Construction And Identification Of Attenuated Vaccine Strain Of Zika Virus Through MicroRNA Targeting

Zika Virus(ZIKV),one of the genus flavivirus in the family flavivirde,is a single-stranded positive-sense RNA virus with a diameter of 50 nm and a genome size of about 10.7 kb.The disease caused by ZIKV is a mosquito-borne virus disease,and mainly transmitted by Aedes aegypti.After 2015,the outbreak of ZIKV in Latin America has lead the onset of microcephaly in a large number of newborns.On February 1,2016,the World Health Organization listed the risk of ZIKV for pregnant women as a public health crisis worthy of global attention.At present,there are no effective vaccines and drugs to prevent and treat ZIKV disease at home and abroad.Therefore,the ZIKV vaccine need to develop urgently.The live attenuated vaccine is one of the currently used types of vaccines.This live attenuated vaccine can fully activate the humoral and cellular immune response,so the body can obtain more extensive and lasting immune protection.The live-attenuated strategy mediated Micro RNA(mi RNA)targeting has had a major breakthrough,and have applied in the field of viral vaccine research.The main mechanism on this strategy is to use the mi RNAs that are specific to cells within a tissue to control the tropism of the virus,thereby reducing or eliminating the replication and pathogenic effect in one certain tissue.The advantage of this strategy is that it not only retains the virus immunogenicity but also reduces the pathogenicity of the virus,providing a completely new means for the development of a new type of attenuated live vaccine.It confirmed that ZIKV infection lead to abnormal proliferation and differentiation of neural stem cells in the embryonic brain,as well as a large number of neuronal deaths,causing microcephaly.Therefore,if ZIKV can be inhibit in the replication of embryonic brain tissue,it is possible to reduce the risk of leading to microcephaly.In this study,we have constructed ZIKV full-length infectious c DNA clones carrying brain tissue-specific mi RNA target sequences by reverse genetics technique.We insert the brain tissue-specific mi RNA target sequences into the 3'UTR region of ZIKV genome to constructed and rescued ZIKV recombinant virus carrying multiple mi RNA target sequences,and identify the attenuated characteristics of the recombinant virus.Finally,we evaluated the immune effect and safety of the recombinant virus in animal models for better to develop the ZIKV live-attenuated vaccine in the future.1.Construction and Identification of Attenuated Vaccine Strain of Zika Virus Through Micro RNA TargetingIn order to design and construct ZIKV attenuated strains rationally,our method based on the principle of specific micro RNAs as an attenuated mechanism in related tissues and organs.We used the single-stranded target sequences of brain tissue-specific mi RT-9,mi RT-124,mi RT-125 and three copies of them,and a mixture of three copies to insert into the front of 3'URT sequence of the ZIKV genome between 10379 bp and10380 bp,respectively.Through reverse genetic manipulation,we successfully obtained6 recombinant viruses carried brain tissue-specific mi RNA target sequences.We further identified the biological characteristics of the rescued recombinant virus through the relevant experiments,such as plaque,growth curve,IFA,structure prediction,genetic stability,interference experiment with RNA mimics in vitro,and animal experiments.We found that the recombinant virus plaque was slightly smaller than the parent strain and could efficiently replicate in BHK-21? C6/36?Vero cell lines,and could efficiently express virus-specific E protein.The structural prediction and sequence analysis revealed that the secondary structure of the recombinant virus strain mi RT-125* 3 was unstable,and 6th generation sequence was mutated.The other 5 strains were all stable and able to stably inherit.RNA mimics interference in vitro can inhibit the replication ability of the mi RT-124*3 recombinant virus strain.The replication of the virus is limited in mouse brain tissue,indicating that mi RNAs can indeed regulate the proliferation of recombinant viruses.More importantly,the recombinant virus has only one-tenth of the neurotoxicity of neonatal rats and its virulence reduced significantly.2.The Evaluation of Immunogenicity and Immunoprotection of Attenuated Vaccine Strain of Recombinant Zika VrusBased on the above experimental results,we selected mi RT-124*3,which has low-virulence,from 6 kinds of recombinant zika viruses and evaluated their immunogenicity and immunoprotective effects using established adult mice and pregnant mice protection models.The results showed the mi RT-124*3 recombinant virus strain can induce high neutralizing antibodies in immunized mice,significantly reduce the viremia produced,and has good immunogenicity.Meanwhile,it can completely protect the pregnant mice after immunized.We cannot detect the virus in the placenta and fetal head.Therefore,the mi RT-124*3 recombinant virus strain can not only effectively protect adult rats,but also protect pregnant mice and fetal rats.In summary,this study used the ZIKV infectious clones as the backbone by reverse genetics technology to construct the recombinant ZIKV strains with the brain tissue-specific mi RNA target sequence.It shows the obvious attenuation characteristics in the small mouse model and it can stably inherited in vitro.At the same time,the virus has a good protective effect on the immunized mice.The above findings provide an indispensable theoretical support for ZIKV live-attenuated vaccine research.