Spatiotemporal Characterizations For Whole Genome Of Dengue Virus And Analysis Of Confirming The Diagnosis, Clinical Characteristics, Prognosis And Prediction Of Dengue And Severe Dengue

BACKGROUND:Dengue fever (DF) is an actue infectious disease caused by dengue viruse (DENV). The clinical spectrum is varify ranged from silent infections with no symptoms to a mild flu-like syndrome, dengue fever (DF), or severe dengue disease (SD), including dengue haemorrhagic fever (DHF) and dengue shock syndrome. The viral genome consists of a single positive sense RNA of 11 000 bp and encods a single polypeptide chain. The signal peptide is dissolved into three structural proteins (C, M/prM and E) and seven nonstructural proteins (NS1, NS2A, NS2B, NS3, NS4A, NS4B and NS5) in the role of host signal peptidase and viral protease enzyme.There are four serotypes referred to as DENV-1, DENV-2, DENV-3 and DENV-4. The disease causes by Aedes aegypti and Aedes albopictus mosquitoes is epidemic in tropic and subtropic areas worldwide, especially in Asia, Pacific islands and South America. It is estimated that 390 million dengue infections in 2010, including 294 million inapparent infections and 100 million apparent infections. Currenlty, no effective antiviral drμgs and vaccines against dengue virus are for patients with DF and normal popμlation. Also, accomply with the social and economic effects, the increased geographies, infections and disease burden of DF spread. Thus, DF has become one of the most important public health problems.Since the first document reported dengue outbreak in Foshan Guangdong province in maliland China, the disease has been epimdemic for about 37 years and been reported in Guangdong, Hainan, Guangxi, Fujian, Zhejiang and Yunnan provinces. Also, the geography of DF spreads to Henan probince in 2013. In 2014, an outbreak of DF has been in Guangdong and leads to 44 894 infections and 6 deaths. All four DENV serotypes have been epidemic. DENV-1 was responsible for dengue fever (DF) epidemics in Guangdong province in 1979 and 1985. Several outbreaks caused by the same virus were also reported in 1991, and from 1995 to 2010; DENV-2 caused DF epidemics in Hainan province in 1985, in Guangxi province in 1988, in Guangdong province in 1993,1998,2001,2013 and 2014 and in Fujian province in 1999. DENV-3 has rarely caused epidemics in mainland China since 1982, and DENV-4 has always been sporadic and has consisted with other serotypes.The E gene of DENV has also been widely used in molecular and evolution analyses in mainland China. However, there is a lack of research evaluaing whether the individual genes, including E gene, are suitable for genotyping the dengue viruses, as well as few analyses of their evolution and selection pressures.Therefore, the characteristics of DENV epidemics in mainland China need to be described according to spatial and temporal analyses.Patients infected with DENV have a wide spectrum of clinical manifestation, ranging from silent infections with no symptoms to a mild flu-like syndrome, dengue fever, severe dengue disease (SD) or deaths. The World Health Organization (WHO) 2009 guidelines identify three diagnostic tests as gold standards for dengue diagnosis: viral isolation and identification, nucleotide detection, and serological tests for IgM or IgG seroconversion. However, these have limitations, such as requests for acute infection (0=5 days post-onset) samples, the time required for viral isolation and identification (more than 1 week), the possibility of false-positive or false-negative final results, and the need for further serum samples to confirm serological tests. An affordable, time-saving, and convenient diagnostic test for confirming dengue infection is thus urgently needed.For the NS1 protein has been detected by ELISA, two forms of NS1 existed and are conservation. The first form is membrance and the other is soluble. It is reported that NS1 protein could be detected in serum of primariy and second infection patients in actue stage (0-6 days after illness). And, it could be found from 9 to 18 days after illness onset. Thus, the detection of NS1 may offer a larger window of opportunity for dengue diagnosis. Recently, two methods (ELISA and Immunochromatography) are for detecting NS1 protein of DENV. According to these methods, the kits of NS1-based detection are used to confirm dengue infection. In lots of clinical trials, these methods could be used to diagnose dengue infection. Thus, we need to perform a meta-analysis to evaluate these methods for confirm the dengue.In 2009, WHO revised the classification system for dengue, defining two major entities-dengue and severe dengue. This new classification also encompasses a set of’warning