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.

A Recombinant Genotype I Japanese Encephalitis Virus Expressing a Gaussia Luciferase Gene for Antiviral Drug Screening Assay and Neutralizing Antibodies Detection

“…In comparison with other reporter genes, the Gluc gene is smaller in size, which will reduce the size of the foreign gene insertion and may make the resultant reporter virus more stable. As a reporter gene, Gluc has been widely applied for the development of reporter viruses [ 42 , 43 , 44 ]. As expected, the GI JEV reporter virus expressing Gluc generated in this study exhibited similar growth properties to its parental virus, and the expression levels of Gluc in the virus supernatants were correlated well with the viral titers.…”

“…The gaussia luciferase assay was performed as previously described [ 42 ]. Briefly, culture supernatants were harvested and cleared by centrifugation at 4 °C.…”

Development of a highly sensitive Gaussia luciferase immunoprecipitation assay for the detection of antibodies against African swine fever virus

“…All serum samples were inactivated at 60°C for 30 min before use. Serum samples collected from pigs infected with a specific pathogen were used to evaluate the diagnostic specificity of the GLuc-p30-based LIPS assay, including 5 porcine reproductive and respiratory syndrome virus-positive ( Li et al., 2022 ), 2 classical swine fever virus-positive provided by Dr. Qin Wang from China Institute of Veterinary Drugs Control, 5 Senecavirus A-positive serum provided by Dr. Zuzhang Wei from Guangxi University, and 3 porcine delta coronavirus-positive and 2 porcine epidemic diarrhea virus-positive provided by Dr. Yongning Zhang from China Agriculture University.…”

Allicin Inhibits Porcine Reproductive and Respiratory Syndrome Virus Infection In Vitro and Alleviates Inflammatory Responses

“…In China, the predominant PRRSV strains in the herd belong to PRRSV-2, although the PRRSV-1 strains have been isolated sporadically. The extremely high mutation rate of the PRRSV genome [ 4 , 5 ] and RNA recombination between different strains [ 6 ] have led to the emergence of new PRRSV strains almost every few years, such as HP-PRRSV strains in Asian countries [ 7 ], NADC30-like strains in China [ 8 ], and NADC34-like strains in China [ 9 ]. Although vaccination is the most effective strategy for PRRS control, commercial vaccines provide limited protection against heterologous infection [ 10 , 11 ].…”