AIM:To investigate the effects of AT-2-inactivated HIV-1 particles on human CD4+T cell activation and cytokine secretion in whole blood (WB) in vitro. METHODS: HIV-1ⅢB particles were inactivated by AT-2 chemical and the concentration of p24 antigen was determined by p24 ELISA. AT-2-inactivated HIV-1ⅢB particles were added to human WB culture system in serial concentrations to stimulate the cells. PHA was used as positive control. After 24 h, all the cultural supernatants were harvested and the concentrations of Th1 (IL-2, IFN-? and TNF-?) and Th2 (IL-4, IL-6 and IL-10) cytokines released to the supernatants were detected by cytometric bead array (CBA). The percentage of CD69 expression on CD4+T cells from WB was detected by immuno-fluorescence staining plus flow cytometry. RESULTS: The concentration of p24 antigen in the AT-2-inactivated specimen was 85.5 ?g/L. 24 h later, the percentage of CD69 expression on CD4+T cells from control group was (1.62?0.63) %, whereas it was (38.82?6.00)%, (3.83?1.07)%, (5.94?0.85)% and (9.30?1.22)% in PHA group, HIV-1 (1/500) group, HIV-1 (1/50) group and HIV-1 (1/5) group, respectively. Cytokines secreted by WB in control group were mainly TNF-? and IL-6. However, all the six cytokines tested were strikingly increased in PHA group, as well as in HIV-1ⅢB groups. CONCLUSION: AT-2-inactivated HIV-1ⅢB particles activate CD4+T cells from WB, and up-regulate both Th1 and Th2 cytokine secretion in WB. Besides the effects of viral proteins, other mechanisms may be proposed that HIV-1 particles act as antigen presenting cell (APC) because many host-derived immune molecules are incorporated into HIV-1 envelop when it is released from infected cells by budding, and exert immune modulation.

Objective To investigate the dynamic changes of dengue viral loads and IgM antibody in patients with dengue fever ( DF) and to analyze their relationships with disease severity. Methods A to-tal of 1 508 serum samples were collected from 1 140 hospitalized patients including 1 050 mild cases and 90 severe cases within 10 days after the onset of DF in Guangzhou in 2014. The viral loads were determined by using fluorescence quantitative RT-PCR. ELISA was performed to measure the dengue virus ( DENV)-spe-cific IgM antibody. Results In general, the DENV viral loads in patients declined gradually from 108 copies/ml on day 1 to 103 copies/ml on day 10 after the onset of DF. The viral loads in severe cases were significantly higher than those in mild cases on days 5 to 7 (P<0. 05). The positive rates of DENV RNA in serum samples also decreased with the disease progression from 100% on day 1 to 40% on day 10. Com-pared with the mild cases, the patients with severe DF showed higher positive rates of DENV RNA on day 6 and day 8 (P<0. 05). The DENV-specific IgM antibody could be detected on day 2 and the secretion of IgM antibody increased gradually with the disease progression. The levels of IgM antibody in mild cases were sig-nificantly higher than those in severe cases (P<0. 001). The positive rates of IgM antibody in patients in-creased form 8% on day 2 to 95% on day 6. Higher positive rates of IgM antibody were detected in mild ca-ses on days 5 and 6 as compared with those in patients with severe DF (P<0. 05). Conclusion High viral load and low level of IgM antibody during the fastigium of DF were closely associated with the disease severity.

Objective To observe the clinical effect ofJiedu-Huayu granule for the Dengue Fever (DF).Methods The patients with DF were randomly divided into the treatment group and control group. Both groups received the conventional treatment, and the treatment group addedJiedu-Huayu granule. The clinical effects were detected.Results At the baseline, the ALT and AST in the treatment were significantly higher than those in the control group (t=2.830,P=0.014;t=2.109,P=0.044). But after treatment, there was no difference between groups. The ALT in the treatment group showed a gradual change and AST recovery time showed short. Compared with control group, the concentration of sVCAM-1 in the treatment group was higher at baseline and the third day (t=2.780,P=0.018;t=2.130,P=0.460), but the concentration of MCP-1 desceased significantly on the 6th day (t=-2.164,P=0.042).Conclusions Compared with the western medicine, the treatment of western medicine andJiedu-Huayu granule could alleviate liver injure, shorten the recovery time, and reduce the concentration of sVCAM-1 and MCP-1.

