DC-SIGN and Galectin-3 individually and collaboratively regulate H5N1 and H7N9 avian influenza A virus infection via interaction with viral envelope hemagglutinin protein.

DC-SIGN and Galectin-3 are two different lectins and have been reported to participate in regulation of several virus infections. WHO has pointed that H5N1 and H7N9 avian influenza viruses (AIVs) play continuous threats to global health. AIV hemagglutinin (HA) protein-a highly glycosylated protein-mediates influenza infection and was proposed to have DC-SIGN and Gal3 interactive domains. This study aims to address the individual and collaborative roles of DC-SIGN and Gal3 toward AIVs infection. Firstly, A549 cells with DC-SIGN expression or Gal3-knockdown, via lentiviral vector-mediated CD209 gene expression or LGALS-3 gene knockdown, respectively were generated. Quantitative reverse transcription PCR (qRT-PCR) results indicated that DC-SIGN expression and Gal3 knockdown in A549 cells significantly promoted and ameliorated HA or NP gene expression, respectively after H5N1 and H7N9-reverse genetics (RG) virus postinfections (P < 0.05). Similar results observed in immunoblotting, indicating that DC-SIGN expression significantly facilitated H5N1-RG and H7N9-RG infections (P < 0.05), whereas Gal3 knockdown significantly reduced both viral infections (P < 0.05). Furthermore, we found that DC-SIGN and Gal3 co-expression significantly enhanced infectivity of both H5N1-RG and H7N9-RG viruses (P < 0.01) and higher regulatory capabilities by DC-SIGN and Gal3 in H5N1-RG than H7N9-RG were noted. The promoting effect mainly relied on exogenous Gal3 and DC-SIGN directly interacting with the HA protein of H5N1 or H7N9 AIVs, subsequently enhancing virus infection. This study sheds light on two different lectins individually and collaboratively regulating H5N1 and H7N9 AIVs infection and suggests that inhibitors against DC-SIGN and Gal3 interacting with HA could be utilized as alternative antiviral strategies.

Identification of Important N-Linked Glycosylation Sites in the Hemagglutinin Protein and Their Functional Impact on DC-SIGN Mediated Avian Influenza H5N1 Infection.

DC-SIGN, a C-type lectin mainly expressed in dendritic cells (DCs), has been reported to mediate several viral infections. We previously reported that DC-SIGN mediated H5N1 influenza A virus (AIVs) infection, however, the important DC-SIGN interaction with N-glycosylation sites remain unknown. This study aims to identify the optimal DC-SIGN interacting N-glycosylation sites in HA proteins of H5N1-AIVs. Results from NetNGlyc program analyzed the H5 hemagglutinin sequences of isolates during 2004-2020, revealing that seven and two conserved N-glycosylation sites were detected in HA1 and HA2 domain, respectively. A lentivirus pseudotyped A/Vietnam/1203/04 H5N1 envelope (H5N1-PVs) was generated which displayed an abundance of HA5 proteins on the virions via immuno-electron microscope observation. Further, H5N1-PVs or reverse-genetics (H5N1-RG) strains carrying a serial N-glycosylated mutation was generated by site-directed mutagenesis assay. Human recombinant DC-SIGN (rDC-SIGN) coated ELISA showed that H5N1-PVs bound to DC-SIGN, however, mutation on the N27Q, N39Q, and N181Q significantly reduced this binding (p < 0.05). Infectivity and capture assay demonstrated that N27Q and N39Q mutations significantly ameliorated DC-SIGN mediated H5N1 infection. Furthermore, combined mutations (N27Q&N39Q) significantly waned the interaction on either H5N1-PVs or -RG infection in cis and in trans (p < 0.01). This study concludes that N27 and N39 are two essential N-glycosylation contributing to DC-SIGN mediating H5N1 infection.

Amino Acid Deletions in p6Gag Domain of HIV-1 CRF07_BC Ameliorate Galectin-3 Mediated Enhancement in Viral Budding.

