Establishment And Application Of The Mouse Model With Zika Virus Infection

Zika virus(ZIKV)is a member of the Flavivirus genus of the Flaviviridae family.ZIKV is an enveloped virus with an approximately 10.7-kb positive-sense RNA genome.Similar to other flaviviruses,the genome of ZIKV encodes a single polyprotein that is cleaved posttranslationally by host and viral proteases into three structural proteins(capsid [C],premembrane [prM],and envelope [E])and seven nonstructural proteins(NS1,NS2 A,NS2B,NS3,NS4 A,NS4B,and NS5).Prior genetic and phylogenetic analyses have identified two classifies ZIKV lineages,African and Asian.At present,all strains currently associated with the outbreak in the Americas are of the Asian genotype.Aedes Aegypti is the main vector of the ZIKV transmission,however increasing evidence showed that the virus can also be transmitted by body liquids,such as semen and blood,and vertically from mother to baby.Since2015,the outbreak of the ZIKV,especially in South America,North America and the Caribbean cost great burden to public health,and the epidemic hasspread to more than 70 Countries and regions,and showed high risk of cross-border spreading.It's expected to infect more than 2 million per year.Since congenital microcephaly,Guillain-Barré syndrome and other serious neurological diseases are closely related to the ZIKV,the World Health Organization announced it as " Public Health Emergency of International Concern".China's State Health Commission also released the " ZIKV disease treatment program(2016 first edition)" on February 3,2016,as more than 20 imported cases have been reported by October 2016.However,there are currently no safe and effective vaccines and specific antiviral drugs.Because of the extensive,complex and seriousness of the outbreak of ZIKV,the basic biological characteristics and pathogenesis of the virus are unknown,and the virulence,transmission and pathogenicity of the virus are not clear.More importantly,lack of vaccine,antibodies and drugs seriously restricted the scientific control of the outbreak of the ZIKV.Mice are not only a useful tool to study the pathogenesis of the virus,but also an important means to evaluate the effectiveness and safety of drugs and vaccines.character of Mouse is gentle and small so that easy to operate,meanwhile mice have susceptibility to a variety of virus.To date,mice has been widely used in the virus pathogenesis research and vaccine and drug evaluation.Therefore,it is urgent to establish an animal model of ZIKV infection,the systematic evaluation of its neurotoxicity and nervous invasion,in vivo replication dynamics and viral shedding,as well as tissue organ replication and dynamic distribution,not only laid the foundation for identifying the ZIKV pathogenesis,but also speed up developing the vaccine and drugs for possible protection and control of ZIKV infection.Despite the relatively short time interval,many mice animal models including C57BL/6 and BALB/c immunocompetent mice or ifnar1-/-,Irf3-/-,Irf5-/-,Irf7-/-,STAT2-/-and other immunodeficient mice have been established to investigate mechanisms of dissemination,pathogenesis,and host immune response to ZIKV.Moreover,these models already are being utilized to evaluate vaccines and drugs.At present,the ZIKV outbreak is still spreading worldwide,and causing serious disease.China also will continue to face long-term imported risk.In order to effectively prevent and control the occurrence and prevalence of ZIKV in China,but also for developing our follow-up vaccine and drugs,we first isolated the contemporary virus strains from the 2016 ZIKV-infected patient's urine by using virus isolation and identification technology platform,then their genomic characteristics,plaque size,one-step growth characteristics and neonatal rat virulence were systematically identified.On this basis,the immunocompetent 129 mice and immunodeficient A129 mice model of ZIKV infection were established.Their changes in body weight,neurological symptoms and death,as well as viremia,tissue organ distribution were dynamically observed.Finally,the protective effect of convalescence serum antibody and the NITD008 were evaluated in vitro and in mice.using the established mouse modelFirst section: Isolation and characterization of ZIKV.In order to obtain the clinical isolates of imported wild ZIKV in our country as soon as possible,we inoculated the blood and urine of the patient and the rat brain and cells.The results showed that the typical neurological symptoms began to appear at 7 days after inoculation in mice.Real-time quantitative RT-PCR was used to detect the large number of swabs in the rat brain.The results