Genome Sequencing Of Dengue Virus GZ2002Strain And Its Infectivity To Human Dendritic Cells*

Dengue virus (DENV), which belongs to flavivirus family, has four distinct diseaseseverity-associated serotypes (DENV-1,2,3,4). Compared with other arbovirus, DENVcan cause severe human disease especially in tropical and subtropical areas. In China, theinputed DENV cases are the primary source that can trigger local pandemic (mainly inguangdong province). Recent years, the DENV epidemic region and the number of infectedpeople are expanded greatly accompanied by the development of urbanization andinternationalization, and some newly isolated strains are also obtained. Although afterdecades of study, there has no unified conclusion about DHF/DSS pathogenesis,so the issueabout DENV prevention and control means should be urgently solved.The whole genome sequencing technology has great significance in understanding theorigin, evolution and variation of the sequenced species. Up to date, more than one hundredgenomes of DENV strains isolated from different regions have been completely sequenced.The bioinformatics analysis of DENV genome can facilitate to find DENV virulence relatedgene locus, and the reverse genetic manipulation technology can be used to constructDENV infectious clone, as well as the mutants,which may contribute to study the DENVprotein functions, pathogenic mechanism and new type attenuated vaccines.The gene polymorphic loci of human molecules that associate with human immuneresponse have been confirmed as principal factors for DENV infection. As the moststrongest professional antigen-processing cells (APCs) in human, dendritic cells (DCs)constitute the most powerful human immunological barrier. It has been confirmed thatDC-SIGN on the surface of DCs can recognize and bind the N-glycosylation sites of DENV E-glycoprotein to mediate DENV infection, and this first step of binding between DCs andDENV virons is crucial for the effective viral infection. The polymorphisms of theDC-SIGN gene, such as-336A/G, may affect the DENV infection outcome because of theimportant roles of DC-SIGN in DENV infection.The DENV-1strain GZ2002was isolated from the serum of a DF patient with positiveIgM antibody, and suckling mice intracerebrally (ic) inoculation assay was performed todetermine its virulence. The GZ2002viral strain was stored in-80℃after propagated inC6/36cells. In the present study, the whole genome of GZ2002was sequenced and theevolutionary analysis was performed. The multiple sequence alignment of coding region/untranslated region (UTR) with three other DENV-1strains with varied virulence was alsoperformed, and some interesting virulence related gene loci were found. We tried toconstruct the GZ2002strain infectious clone that may be useful for characterization ofstrain-specific protein functions. Furthermore, we collected700cases of healthy humanblood samples, and the gene polymorphisms of DC-SIGN [CD209-139(G/A)/-336(A/G)]were confirmed in203cases, and the infectivity of GZ2002strain to human DCs (CD209,-139A/-336A) were conducted.RESULTS1. The whole genome sequencing of GZ2002strain.The DENV GZ2002strain was propagated in C6/36cells, and the infected cells andsupernatant were collected when cytopathic effect (CPE) phenomenon appeared. Wedesigned six pairs of overlapping primers according to the whole sequence of five NCBIpublished DENV-1stains: AY145123、FJ176780、FJ176779、DVU88536、EF025110. SixGZ2002strain covering cDNA fragments were amplificated by RT-PCR, and cloned intopMD-18T vector, respectively. The whole genome sequence of GZ2002strain was obtainedby sequencing the frgments carrying recombinant plasmids and splicing by DNAstarsoftware. The spliced genome sequence is then annotated and submitted to GenBank bySequin software, and the accession number is JN205310.2.The bioinformatics analysis of GZ2002genome sequence.The complete genome of GZ2002strain is composed of10735nucleotides (nt) with asingle long open reading frame of10176nt that encodes a polyprotein of3392amino acids(aa).The5′and3′untraslated regions (UTR) contain94nt and462nt, respectively. Theanalysis of evolutionary tree showed that GZ2002belonges to DENV-1genotype IV, and it may associated with DENV-1Indonesia strain isolated in1998s. Comparison of thegenomic sequences of different pathogenic strains (16007, Cambodia,98901530), we foundnumbers of amino acid mutational sites in coding region that are associated with thepathogenicity of virus strain. Furthermore, the5′noncoding regions are highly conserved,and the3′noncoding regions of16007and Cambodia strains have a hypervariable regionthat deserves further investigation.3. Partial construction of the GZ2002infectious clone.Big fragment cloning is the major aporia. In our study, we chose the large loadingplasmid pFastBac-HTA as cloning vector, and three pairs of cloning primers were designedto obtain the whole genome of DENV strain GZ2002. Now, the infectious cloneconstructing process is partially completed: three big fragments (pFast1, pFast2, pFast3)based on pFastBac-HTA were successfually amplified and cloned into pMD-18T vector,respectively, and the inserted sequences were validated through direct DNA sequencing.One of the three fragments (pFast2) was successfully cloned into pFastBac-HTA vector.4. Different CD209-139/-336locus polymorphisms combinations exist in thetested people.We designed CD209amplified primers, right28and423, according to NCBI releasedsequence, and the amplified fragment include CD209-139and-336loci. We collected700cases of healthy human blood samples and extract their whole blood genome (successfullyextracted605cases), and PCR was performed to obtain the PCR product (about457bp).TheSpeI restriction enzyme was chosen to identify the-139locus polymorphisms of605cases,and the results revealed that280cases are of-139A,86cases of-139G and239cases with-139A/G. A mismatch PCR was performed to identify-336locus polymorphisms of300cases, and203cases were successfully tested. Among203cases,154were tested for-336A,6for-336G and43for A and G. Combination with-139locus polymorphisms,203cases intotal were achieved for both polymorphism loci (CD209-139/-336), and we found that theCD209-139A/-336A is the major phenotype (36.5%) in our tested samoles,while the CD209-139AG/-336A goes second (30%)(please see the following table). 5. Culture of human dendritic cells from peripheral blood samples.Thirty milliliters of human peripheral blood samples were collected, and the peripheralblood monouclear cells (PBMC) were obtained by ficoll-gastrografin density gradientcentrifugation, and cultured in bottles with fresh medium supplemented by cytokines(GM-CSF, IL-4). The DCs were verified by determining the cell morphology, CD1aexpression level and mixed lymphocyte proliferation ability. The results showed that humandendritic cells can be obtained post7d culture in our experimental condition. Dendritic cellshave a large and nonuniform micro-morphology, and there are many branche-like processeson their surface. Furthermore, the cultured DCs showed dark blue, dark-stained nucleus andmany burr sample bumps under microscope after Wright stain. Flow cytometry (FCM) alsorevealed the CD1a expression level of the obtained cells is91%and the MTT lymphocyteproliferation experiment showed the obtained cells have strong ability to stimulate theproliferation of lymphocytes.6. DENV GZ2002can infect human DCs with CD209-139A/-336A polymorphismcombinations.The dendritic cells that express CD209(-139A/-336A) polymorphism combinationwere cultured. The DENV infection to dendritic cells was performed using routing protocol.The post-infected (p.i) dendritic cells are used to test CD1a,CD83,HLA-DR expressionlevels by flow cytometry assay and morphological changes by Wright stain.The resultsshowed that the post-infected cells have a round ball like and shorter branche-like processesin morphology, and the expression levels of CD1a、CD83、HLA-DR are also changed postDENV infection: CD1a (66.9%→94.8%)，HLA-DR (34.6%→77.4%)，CD83(14.9%→66.8%) when compared to the normal DCs. Furthermore, the collected supernate wasused to infect Vero cells, and the existence of DENV particles in the supernate was verifiedby testing DENV specific E and NS1protein with indirect immunofluorescent assay (IFA).