Establishment Of QPCR Detection Method For Yunnan Mosquito Vector Chikungunya Virus And Research On Immunization Of Recombinant Adenovirus Vaccine

Chikungunya virus is a mosquito-borne virus,which is transmitted by Aedes mosquitoes.It was first discovered and isolated in the serum of Tanzanian patients in 1952.CHIKV mainly causes Chikungunya fever(CHIKF),which is an acute febrile disease.The main manifestations of infected patients were arthritis,joint pain,fever,muscle pain and rash.In 1987,CHIKV was first discovered and isolated in Xishuangbanna,Yunnan,China.With the exchange of global trade and tourism,the number of imported CHIKV infections in China are increasing year by year,which is prone to epidemic situation.There is no candidate vaccine that can effectively target CHIKV infection.In this study,a recombinant plasmid containing the E1 gene of Chikungunya virus was used as a positive standard.By optimizing the reaction conditions,a CHIKV qPCR detection method was established,and its specificity,sensitivity and reproducibility were evaluated.This method was used detect the prevalence of CHIKV carried by mosquito vectors in some areas of Yunnan Province,and the genetic evolution of positive samples was analyzed.The recombinant adenovirus carrying the CHIKV E2 gene was amplified and purified in laboratory,and the immunogenicity of the candidate vaccine was evaluated by immunizing mice.The main findings are as follows:(1)qPCR results showed that the Ct value and the positive standard had a good linear relationship in the range of 6.94?6.94× 108 copies/?L,the correlation was 0.997,the slope was-3.571;the detection limit was 0.694 copies/?L.No cross-reactivity with ZIKV,DENV and JEV;the coefficient of variation within and between groups is less than 2%.51 mosquito samples were detected by SYBR green polymerase chain reaction,and the positive rate was 3.9%.The virus was mainly carried and transmitted by Culex tritaeniorhynchus and Aedes aegypti.In summary,the established qPCR method can be used to detect Chikungunya virus.(2)A molecular epidemiological investigation of mosquito-borne CHIKV was carried out in some areas of Yunnan.Genetic evolution analysis showed that the strain belonged to ECSA subtype,belonging to the same family as the strain isolated in Guangzhou,China in 2010.The result of homology analysis shows that the two amplified CHIKV strains have high homology with Sri Lankan strains(SL-CK-1,SL11131),and it is suspected that the virus may spread to Yunnan in China through two ways:1.The virus was spread from Sri Lanka to Yunnan,China via mosquito vectors;2.The mosquito bites a patient who has been infected with CHIKV and carries the virus.The mutation analysis of major amino acid sites showed that there was no A226V mutation in two amplified CHIKV E 1 genes.It is speculated that the CHIKV detected may not be transmitted by Aedes albopictus.(3)Purify the recombinant adenovirus expressing the CHIKV E2 gene previously constructed in the laboratory and immunize mice with adjuvant A+D.Collect mouse blood and separate serum every week,and use serum to detect the following 5 indicators;detection of specific antibodies,detection of neutralizing antibodies,detection of T lymphocyte proliferation,detection of T lymphocyte subtypes,and IgG antibody analysis.Type tests(IgG 2a and IgG 2b)were used to evaluate the immune effect of the purified vaccine.The results of the specific antibody test showed that the antibody level of the adjuvant(A+D)+rAd-CHIKV-E2 group was 1.18 times that of the rAd-CHIKV-E2 group.The test results of neutralizing antibody levels indicate that the adjuvant(A+D)+rAd-CHIKV-E2 immunization group can achieve a maximum level of neutralizing antibody of 1:4096,and the rAd-CHIKV-E2 group can achieve a maximum of 1:1024.The comprehensive results show that the recombinant adenovirus candidate vaccine carrying the CHIKV E2 gene can induce humoral and cellular immune responses in mice.The effect of immunization with adjuvant is better,indicating that adjuvant A+D can be used as a candidate adjuvant in the development of recombinant adenovirus vaccine.In summary,this study investigated and analyzed CHIKV carried by mosquitoes in some areas of Yunnan province by using the established qPCR,which provided a reference for the prevention and control of the CHIKV epidemic in Yunnan province;After immunizing mice with purified recombinant adenovirus,highly specific antibodies and neutralizing antibodies were detected,which can induce humoral and cellular immune responses in mice.To lay the foundation for research on the development of CHIKV recombinant adenovirus vaccine and the use of adjuvants.