signs’ intended to help clinicians identify patients likely to develop complications during the critical phase of the illness. The warning sings could help clinicians to improve case management and to facilitate appropriate use of limited resources. During the early stage of dengue, it can be confirmed by detection of the nucleotide or the specific antigien. However, the clinical manifinastions for the severe dengue occurred in the late stage. The nucleotide or the specific antigien was not detected in the blood. Also, no specifical laboratory indicators were existed. But, the deaths rates could be less than 1% for severe dengue, if these patients achieved good clinical support and care. Thus, researchers have focused on a number of risk factors for more severe dengue disease. Dengue was also clarisified as two kinds:dengue with waring sings, such as abnimial pain, bleeding and hepatomegaly and dengue without waring sings. As patients with dengue with waring sings might inditicate more severe and associated with deaths, the cinical manifestions were the early indicators. The frequencies of symptoms/signs of vomiting/nausea, abdominal pain, skin rashes, and bleeding were also found to be correlated with severe dengue, he published studies were not able to conclude that these symptoms/signs are associated with severe dengue. Clinical bleeding manifestations are highly variable, such as skin petechiae, gum and gastrointestinal bleeding etc. The tourniquet test, one of the original diagnostic criteria for SD, has been shown to differentiate poorly between DF and SD. Based on the controversies were existed in the published studies, we conducted the meta-analysis to identify which clinical symptoms/signs areassociated with SD and to help find better methods to predict the development of SD in patients with DF.A number of blood parameters have been considered as potential predictors, most commonly the platelet count. However, the association between thrombocytopenia and haemorrhage is weak, with lower platelet counts correlating more closely with the severity of vascular leakage in one study. Hepatic involvement is also common in dengue, and liver enzymes are frequently elevated in infections of any kinds of severity. More marked derangements are usually associated with more severe disease profiles. Hypoproteinaemia is also recognized that it correlates with the severity of leakage. The meta-analysis coagulators showed a positive association between DSS and prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT). These results were derived in part from a dataset with dengue across Asia and Latin America. Howevere, it was lack of the data from mainland China. In 2014, an outbreak of DF has been in Guangdong and leads to 44 894 infections and 6 deaths. We analyzed the clinical manifestions, signs and blood parameters in dengue patients to predict or diagnose severe dengue.Part I:Spatiotemporal characterizations of dengue virus in mainland ChinaMETHODS:The complete sequences of dengue viruses and four standard strains (DENV-1: Hawaii, DENV-2:New guinea-C, DENV-3:H-87, DENV-4:H241) epidemic in mainland China from 1978 to 2011 were downloaded from GenBank (http://www.ncbi.nlm.nih.gov/genbank/). Phylogenetic analysis and overall evolutionary distance of DENV of DENV-1 and DENV-2 were performed on a gene-by-gene using the Mega 5.05 software.The number of amino acid changes was analyzed using the SPSS software package (version 13.0). The continuous variables were summarized as mean± standard deviation, and comparisons were performed with One-Way ANOVA and Student-t Test. When a significant difference was observed in the equality of variances (p<0.1), the Kruskal-Wallis test was used. A P value<0.05 was considered statistically significantly. All P values were two-sides.RESULTS:1. There were 19 DENV-1,11 DENV-2,6 DENV-3 and 4 DENV-4 viruses. The results of analyzing DENV-1 and DENV-2 showed that none of the phylogenetic trees created from each individual gene were similar to the trees created using the complete genome and the evolutionary distances were variable with each individual gene.2. There were four groups based on different dengue serotype. The AA mutations were 85.211±13.252,72.727±21.448,59.167±22.6 and 82±18.129 for DENV-1, DENV-2, DENV-3 and DENV-4 respectively. The significantly difference was existed in the four groups (F=5.661,p=0.003). The results of comparions among groups showed that significantly amino acide variations between DENV-1, DENV-2 and DENV-3 (p=0.002,0.003). It indicated that the amino acide variations frequency of DENV-1 was highest in mainland China.3. The four groups based on the serotypes (DENV-1 and DENV-2) and the isolation year. The AA mutations of ORF