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

2020 ◽  
Author(s):  
Ujike Makoto ◽  
Etoh Yuka ◽  
Urushiyama Naoya ◽  
Taguchi Fumihiro ◽  
Enjuanes Luis ◽  
...  
Keyword(s):  
Cell Growth ◽  
Cdna Clone ◽  
Recombinant Virus ◽  
Reverse Genetics ◽  
Infectious Cdna Clone ◽  
Full Length ◽  
Egfp Expression ◽  
Egfp Gene ◽  
Viral Growth ◽  
Recombinant Viruses

AbstractTorovirus (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.

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

2021 ◽  
Author(s):  
Makoto Ujike∗ ◽  
Yuka Etoh ◽  
Naoya Urushiyama ◽  
Fumihiro Taguchi ◽  
Hideki Asanuma ◽  
...  
Keyword(s):  
Viral Replication ◽  
Cdna Clone ◽  
Recombinant Virus ◽  
Reverse Genetics ◽  
Full Length ◽  
Egfp Expression ◽  
Egfp Gene ◽  
Virus Growth ◽  
Recombinant Viruses ◽  
Gene Retention

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.

Effects of the nsP2-726 Pro mutation on infectivity and pathogenesis of Sindbis virus derived from a full-length infectious cDNA clone

2009 ◽  
Vol 142 (1-2) ◽  
pp. 204-207 ◽  
Author(s):  
Wu-yang Zhu ◽  
Shi-hong Fu ◽  
Jing-lin Wang ◽  
Ying He ◽  
Qing Tang ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Sindbis Virus ◽  
Infectious Cdna Clone ◽  
Full Length

scholarly journals Synthesis of a Full-Length Infectious cDNA Clone of Cucurbit Aphid-Borne Yellows Virus and Its Use in Gene Exchange Experiments with Structural Proteins from Other Luteoviruses

Virology ◽  
1995 ◽  
Vol 214 (1) ◽  
pp. 150-158 ◽  
Author(s):  
D. PRÜFER ◽  
CATHERINE WIPF-SCHEIBEL ◽  
K. RICHARDS ◽  
H. GUILLEY ◽  
H. LECOQ ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Infectious Cdna Clone ◽  
Full Length ◽  
Structural Proteins ◽  
Gene Exchange

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

2021 ◽  
Vol 3 (1) ◽  
Author(s):  
Ziyue Ma ◽  
Hui Zhang ◽  
Ming Ding ◽  
Zhongkai Zhang ◽  
Xiuling Yang ◽  
...  
Keyword(s):  
Cdna Clone ◽  
Nicotiana Benthamiana ◽  
Complete Genome ◽  
Yunnan Province ◽  
Systemic Infection ◽  
Infectious Cdna Clone ◽  
Full Length ◽  
Biologically Active ◽  
Reverse Genetic ◽  
Tomato Plants

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.

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

2006 ◽  
Vol 81 (4) ◽  
pp. 1813-1820 ◽  
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.

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

2002 ◽  
Vol 76 (12) ◽  
pp. 5847-5856 ◽  
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.

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

Viruses ◽  
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.

scholarly journals Construction of a full-length infectious cDNA clone of swine vesicular disease virus strain NET/1/92 and analysis of new antigenic variants derived from it

2000 ◽  
Vol 81 (11) ◽  
pp. 2763-2769 ◽  
Author(s):  
J. M. J. Rebel ◽  
C. H. Leendertse ◽  
A. Dekker ◽  
F. van Poelwijk ◽  
R. J. M. Moormann
Keyword(s):  
Amino Acid ◽  
Disease Virus ◽  
Cdna Clone ◽  
Parent Strain ◽  
Biological Properties ◽  
Infectious Cdna Clone ◽  
Full Length ◽  
Swine Vesicular Disease Virus ◽  
Vesicular Disease

The Dutch swine vesicular disease virus (SVDV) isolate NET/1/92 was one of the first isolates belonging to a new SVDV antigenic group. This strain was completely sequenced and was shown to have 93% similarity with the UKG/27/72 isolate. To enable antigenicity, replication, maturation and pathogenicity studies of NET/1/92, an infectious full-length cDNA clone, designated pSVD146, was prepared. The in vitro and in vivo biological properties of the virus derived from pSVD146 were studied by analysing antigenicity, plaque morphology, growth curves and virulence in pigs. The epitopes of newly prepared monoclonal antibodies were roughly mapped by fusion-PCR. Fine mapping of epitopes at the amino acid level was achieved by introducing single amino acid mutations in pSVD146. Two new amino acids important in epitope formation were located in VP1; one was mapped in the C-terminal end and the second is thought to be located in the H–I loop. Growth curve and plaque sizes in vitro were similar between virus derived from pSVD146 and the parent wild-type virus. In virulence studies in pigs, the lesions score, neutralization titres and the seroconversion rates were comparable between virus derived from pSVD146 and the parent strain. Since virus derived from pSVD146 had the same biological properties as the parent strain NET/1/92, the full-length infectious cDNA clone pSVD146 will be very useful in studies of the antigenicity, virulence, pathogenesis, maturation and replication of SVDV.

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