A cDNA-based reverse genetics system for feline calicivirus identifies the 3′ untranslated region as an essential element for viral replication

Cheng, Jie; Tang, Aoxing; Chen, Jing; Zhang, Da; Chen, Meng; Li, Chuan‐Feng; Wei, Hulai; Liu, Guangqing

doi:10.1007/s00705-022-05695-1

Cited by 3 publications

(3 citation statements)

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“…The resulting viral particles are capable of infecting a cell line susceptible to FCV, starting a new cycle of replication and expressing the fluorescent protein [76]. Later on, FCV LC region was found to tolerate foreign insertions such as AcGFP, DsRedFP [74], or mCherry [90] without hampering viral viability. In human norovirus, a GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPglinked RNA, establishing a complete reverse genetics system expressed from cDNA with EF-1α mammalian promoter and without the need of a helper virus [91].…”

Section: Year Of Publication [Reference]mentioning

confidence: 99%

“…The insertion of the GFP gene at the start of ORF1 of TV completely abolished the infectivity of the transcribed RNA, which is not capable of expressing the fluorescent protein either [77]. This is why, in recent years, studies have been conducted to identify regions that tolerate insertions [88] and allow greater effectiveness in obtaining labeled replicon reporter genes [74,90]. The case of Norwalk virus replicons require special mention since a cell line stably expressing the replicon has been established [92].…”

Section: Year Of Publication [Reference]mentioning

confidence: 99%

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Highs and Lows in the Calicivirus Reverse Genetics

Álvarez,

Arboleya,

Abade dos Santos

et al. 2024

Preprint

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In Virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an “infectious clone”. This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate RNA polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward, since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse transcribed into cDNA before any modification -such as restriction enzyme digestion, gene knock-out or knock-in, site directed mutagenesis, ligation, etc.- can be done. Establishing reverse genetics systems for members of the Caliciviridae has proven exceptionally challenging, due to the low number of members of this family that propagate in cell cultures. Despite the successful rescue of Feline calicivirus (FCV) from cDNA containing plasmid more than 20 years ago, reverse genetics methods are not routine procedures than can be easily extrapolated to other caliciviruses. Reports of calicivirus reverse genetics systems have been few and far between, in this review, we discuss the main pitfalls, failures, and delays behind several successful calicivirus infectious clones.

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Section: Year Of Publication [Reference]mentioning

confidence: 99%

Section: Year Of Publication [Reference]mentioning

confidence: 99%

Highs and Lows in the Calicivirus Reverse Genetics

Álvarez,

Arboleya,

Abade dos Santos

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Later on, the FCV LC region was found to tolerate foreign insertions, such as AcGFP, Discosoma sp. red fluorescent protein (DsRed) [ 90 ], or mCherry [ 104 ], without hampering viral viability. In human norovirus, a GFP reporter construct containing the GFP gene in ORF1 produced complete virions that contain VPg-linked RNA, establishing a complete reverse genetics system expressed from cDNA with the EF-1α mammalian promoter and without the need for a helper virus [ 105 ].…”

Section: Chronology Of the Calicivirus Reverse Geneticsmentioning

confidence: 99%

Highs and Lows in Calicivirus Reverse Genetics

Álvarez,

Arboleya,

Abade dos Santos

et al. 2024

Viruses

View full text Add to dashboard Cite

In virology, the term reverse genetics refers to a set of methodologies in which changes are introduced into the viral genome and their effects on the generation of infectious viral progeny and their phenotypic features are assessed. Reverse genetics emerged thanks to advances in recombinant DNA technology, which made the isolation, cloning, and modification of genes through mutagenesis possible. Most virus reverse genetics studies depend on our capacity to rescue an infectious wild-type virus progeny from cell cultures transfected with an “infectious clone”. This infectious clone generally consists of a circular DNA plasmid containing a functional copy of the full-length viral genome, under the control of an appropriate polymerase promoter. For most DNA viruses, reverse genetics systems are very straightforward since DNA virus genomes are relatively easy to handle and modify and are also (with few notable exceptions) infectious per se. This is not true for RNA viruses, whose genomes need to be reverse-transcribed into cDNA before any modification can be performed. Establishing reverse genetics systems for members of the Caliciviridae has proven exceptionally challenging due to the low number of members of this family that propagate in cell culture. Despite the early successful rescue of calicivirus from a genome-length cDNA more than two decades ago, reverse genetics methods are not routine procedures that can be easily extrapolated to other members of the family. Reports of calicivirus reverse genetics systems have been few and far between. In this review, we discuss the main pitfalls, failures, and delays behind the generation of several successful calicivirus infectious clones.

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