| Human enterovirus 71(EV71), a member of Enterovirus genus within the Picornaviridae family, is a positive single-stranded RNA virus. The children’s infection of EV71 is usually association with the symptoms such as herpangina, aseptic meningitis, brainstem or cerebellar encephalitisand acute flaccid paralysis, myocarditis, rapid fatal pulmonary edema with hemorrhage, and hand, foot, and mouth disease(HFMD). And infant under 5 years old is particularly susceptible to severe EV71-associated neurological complications. Since the first case of foot, hand and mouth disease caused by EV71 was reported in 1969, its rapid expansion in the Asia-Paci?c region, especially in Southeast Asia, has been an important public health issue. From 1999 to 2009, there were more than 50 million children affected by HFMD outbreak in Beijing, Shenzhen and Guangzhou, which at least led to 200 deaths. After the eradication of the poliovirus, EV71 has now been regarded as the most concerned neurotropic enterovirus.Currently, the most promising vaccine type of EV71 is inactivated vaccine. Currently, the inactivated EV71 vaccines independently developed by three domestic companies have passed III clinical trials, and are now under the approval stage. However, there are still some concerns related to the inactivated vaccines. EV71 immune protection mechanism is more complex: humoral and cellular immunity plays equal important role in immunologic protection of EV71. Due to the limited ability of inactivated vaccines to induce cellular immunity, they may be not the optimal vaccine type of EV71. Live attenuated vaccine is another type of successful vaccine, which is costless and can induce a comprehensive, long-lasting immune response. Till now, human has successfully developed the attenuated live vaccines for poliomyelitis, epidemic encephalitis, measles and other diseases, which provide reference for the development of live attenuated EV71 vaccine. However, the traditional method for developing live attenuated vaccines by passaging the virus in animals and cells may take long time along with uncertainty. With the development of genetic engineering technology, to obtain the attenuated strain of viruses by modifying the viral genome using reverse genetics has become reality, and a variety of genetically engineered liveattenuated vaccine has come out.Micro RNAs(mi RNAs) are small noncoding RNA molecules that widely present in eukaryotes. Kinds of natural and engineered mi RNA have been shown to down-regulate target gene m RNAs specifically. Meanwhile, mi RNAs show their tissue- and process-specific expression pattern, and they are relative conserved across different species. Recently, some mi RNA targeting sequences have been engineered into the viral genome to control virus tissue tropism of both RNA and DNA viruses, and this approach may provide a novel strategy for developing live attenuated viral vaccines. EV71 infection mainly cause nervous system damage, with brain edema, brain stem and spinal cord inflammation as the most serious damage in EV71 infection death cases. Therefore, we may be able to reduce the risk of the appearance of severe neurological complications or even death caused by EV71 infection, if we can inhibit the replication of EV71 in the nervous system.In this study, we constructed an infectious full-length clone of EV71 pandemic strain by using reverse genetics technology, and identified that the recovered virus exhibited the similar biological properties to the parental strain. To suppress the viral replication in neuronal tissue, we inserted the complementary target elements of the neuron-specific mi R124 and mi R125 into 162 or 729 site of 5′ UTR in the EV71 genome, respectively. We constructed and rescued the recombinant EV71 viruses carrying brain tissue-specific mi RNA target sequence, and examined the attenuated features of recombinant viruses in vitro and in vivo. Finally, we evaluated the ability of recombinant viruses to elicit immunogenicity and protective efficacy in mouse model.1. The construction and identification of an infectious full-length EV71 clone. In order to obtain the infectious clones of EV71 H strain, its full-length c DNA was amplified by RT-PCR and cloned into p EV plasmid. The in vitro transcriptional RNA products were transfected into RD cells to produce the progeny virus. The replication and expression of viral protein, and plaque morphologies of recovered EV71 was identified by using indirect immunofluorescence assays, RT-PCR and plague assays. Our results showed that the plaque morphology of recombinant virus was similar to that of WT one. Further experiments showed that the recovered virus could induce the same neurological symptoms in suckling mice as the WT virus did,and the fatality rate was similar. In summary, we have successfully constructed a full-length infectious clone of EV71 H strain, providing the basis for the following mechanism studies of viral pathogenicity and attenuation, as well as the development of new live attenuated vaccines.2. The construction of recombinant EV71 viruses with brain tissue-specific mi RNA target sequence.We inserted the targeting sequences of neuron-specific mi R124 and mi R125 into 162 or 729 site of 5′ UTR in the EV71 genome respectively, and rescued the recombinant virus. Biochemical assays revealed that these two recombinant viruses carrying the target sequences of brain tissue-specific mi RNA were recovered, and they exhibited similar viral protein expressing profiles and plaque morphologies in Vero cell to those of wild type(WT) virus. Their one-step growth curves showed that recombinant and WT viruses exhibited similar replication kinetics in RD cells, while the replication of recombinant viruses in the neurons and exhibited was significantly reduced. In the presence of individual or both brain-specific mimics of mi R-124 and mi R-125, the titers of recombinant viruses were substantially decreased comparing with that of WT virus. And the co-presence of mi R-124 and mi R-125 mimics more effectively inhibited the expression of recombinant viruses than the addition of either individual mimic. Meanwhile, the protein expression of recombinant virus also was efficiently blocked by mi RNAs. Animal experiment results also suggested that the insertion of mi RNA targets in viral genome strongly attenuates the neurovirulence of EV71. Above all, the recombinant virus was highly attenuated in vitro and in vivo.3. The potentials of recombinant EV71 virus to the induce immunoprotection. We examined the immunogenicity of the recombinant virus in mice, and the results shown that the recombinant virus can induce comparable humoral immunity as that WT virus. The recombinant virus could induce high levels of EV71-speci?c neutralizing antibodies, and the neutralizing titers reach 1:90. More importantly, the inoculation of neutralizing antibodies in suckling mice could protect all them away from the lethal dose of EV71 challenge. These results revealed that the recombinant virus could induce well immunogenicity and immunoprotection in mice. Overall, we successfully constructed recombinant EV71 viruses carrying target sequences of brain tissue-specific mi RNA by using reverse genetics technology, and the recombinant virus was genetically stable, and highly attenuated in sulking mice model. Meanwhile, the recombinant virus elicited satisfactory immunogenicity in mice, and the neutralizing antibodies could protect all suckling mice away from the lethal dose of EV71 challenge. This study may provide an innovative ideas and theoretical support for constructing live-attenuated viral vaccine with mi RNA. |
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