HIV-1 CRF07_BC is a recombinant virus with amino acid (a.a.) deletions in p6Gag, which are overlapped with the Alix-binding domain. Galectin-3 (Gal3), a ß-galactose binding lectin, has been reported to interact with Alix and regulate HIV-1 subtype B budding. This study aims to evaluate the role of Gal3 in HIV-1 CRF07_BC infection and the potential effect of a.a. deletions on Gal3-mediated regulation. A total of 38 HIV-1+ injecting drug users (IDUs) were enrolled in the study. Viral characterization and correlation of Gal3 were validated. CRF07_BC containing 7 a.a. deletions and wild-type in the p6Gag (CRF07_BC-7d and -wt) were isolated and infectious clones were generated. Viral growth kinetic and budding assays using Jurkat-CCR5/Jurkat-CCR5-Gal3 cells infected with CRF07_BC were performed. Results indicate that 69.4% (25/38) of the recruited patients were identified as CRF07_BC, and CRF07_BC-7d was predominant. Slow disease progression and significantly higher plasma Gal3 were noted in CRF07_BC patients (p < 0.01). Results revealed that CRF07_BC infection resulted in Gal3 expression, which was induced by Tat. Growth dynamic and budding assays indicated that Gal3 expression in Jurkat-CCR5 cells significantly enhanced CRF07_BC-wt replication and budding (p < 0.05), while the promoting effect was ameliorated in CRF07_BC-7d. Co-immunoprecipitation found that deletions in the p6Gag reduced Gal-3-mediated enhancement of the Alix-Gag interaction.

Dengue overview: An updated systemic review.

Dengue is caused by the dengue virus (DENVs) infection and clinical manifestations include dengue fever (DF), dengue hemorrhagic fever (DHF), or dengue shock syndrome (DSS). Due to a lack of antiviral drugs and effective vaccines, several therapeutic and control strategies have been proposed. A systemic literature review was conducted according to PRISMA guidelines to select proper references to give an overview of DENV infection. Results indicate that understanding the virus characteristics and epidemiology are essential to gain the basic and clinical knowledge as well as dengue disseminated pattern and status. Different factors and mechanisms are thought to be involved in the presentation of DHF and DSS, including antibody-dependent enhancement, immune dysregulation, viral virulence, host genetic susceptibility, and preexisting dengue antibodies. This study suggests that dissecting pathogenesis and risk factors as well as developing different types of therapeutic and control strategies against DENV infection are urgently needed.

Virus Hijacks Host Proteins and Machinery for Assembly and Budding, with HIV-1 as an Example.

Viral assembly and budding are the final steps and key determinants of the virus life cycle and are regulated by virus-host interaction. Several viruses are known to use their late assembly (L) domains to hijack host machinery and cellular adaptors to be used for the requirement of virus replication. The L domains are highly conserved short sequences whose mutation or deletion may lead to the accumulation of immature virions at the plasma membrane. The L domains were firstly identified within retroviral Gag polyprotein and later detected in structural proteins of many other enveloped RNA viruses. Here, we used HIV-1 as an example to describe how the HIV-1 virus hijacks ESCRT membrane fission machinery to facilitate virion assembly and release. We also introduce galectin-3, a chimera type of the galectin family that is up-regulated by HIV-1 during infection and further used to promote HIV-1 assembly and budding via the stabilization of Alix-Gag interaction. It is worth further dissecting the details and finetuning the regulatory mechanism, as well as identifying novel candidates involved in this final step of replication cycle.

The first case of monkeypox virus infection detected in Taiwan: awareness and preparation.

OBJECTIVES: Monkeypox has recently been detected outside African countries. This study aimed to report and analyze the first case of monkeypox virus infection in Taiwan. METHODS: The global epidemiological information was collected from the World Health Organization (WHO) and US Centers for Disease Control and Prevention (CDC). The data from the first confirmed Taiwanese monkeypox case was obtained from Taiwan Centers for Disease Control. Monkeypox diagnosis and prevention strategies were obtained from WHO guidelines on monkeypox. Phylogenetic tree analysis and sequence alignment and comparison were used to identify the phylogeny and single nucleotide polymorphism (SNP) characterization. RESULTS: Epidemiological data indicated that since 2013, monkeypox has caused outbreaks outside African countries through contact with infected animals and international travels. Recently, two confirmed monkeypox cases were reported in Singapore and South Korea. On June 24, 2022, Taiwan CDC reported the first confirmed case of monkeypox virus infection in a 20-year-old man who returned from Germany, from January to June 2022. This is the third confirmed case of an imported monkeypox infection in Asia. Phylogenetic analysis demonstrated that this imported monkeypox virus belonged to the West African clade and is clustered with the 2022 European outbreak monkeypox isolates. Full-length sequence analysis indicates that this virus contains 51 SNPs, and has five variant SNPs compared with the recent outbreak strains. CONCLUSION: This study suggests that active surveillance, enhancing border control, and the development of vaccines and antiviral drugs are urgently required to prevent and control the burden of monkeypox disease.

