Identification Of The Critical Determinants Of Increased Neurovirulence Of ZIKA Virus

The recent global outbreak of the ZIKA virus(ZIKV)epidemic from 2015 to2016 has shown that this viral infection is associated with an elevated incidence of certain neurological diseases,such as congenital ZIKA syndrome(containing microcephaly)and adult Guillain-Barre syndrome.However,the critical factors that determine increased neurovirulence of ZIKV remain unknown.We hypothesized that viral genomic adaptations during ZIKV infection of the nervous system may enhance the severity of neurological disease.Previous results have shown that ZIKV can infect newborn mice and cause neurological disorders such as microcephaly,paralysis and seizures.Mechanistically,these neurological manifestations are related to susceptibility of newborn mouse neural progenitor cells and other nerve cells to the ZIKV infection.Therefore,newborn mice could be used as a model to study whether and how the genetic features of ZIKV would affect pathogenesis in the nervous system.To test this hypothesis,we propagated a 2016 clinical isolate of ZIKA virus(SW01)in the brains of newborn mice for 11 consecutive times,then obtained a mouse CNS adapted ZIKA virus(MA-SW01).The neurotoxicity and replication capacity of MA-SW01 were significantly higher in vivo than its parental strain,and MA-SW01 target areas in brain were the cerebral cortex and hippocampus.Through high-throughput next-generation sequencing analysis of MA-SW01,we found that MA-SW01 had four high-frequency missense mutations higher than 80% on the whole genome,corresponding to3 amino acid mutations on virus E protein(D67N,M68 I,N154D)and 1 on NS2 A protein(A117T).Further reverse genetic experiments of the virus in combination with in vivo experiments in mice,we found that a guanine nucleotide(G)to adenine nucleotide(A)replacement at the 1069 site of virus open reading frame,corresponding to an aspartic acid(D)to asparagine(N)change on viral E protein,was necessary and sufficient to render the MA-SW01 virus with increased neurotoxicity.Mechanism study revealed that this mutation did not form a glycosylation modification appeared on dengue virus,and the virulence changes did not depend on the glycosylation function.In order to study whether this E protein mutation was a result of rapid viral adaptation during replication in the central nervous system,we thoroughly analyzed the ratio of 1069 A variant before and after infection in mice.It showed that the rate of 1069 A in SW01 was 0.055% before inoculation.This rate rapidly increased to 22.7% after one passage and maintained above 92%after 5 passages.These findings indicated that the 1069 A variant within original clinical strain has growth advantage during infection of the CNS in mice.Mechanistically,the molecularly cloned virus carrying 67 N mutation on E protein replicated much more efficiently than the wild type 67 D virus in center nervous system.These findings have improved our understanding of genetic evolution of ZIKA virus and its association with neurological pathogenesis,and provided clues to the development of new antiviral strategies against potential epidemics in the future.