Characterization and phylogenetic analysis of a neurovirulent Zika virus isolated from Cambodia in 2019

The geographic range of Zika virus (ZIKV) has expanded from Asia to the Americas, leading to the 2015-2016 pandemic with enhanced neurovirulence. At present, ZIKV is continuously circulating in many Southeast Asian countries. Unfortunately, the persistent evolution of ZIKV in Southeast Asia and its influence on the biological characteristics of the virus remain incompletely understood. In this study, the in vitro and in vivo properties of a new ZIKV isolate obtained from Cambodia in 2019 (CAM/2019) were characterized and compared with those of the Cambodian strain (CAM/2010). Compared with CAM/2010, the CAM/2019 virus showed similar plaque morphology and growth curves in cell cultures and induced comparable viremia and organ viral loads profiles in both BALB/c and A129 (IFNAR1^-/- ) mice upon intraperitoneal (i.p.) inoculation. Remarkably, the CAM/2019 virus exhibited enhanced neurovirulence in neonatal mice compared with CAM/2010, with a 74-fold reduction in the 50% lethal dose (LD₅₀ ). Consistently, CAM/2019 produced higher viral loads in the brains of BALB/c neonatal mice than CAM/2010 did. Sequence alignment showed that the CAM/2019 virus has acquired 12 amino acid substitutions, several of which were found to be associated with neurovirulence. In particular, the CAM/2019 virus shared an A1204T substitution in NS2A with the Thai isolate SI-BKK02 that was isolated from a microcephaly case. Taken together, our results indicate that a ZIKV strain isolated with specific mutations has emerged in Cambodia, highlighting the need for extensive molecular and disease surveillance in Cambodia and other Asian countries.