The Establishment Of Adaptive Strains Of Zika Virus And Its Neurovirulence In Neonatal Mice

Background Zika virus has evolved into a public health emergency due to its unprecedented causal link to microcephaly,and rapidly spread to dozens of countries and regions of the world.Zika virus could be classified into two genotypes:African subtype and Asian subtype.Zika virus in the Americas outbreak evolved from Asian subtype.Zika virus is coated on the surface with M and E proteins in the envelope.E protein is the main component participated in the receptor binding and membrane fusion,and the only N-glycosylation site Asn-154 in the presence of E protein can affect this process.All the comtemporary epidemic and some Zika strains of African subtype contained the potential glycosylation site,whereas some hiatorical African subtype Zika virus lack the glycosylation motif.There are also reports of imported Zika virus in our country.Objectives The purpose of this study is to screen the gene mutation enhanced Zika virus virulence through serial passages using a virus strain of Zika virus ZKC2/2016 in newborn mice brain.It is to provide theoretical basis and experimental basis,for the in-depth study of Zika virus pathogenic factors and the research on the pathogenic mechanism of the virus,that the mechanism of the enhanced neurovirulence was further analyzed.Furthermore,this study may have far-reaching significance in clinical disease warning,attenuated vaccine and development of special effects drugs.Methods 1.The gene mutation enhancing Zika virus virulence was screened through serial passages in newborn mice brain.(1)The Zika virus strain was screened for phenotype enhancement by using the Zika strain ZKC2/2016 in the neonatal 1 day BALB/c micet brain.(2)The primers for ZKC2/2016 whole genome were designed according to the ZKC2/2016 sequence obtained through the second generation sequencing.(3)The whole genome sequence of Zika were amplificated,the genome sequences of all generations of viruses were spliced by software SeqMan,and the mutation site were determined by using software MEGA.(4)Viruses were purified three times by the plaque assay,it wasto ensure that there was no gene mutation in the purification process that the whole genome was sequenced,and the relevant sequence was uploaded by Bankit.(5)To compare and genetic evolution analysis of download sequences and mutant Zika sequences,Sequence alignment and phylogenetic analysis were conducted using MEGA.(6)The corresponding protein structure of gene mutation site were predicted through the online tool swiss-model;(7)The protein structure was analyzed by PyMol and Chimera,and the effect of mutation site on the corresponding protein structure was analyzed.2.The neurovirulence of the strain was further confirmed in neonatal mice.(1)It was established that a fluorescence quantitative RT-PCR detection method for Zika virus,and to prepare RNA reference materials by vitro transcription.(2)A large number of amplification of the purified ZKC2P4 and ZKC2P6 were conducted in the brain of one-day-old BALB/c,and quantified by quantitative RT-PCR.(3)The body weight and survival rate of the newborn mice injected with ZKC2P6 virus were compared with that of ZKC2P4 virus.(4)The brain weight,width of the brain,width of the ventricle and the thickness of cerebral cortex in the two groups were compared in one-day-old BALB/c.(5)The pathological changes,of small mouse parts(the olfactory bulb,the cerebral cortex,hippocampus,cerebellum,brainstem)and cervical spinal cord,were compared between ZKC2P4 and ZKC2P6 group in one-day-old BALB/c.(6)Zika virus replication was compared with ZKC2P4 and ZKC2P6 in one-day-old BALB/c brain,including viral RNA quantified by fluorescence quantitative PCR,and virus particles in brain tissues determined by immunofluorescence;The replication curve of ZKC2P4 and ZKC2P6 were drawn in in African green monkey kidney cells Vero and human neuroblastoma cells SY5Y.(7)The differences in inducing cell apoptosis between ZKC2P6 and ZKC2P4 were compared by detecting activated Cas3 using immunofluorescence,and activated Cas3 and cleaved PARP using western blotting.Results 1.The neurovirulent enhancement strain ZKC2P6 was screened.(1)The neonatal mice didn't began to appear a series of symptomsat 3 days post infection until ZKC2/2016 come to the fifth generation,similar symptoms did arise in the sixth to eighth generation.(2)By sequencing and alignment,E143K and R3394K were found in the fourth generation,and seven amino acids(aa)(from 445 to 451)were further deleted in the fifth to eighth generation.(3)11 other strains with a loss of N-linked glycosylation by amino acids deletion,and 7 strains with a loss of N-linked glycosylation by point mutation,were identified from 213 Zika virus complete sequences in Genbank.(4)Phylogenetic analysis revealed that the strain ZKC2P6 is the only one non-glycosylated E protein strain by amino acids deletion in the Asian lineage.(5)By structure predicting,we found the deletion of 7 amino acids affect the local alpha helix of Zika virus E protein structure domain I,and resulted in of "150 loop" of the E protein shortened,and the fusion ring of E proteindimer exposed.2.The neurovirulence of the strain was further confirmed in neonatal mice.(1)Clinical performance:(1)Compared with group ZKC2P4,ZKC2P6 group mice showed more severe disease states,such as the earlier weight loss(P<0.05)and death(P<0.05),and gastric emptying,decreased activity,and hind legs lame.(2)Apparent changes of the brain:Compared with ZKC2P4 group,the BALB/c newborn mice in the ZKC2P6 group showed lighter brain weight(P<0.05),the width of the brain and the width of the ventricle were smaller.(3)Cortex and ventricle:compared with the ZKC2P4 group,the cerebral cortex of BALB/c in the ZKC2P6 group was thinner and the ventricle enlarged.(4)Pathological examination:ZKC2P6 infection resulted in the death of a large number of nerve cells and keratinocytes in various parts of the brain(olfactory bulb,cerebral cortex,hippocampus,cerebellum,brain stem)and cervical spinal cord.(5)Viral replication:1)ZKC2P6 showed significantly replication in the brain compared with ZKC2P4:? ZKC2P6 viral RNA was 125 and 4 times of ZKC2P4 on 3 and 5 days post infection,respectively(P<0.05).?ZKC2P6 virus particles were significantly more than ZKC2P4 on 5 days post infection.2)ZKC2P6 showed significantly replication in SY5Y compared with ZKC2P4:ZKC2P6 viral RNA was 5 and 4.7 times of ZKC2P4 on 48 and 72 hours post infection,respectively(P<0.05).(6)We found that ZKC2P6 induced more apoptosis:?By immunofluorescence detecting,the activated Cas3 of ZKC2P6 group was obviously more than that of ZKC2P4 in cerebral cortex on 5 days post infection.?By western blotting,the cleaved PARP and activated Cas3 of ZKC2P6 group were more than that of ZKC2P4 group in the brain on 5 days post infection(P<0.05).Conclusions 1.Adaptive strain of Zika Virus with E protein glycosylation site deletion was successfully constructed through a series of passages in mouse brain.2.Compared with ZKC2P4,ZKC2P6 increased the ability of replication in nerve cells and accelerated the apoptosis of nerve cells,and accelerated the disease progression in clinical symptoms.3.Collectively,the absence of E protein glycosylation sites increases the neurovirulence of Zika virus.