The Prevalence Risk Of Zika Virus In Yunnan And The Establishment Of Tree Shrew Animal Model

Zika virus is an insect-borne virus that has spread in more than 80 countries and regions around the world.In view of the widespread distribution of Zika virus vectors and infections leading to serious neurological diseases such as microcephaly,the World Health Organization announced the Zika epidemic as a“public health emergency of international concern”.As the core node of the national“Belt and Road”development strategy,Yunnan Province is bordered by Myanmar,Vietnam and Laos.There are numerous border crossings,and the economic and trade exchanges are frequent.Simultaneously,Yunnan Province is the hardest-hit area for the transmission and spread of various infectious diseases.And the popularity of these diseases are characterized by the typically cross-border intrusion,and several pathogens concurrently prevail with high incidence in the territory.Thus,Yunnan Province is exposed to the huge risks of ZIKV input and local popularity.However,there is no investigation on on the effect factors of prevalence and spread of ZIKV in Yunnan Province.South Asia and Southeast Asia,which are adjacent to Yunnan,have been the main epidemic areas of ZIKV,but before the first case of ZIKV imported in Guangzhou in January 2016,the existence of imported cases of ZIKV virus in Yunnan is still unclear.In this study,a retrospective analysis of ZIKV epidemiology was conducted on 608 samples of unexplained fever in various ports in Yunnan Province from August 2014 to February 2016.A case of ZIKV importation was found by qualitative and quantitative detection of ZIKV nucleic acid.In this case,it was imported from Bangladesh in Kunming Airport in 2014,and the patient had a fever.The whole genome sequence of the strain was obtained by PCR amplification and named ZK-YN001.The evolutionary analysis of the origin of this strain found that it belongs to the Asian strain,and its origination time is about 2006.The evolution rate is 6.819?5.929-7.709?×10^-4substitutions/site/year.Compared with the sequence of major Asian strains,ZK-YN001 is in the middle transition state of the low-Prevalence stage and explosive pandemic stage of ZIKV in Asia.The discovery of ZK-YN001corrected the epidemic history of ZIKV in China,which suggested that Yunnan Province has the potential risk of importing ZIKV infection from neighboring countries.ZIKV's media-Aedes mosquitoes are widely distributed in Yunnan Province,while the annual average temperature of Yunnan Province is 18.8°C,the average relative humidity is 79.2%,and the monthly cumulative average precipitation is 104.3 mm.The climatic conditions are particularly suitable for Aedes mosquito breeding,and the distribution density and activity level of mosquito in Yunnan Province indicated from June to September is the high-risk month of Zika,and should be prepared and prevented in advance.The clinical symptoms of human infection with ZIKV are complicated,the pathogenesis of the disease is still unclear.There are no effective vaccines and therapeutic drugs.The animal model is a necessary technical platform for researching the pathogenesis,drug and vaccine.Since the outbreak of ZIKV,Researchers from all over the world have established experimental models of Zika fever disease using animals such as mice and monkeys,which lays an important foundation for clarifying the pathogenesis of Zika virus-induced microcephaly/neural injury and preclinical evaluation of candidate vaccine drugs.However,although the infected mice have symptoms such as neurological diseases and even death,it's too different from human infection symptoms;NHPs infections failed to reproduce the main clinical human symptoms,and the research cost is high and the cycle is too long.There is an urgent need to find an ideal animal model that can simulate human natural infections and related clinical symptoms.The tree shrews are mainly distributed in southwestern China,which is mainly in Yunnan Province.It is a small primate mammal that is easy to breed and raise.In recent years,researchers have widely used tree shrews as model animals in the study of human disease development and human viral infections,and have achieved a series of remarkable research results,but there is no research on Zika virus-infected tree shrews report.In order to establish an animal model of ZIKV-infected tree shrews,this study simulates a mosquito bite for subcutaneous injection of an adult infected tree shrew.There have been similar clinical symptoms of human infection:typical skin disease rash,and ZIKV infection can cause pathological damage to the skin.Infiltration of inflammatory cells,presence of viral particles and hemorrhage under the skin;viremia and regular detoxification of saliva;systemic infection involving multiple tissues and organs can be established,in which testicular tissue is sensitive and obvious pathological damage.Further,various primary cells derived from different tissues of tree shrews were infected with ZIKV to determine the tropism and infection characteristics of the virus against different primary cells.The detection of viral nucleic acid and antigen found that the primary cells of artery and skin are most sensitive to ZIKV,and the viral nucleic acids and proteins are efficiently replicated and expressed,releasing a large number of infectious progeny viruses;the infection causes cell death and other serious cytopathic diseases.In summary,tree shrews can be used as an animal model of ZIKV infection from in vitro cell levels.Finally,in order to investigate the immune response induced by ZIKV infection in tree shrews,we sequenced the PBMC transcriptome of tree shrews and found that ZIKV infection can stimulate the expression of various anti-virus related genes and inflammatory factors in varying degrees.The S100A12 gene,which characterizes the severity of skin diseases,is significantly up-regulated during infection.ZIKV infection also stimulates a strong humoral immune response in tree shrews.The titer of neutralizing antibodies in tree shrews can reach 534.8 at 20 day post infection.Neutralizing antibodies have protective effects on suckling mice and effectively prevent secondary infection of tree shrews.In summary,this study reveals the potential risks of ZIKV input and epidemic in Yunnan by analyzing the epidemiology and environmental factors of ZIKV in Yunnan Province,and provides a research basis for the scientific prevention and control of ZIKV.Then the establishment of ZIKV in vivo infection tree shrew disease model and the determination of infectivity and different tropism of primary cells in vitro supplemented the deficiency of existing animal models.ZIKV-infected tree shrews stimulated the body to have a strong immune response,and the resulting neutralizing antibodies have a strong protective effect,which may provide new ideas for studying the antigenic target of ZIKV and drug treatment.