Cross Reactivity Of Antibody Response In Dengue Fever Between Different Genotypes Or In Serum From Patients With DENV2 Or ZIKV Infection

7Background: In 2014,the outbreak of type 1 dengue fever in Guangdong province,and type 2 dengue virus(DENV2)epidemic in 2015.In 2016,with the outbreak of the ZIKV virus in the Americas,China has also found imported cases.At present,the ADE effect is considered to be an important risk factor for severe dengue,but the host immune status after DENV or ZIKV infection is still unclear.Dengue virus has four serotypes(DENV1-4),and the same serotype can be divided into different genotypes due to the difference of the sequence.Our previous study found that there were two different genotypes in the outbreak of dengue fever in 2014,and there was no cross reaction between the different genotypes.New ZIKV infections often occur in the same region as DENV.ZIKV has a high homology with DENV in sequence and structure,in which DEVN2 E protein sequence is consistent with ZIKV,which can induce immunological cross reaction of 54%.Content and objectives: This paper first discusses the characteristics of DENV1 serotypes of different genotypes on serum antibody response in Guangdong Province in 2014 to reveal the changes of dengue fever epidemic,the same serotype in different genotypes of serum cross reactivity and antibody neutralizing activity;and in 2015 DENV2 patients in Chaozhou area and ZIKA patients admitted to our hospital as the research object,analysis of the mechanism of dengue fever and Zika virus induced host antibody and cross reaction.Methods: 1.Construction and expression of DENV1,2 and ZIKV recombinant E protein C tagged recombinant DENV1(Genotype IV and Genotype I),DENV2 and ZIKA E protein were expressed in mammalian cell expression system.The virus RNA was extracted from the acute phase of the patient,and the outer membrane region of the E protein was amplified by RT-PCR or two step PCR method,and then the plasmid pc DNV3.1 was cloned to construct the expression plasmid.The plasmid was transfected into 293 T cells,and the supernatant containing recombinant E protein was harvested,and the next step was to capture ELISA.2.Acquisition of ELISA The E protein of 293 T cells was transfected with anti label antibody and the ELISA method was established.3.Analysis of the binding of serum antibody to recombinant virus E protein 1)Using the established ELISA method to detect the binding reaction of 36 cases of DENV1 infection iincluding different stages of disease combined with Genotype IV,Genotype I E protein.Serum samples were diluted with 1:1000 and 10 times diluted,with a total of 5 dilutions.2)the serum antibody of 30 DENV2 and 3 ZIKA infected patients were combined with ZIKV,DENV1 and DENV2 E protein.Serum samples were diluted with 1:100 and 3 times diluted,with a total of 8 dilutions.Results: 1.ZIKV,DENV1 and DENV2 recombinant expression plasmid of E were successfully constructed and verified by sequence,the expression protein was about 56 KD.The capture ELISA method for the detection of serum antibody was established with the expressed E protein.2.2.In the 36 cases of type DENV1,the serum levels of Genotype I and IV E protein were significantly increased in the different stages of disease and the stronger antibody binding reaction(P=0.038)was observed in the late course of disease.3.In the 30 cases of type DENV2 hospitalized cases,most of the cases(24/30)serum has obvious binding reaction with DENV1 and ZIKV E protein.there are 3 kinds of combination forms,DENV1>ZIKA(4/30),DENV1 < ZIKA(11/30)and DENV=ZIKA(9/30).Of the 3 patients with ZIKV,there were two cases have strong antibody responses in the acute phase and recovery phase.and had significant cross-reaction with DENV1 and DENV2.Conclusions: 1.the recombinant E protein was successfully expressed in mammalian cell lines,and the capture ELISA method was developed for the detection of serum antibodies.2.the serum of different genotypes of DENV1 showed antibody cross binding reaction,and there was a tendency to increase with the course of disease.3.Both DENV and ZIKV could induce the production of host serum antibody and showed a high degree of cross reactivity.