Infectious recombinant Senecavirus A expressing novel reporter proteins

Abstract

Senecavirus A (SVA) is an emerging picornavirus that has been associated with vesicular disease and neonatal mortality in swine. The construction of SVA virus carrying foreign reporter gene provides a powerful tool in virus research. However, it is often fraught with rescuing a recombinant picornavirus harboring a foreign gene or maintaining the stability of foreign gene in the virus genome. Here, we successfully generated recombinant SVA GD05/2017 viruses (V-GD05-clone) expressing the green fluorescent protein (iLOV), red fluorescent protein (RFP), or NanoLuc luciferase (Nluc). These recombinant viruses have comparable growth kinetics to the parental virus. Genetic stability analysis indicated that V-GD05-iLOV was highly stable in retaining iLOV gene for more than 10 passages, while V-GD05-RFP and V-GD05-Nluc lost the foreign genes in five passages. In addition, high-intensity fluorescent signals were found in the V-GD05-RFP- and V-GD05-iLOV-infected cells by fluorescence observation and flow cytometry analysis, and the luciferase activity assay could quantitatively monitor the replication of V-GD05-Nluc. In order to identify the porcine cell receptor for SVA, anthrax toxin receptor 1 (ANTXR1) was knocked out or overexpressed in the ST-R cells. The ANTXR1 knock-out cells lost the ability for SVA infection, while overexpression of ANTXR1 significantly increased the cell permissivity. These results confirmed that ANTXR1 was the receptor for SVA to invade porcine cells as reported in the human cells. Overall, this study suggests that these SVA reporter viruses will be useful tools in elucidating virus pathogenesis and developing control measures. Key points • We successfully generated SVA viruses expressing the iLOV, RFP, or Nluc. • The iLOV was genetically stable in the V-GD05-iLOV genome over ten passages. • ANTXR1 was the receptor for SVA to invade porcine cells. Supplementary Information The online version contains supplementary material available at 10.1007/s00253-021-11181-6.

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

“…Viruses containing reporter genes within their genomes are a useful tool for detection and/or quantification of viral replication and screening antiviral agents. In recent years, the SVA reverse genetic systems have been widely used to construct recombinant viruses to express foreign genes (Liu et al, 2020a(Liu et al, , 2021Wang et al, 2021). A high-throughput neutralization test and screening of antiviral drugs based on an EGFP reporter SVA virus have been established to measure Nabs and antiviral drugs 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] method is used to detect BHK-21 cell cytotoxicity.…”

Senecavirus A as an Oncolytic Virus: Prospects, Challenges and Development Directions

“…Unfortunately, the foreign genes in these viral genomes became unstable after several passages. In order to circumvent this problem, Wang et al ( 100 ) inserted the green fluorescent protein (iLOV), red fluorescent protein (RFP) or nanoluciferase (Nluc) gene at the junction between SVA 2A and 2B and, in addition, a stop-restart translation element T2A was inserted into the side of the 2B product of SVA to retain the natural coding sequence of the viral protein surrounding the foreign gene. The author found that SVA with the iLOV tag can maintain a high level of stable passage in cells.…”

Novel putative pathogenic viruses identified in pangolins by mining metagenomic data

“…The polyprotein of senecavirus could be cleaved into twelve polypeptides, namely the L, VP1, VP2, VP3, VP4, 2A, 2B, 2C, 3A, 3B, 3C, and 3D. 28 Currently, senecavirus belongs to the family Picornaviridae, and there is only one species Senecavirus A in the Senecavirus genus. It is reported that senecavirus may cause vesicular disease in pigs.…”