Comparison of chikungunya viruses generated using infectious clone or the Infectious Subgenomic Amplicons (ISA) method in Aedes mosquitoes

Abstract

Reverse genetics systems provide the opportunity to manipulate viral genomes and have been widely used to study RNA viruses and to develop new antiviral compounds and vaccine strategies. The recently described method called ISA (Infectious Subgenomic Amplicons) gives the possibility to rescue RNA viruses in days. We demonstrated in cell culture that the use of the ISA method led to a higher genetic diversity of viral populations than that observed using infectious clone technology. However, no replicative fitness difference was observed. In the present study, we used the chikungunya virus as a model to compare in Aedes aegypti and Aedes albopictus mosquitoes the genotypic and phenotypic characteristics of viruses produced either from an infectious clone or using the ISA method. We confirmed the results found in cellulo corroborating that the use of the ISA method was associated with higher genetic diversity of viral populations in mosquitoes but did not affect the vector competence validating its use for in vivo experiments.

Fig 1. Genetic diversity in Aedes mosquitoes.

Panels A and B represent the number of minority and majority mutations respectively detected in mosquito heads (substitution frequency < and ≥ 50% respectively). Mutation characteristics are represented in panels C (Non synonymous; Synonymous mutations) and D (Transition; Transversion). In Panels A and B, the bottom and top of the box represent first and third quartiles, the band inside the box represents median value and the ends of the error bars represent minimum and maximum values. IC and ISA mean group of mosquitoes infected by IC_virus and ISA_virus respectively. “dpi” means days post-infection. * means that a significant difference (p value <0.05) was observed between mosquitoes infected by IC_virus and ISA_virus.

Fig 2. Vector competence in Aedes mosquitoes.

Dissemination efficiency (DE; proportion of mosquito with infectious virus in the head) was determined by testing mosquito heads with a TCID₅₀ assay (Panel A). The intensity of viral dissemination was assessed by comparing amounts of infectious particles in collected heads (Panel B; only positive heads were taken into account). Transmission efficiency (TE; proportion of mosquitoes with infectious virus in the saliva) was determined by testing mosquito saliva with a TCID₅₀ assay (Panel C). The intensity of viral transmission was assessed by comparing amounts of infectious particles in collected saliva (Panel D; only positive saliva were taken into account). In panels B and D, the average values are shown and error bars represent standard deviation. IC and ISA mean group of mosquitoes infected by IC_virus and ISA_virus respectively. “dpi” means days post-infection.