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

Abstract

Coxsackievirus A16 (CA16) belongs to the Human Enterovirus A species, which is a common pathogen causing hand, foot, and mouth disease in children. Currently, specific vaccines and drugs against CA16 are unavailable, and there is an unmet need to further understand the virus and invent effective treatment. Constructing a CA16 infectious clone with a reporter gene will greatly facilitate its virological studies. Here, we first reported the construction of a CA16 infectious clone (rCA16) whose progeny is highly replicative and virulent in suckling mice. On the basis of rCA16, we further inserted a NanoLuc (Nluc) reporter gene and made the rCA16-Nluc clone. We found that the Nluc gene in rCA16-Nluc is stable during continuous growing in Vero cells and thus allowed detection of a steady luciferase signal in rCA16-Nluc-infected Vero cells over 10 passages. Its application in antivirals characterization and high-throughput screening is exemplified by measuring IC50, CC50, and selection index of guanidine hydrochloride, ribavirin, chloroquine, and ammonium chloride against CA16. Finally, we showed that rCA16-Nluc based assay greatly simplified the CA16 neutralizing antibody tests. Thus, these two CA16 infectious clones will be robust tools for future enterovirus studies and antivirals development.

Reporter Coxsackievirus A5 Expressing iLOV Fluorescent Protein or Luciferase Used for Rapid Neutralizing Assay in Cells and Living Imaging in Mice

“…Previous research has shown that influenza A virus, foot and mouth disease viruses, Senecavirus A, West Nile virus and dengue virus have been engineered to carry a reporter gene for high throughput monoclonal antibody and antiviral assays [ 25 , 26 , 27 , 28 , 29 , 30 ]. Tagged EV-A71 and CV-A16 of the Enterovirus A are constructed by the same strategy to insert the GFP, nano luciferase, or gaussia luciferase gene between the 5′-UTR and the N-terminus of VP4, with the addition of a 2A protease cleavage site (ITTLG) at C-terminus of the 5′-UTR [ 13 , 14 , 15 , 16 , 17 ]. CV-B3 was constructed as a stable expression vehicle for GFP released from the viral polyprotein by 2A pro cleavage.…”

“…Many researchers have shown that poliovirus, enterovirus 71, coxsackievirus A16, coxsackievirus B3, coxsackievirus B5 and Rhinovirus A within the Picornaviridae family have been engineered to carry reporter genes expressing fluorescent proteins and different kinds of luciferases [ 13 , 14 , 15 , 16 , 17 ]. These reporter viruses have been used to facilitate assays of antiviral reagents and in vivo imaging of mice [ 14 , 16 , 17 ].…”

“…Many researchers have shown that poliovirus, enterovirus 71, coxsackievirus A16, coxsackievirus B3, coxsackievirus B5 and Rhinovirus A within the Picornaviridae family have been engineered to carry reporter genes expressing fluorescent proteins and different kinds of luciferases [ 13 , 14 , 15 , 16 , 17 ]. These reporter viruses have been used to facilitate assays of antiviral reagents and in vivo imaging of mice [ 14 , 16 , 17 ]. However, one of the major challenges is that recombinants with large tagged genes are difficult to rescue and stably passaged in cells because of the limitation of insertion capacity of enteroviruses with a small genome of approximately 7.4 kilobases.…”

“…Recently, iLOV (light, oxygen or voltage sensing) and nano luciferase (Nluc) have been exploited to construct reporter viruses for establishing detection methods [ 16 , 18 , 19 ]. iLOV has been applied to rhinovirus A and tobacco mosaic virus [ 20 , 21 ].…”