Objective: To analyze the clinical features of death cases of dengue fever and the causes of their deaths. Methods: The clinical data and death reports of nine death cases of dengue fever in Guangdong Province from June 23, 2014 to September 10, 2019 were retrospectively analyzed. All of nine cases were positive for serum dengue virus RNA as confirmed by reverse transcription polymerase chain reaction, and some of the virus strains were serotyped. Results: The median age of the nine patients was 57.5 (range: 18-80) years. Among them, six patients were females; eight patients were local cases and one was imported case; all of nine cases occurred in September and October. The median time from onset to visit hospital and diagnosis was three and four days, respectively. Three of the nine patients had underlying diseases. All of nine cases had fever, including three with double-peak fever. Eight of the cases had three or more severe dengue fever warning indexes before admission. Three cases had severe bleeding upon admission, one case had shock, and six cases had organ failure. Three cases underwent invasive examination, including arterial puncture catheterization, endoscopic titanium clip hemostasis, and percutaneous transluminal coronary angiography. Two cases developed malignant arrhythmia and one had massive hemorrhage of subcutaneous soft tissue after operation. The death cases included four cases of cardiogenic shock and fatal arrhythmia, three cases of encephalitis and encephalopathy, one case of refractory shock, and one case of acute liver failure. Among the six cases that underwent serological typing, four were identified as dengue virus type 1 (DEN-1) and two were DEN-2. Conclusions Severe organ failure is the major cause of dengue fever-related death, especially fulminant myocarditis, and DEN-1 is most common. Early diagnosis and treatment, and avoidance of invasive procedures can effectively reduce the mortality rate of the severe dengue fever patients.

Objective:To investigate the serotype distribution and phylogenetic analysis of virus complete genome from indigenous dengue patients in Guangzhou in 2019 and provide evidence for the development of prevention and treatment strategies.Methods:Dengue virus serotypes of indigenous dengue cases in 2019 were detected using serotype specific fluorescent PCR kits. Complete genome in the culture was performed on Illumina platform. Phylogenetic analysis was conducted on complete genomes extracted from ViPR and the isolates from this study with MEGA7.0 software.Results:In 2019, three prevalent serotypes of dengue virus were found in Guangzhou, among which serotype 1 accounted for 80.35%, serotype 2 accounted for 12.97% and serotype 3 accounted for 6.68%. There were no significant differences in gender, age and severity among three serotypes. Phylogenetic analysis of virus complete genome showed that serotype 1 belonged to genotypeⅠand had two origins, which was close to the Cambodian strain; serotype 2 belonged to genotype cosmopolitan, which was close to the epidemic strain in Southeast Asia; serotype 3 belonged to genotypeⅢ, which was in the same branch as the Indian strain.Conclusions:The dengue epidemic was caused by dengue virus serotypes 1, 2 and 3 in Guangzhou in 2019. Each serotype belonged to a genotype.

Objective:To compare the levels of cytokines in patients with dengue fever (DF) and severe dengue (SD) at different time points during the course of disease, to seek the cytokines that can be used as early warning signs of SD, and to explore the relationship between the immune response and the pathogenesis of SD.Methods:Plasma samples at different time points were collected from 60 hospitalized patients including 48 mild cases and 12 severe cases in Guangzhou Eighth People′s Hospital during June to December, 2014. Levels of 19 cytokines including TNF-α, IL-6, IL-8, IL-10, IL-15, IL-17A, IFN-α2, IP-10, MCP-1, RANTES, GRO-α, PDGF-AA, PDGF-AB/BB, MIF, VEGF, sVCAM-1, sICAM-1, sFas and sFasL were determined by a multiplex Luminex system.The viral loads were determined by using fluorescence quantitative RT-PCR and the correlation between viral loads and cytokine level were analyzed.Results:The levels of TNF-α, IL-6, IL-8, IL-10, IL-15, IFN-α2, IP-10, MCP-1 and sVCAM-1 increased in dengue patients, while RANTES, GRO-α and PDGF decreased.The levels of IL-17A, MIF, VEGF, sICAM-1, sFas and sFasL did not change. The levels of TNF-α, IL-6, IFN-α2, IP-10 and sVCAM-1 in SD patients were significantly higher than those in DF patients at the early stage (Day 2-5 after disease onset), and the differences were statistically significant. On day 6-10, the levels of TNF-α, IL-6, IL-8, IL-10, IL-15, PDGF, RANTES, IFN-α2, IP-10 and sVCAM-1 were significantly different between DF and SD patients. The results of correlation analysis showed that the level of IL-15 was moderately correlated with viral load, while other cytokines was only weakly correlated or not.Conclusions:TNF-α, IL-6, IFN-α2, IP-10 and sVCAM-1 can be used as early warning signs of SD. The level of cytokines is related to the severity of dengue fever.