of indirect immunofluorescence showed that serum and separation The resulting virus has a strong specific binding.The plaque assayshowed that the newly isolated virus could infect BHK-21 cells and form a typical plaque with uniform size and clear edges.Electron microscopy results show that the ZIKV particles were round,the surface is relatively smooth,the diameter of about 40 nm.The whole genome sequence analysis showed that the isolated SZ01 strain was highly homologous to the H/PF/2013 strain isolated from French Polynesia.Phylogenetic analysis based on complete CDS showed that we isolated the SZ01 strain Asian pedigree.Finally,the neurotoxicity of the newly isolated strain was evaluated on 1-day-old suckling mice.The results showed that the neonatal mice had symptoms such as arch,limb paralysis and drowsiness before the 103 PFU virus was vaccinated and began to die.At the same time,the brain tissue,heart,liver,lung and kidney after 12 days of inoculation were detection of higher titers of viral nucleic acids,indicating that the ZIKV has a typical neonatal nerve virulence,and can be in a variety of tissues and organs in the effective replication and amplification.The results of the above experiments show that we have successfully isolated and obtained the imported plant virus virus in China,which provides reliable toxic resources for the establishment and evaluation of subsequent animal models.Second section: Establishment of novel mouse model of ZIKV infectionIn order to establish a mouse model of ZIKV infection,129 mice and IFN-?/? receptor deficient A129 mice were selected as the research object,and105 PFU virus was inoculated by intraperitoneal route.The results showed that129 mice had a virulence peak at day 1 post infection(pi)with peak level of 106 RNA copies / ml,but persisted for 2 days and disappeared at day 3 pi,however the viremia of A129 mice could maintain about 7 days and the peak value occurred at day 2 pi,and the viral load(5.13×108 RNA copies/ml)was significantly higher than that of 129 mice.The results of real-time quantitativeRT-PCR analysis showed that the virus nucleic acid was detected in the brain,heart,liver,spleen,kidney and testis on 1 and 2 dpi.A129 mice were still able to detect in different tissues on 7 dpi,high titer of viral nucleic acids,and viral load was significantly higher than that of 129 mice.These results suggest that we have successfully established a mouse model of ZIKV infection,the relevant data for the next step in the evaluation of vaccines and antiviral drugs to provide a useful reference.Third section: Evaluation of antibodies and drugs in the established mouse modelFinally,using the established mice model,we evaluated the therapeutic effect of human convalescent phase serum and anti-viral drug NITD008.First,we determined the antiviral activity of antibodies and drugs in vitro.The results showed that the recovery of human convalescent phase serum antibody could effectively inhibit the replication of ZIKV,the neutralization titer of ZIKV GZ01 strain was 1: 161;NITD008 drug was dose-dependent in the inhibition of GZ01 and FSS13025 strains.Compared with the control group,NITD008 with concentration of 5?M could decrease the virus titer by 400 times and 1000 times,respectively.The EC50 values were 241 and 137 nM,respectively,and the viral genome was also inhibited by real-time quantitative RT-PCR method,the EC50 values were 950 and 283 n M,respectively.Then,we used 129 mice to evaluate the efficacy of antibodies in the treatment of ZIKV.The results showed that compared with the control group,the antibody could completely remove the viremia of 126 mice infected with the ZIKV and significantly reduce the viral load in the spleen and testis of the treated group.Finally,we used A129 mice to evaluate the efficacy of NITD008 in the treatment of ZIKV.Compared with the control group,the NITD008 treatment group was significantly reduced 2.6times the viremia.More importantly,the treatment group can significantly reduce the incidence of infection in mice and mortality,50mg/kg of drugs can make 70% of mice survive.These results will provide a new perspective and data support for the future development of Zika vaccine and antiviral drugs.In conclusion,this study successfully isolated and characterized a ZIKV strain,established a new mouse model of ZIKV infection,and evaluated the protective and therapeutic effects of neutralizing antibodies and drugs in the model.Present work provides a reliable model for future researches on ZIKV virulence and transmission,and laid a solid foundation for vaccine research and development,antibody and drug treatment effectiveness evaluation.