Construction and Generation of a Recombinant Senecavirus a Stably Expressing the NanoLuc Luciferase for Quantitative Antiviral Assay

Abstract

Senecavirus A (SVA), also known as Seneca Valley virus, is a recently emerged picornavirus that can cause swine vesicular disease, posing a great threat to the global swine industry. A recombinant reporter virus (rSVA-Nluc) stably expressing the nanoluciferase (Nluc) gene between SVA 2A and 2B was developed to rapidly detect anti-SVA neutralizing antibodies and establish a high-throughput screen for antiviral agents. This recombinant virus displayed similar growth kinetics as the parental virus and remained stable for more than 10 passages in BHK-21 cells. As a proof-of-concept for its utility for rapid antiviral screening, this reporter virus was used to rapidly quantify anti-SVA neutralizing antibodies in 13 swine sera samples and screen for antiviral agents, including interferons ribavirin and interferon-stimulated genes (ISGs). Subsequently, interfering RNAs targeting different regions of the SVA genome were screened using the reporter virus. This reporter virus (rSVA-Nluc) represents a useful tool for rapid and quantitative screening and evaluation of antivirals against SVA.

Establishment of the Foot-and-Mouth Disease Virus Type Asia1 Expressing the HiBiT Protein: A Useful Tool for a NanoBiT Split Luciferase Assay

“…The authors demonstrated that RvJX-Nsp2325-HiBiT can be used as a highly efficient platform for the screening and evaluation of anti-PRRSV therapies by showing that RvJX-Nsp2325-HiBiT is a convenient and stable tool for evaluating viral propagation both in vitro and in vivo. In addition, a recombinant senecavirus A (rSVA) expressing the Nano-Luc gene between SVA 2A and 2B has been established [32]. Based on a comparison of rSVA Nano-Luc with the traditional PRNT50 assay, a new neutralization assay was proposed, and the applicability of rSVA Nano-Luc to antiviral interferon-stimulated gene screening was demonstrated.…”

The development and characterization of a stable Coxsackievirus A16 infectious clone with Nanoluc reporter gene

“…Nluc has been inserted to an EV71 infectious clone and this EV71-Nluc virus is genetically stable and confers stable luciferase activity over 10 passages in cultured cells ( Yang et al, 2021 ), overcoming its shorter lifetime in EV71-Gluc virus ( Xu et al, 2015 ). In another reverse genetic study of Senecavirus A with a insertion of Nluc gene between 2A and 2B, this reporter virus is also stable over 10 passages in BHK-21 cells ( Guo et al, 2021 ). Consistent with these earlier reports, we report here that the rCA16-Nluc virus has been confirmed stable in Vero cells by multiple assays.…”

Engineering Non-Human RNA Viruses for Cancer Therapy

“…Engineered SVV (eSVV) rescue system: The SVV genome has a length of 7.2 kb containing a unique orf flanked by a 5 untranslated region (UTR) and 3 UTR followed by a poly(A) tail [55]. The orf codifies for a single polyprotein that undergoes post-translational modifications by virus-encoded proteases to generate the final protein products (5 -L-VP4-VP2-VP3-VP1-2A-2B-2C-3A-3B-3C-3D-3 ) (Figure 2B) [56].…”