ABSTRACT
Objective:To analyze the first suspected dengue fever case in Dapeng New District by characterizing the virus and exploring its origin. Methods:Dengue virus nucleic acid in blood sample was identified by real-time fluorescent RT-PCR. The E gene of dengue virus was amplified and sequenced. Homology and phylogenetic tree of this dengue virus were compared with the isolates from other regions. Results:Nucleic acid of dengue virus type 1 was detected in the serum sample from the suspected dengue fever patient. Phylogenetic tree showed that the homology based on nucleotide sequence of E gene of DP-DGR19001 was very similar with the isolates of MG894965/TW/CHN/2016 （99.87%）， LC428079/VNM/2017（99.80%）， MG894867/TW/CHN/2015 （99.60%） and MN512242/GX/CHN/2014 （99.46%）. The deduced amino acid sequence of 5 isolates was 100% identical. The patient had traveled to high-incidence country Cambodia in 14 days before infection. Conclusions:The first suspected dengue fever case in Dapeng New District is caused by dengue virus type 1. The case is an imported dengue fever .
ABSTRACT
Objective To construct a recombinant expression vector for expression of the function-al domains of dengue virus serotype 1 ( DENV1 ) envelope ( E ) protein in native soluble form. Methods The genes encoding the functional domains of DENV1-E protein (1-394 aa) were amplified with PCR and then cloned into the Psectag2B-Fc eukaryotic expression vector.The 293T cells were transfected with the recombinant vector by cationic lipid-based delivery.The cell clones expressing the fusion DENV1-E-Fc protein were screened out with 2 mg/ml of Zeocin.Immunofluorescence assay ( IFA) was performed to analyze the antigenicity and integrity of the fusion protein.The fusion proteins were purified from cell lysate with Protein-G and further identified by Western blot assay.Results The soluble form of fusion protein with a molecular weight of about 90×103 was obtained at a yield of about 25 μg per 1×107 cells.The results of IFA indicated that the fusion protein kept its integrity with right conformational epitopes.The fusion protein was successfully expressed with the advantage of good specificity as indicated by IFA and Western blot assay. Conclusion The recombinant fusion protein in soluble form was successfully expressed in eukaryotic ex-pression system, which paved the way for further investigation on the function of DENV1 E protein and its protective epitopes.
ABSTRACT
Dengue fever is the most important arbovirus infection found in tropical regions around the world. Dispersal of the vector and an increase in migratory flow between countries have led to large epidemics and severe clinical outcomes, such as dengue haemorrhagic fever and dengue shock syndrome. This study analysed the genetic variability of the dengue virus serotype 1 (DENV-1) in Brazil with regard to the full-length structural genes C/prM/M/E among 34 strains isolated during epidemics that occurred in the country between 1994-2011. Virus phylogeny and time of divergence were also evaluated with only the E gene of the strains isolated from 1994-2008. An analysis of amino acid differences between these strains and the French Guiana strain (FGA/89) revealed the presence of important nonsynonymous substitutions in the amino acid sequences, including residues E297 (Met→Thr) and E338 (Ser→Leu). A phylogenetic analysis of E proteins comparing the studied isolates and other strains selected from the GenBank database showed that the Brazilian DENV-1 strains since 1982 belonged to genotype V. This analysis also showed that different introductions of strains from the 1990s represented lineage replacement, with the identification of three lineages that cluster all isolates from the Americas. An analysis of the divergence time of DENV-1 indicated that the lineage circulating in Brazil emerged from an ancestral lineage that originated approximately 44.35 years ago.
Subject(s)
Humans , Dengue Virus/genetics , RNA, Viral/genetics , Amino Acid Sequence , Brazil , Dengue Virus/classification , Dengue Virus/isolation & purification , Genotype , Molecular Sequence Data , PhylogenyABSTRACT
Aedes albopictus C6/36 cells are susceptible to Dengue virus. The C6/36 cell line was infected with type 1 Dengue virus (DEN 1). At different time after these cells infected, morphological observations with electron microscope were conducted with super thin slicing method and the ultrastructural characteristics of DEN 1 virus in infected cells were recorded.