THE FEASIBILITY OF CONSTRUCTING A PORCINE TESCHOVIRUS INFECTIOUS cDNA CLONE TAGGED WITH iLOV FLUORESCENT PROTEIN RESCUED AS A VISIBLE MARKER VIRUS

Abstract

Viral vectors serve as promising tools for the development of novel multivalent or multipathogen vaccines. Poliovirus (of the family Picornaviridae) vectors offer the advantages of small genome size, ease of manipulation, inherent stability in the intestinal tract, and induction of potent mucosal immunity through oral administration. Porcine teschovirus (PTV), also belonging to Picornaviridae, generally causes asymptomatic infections in pigs. PTV’s wide tissue tropism suggests that it can act as a potential vector for vaccine development; however, no infectious PTV cDNA clone has been reported yet. In this study, infectious PTV cDNA was cloned and recombinant porcine teschovirus (rPTV) was constructed with a unique XhoI site introduced into 2A, as well as substitution of the G–H loop sequence “RNNQIPQDF” of VP1 by an 8-histidine marker which helps to differentiate it from the parental PTV. Subsequently, the coding sequence of a small fluorescent protein, iLOV, was incorporated into the XhoI site to rescue the recombinant rPTV-iLOV virus, allowing for direct visualization of the viral infection. These rescued viruses were replication-competent and antigenically identical to the parental virus, but showed attenuation due to an impaired self-cleaving function caused by the insertion of iLOV into the 2A protease site, as assessed by a double reporter expressing system. This rescued recombinant virus shows potential for the development of attenuated vaccines with few safety concerns and may serve as an important tool to visually study virus–cell interactions.