Molecular characterization of the envelope gene of dengue virus type 3 newly isolated in Guangzhou, China, during 2009-2010.

BACKGROUND: After an absence of 29 years, dengue virus type 3 (DENV-3) re-emerged in Guangzhou in 2009 and again in 2010. However, the geographical route by which the virus entered the city, and how it has changed genetically, remain unclear. Therefore, we carried out a comprehensive investigation into the molecular characteristics of the DENV-3 involved. METHODS: The envelope (E) genes of viruses isolated from dengue patients during the 2009-2010 epidemics were sequenced and compared with previously published E gene sequences of global representative DENV-3 strains available in GenBank, including isolates circulating in other provinces of China. RESULTS: A total of 13 isolates (seven from 2009 and six from 2010) were obtained from human serum samples. Phylogenetic analysis revealed that the isolates were grouped into three genotypes (I, III, and V) and then two clades within genotype III (genotype I from Indonesia, genotype III clade A from Côte d'Ivoire, genotype III clade B from Tanzania, and genotype V from Philippines). In addition, there were 1.3-9.0% and 0.5-3.9% differences in the nucleic and deduced amino acid sequences between the 2009 and 2010 strains, respectively. CONCLUSIONS: The DENV-3 viruses from the period 2009-2010 were not from the continuous spread of an epidemic strain or the re-emergence of the 2009 strains in the 2-year period. The introduction of different DENV-3 genotypes following more than one geographical route was an important contributing factor to the 2009-2010 dengue epidemics in Guangzhou.

Defective activities, but not secretions, resulting from gene point mutations of human mannan-binding lectin.

Human mannan-binding lectin (MBL) plays a pivotal role in innate immunity. Substantial literature supports the belief that three point mutations, CGT52TGT, GGC54GAC and GGA57GAA, in the collagen-like region (CLR) of the human MBL gene, are associated with increased susceptibility to infection, autoimmunity and carcinogenesis. To investigate the mechanisms of MBL deficiency, human wild-type and three variant MBL genes were expressed in COS-7 and Chinese hamster ovary (CHO) cells. Results showed that no apparent differences were found among the levels of gene transcription and protein secretion of four forms of MBL. However, the degree of oligomerization of variant forms of MBL was found to be much lower than that of recombinant human wild-type MBL. The ability of variant MBL proteins to bind mannan was much weaker than that of the wild-type MBL protein, and the MBL variants failed to effectively activate the complement lectin pathway. These data suggested that a lower order oligomer, but not decreased plasma levels of MBL, may be the main result of MBL gene mutations and may be associated with immunodeficiency.

[Molecular epidemiologic analysis on new emerged type 3 dengue virus in Guangzhou in 2009].

OBJECTIVE: To analyze and trace the infection source the envelope (E) gene of the new emerged type 3 dengue virus in Guangzhou in 2009. METHODS: Sera were collected from patients infected with local dengue fever. Dengue virus was cultured and isolated by C6/36 cells. The whole length E gene was amplified from the positive specimen by RT-PCR, thereby sequenced and phylogenetic tree drawn by neighbor-joining method. Both data on epidemiologic and molecular studies were processed and analysed. RESULTS: 7 strains of type 3 dengue virus were isolated from samples of the 19 patients. E gene of these strains was amplified. The complete E genes of 7 strains belonged to 1479 nucleotides in length, encoding a polyprotein of 493 amino acids. Data from the phylogenetic analysis showed that 09/GZ/1081, 09/GZ/1483 and 09/GZ/10806 strains fell within the Southeast Asia/South Pacific group. 09/GZ/10616, 09/GZ/11144, 09/GZ/11194 while 09/GZ/13105 strains fell within the India group. CONCLUSION: The type 3 dengue virus identified in Guangzhou area in 2009 was imported and could be decided into two genotypes.

[Relation between clinical detection rates of the novel influenza virus A/H1N1 RNA in confirmed patients in Guangzhou and the disease course].

OBJECTIVE: To study the relation of the detection rates of the novel influenza virus A/H1N1 RNA in clinically confirmed patients in the 2009 pandemic with the age distribution of the patients and the disease course. METHODS: A total of 151 clinical patients with H1N1 infection were enrolled in this study, from whom 833 dynamic throat swab samples were obtained for detecting the H1N1 RNA using real-time PCR. A statistical analysis of the age distribution was performed among the patients with different disease courses. Chi-square for trend test was used to study the correlation between the detection rates of H1N1 RNA and the time of disease onset. RESULTS: The majority of patients were young with their ages ranging from 10 to 20 years (57.26%) and 20 to 30 years (22.18%). Chi-square for trend test revealed that the positivity rates of the throat swabs in the patients decreased with the prolongation of the disease course (chi(2)=9.784, P=0.002). CONCLUSION: Most of the H1N1 patients are young within the age range of 10-30 years, and the longest disease course can exceed 10 days. The positivity rates of throat swabs from the H1N1 patients decreases with the prolongation of the disease course.

[Study on E protein epitopes and primary identification of main yellow virus].

