The Relationship Between Genome Variation Of Dengue Virus,Viral Load And Innate Immunity And Severity Of Dengue Fever

Dengue fever(DF),caused by dengue virus(DENV)infection,is an acute mosquito-borne viral disease.DENV can be classified into four serologically-distinct but genetically-related serotypes(DENV-1~ DENV-4),and infection with anyone serotype results in a wide spectrum of clinical illness ranging from asymptomatic to life-threatening disease.Symptomatic disease includes two entities: DF and severe dengue(SD)according to the 2009 World Health Organization(WHO)classification.DF is a self-limited mild form of disease characterized by high fever,headache,fatigue,skin rashes and so on lasting about 7 days.However,SD,characterized by severe plasma leakage,severe bleeding and severe organ impairment may be fatal.Although many efforts have been made to elucidate the underlying mechanisms of SD,it still remains unclear.Secondary heterotypic DENV infection,virus variation and immunopathology have been postulated and implicated in the pathogenesis of severe dengue.Guangdong,especially Guangzhou city,remains the main epidemic area and has the highest incidence of dengue in mainland China.There has been near-annual dengue cases reported in Guangzhou in past decades,with a peak every 3-5 years.Dengue in China is still characterized as an imported disease,although a number of large dengue outbreaks were reported.Recent dengue epidemics in Guangdong have become more severe in addition to being more frequent.All four dengue serotypes were reported during these epidemics,but DENV-1 has become predominant in recent years Dengue outbreaks have occurred continuously from 2013 to 2014 in Guangzhou.Dengue epidemic in 2014 caused approximately 40,000 cases of infection,making this the largest outbreak with more severe and death cases in the history of Guangzhou.But the transmission sources in this dengue epidemic were still unelucidated.In Southeast Asia and South America,dengue is viewed as a pediatric illness and children have greater risk of severe disease while adults show a mild disease.Much of our understanding of severe dengue pathogenesis originated from child cases in the Southeast Asia.However,dengue predominantly affects adults and the elderly are at higher risk for severe dengue in China.The clinical features were also different from that in endemic areas.At present,limited data about DENV and innate immunity in the pathogenesis of SD was available in China.It is an urgent problem to be solved to study the relationship between DENV genome variation and immune response and severity of disease to explore the pathogenesis of SD.This study will help to early diagnosis and treatment of SD to reduce the mortality.Objective: To look for molecular evedience to support the conclusion that DENV-1 has circulated locally in Guangzhou by analyzing the molecular characterization of DENV-1 circulating in Guangzhou,2013-2014 and elucidate the pathogenesis of SD in Chinese adult dengue patients by studying the relationship between genome variation of dengue virus,viral load and innate immunity and severity of dengue fever.Methods: Genome-wide RNA sequencing in the sera from indigenous dengue cases in 2013-2014 epidemics in Guangzhou was performed on Ion Torrent Proton platform.A phylogenetic analysis was conducted on complete genomes extracted from GenBank and the isolates from this study to determine the characteristics and origin of DENV isolates responsible for the 2013-2014 epidemics in Guangzhou.Polymorphism site composition,gene expression levels and genetic diversity of DENV-1 were compared between DF and SD patients to analyze the relationship between genome variation of DENV and severity of disease.Levels of IgM and IgG antibodies were detected using capture ELISA kits to investigate the percentage of patients with primary or secondary infection.Difference analysis of secondary infection rate was done between DF and SD patients to explore secondary heterotypic DENV infection in the pathogenesis of SD.The serotype and viral loads were determined by using fluorescence quantitative RT-PCR.The dynamic changes and difference between DF and SD patients during day 1-10 after disease onset was investigated to analyze their relationships with disease severity.Levels of inflammation mediators in plasma collected from patients at different time points were determined by a multiplex Luminex? system.Different trends of inflammation mediators throughout the disease were compared between DF and SD patients to understand the contribution of inflammation to disease severity.Expression of pattern recognition receptors pathway-related genes in peripheral blood mononuclear cells from dengue patients at different time points was detected using PCR array.Results:(1)Two genotypes including genotype I and III DENV-1 co-circulated in Guangzhou in 2013-2014.The predominant epidemic genotype-III lineage DENV-1 characterized with a 21 nucleotide deletion in the 3'-untranslated region has been circulating locally since 2013,which mainly responsible for 2014 large outbreak in Guangzhou and originated most likely from Singapore.(2)No significant differences were found in the genotype-III groups between DF and SD.In contrast,the genotype-I clade displayed more variations relative to dengue severity.The nucleotide substitutions among variants of the DENV-1 genotype-I were predominantly observed in E and NS1 protein.(3)The DENV-specific Ig M antibody increased gradually with the disease progression.The positive rates of IgM antibody in patients increased to 95% on day 6.IgG antibody increased relatively slow.There was no difference of secondary infection rate between DF and SD patients.(4)The viral loads were significantly higher in severe patients than those in mild cases during critical stage of disease.And contrary to traditional views,higher viral loads were observed in patients with primary infection than secondary infection.(5)Inflammation mediators displayed significant differences on day 8-10 of illness between mild and severe dengue patients.Their concentrations were higher in severe patients than mild ones at the same time points.Moreover,those cytokines decreased gradually in mild patients but not in severe patients.(6)Host recognize and bind dengue viral RNA through TLR7/8,RIG-I and MDA5,activating downstream signaling to induce the expression of inflammatory cytokines and and beta interferons-?/?.Pattern recognition receptors related genes expression in dengue patients was strongly enhanced.Under-regulated genes expression was observed in severe than mild patients during the critical and recovery stage of disease.Conclusions: DENV-1 has circulated locally in Guangdong probably since 2013,and that epidemic dengue might have changed from being imported to endemic,or from a simple imported mode to a mixture of imported and endemic.Genetic diversity of DENV-1 and secondary infection might have roles in severity of disease,but not be the most important risk factors for severe dengue in our patients.Innate immune response played an important role in the pathogenesisi of SD.