Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Li Y

PROVIDER: S-EPMC9136234 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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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.

Li Yanhua Y Ren Cicheng C Li Chenxi C Xiao Yihong Y Zhou Yanyang Y

Frontiers in microbiology 20220513

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) <i>via</i> an additional sub ...[more]

PMID: 35633700

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Project description:Porcine reproductive and respiratory syndrome virus (PRRSV) can subvert early innate immunity, which leads to ineffective antimicrobial responses. Overcoming immune subversion is critical for developing vaccines and other measures to control this devastating swine virus. The overall goal of this work was to enhance innate and adaptive immunity following vaccination through the expression of interferon (IFN) genes by the PRRSV genome. We have constructed a series of recombinant PRRS viruses using an infectious PRRSV cDNA clone (pCMV-P129). Coding regions of exogenous genes, which included Renilla luciferase (Rluc), green and red fluorescent proteins (GFP and DsRed, respectively) and several interferons (IFNs), were constructed and expressed through a unique subgenomic mRNA placed between ORF1b and ORF2 of the PRRSV infectious clone. The constructs, which expressed Rluc, GFP, DsRed, efficiently produced progeny viruses and mimicked the parental virus in both MARC-145 cells and porcine macrophages. In contrast, replication of IFN-expressing viruses was attenuated, similar to the level of replication observed after the addition of exogenous IFN. Furthermore, the IFN expressing viruses inhibited the replication of a second PRRS virus co-transfected or co-infected. Inhibition by the different IFN subtypes corresponded to their anti-PRRSV activity, i.e., IFNω5 ° IFNα1 > IFN-β > IFNδ3. In summary, the indicator-expressing viruses provided an efficient means for real-time monitoring of viral replication thus allowing high‑throughput elucidation of the role of host factors in PRRSV infection. This was shown when they were used to clearly demonstrate the involvement of tumor susceptibility gene 101 (TSG101) in the early stage of PRRSV infection. In addition, replication‑competent IFN-expressing viruses may be good candidates for development of modified live virus (MLV) vaccines, which are capable of reversing subverted innate immune responses and may induce more effective adaptive immunity against PRRSV infection.

Dataset Information

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

Publications

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

Similar Datasets

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