were 82±6.7 in the 5 DENV-1 strains from 1991 to 2000; The AA mutations of ORF were 89.2±14.2 in the 12 DENV-1 strains from 2001 to 2010. Also, The AA mutations of ORF were 74.8±7.8 in the 4 DENV-2 strains from 1991 to 2000; The AA mutations of ORF were 70.0±5.7 in the 4 DENV-2 strains from 2001 to 2010. The significant difference was existed in these four groups (F=3.671, p=0.029). Futher analyzing result showed that the AA changes in the E (Z =2.961, p=0.003) and NS1 (t=4.89, p<0.001) genes were significantly different between the DENV-1 and DENV-2 isolates from 2001-2010.4. According to the alignment of these DENV-1 and DENV-2 isolates, the N290D, L402F and A473T mutations in the E gene and the R101K, G105R, D340E and L349M mutations in the NS1 gene region of the DENV-1 isolates may be significant, while these AAs in the DENV-2 isolates have not been changed.5. The two groups based on the geography of dengue epidemic (Guangzhou and Non-Guangzhou). For the 14 Guangzhou strains of DENV-1, The AA mutations of NS3 were 12.1±2.0, while for the 5 Non-Guangzhou statins, they were 9.8±1.8. These two groups were siginifcantly difference (t=2.3, p=0.034). For the 4 Guangzhou strains of DENV-2, The AA mutations of NS3 were 12.8±1.5, while for the 7 Non-Guangzhou statins, they were 8.4±4.5. These two groups were siginifcantly difference (Z=2.2, p=0.042).Part Ⅱ:NSl-based tests with diagnostic utility for confirming dengue infectionMETHODS:We have devised the criteria for research inclusion and exclusion. The confirmed for dengue infection was needed laboratory detection, as one of the three methods (viral isolation and identification, nucleotide detection, and serological tests for IgM or IgG seroconversion) and the research also have the information for the results of detecting NS1 protein. The sample of included study was at least 50. Search database as NCBI PubMed, ISI Web of Science, Google Scholar and the Chinese National Knowledge Infrastructure databases (CNKI) were searched untilto October 2012. Key words for searching were dengue, NS1, non-structure 1, diagnosis. The included studies were retrieved and the quality of study was also evaluated. This meta-analysis was conducted with STATA 11.0 software.RESULTS:Totals of 18 observed studies which contained 12 313 patients were included in this analysis. For the single NS1-based tests-ELISA (Panbio Dengue Early ELISA Kit, Dengue NS1 Ag ELISA Kit, and Platelia Dengue NS1 Ag-ELISA Kit) and immunochromatography (Dengue NS1 Ag STRIP Kit and SD BIOLINE Dengue Duo Strip Kit)-the summarized sensitivities, specificities and diagnosis odds ratio (DOR) were 67%(95% confidence interval (CI) 59-74%) and 99%(95% CI 97-99%),71% (95% CI 61-79%) and 99%(95% CI 98-100%), and 148 (95% CI 51-429) and 328 (95% CI 103-1046), respectively. The hierarchical summary receiver operating characteristics (HSROCs) were 0.92 and 0.96, respectively. For NS1 combined with an anti-dengue-specific IgM test, the summarized sensitivity, specificity, and HSROC were 83%(95% CI 68-92%),86%(95% CI 79-91%), and 0.91 (95% CI 0.89-0.93), respectively. The accuracy for serotypes was 50.0-90.9% for DENV-1,38.5-85.7% for DENV-2,46.7-91.3% for DENV-3, and 21.7-87.0% for DENV-4.Part III:Predictive Symptoms and Signs of Severe Dengue Disease for Patients with Dengue FeverMETHODS:We have devised the criteria for research inclusion and exclusion. The criteria of dengue were WHO criteria. And we have not limited the type of study, such as obsearved studies. The included studies must have two groups with dengue and severe dengue (dengue haemorrhagic fever or dengue shock syndrome) and be retrived the information of clinical manifestions and signs. When two or more publications reported the same study, we chose the most recent one. Reports providing inadequate information were excluded. Search database as NCBI PubMed, Armed Forces Pest Management Board Literature Retrieval System and Google Scholar were searched from January 2000 to August 2012. Key words for searching were dengue, DF, dengue haemorrhagic fever, DHF, dengue shock syndrome, DSS and clinical. The included studies were retrieved and the quality of study was also evaluated. This meta-analysis was conducted with STATA 11.0 software.RESULTS:Totals of 16 observed studies were included in this analysis. There were 11 factors which were vomiting/nausea were included in 13 studies, abdominal pain in 13, skin rashes in 10, bleeding (hematemesis, melena, gum bleeding, and epistaxis) in 13, headache in 13, lethargy in 6, retroorbital pain in 9, diarrhea in 7, hepatomegaly in 8, and tourniquet test in 4 studies. Among the 11 factors studied, five symptoms demonstrated an increased risk for SDD, including bleeding [OR:13.617; 95% confidence