Critical reappraisal of the A226V mutation in Chikungunya outbreaks: possible role in increased pathogenesis?

CHIKV is a mosquito-transmitted alphavirus responsible for the first autochthonous Italian outbreak in 2007.We previously analyzed 7 CHIKV isolates (5 imported and 2 autochthonous) with respect to the presence of A226V mutation in E1gp. All the isolates showed this mutation except the one imported from India in 2006. Since this mutation has been associated with enhanced replication and fitness in A. albopictus vector, we investigated the possible involvement of A226V mutation in enhanced infection capability in primate cells. To this aim,Vero E6 and C6/36 cells were infected with two CHIKV isolates, one carrying the A226V mutation and one wild type, using single replication cycle conditions. Progeny virus was measured by both quantitative real time RT-PCR and viral infectivity assay. No significant differences were observed between the two isolates either in terms of replication kinetic or in virus yield, on both Vero E6 and C6/36 cells. Moreover, experiment of inhibition of virus replication were performed for both isolates on Vero E6 cells using increasing amounts of recombinant IFN-alpha and virus yield was measured. A dose-dependent inhibition of virus yield for both CHIK isolates was observed, with a different sensitivity to IFN-alpha between the isolate carrying the A226V mutation and the wild type one. Our results suggest i) that A226V mutation does not influence replication ability in both host species, when using single replication cycle conditions; ii) the differences between wild type and mutated strains may be due to different sensitivity and/or activation ability of innate immune mechanisms.