Isolation And Culture Of Imported Chikungunya Virus And Its Infectivity To Tree Shrews In Yunnan Province

Chikungunya virus(CHIKV)is an insect-borne virus belonging to the phleboviridae family and the genus of alphavirus with a single strand positive RNA.In humans CHIKV infection can lead to Chikungunya disease with sudden high fever,rash,joint pain and other typical clinical symptoms.At present,Chikungunya fever has spread in many countries and regions around the world,and has become one of the major global public health problems.The epidemic of Chikungunya fever in China is mainly transmitted by import.Yunnan province is located in the southwest border of China and adjacent to southeast Asian countries.It is the radiation center of China to south and southeast Asian countries.In recent years,outbreaks of Chikungunya fever have been frequent in southeast Asian countries.With the process of economic globalization,the frequent cross-border exchanges of people have brought great challenges to the prevention and control of CHIKV in Yunnan.In addition,there is no specific clinical drug or protective vaccine for people with CHIKV.Among them,the lack of a real simulation of human CHIKV infection after the development of small animal model is one of the key factors restricting CHIKV drug development and vaccine research.As a unique animal resource in yunnan province,tree shrews have the advantages of short reproductive cycle and easy breeding,so it has been widely accepted as an animal model.At present,animal models of various viruses have been successfully established in tree shrews,but there is no report on the establishment of animal models of tree shrews to simulate the clinical symptoms of CHIKV infection.Based on this,this paper isolated and cultured CHIKV virus from imported CHIKV cases in Yunnan province,and carried out in vitro and in vivo infective studies on tree shrews with this isolated strain,providing important scientific basis for tree shrews to become animal models of CHIKV infection.First,10 plasma samples of imported CHIKV from southeast Asian countries were collected from may to August 2019 for nucleic acid extraction,whole-genome amplification and analysis of the virus,as well as in vitro isolation and culture of the virus.The results showed that the 10 strains were 99.61%to 99.74%nucleotide consistent with the east/middle/South Africa(ECSA)lineage,and the analysis of the homology relationship evolutionary tree showed that these CHIKV strains all belonged to the Indian Ocean subtype in the ECSA lineage.The 10 strains were K211E mutations in E1,V264A,G60D and I211T mutations in E2.C6/36 and BHK cells were used to isolate and culture CHIKV in vitro.Real-time PCR,plaque assay,IFA,and CHIKV positive and negative chains were used to detect CHIKV.The results showed that we successfully isolated and cultured 9 cases of CHIKV strains in vitro with virus titer of 10⁶-10⁷pfu/ml.Secondly,we used the isolated CHIKV strain to conduct infective studies on primary skin fibroblasts(TSDF)and kidney cells(TSKC)of tree shrews,respectively.The results showed that after CHIKV infected primary cells,the supergene of the virus was detected by real-time PCR,plaque assay,IFA,CHIKV positive and negative chains,and cytokines.It was found that the isolated and cultured CHIKV strain had certain infectivity to TSDF and TSKC.Finally,in order to further establish the animal model of CHIKV infected tree shrews,we used the isolated CHIKV strain to infect the adult tree shrews with subcutaneous injection and left injection.The results showed that the shrews developed persistent viremia and clinical symptoms very similar to those seen in humans:rashes and pad swelling.Real-time PCR was used to detect 11 kinds of tissue grinding fluids in brain,liver,skin,left muscle,right muscle,bone,prostate,spleen,ankle joint,knuckle joint and elbow joint.The results showed that viral nucleic acid could be detected in 11 tissues during the 16w period,among which the highest viral load was found in the spleen and knuckle tissues,reaching 10^8.5 copies/ml.To sum up,this study revealed the main genotypes of CHIKV cases and their infectivities to tree shrews through the isolation and culture of 10 imported CHIKV strains from Yunnan province and the study on their infectivities to tree shrews,providing important scientific basis for tree shrews to become animal models of CHIKV infection.At the same time,it also provides an important theoretical basis for future research on the pathogenesis of CHIKV clinical symptoms.