Stabilization of a full-length infectious cDNA clone for duck Tembusu virus by insertion of an intron

AbstractTomato 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.

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Genome Sequence, Full-Length Infectious cDNA Clone, and Mapping of Viral Double-Stranded RNA Accumulation Determinant of Hypovirus CHV1-EP721

Journal of Virology ◽

10.1128/jvi.01625-06 ◽

2006 ◽

Vol 81 (4) ◽

pp. 1813-1820 ◽

Cited By ~ 51

Author(s):

Haiyan Lin ◽

Xiuwan Lan ◽

Hong Liao ◽

Todd B. Parsley ◽

Donald L. Nuss ◽

...

Keyword(s):

Genome Sequence ◽

Cdna Clone ◽

Amino Acid Level ◽

Cryphonectria Parasitica ◽

Infectious Cdna Clone ◽

Full Length ◽

Cdna Clones ◽

Coding Region ◽

Viral Dsrna ◽

Double Stranded Rna

ABSTRACT Cryphonectria parasitica strain EP721 is infected with a strain of hypovirus CHV1, CHV1-EP721, and exhibits typical hypovirulence-associated traits such as reduced pigmentation and reduced asexual sporulation. However, the accumulation of the viral double-stranded RNA (dsRNA) in this hypovirus-infected C. parasitica strain is atypically low. We now report the complete nucleotide sequence and construction of a full-length infectious cDNA clone for hypovirus CHV1-EP721. The genome sequence of CHV1-EP721 was determined to be 12,724 bp in length and to share extensive homology with two other hypovirus strains, CHV1-Euro7 and CHV1-EP713, with an average of 99% and 90% identities at the nucleotide level and 99% and 92% identities at the amino acid level, respectively. CHV1-EP721 was successfully introduced into virus-free fungal host strain EP721(-v) by transfection with transcripts derived from a full-length viral cDNA. The transfected strain had a phenotype indistinguishable from that of EP721, and the accumulation of CHV1-EP721 dsRNA in the transfectant was lower than those transfected by CHV1-Euro7 and CHV1-EP713 transcripts. Through the construction of chimeric viruses by domain swapping using infectious cDNA clones of CHV1-EP721, CHV1-EP713, and CHV1-Euro7 hypoviruses, the determinant for the low level of viral dsRNA accumulation in CHV1-EP721 was mapped to the second of two CHV1-EP721 open reading frames (ORFs), ORF B. Further refined swapping of domains within ORF B identified a 2.5-kb coding region between p48 and the polymerase domain of CHV1-EP721 as being responsible for the low viral dsRNA accumulation. Evidence is also provided that low rates of hypovirus transmission through conidial spores correlates with low viral dsRNA accumulation.

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Infectious cDNA Clone of the Epidemic West Nile Virus from New York City

Journal of Virology ◽

10.1128/jvi.76.12.5847-5856.2002 ◽

2002 ◽

Vol 76 (12) ◽

pp. 5847-5856 ◽

Cited By ~ 158

Author(s):

Pei-Yong Shi ◽

Mark Tilgner ◽

Michael K. Lo ◽

Kim A. Kent ◽

Kristen A. Bernard

Keyword(s):

New York ◽

New York City ◽

West Nile Virus ◽

Cdna Clone ◽

York City ◽

Infectious Cdna Clone ◽

Full Length ◽

Progeny Virus ◽

West Nile ◽

Full Length Cdna

ABSTRACT We report the first full-length infectious clone of the current epidemic, lineage I, strain of West Nile virus (WNV). The full-length cDNA was constructed from reverse transcription-PCR products of viral RNA from an isolate collected during the year 2000 outbreak in New York City. It was cloned into plasmid pBR322 under the control of a T7 promoter and stably amplified in Escherichia coli HB101. RNA transcribed from the full-length cDNA clone was highly infectious upon transfection into BHK-21 cells, resulting in progeny virus with titers of 1 × 109 to 5 × 109 PFU/ml. The cDNA clone was engineered to contain three silent nucleotide changes to create a StyI site (C to A and A to G at nucleotides [nt] 8859 and 8862, respectively) and to knock out an EcoRI site (A to G at nt 8880). These genetic markers were retained in the recovered progeny virus. Deletion of the 3′-terminal 199 nt of the cDNA transcript abolished the infectivity of the RNA. The plaque morphology, in vitro growth characteristics in mammalian and insect cells, and virulence in adult mice were indistinguishable for the parental and recombinant viruses. The stable infectious cDNA clone of the epidemic lineage I strain will provide a valuable experimental system to study the pathogenesis and replication of WNV.

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

Viruses ◽

10.3390/v13061050 ◽

2021 ◽

Vol 13 (6) ◽

pp. 1050

Author(s):

Liqin Tu ◽

Shuhua Wu ◽

Danna Gao ◽

Yong Liu ◽

Yuelin Zhu ◽

...

Keyword(s):

Mosaic Virus ◽

Infection Rate ◽

Cdna Clone ◽

Infectious Clone ◽

Infectious Cdna Clone ◽

Full Length ◽

Economic Losses ◽

Chinese Isolate ◽

Solanum Lycopersicum L ◽

Rigid Rod

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.

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