Identification Of Key Neurovirulence Determinants Of The African Zika Virus Lineage

Zika virus got its name in 1947 when it was discovered in a sentry monkey in the Zika forest in Uganda,Africa.The cases of infected humans were few and the virus was ignored by public until 2007.In 2015,the largest ZIKV epidemic outbreak in the history occurred in South America,and it was listed as an "international public health emergency" by WHO because its infection caused microcephaly in newborns.Zika virus has clear neurotropic,which can be divided into African lineage and Asian lineage.Previous studies found that the neurovirulence of representative strains MR766,IbH in African lineage was significantly stronger than that in Asian lineage.However,the mechanism of the difference in neurovirulence between the two strains remains unclear.We hypothesized that there may be one or more conserved amino acid site mutations in different zika virus lineages,leading to different characteristics of neurovirulence.In order to explore the reasons for the difference in neurovirulence between African and Asian lineages of Zika virus,we first analyzed and screened potential virulence sites on African lineages of Zika virus through bioinformatics methods,and constructed and rescued mutated viruses carrying potential virulence sites by reverse genetics.The basic biological characteristics of the mutant virus were identified at the cellular and animal levels,and the neurotropism of the K101R mutant was evaluated.Finally,the protein structure and function changes of the mutant virus were analyzed based on biochemistry and other methods,and the molecular mechanism of enhanced neurovirulence of Zika virus induced by K101R mutation was preliminarily explored.The main research contents and findings of this topic are as follows:1.Design and rescue of recombinant ZIKVs carrying potential virulence site mutations.For screening of African lineage ZIKV potential sites of neurovirulence,we first BLAST 32 ZIKVs strains of the amino acid sequences by MEGA software.Then selected representative strains of African and Asian lineage.By conservative sequence alignment and amino acid analysis,7 sites in structural proteins were identified as mutation targets based on existing literature on virulence region analysis.In order to explore the specific impact of each potential virulence sites,we introduced the corresponding mutation into the infectious clone of an Asian strain by reverse genetics method and site-directed mutation technique.6 mutants K101R,V110I,V125I,P148A,1459V,and L728F were obtained by reverse genetics rescue.All the above-mentioned viruses could form plaques on BHK-21 cells,effectively express the viral proteins,and effectively replicate in BHK-21 cells,reaching the peak value 72h after infection.2.Neurotropic evaluation of recombinant ZIKV K101R mutant.In order to screen out the key neurovirulence sites,we compared the difference of neurovirulence among the mutant viruses,and injected each mutant virus into neonatal mice by neurovirulence test in neonatal mice.Survival curves of neonatal mice were compared among each group,and a mutant virus K101R with strong virulence characteristics was found.In order to explore the specific effects of mutations at this site on neurotropic of Zika virus,we compared the differences between K101R mutant and WT by measuring the growth curve and intracranial replication of the virus.In vitro experiments,we found that K101R mutant had stronger proliferation ability on nerve cells,and in vivo experiments showed that K101R had stronger intracranial replication and stronger neurotropism.The above studies indicated that amino acid 101 is a key site affecting the neurovirulence of Zika virus,and K101R mutation can significantly enhance the neurovirulence of Zika virus,showing stronger proliferation ability on nerve cells and high pathogenicity and neurovirulence in vivo.3.Studies on the mechanism of enhanced virulence of K101R mutant on capsid proteinIn order to further explore how this site affects viral virulence,we determined the effects of K101R mutation on viral RNA replication,C protein expression and nucleic acid binding ability of C protein by analyzing the structure and functional characteristics of C protein at site 101,RNA replication dynamics analysis,protein expression and function evaluation.The results showed that the mutation had no significant effect.In addition,the transcriptome sequencing of infected mouse brain was used to explore the effect on the transcriptome of neonatal mouse brain.It was found that the transcription level of inflammatory factors in the brain of mice infected with this virus was significantly increased,indicating that the K101R mutant caused more severe inflammatory response.This part of the study showed that K101R mutation did not affect the viral RNA replication and protein synthesis and other viral maturation mechanisms,and its strong virulence was mainly manifested in the interaction with the host,leading to stronger inflammatory response in the host brain tissue.Above all,we through bioinformatics screen potential sites of neurovirulence,of a series of reverse genetics build and save the mutant viruses,screened out the K101R mutant of enhanced neurovirulence in animal models,and further evaluation of the mutant strains of nerve toxicity,found that the mutation to strengthen the infectivity of hNPCs.Moreover,it enhanced the ability of intracranial proliferation in Suckling mice.We further evaluated the effect of the mutation on the structure and function of capsid protein,and found that the mutation did not affect the packaging and maturation process of the virus.The mouse brain transcriptome showed that the mutation mainly enhanced the interaction between the virus and the host nervous system,and induced a stronger inflammatory response in the brain tissue.In this study,we identified a new key site 101 on the Zika virus genome,which deepens our understanding of the African and Asian lineages of Zika virus.In addition,the discovery of this site also provides a new reference for pathogen monitoring.Although no mutation of K101R has been reported in the current epidemic strains of Zika virus,the monitoring of this site is conducive to timely warning,which is of great significance to public health undertakings.