OBJECTIVE: To analysis the E protein epitopes of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus and to distinguish the shared or specific epitopes among them. METHODS: Bioinformatic software DNAStar was used to analyze the hydrophilicity, flexibility, surface probability and antigenicity of dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E protein amino acid sequences. The influence of secondary structure was also considered. Based on the bio-informatic analysis of E protein epitopes, 6 specific epitopes were amplified and inserted into prokaryotic expression vector pMAL-c2x. The vectors was then transferred into E. coli BL21 (DE3) and Rosetta (DE3). Isopropyl-beta-D-thiogalactoside (IPTG) was used to induce the expression of gene segments and SDS-PAGE were used identify the expression proteins. The antigenicity was tested, using Western blot. RESULTS: 15 shared epitopes and 47 specific epitopes were forecasted by bioinformatic analysis, and 6 specific epitopes from dengue virus type 1-4, Japanese encephalitis virus and yellow fever virus E protein were expressed in E. coli successfully. Two specific antigenic determinant from dengue virus type 1 and dengue virus type 2 were confirmed using Western blot, while the others epitopes shown no antigenic reaction property. CONCLUSION: Two specific antigenic determinant were confirmed, under Western blot.

[A genetic optimization designing method for microorganism detection genechip probe based on genetic algorithm].

A new automatic selection approach of microorganism specific fragment combination is presented in this paper. Genetic algorithm is used to search optimal solution on the basis of classification ability of SNP combination, which is evaluated by the rough set theory. Other related experimental parameters are also been incorporated. Experimental results show that the method can find the best SNP combination pattern efficiently and accurately, which implies that it is a reliable approach to the genechip probe design.

[Expression of human CGT52TGT MBL mutant in CHO cells and analysis of the expression product].

AIM: To explore preliminarily the mechanisms of immunodeficiency resulted from the CGT52TGT point mutation of mannan-binding lectin(MBL) gene. METHODS: The MBL gene containing CGT52TGT point mutation was amplified from the plasmid pMBLm52 by PCR, and then inserted into the eukaryotic expression vector pcDNA4/HisMax C. After confirmed by DNA sequencing, the recombinant expression vector was transfected into Chinese-hamster ovary(CHO) cells by electroporation. Zeocin of 800 mg/L had been used for 30 days to select electroporated CHO cells, and then 200 mg/L for another 30 days to obtain stable transfectant. The expression of mRNA was analyzed by RT-PCR, the recombinant protein was purified from the culture supernatant by Ni-NTA agarose chromatography and analyzed by SDS-PAGE under nonreducing condition and Western blot. RESULTS: The cDNA fragment amplified from pMBLm52 plasmid by PCR was about 750 bp and the recombinant plasmid pcDNA4/HisMax C-MBLm52 was constructed and transfected into CHO cells. The expression product purified from the culture supernatant appeared mainly at the site of M(r) 60,000, indicating a much lower oligomerization level than that of the recombinant human wild MBL and human plasma-derived MBL. CONCLUSION: The CGT52TGT point mutation of MBL gene does not affect the secretion of its product, but a Cys introduced by the mutation could form another disulfide bond which may disrupt the structure of MBL molecule as well as its function.

[Construction of the expression vector PcDNA4/His C-MBL and its expression in Chinese-hamster ovary cells].

OBJECTIVE: To construct pcDNA4/His C-MBL recombinant eukaryotic expression plasmid and examine its expression of mannan-binding lectin (MBL) in mammary cells. METHODS: The target sequence was amplified by PCR from pGEM-MBL plasmid that contains wild-type human MBL cDNA, and inserted into eukaryotic expression vector PcDNA4/His C followed by restriction mapping and sequencing. The recombinant plasmid PcDNA4/His C-MBL was transformed into Chinese-hamster ovary (CHO) cells by electroporation, and the Zeocin-resistant clones were selected for analysis of mRNA expression by reverse transcription (RT)-PCR. The expressed product was purified by immobilized metal affinity chromatography (IMAC) and identified by SDS-PAGE and Western-blot analysis, and its immunoreactivity was detected by an indirect enzyme-linked immunosorbent assay ELISA using the anti-serum from Balb/C mice immunized with the recombinant protein. RESULTS: The cDNA fragment of 750 bp was amplified from pGEM-MBL plasmid, which was shown by restriction enzyme digestion and DNA sequencing. The mRNA expression of Zeocin-resistant CHO cell clones was detected by RT-PCR. Three components of 29, 58 and 87 kD in the purified recombinant product were found by SDS-PAGE and the 29 kD component could be recognized by anti-6His antibody in Western blot analysis. The titers of the anti-serum from immunized mice were 1: 819 200 against the recombinant protein and 1:25 600 against both the natural human MBL and the recombinant trimeric carbohydrate-recognition domain (CRD) of human MBL, as determined by the indirect ELISA. CONCLUSION: The cell strains that express recombinant human MBL (rhMBL) and rhMBL protein have been obtained successfully, which provides the basis for further research of MBL molecule.