| Dengue virus (DENV) belongs to the genus Flavivirus. Viral genome is a positive-sense single-stranded RNA approximately 11,000 nucleotides long, encoding three structural proteins:capsid (C), membrane (M) and envelope (E) protein and seven nonstructural (NS) proteins. There are four closely related, but antigenically distinct, virus serotypes (DENV-1, DENV-2, DENV-3 and DENV-4). In recent years, with the development of the international trade, urbanization and the fluidity of population, DENV kept spreading wider, and the number of infected people began to increase.Phylogenetic analysis based on the full length DENV genomic sequence can reveal genetic rules and variations of DENV. Moreover, Phylogenetic analysis can help us to trace the source of virus, and provide the biological basis for the pathogenic mechanism exploration and vaccines development of DENV. The pathogenesis of dengue is likely to be a complex interplay with host immunity and characteristic of virus.Cytokines play key roles in the process of immune response, and cytokine profile is association with severity of many diseases. Development of prognostic makers in cytokines can timly find patients with severe dengue and take treatment.65 dengue patients’s sera, including 25 severe dengue patients and 40 dengue patients, were collected from Guangzhou Eighth People’s Hospital in the epidemic period of dengue fever in Guangzhou. All samples were serology and etiology detected and analysis the correlation between disease severity and expression levels of cytokines.We isolated 7 strains from 56 strains of dengue virus type 1 and 4 strains from 9 of Dengue virus type 2 strains.The complete genome of seven dengue virus type1 strains contain a single long open reading frame of 10176 nt that encodes a polyprotein of 3392 amino acids (aa). The analysis of phylogenetic tree showed that three dengue viruses belong to DENV-1 genotype I,and it may associated with Guangdong DG14 strain isolated in 2011, DG27 strain and HuangPu01 isolated in 2013. Four DENV-1 genotype V may associated with Guangdong GZ9104 strain and Indian RR57 strain isolated in 2009. Alignment of nucleotide and amino acid sequences indicated that the nucleotide homology between genotype I trains isolated from severe dengue patients and mild dengue patients was more than 98%. Amino acid sequences were identical between genotype V trains from different severity disease with more than 99% homology. So we think dengue virus type 1 in Guangdong may develop to circulating strains and case dengue fever in Guangdong province. We need more epidemiological character of dengue virus to control and prevent dengue fever.The complete genome of four dengue virus 2 type with a single long open reading frame of 10173 nt encodes a polyprotein of 3391 amino acids (aa). The analysis of phylogenetic tree showed that four dengue 2 type viruses belonge to DENV-2 genotype cosmopolitan, and it may associated with DENV-2 Guangdong strain GZ40 isolated in 2010, Viet Nam strain BID/V703 isolated in 2006 and Indonesia strain BA05i isolated in 2004. Homology of nucleotide sequences were 99.9%～100% among four dengue virus 2 type strains and amino acid sequences were identical. So dengue viruses isolated from patients with different sevrtity shared same sequences. There were dengue fever caused by other genotype DENV2 before. So DENV2 may casued by imported virus.To detect the expression of serum cytokines in infected patients with dengue virus and their relationship with disease severity, peripheral blood was collected and serum was separated including twenty-five severe dengue patients(severe group), forty mild dengue patients (mild group) and eighteen controls(control group). The expression of twenty-seven cytokines were detected using a multiplex Luminex system. The relationship between cytokines and disease severity was investigated by Kruskal Wallis and Logistic regression analysisThe result showed that expression levels of IL-1β, IL-6, IFN-γ, TNF-α, IL-1Ra, IL-10, Eotaxin, IL-8, IP-10, MCP-1, MlP-1α, G-CSF in infected patients with dengue virus (severe and mild)were higher than non-infected(control). Expression levels of IL-2, IL-9, RANTES, GM-CSF, IL-15, PDGF-BB and VEGF in infected patients with dengue virus were lower than non-infected. The level of IL-5, IL-12, IL-13, IL-17 and basic FGF in three groups had no significant difference (P>0.05). Logistic regression analysis showed that IL-4 (β=0.946, OR=2.576, 95%CI (1.322,5.021),P=0.005) and GM-CSF (β=-0.105, OR=0.900,95% CI (0.838,0.967), P=0.004) entered into the regression equation and the precision reached 95.4%. Cluster analysis of patients by expression levels of cytokines agreed with the observed severity. It showed that expression levels of cytokines could predicted dengue severity.In conclusion, we think the epidemical dengue viruses were multiple in serotype and genotype and DENV1 associated with previous epidemic strains. The same strain caused different disease severity. Moreover, IL-4 and GM-CSF could be the bio-markers to predictive disease severity. |
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