interval (CI):3.281-56.508], vomiting/nausea (OR:1.692; 95% CI: 1.256-2.280), abdominal pain (OR:2.278; 95% CI:1.631-3.182), skin rashes (OR: 2.031; 95% CI:1.269-3.250), and hepatomegaly (OR:4.751; 95% CI:1.769-12.570). Among the four bleeding-related symptoms including hematemesis, melena, gum bleeding, and epistaxis, only hematemesis (OR:6.174; 95% CI:2.66-14.334; P< 0.001) and melena (OR:10.351; 95% CI:3.065-34.956; P< 0.001) were significantly associated with severe dengue. No significantly associations with severe dengue were found for gender, lethargy, retroorbital pain, diarrhea, or tourniquet test, whereas headache appeared protective (OR:0.555; 95% CI:0.455-0.676).Part Ⅳ:Analysis of dengue outbreak in Guangdong province 2014METHODS:We retrospectively analyzed the dengue patients with hospitalizing in Nanfang Hospital, Southern Medical Universtiy and the 8th People’s Hospital in Guangzhou, Guangdong province from June to October 2014. The criteria of diagnosing dengue infection were according to dengue guideline published by the WHO in 2009.The SPSS software (version 13.0) was used to analyze the data. If the continuous variables were suitable for parameters test, the variables were summarized as mean± stabdard deviation (SD), and comparisons were performed with the two-sample test (Students-t test); if the continuous variables were not suitable for parameters test, the variables were summarized as media (Minumum and Maxiumum), the comparisons were performed with Kruskal-Wallis test. Also, the Receiver Operating Characteristic Curve (ROC) were graphed by MedCalc software and the areas under the ROC (AUC) were analyzed. A P value <0.05 was considered statistically significantly. All P values were two-sides.RESULTS:A total of 212 hospitalized patients with dengue infection were included. The mean age was 40.6±16.0. The male and female patients were 102 and 110. According to the servity of dengue, two groups, dengue and severe dengue, were existed. Totals of 174 were in dengue and 38 in severe dengue. It was significantly difference existed in these two groups in the gender (p=0.003). This reulst showed that females were more likely to progress into severe dengue. Also, in the clinical manifesntions with these two groups, the significantly differences of conscious disturbance, lethargy, jaundice, pleural effusion, ascites, hematocrit rise more than 20% together with a concurrent rapid drop in platelets and vaginal bleeding were existed. All the p values were less than 0.05. The blood parameters results showed that ALT, AST, WBC count, APTT and PT in severe dengue were significantly higher than the dengue, while the ALB and PLT count in severe dengue were significantly lower than dengue. As the AUC was between 0.5-0.7, AST and ALB has lower value for diagnosing and predicting severe dengue. Also, ALT, WBC count and APTT had the lower values as the 95% CI contains 0.5. PLT and PT had some value in diagnosing severe dengue in mainland China. Thes AUCs were 0.775 and 0.791.CONCLUSIONS:1. According to this study, the complete sequence of DENV-1, as well as the complete genome or ORF sequence of DENV-2, is suitable or use in analyzing viral evolution and selection pressure, whereas the E genes are not. Additionally, the mutations in the E and NS1 regions may have had effects on the DENV-1 epidemics since 2001, and mutations in the NS3 region might affect the DENV-1 and DENV-2 epidemics in different regions.2. Our analysis suggests that the single NS1-based capture method could be used as a good dengue diagnosis method with a high summarized specificity, and if combined with an IgM test with a high sensitivity, it could be used as a screening method. The NS1-based capture tests can be applied to distinguish DENV-1 and DENV-3 from other serotypes. Moreover, the Dengue NS1 Ag STRIP Kit may be the best kit for confirming and serotyping dengue infection.3. Dengue fever patients with vomiting/nausea, abdominal pain, skin rashes, bleeding (hematemesis/melena), and hepatomegaly were more likely to develop severe dengue, while patients with headache had a lower risk of progression into severe dengue. Other factors such as gender, lethargy, retroorbital pain, diarrhea, and positive tourniquet test are not associated with severe dengue.4. In mainland China, females are more likely to progressing into severe dengue. The factors associated with severe dengue are conscious disturbance, lethargy, jaundice, pleural effusion, ascites, hematocrit rise C20% together with a concurrent rapid drop in platelets and vaginal bleeding. There is some valule of ALT, AST, ALB, WBC, PLT, APTT and PT for predicting and diagnosing severe dengue. However, these need futher research to confirm the conclusions.