to the use of a midiprepped plasmid in terms of viral yield, albeit with a slight delay in virus recovery kinetics. RCA, however, has lower cost and time requirements and amplifies DNA with high fidelity and with no chance of unwanted mutations due to toxicity. We show that sequential RCA reactions do not introduce mutations into the viral genome and, thus, can replace the need for glycerol stocks or bacteria entirely. These results indicate that RCA is a viable alternative to traditional plasmid-based approaches to viral rescue.ImportanceThe development of infectious cDNA clones is critical to studying viral pathogenesis and for developing vaccines. The current method for propagating clones in bacteria is limited by the toxicity of the viral genome within the bacterial host, resulting in deleterious mutations in the viral genome, which can only be detected through whole-genome sequencing. These mutations can attenuate the virus, leading to lost time and resources and potentially confounding results. We have developed an alternative method of preparing large quantities of DNA that can be directly transfected to recover infectious virus without the need for bacteria by amplifying the infectious cDNA clone plasmid using rolling circle amplification (RCA). Our results indicate that viral rescue from an RCA product produces a viral yield equal to bacterial-derived plasmid DNA, albeit with a slight delay in replication kinetics. The RCA platform, however, is significantly more cost and time-efficient compared to traditional approaches. When the simplicity and costs of RCA are combined, we propose that a shift to an RCA platform will benefit the field of molecular virology and could have significant advantages for recombinant vaccine production.