Boosting the detection performance of severe acute respiratory syndrome coronavirus 2 test through a sensitive optical biosensor with new superior antibody.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) virus emerged in late 2019 leading to the COVID-19 disease pandemic that triggered socioeconomic turmoil worldwide. A precise, prompt, and affordable diagnostic assay is essential for the detection of SARS-CoV-2 as well as its variants. Antibody against SARS-CoV-2 spike (S) protein was reported as a suitable strategy for therapy and diagnosis of COVID-19. We, therefore, developed a quick and precise phase-sensitive surface plasmon resonance (PS-SPR) biosensor integrated with a novel generated anti-S monoclonal antibody (S-mAb). Our results indicated that the newly generated S-mAb could detect the original SARS-CoV-2 strain along with its variants. In addition, a SARS-CoV-2 pseudovirus, which could be processed in BSL-2 facility was generated for evaluation of sensitivity and specificity of the assays including PS-SPR, homemade target-captured ELISA, spike rapid antigen test (SRAT), and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Experimentally, PS-SPR exerted high sensitivity to detect SARS-CoV-2 pseudovirus at 589 copies/ml, with 7-fold and 70-fold increase in sensitivity when compared with the two conventional immunoassays, including homemade target-captured ELISA (4 × 103 copies/ml) and SRAT (4 × 104 copies/ml), using the identical antibody. Moreover, the PS-SPR was applied in the measurement of mimic clinical samples containing the SARS-CoV-2 pseudovirus mixed with nasal mucosa. The detection limit of PS-SPR is calculated to be 1725 copies/ml, which has higher accuracy than homemade target-captured ELISA (4 × 104 copies/ml) and SRAT (4 × 105 copies/ml) and is comparable with qRT-PCR (1250 copies/ml). Finally, the ability of PS-SPR to detect SARS-CoV-2 in real clinical specimens was further demonstrated, and the assay time was less than 10 min. Taken together, our results indicate that this novel S-mAb integrated into PS-SPR biosensor demonstrates high sensitivity and is time-saving in SARS-CoV-2 virus detection. This study suggests that incorporation of a high specific recognizer in SPR biosensor is an alternative strategy that could be applied in developing other emerging or re-emerging pathogenic detection platforms.

Emerging and Re-Emerging Diseases.

Throughout history, infectious diseases have vastly impacted human civilization [...].

A Retrospective Survey among SARS-CoV-1 Infected Healthcare Workers after Three Years Post-Infection.

Healthcare workers (HCWs) are on the frontline fighting several infectious diseases including SARS-CoV-1 and COVID-19. Coronavirus neutralizing antibodies (nAbs) were recently reported to last for a certain period. The factors affecting nAbs' existence remain unclear. Here, we retrospectively analyzed the factors correlating with nAbs' from SARS-CoV-1 long-term convalescence HCWs in Taiwan. One hundred and thirty SARS-CoV-1 convalescent patients were recruited between August 2006 and March 2007. Blood samples were collected to determine the anti-nucleocapsid (N) and anti-spike (S) antibodies' existence status and neutralization ability. Neutralization ability was measured using SARS-CoV-1 pseudotyped viruses. Statistical analysis of factors associated with anti-SARS-CoV-1 antibodies' existence status was determined using SAS software. 46.2% SARS-CoV-1 convalescent patients presented anti-N antibody after three years post-infection. Among sixty participants, ten participants co-presented anti-S antibodies. Eight participants with anti-S antibody displayed neutralization ability to SARS-CoV-1. The gender, age, and disease severity of participants did not affect the anti-N antibody existence status, whereas the anti-S antibody is significantly reduced in participants with old age (>50 years, p = 0.0434) after three years post SARS-CoV-1 infection. This study suggests that age is an important factor correlated with the duration of SARS-CoV-1 protective antibody existence status.

The Antiviral Role of Galectins toward Influenza A Virus Infection-An Alternative Strategy for Influenza Therapy.

