Rediscovery Of Imported Zika Virus Infection Case And Establishment Of Tree Shrew Animal Model Of Vaginal Infection

Zika virus（ZIKV）is a single-stranded positive chain RNA virus of the flavivirus genus,flavivirus family,and belongs to the insect-borne transmission virus.Aedes aegypti and Aedes albopictus are the main vectors of ZIKV,divided into Asian and African genotypes.Currently,ZIKV has spread in many countries and regions around the world.In 2016,the WHO defined the ZIKV outbreak as a"public health emergency of international concern"due to its widespread vectors and severe neurological diseases such as microcephaly.Yunnan province is adjacent to many countries in Southeast Asia,with numerous border ports and border residents’exchanges,and frequent cross-border personnel exchanges.Both Aedes aegypti and Aedes albopictus are distributed in the province,which may easily cause a variety of infectious diseases to cross the border into the Yunnan province.South Asia/Southeast Asia have been the main epidemic areas of ZIKV in Asia.In 2014,China’s first imported ZIKV case was imported from Bangladesh to Yunnan Province,and in 2019,the second imported ZIKV case was imported from Myanmar.Accompanied by fever（37.7°C）,headache,maculopapular rash.The entire genome sequence of the virus was obtained by extracting and PCR amplification of the patient’s serum viral nucleic acid and named YNZIKV02（Gen Bank Number:MN245143）.The evolutionary analysis of the origin of the virus strain found that YNZIKV02 belongs to the Asian strain,and the origin time was about 1993.Comparison with major Asian strains found that YNZIKV02 is a typical SVM genotype in Southeast Asia.It is worth noting that mutations in the A188V site of the NS1 protein of this strain help to increase the secretion of NS1 into the circulatory system,increase the virus uptake by mosquitoes,and also lead to the enhancement of interferon by NS1.In addition,a suitable climate environment,a wide range of active media,a large number of susceptible people,and frequent economic and trade exchanges with neighboring countries have caused Yunnan Province to face huge risks of ZIKV imports and local prevalence.Human infection with ZIKV generally manifests as a self-limiting disease,the clinical symptoms are more complicated,the pathogenesis is still unclear,and there are no effective antiviral drugs and approved vaccines for marketing.Animal models are a basic tool for studying the mechanism of disease occurrence and development,and a necessary platform for vaccine drug development.Researchers from all over the world have used mice and non-human primates to establish a variety of ZIKV models,which provide technical support for elucidating the pathogenesis of ZIKV infection and developing drugs and vaccines.However,both mice and non-human primates failed to reproduce the main clinical symptoms after ZIKV infection in humans.Mice differ greatly from humans,and non-human primates are similar to humans in the development of the nervous system,but the cost is high and the experimental period is long.Therefore,there is an urgent need to establish a more ideal animal model that can reproduce the symptoms of human infection with ZIKV.The tree shrew is an experimental animal with characteristics in Yunnan Province.It is a small primate mammal with small size and easy to reproduce.It has been widely used in the research of various virus infection models.In this study,a stable ZIKV GZ01 strain culture system and an efficient detection method were established,and ZIKV vaginal infections of mature tree shrews were carried out through simulated sexual transmission.It was found that vaginal infections of tree shrews did not show rashes.Some tree shrews showed transient low-load viremia at 5-7 dpi,and 5 dpi can reach 10^5.6 copies/ml.The regular detoxification of vaginal lavage fluid and urine can continue to 15 dpi,and the detected viral load ranges are 10^4.46-10^7.35 and 10^4.40-10^5.92 copies/ml,respectively.Urine is an order of magnitude higher.Viral nucleic acid can be detected in multiple tissues and organs,which can establish systemic multiple tissue and multiple organ infections of tree shrews;spleen is the main infected organ,which can be detected continuously at 5-10dpi,and efficient replication and expression occur;Reproductive organs mainly include ovary,vagina and vulva,3-5 dpi can be detected continuously.3 dpi can detect viral RNA in the ovary,vagina,vulva and bladder,but only 5 dpi viremia and ZIKV RNA in liver,spleen,lung and muscle,indicating that ZIKV replication in reproductive organs takes priority over blood and other organs.To sum up,through the analysis of the second imported case of ZIKV in Yunnan province,this study reveals that Yunnan province is facing huge risks of ZIKV import and local popularity,providing direction and research basis for ZIKV prevention and control.The establishment of the mature tree shrew model with vaginal infection of ZIKV confirmed the infection characteristics and tissue addiction of the tree shrew with vaginal infection,revealed and verified the transmission of ZIKV through sexual contact.