Objective:To analyze the clinical features of patients with severe dengue (SD) in Guangdong Province, and to improve the understanding of the diagnosis and treatment of SD in China.Methods:The clinical data, laboratory examination and etiological test results of 257 SD cases from 29 dengue fever designated hospitals in Guangdong Province from January 1, 2013 to December 31, 2019 were respectively collected. The relevant indicators of the criteria for severe organ involvement were quantified. Logistic regression analysis was performed to analyze the risk factors for the development of multiple organ failure in SD patients.Results:Among the 257 SD patients, age was (64.1±20.1) years old, with 65.4%(168/257) of them ≥60 years old, 142 were male and 115 were female. One hundred and fifty-two (59.1%) patients had underlying conditions, including 115(44.7%) patients with hypertension. The clinical manifestations were mainly fever (98.4%(253/257)), fatigue (70.0%(180/257)), cough or expectoration (44.4%(114/257)), lethargy or irritability (39.3%(101/257)), vomiting (30.4%(78/257)), abdominal pain or tenderness (20.6%(53/257)), hepatomegaly (2.3%(6/257)), bleeding tendency (59.5%(153/257)), and pleural effusion or ascites (43.6%(112/257)). Platelet count levels were decreased in 90.9%(231/254) of the cases, and 97.1%(234/241) of patients had normal or decreased hematocrit. The most common of severe manifestations were severe organ involvement (61.1%(157/257)), followed by severe bleeding (37.0%(95/257)) and severe plasma leakage (30.0%(77/257)). Severe organ involvements were more common in the kidney (27.6%(71/257)) and heart (26.8%(69/257)). Multivariate logistic regression analysis showed that age (odds ratio ( OR)=1.051, 95% confidence interval ( CI) 1.004 to 1.100, P=0.035), hypertension ( OR=5.224, 95% CI 1.272 to 21.462, P=0.022), elevated aspartate aminotransferase (AST) level ( OR=1.002, 95% CI 1.001 to 1.003, P=0.001), blood urea nitrogen (BUN) ( OR=1.050, 95% CI 1.005 to 1.098, P=0.030), and international normalized ratio (INR) ( OR=4.604, 95% CI 1.601 to 13.238, P=0.005) were risk factors for the development of multiple organ failure in SD patients. The detection results of serum samples form 113 SD patients in acute phase showed that dengue virus (DENV)-1 accounted for 89.4%(101/113), DENV-2 accounted for 9.7%(11/113), and DENV-3 accounted for 0.9% (1/113). Conclusions:Elderly and those with co-existing conditions such as hypertension in SD patients in Guangdong Province are more common. Severe organ involvement such as kidney and heart is the main cause of SD. DENV-1 infection is predominant. Significant elevated levels of AST, BUN and INR may be related to a poor prognosis.

Objective: To investigate the sensitivity and specificity of commercial nonstructural protein 1 (NS1) testing kits for Dengue fever diagnose, and provide the evidence for diagnostic criteria revision. Methods: 300 PCR or virus isolation positive blood samples for dengue virus were collected from sentinel hospitals for dengue surveillance in Guangzhou, Dongguang and Zhongshang from May 2015 to Nov. 2016. At the same time, 308 PCR negative samples for Dengue virus were collected as control group. The information of the sample was collected using questionnaires. These samples were tested using imported and domestic ELISA and the colloidal gold-labeled kits that were widely used for detecting dengue NS1. Sensitivity, specificity and coincidence were calculated and analyzed, and Z hongshan's result was regarded as the reslut of the third part. Results: The positive group includes 133 males and 167 females, average ages are 47.2±13.3, 179, 110 and 11 of them is Dengue Ⅰ, Ⅱ and Ⅲ respectively. The negative group includes 154 males and 154 females, average ages are (40.1±11.6) years old. The sensitivity of domestic ELISA Kits (94.5%) is less than imported (99.5%), and the result has statistical significance (χ²=8.59, P=0.030), the specificity is 99.7% and 97.7% respectively; The sensitivity of imported and domestic the colloidal gold-labeled Kits is 97.5% and 96.5% respectively, both of specificities are 100%. The sensitivity and specificity of Dengue Ⅰ for NS1 test are more than 97.0%. The sensitivity of domestic ELISA and gold-labeled Kits is 90.0% and 95.0%, and the specificity is 96.8% and 100% respectively for Dengue Ⅱ test. The sensitivity of imported ELISA and gold-labeled Kits is 100% and 98.0%, and the specificity is 99.4% and 100% respectively for Dengue Ⅱ test. The result of the third party show the sensitivity and specificity of domestic ELISA and gold-labeled Kits are 90.0% and 98.0%, the differences has statistical significance (χ²=5.67, P=0.020). Conclusion NS1 testing can be used as early dengue fever diagnose for higher sensitivity and specificity.

Objective: To investigate the roles of pattern recognition receptors (PRRs) including Toll-like receptors (TLRs), retinoic acid-inducible geneⅠ-like receptors (RLRs) and nucleotide binding oligomerization domain-like receptors (NLRs) in the pathogenesis of dengue fever (DF) and the induction of immune responses to dengue virus. Methods: Blood samples were collected from patients with DF at three different time points to isolate peripheral blood mononuclear cells (PBMCs) by density gradient centrifugation. Then PBMCs were used to extract ribonucleic acid (RNA). Expression of genes was detected by polymerase chain reaction (PCR) array. Results: Expression of genes relating to PRRs signaling pathways in DF patients increased significantly in the early stage of the disease as compared with those in healthy controls, but decreased gradually during the recovery period. Expression of genes encoding TLR7 and TLR8 was enhanced at the early stage of DF. No significant changes in the expression of TLR3 and TLR9 genes were observed during the course of the disease. The genes encoding RIG-Ⅰ, MDA5 and LGP2 of RLRs family as well as NOD2 and OAS2 of NLRs family were all up-regulated. Conclusion Signaling pathways mediated by PRRs including TLR7/8, RIG-Ⅰ, MDA5 and so on play an important role in the pathogenesis of DF and the induction of immune responses to dengue virus.