Generation of a porcine reproductive and respiratory syndrome virus expressing a marker gene inserted between ORF4 and ORF5a

Wang, Yuxu; He, Wei; Li, Qingqing; Xie, Xiaoyan; Qin, N.; Wang, Hao; Huang, Jia-Bin; Lin, Siyuan; Ouyang, Kang; Chen, Ying; Huang, Weijian; Wei, Zuzhang

doi:10.1007/s00705-020-04679-3

Cited by 12 publications

(12 citation statements)

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“…Taking advantage of this system, the research on PRRSV molecular biology has made significant progress in understanding the mechanisms of viral RNA synthesis, the function study of viral proteins, and the identification of viral virulence factors and viral determinants of cell tropism (reviewed in (Chaudhari and Vu (2020)). With the reverse genetics of PRRSV available, PRRSV was also explored as a viral vector for the expression of a foreign gene of interest (Fang et al, 2006;Pei et al, 2009;Zheng et al, 2010;Guo et al, 2016;Gao et al, 2018;Huang et al, 2018;Wang et al, 2020). In this study, an infectious cDNA clone of the HP-PRRSV TA-12 strain was established via homologous recombination in vitro.…”

Section: Discussionmentioning

confidence: 99%

“…With the availability of reverse genetics, PRRSV has been explored as a viral vector to express a foreign gene of interest, such as reporter genes (Fang et al, 2006;Guo et al, 2016;Huang et al, 2018) for tracking viral infection and antigens of other pathogens for vaccine development (Pei et al, 2009;Zheng et al, 2010;Gao et al, 2018). Two main strategies have been employed to deliver the foreign gene, including as a fusion protein within nsp2 and as an additional subgenomic RNA inserted at three potential locations in the PRRSV genome: between ORF1b and ORF2a, between ORF4 and ORF5a, and between ORF7 and 3′UTR (Pei et al, 2009;Wang et al, 2013Wang et al, , 2020Tian et al, 2017). The intergenic junctions between ORF1b and ORF2a and between ORF7 and 3′UTR are the most commonly used locations for gene insertion.…”

Section: Introductionmentioning

confidence: 99%

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A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing a Gaussia Luciferase for Antiviral Drug Screening Assay and Luciferase-Based Neutralization Assay

Ren

et al. 2022

Front. Microbiol.

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The reverse genetics system is a valuable tool in the virological study of RNA viruses. With the availability of reverse genetics, the porcine reproductive and respiratory syndrome virus (PRRSV) has been utilized as a viral vector for the expression of foreign genes of interest. Here, we constructed a full-length cDNA clone of a highly pathogenic PRRSV (HP-PRRSV) TA-12 strain. Using this cDNA clone, we generated a reporter virus expressing a gaussia luciferase (Gluc) via an additional subgenomic RNA between ORF7 and 3′UTR. This reporter virus exhibited similar growth kinetics to the wild-type (WT) virus and remained genetically stable for at least ten passages in MARC-145 cells. In cells infected with this reporter virus, the correlation between the expression levels of Gluc in culture media and the virus titers suggested that Gluc is a good indicator of the reporter virus infection. With this reporter virus, we further established the Gluc readout-based assays for antiviral drug screening and serum neutralizing antibody detection that exhibited comparable performance to the classical assays. Taken together, we established a reverse genetics system of HP-PRRSV and generated a novel reporter virus that could serve as a valuable tool for antiviral drug screening and serum neutralizing antibody detection.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing a Gaussia Luciferase for Antiviral Drug Screening Assay and Luciferase-Based Neutralization Assay

Ren

et al. 2022

Front. Microbiol.

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“…The infectious PRRSV-2 cDNA clone pGXAM was derived from a cell adapted PRRSV strain, GXNN1396-P96 (Genbank accession no. MN660068) (33). Figure 1 shows the cloning strategy for the infectious PRRSV-2 cDNA clone pGXAM carrying the RFP and GFP genes.…”

Section: Cells Viruses and Plasmidsmentioning

confidence: 99%

Generation of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing Two Marker Genes

Wang

Xie

et al. 2020

Front. Vet. Sci.

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Porcine reproductive and respiratory syndrome virus (PRRSV) has been used as a gene expression vector in the development of vaccines. Most of these recombinant PRRSV vectors express only a single foreign gene through either an internal insertion in the hypervariable region of nsp2 or expression cassette and some of these recombinant vectors are genetically unstable. Here, we combined internal insertion in nsp2 and expression cassette methods to generate a novel recombinant PRRSV stably expressing the red fluorescence protein (RFP) and the green fluorescence protein (GFP) genes. Biological characteristic analysis of the recombinant PRRSV carrying the two marker genes, rGX-RFP-GFP, showed that it displayed similar growth kinetics and yet it yielded less infectious viruses when compared to the parental virus rGXAM. Co-expression of both the RFP and GFP was observed using confocal fluorescence microscopy when the rGX-RFP-GFP viruses infected MARC-145 cells. Furthermore, the PRRSV-based two-marker gene expression vector is genetically stable during 20 serial passages in MARC-145 cells. These data demonstrate that it is possible to express two interested immunogens from a single PRRSV vector.

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“…Cloned viruses expressing fluorescent reporters are widely used in molecular biology (Cheng et al, 2020; Mei et al, 2019; Wang et al, 2020; Xie et al, 2020). These viruses have been used to assess virus replication in a range of cells, tissues, and organisms, greatly advancing our understanding of virus biology and virus‐host interactions.…”

Section: Introductionmentioning

confidence: 99%

Beeporter: Tools for high‐throughput analyses of pollinator‐virus infections

Evans

Banmeke

Palmer‐Young

et al. 2021

Molecular Ecology Resources

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Pollinators are in decline thanks to the combined stresses of disease, pesticides, habitat loss, and climate. Honey bees face numerous pests and pathogens but arguably none are as devastating as Deformed wing virus (DWV). Understanding host‐pathogen interactions and virulence of DWV in honey bees is slowed by the lack of cost‐effective high‐throughput screening methods for viral infection. Currently, analysis of virus infection in bees and their colonies is tedious, requiring a well‐equipped molecular biology laboratory and the use of hazardous chemicals. Here we describe virus clones tagged with green fluorescent protein (GFP) or nanoluciferase (nLuc) that provide high‐throughput detection and quantification of virus infections. GFP fluorescence is measured noninvasively in living bees via commonly available long‐wave UV light sources and a smartphone camera, or a standard ultraviolet transilluminator gel imaging system. Nonlethal monitoring with GFP allows continuous screening of virus growth and serves as a direct breeding tool for identifying honey bee parents with increased antiviral resistance. Expression using the nLuc reporter strongly correlates with virus infection levels and is especially sensitive. Using multiple reporters, it is also possible to visualize competition, differential virulence, and host tissue targeting by co‐occuring pathogens. Finally, it is possible to directly assess the risk of cross‐species “spillover” from honey bees to other pollinators and vice versa.

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Generation of a porcine reproductive and respiratory syndrome virus expressing a marker gene inserted between ORF4 and ORF5a

Cited by 12 publications

References 50 publications

A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing a Gaussia Luciferase for Antiviral Drug Screening Assay and Luciferase-Based Neutralization Assay

A Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing a Gaussia Luciferase for Antiviral Drug Screening Assay and Luciferase-Based Neutralization Assay

Generation of a Recombinant Porcine Reproductive and Respiratory Syndrome Virus Stably Expressing Two Marker Genes

Beeporter: Tools for high‐throughput analyses of pollinator‐virus infections

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