Animal lectins are proteins with carbohydrate recognition activity. Galectins, the ß-galactoside binding lectins, are expressed in various cells and have been reported to regulate several immunological and physiological responses. Recently, some galectins have been reported to regulate some viral infections, including influenza A virus (IAV); however, the mechanism is still not fully understood. Thus, we aim to review systemically the roles of galectins in their antiviral functions against IAVs. The PRISMA guidelines were used to select the eligible articles. Results indicated that only Galectin-1, Galectin-3, and Galectin-9 were reported to play a regulatory role in IAV infection. These regulatory effects occur extracellularly, through their carbohydrate recognition domain (CRD) interacting with glycans expressed on the virus surface, as well as endogenously, in a cell-cell interaction manner. The inhibition effects induced by galectins on IAV infection were through blocking virus-host receptors interaction, activation of NLRP-3 inflammasome, augment expression of antiviral genes and related cytokines, as well as stimulation of Tim-3 related signaling to enhance virus-specific T cells and humoral immune response. Combined, this study concludes that currently, only three galectins have reported antiviral capabilities against IAV infection, thereby having the potential to be applied as an alternative anti-influenza therapeutic strategy.

Regulatory roles of galectins on influenza A virus and their potential as a therapeutic strategy.

Galectins, are ß-galactoside binding lectins expressed in numerous cells and are known to regulate various immune responses and cellular physiological functions. Galectins have been reported to participate in the regulation of several viral infections via carbohydrate­dependent/independent manner. Galectins have displayed various regulatory functions on viral infection, however, the detailed mechanism remains unclear. More recently, some members of galectins have been reported to regulate influenza A virus (IAV) infection. In this review, we aim to analyze and summarize current findings regarding the role of galectins in IAV infection and their antiviral potential therapeutic application in the treatment of IAVs. The eligible articles were selected according to the PRISMA guidelines. Results indicate that Galectin-1(Gal-1), Galectin-3(Gal-3) and Galectin-9 (Gal-9) were found as the predominant galectins reported to participate in the regulation of IAVs infection. The inhibitory regulation of IAVs by these galectins occurred mainly through extracellular binding to glycosylated envelope proteins, further blocking the interaction between influenza envelope and sialic acid receptor, interacting with ligands or receptors on immune cells to trigger immunol or cellular response against IAVs, and endogenously interacting cellular components in the cytoplasm to activate inflammasome and autophagy. This study offers information regarding the multiple roles of galectins observed in IAVs infection and suggest that galectins has the potential to be used as therapeutic agents for IAVs.

Targets and strategies for vaccine development against dengue viruses.

Dengue virus (DENV) is a global health threat causing about half of the worldwide population to be at risk of infection, especially the people living in tropical and subtropical area. Although the dengue disease caused by dengue virus (DENV) is asymptomatic and self-limiting in most people with first infection, increased severe dengue symptoms may be observed in people with heterotypic secondary DENV infection. Since there is a lack of specific antiviral medication, the development of dengue vaccines is critical in the prevention and control this disease. Several targets and strategies in the development of dengue vaccine have been demonstrated. Currently, Dengvaxia, a live-attenuated chimeric yellow-fever/tetravalent dengue vaccine (CYD-TDV) developed by Sanofi Pasteur, has been licensed and approved for clinical use in some countries. However, this vaccine has demonstrated low efficacy in children and dengue-naïve individuals and also increases the risk of severe dengue in young vaccinated recipients. Accordingly, many novel strategies for the dengue vaccine are under investigation and development. Here, we conducted a systemic literature review according to PRISMA guidelines to give a concise overview of various aspects of the vaccine development process against DENVs, mainly targeting five potential strategies including live attenuated vaccine, inactivated virus vaccine, recombinant subunit vaccine, viral-vector vaccine, and DNA vaccine. This study offers the comprehensive view of updated information and current progression of immunogen selection as well as strategies of vaccine development against DENVs.

Dengue hemorrhagic fever - A systemic literature review of current perspectives on pathogenesis, prevention and control.

BACKGROUND: Dengue is an arboviral disease caused by dengue virus. Symptomatic dengue infection causes a wide range of clinical manifestations, from mild dengue fever (DF) to potentially fatal disease, such as dengue hemorrhagic fever (DHF) or dengue shock syndrome (DSS). We conducted a literature review to analyze the risks of DHF and current perspectives for DHF prevention and control. METHODS: According to the PRISMA guidelines, the references were selected from PubMed, Web of Science and Google Scholar database using search strings containing a combination of terms that included dengue hemorrhagic fever, pathogenesis, prevention and control. Quality of references were evaluated by independent reviewers. RESULTS: DHF was first reported in the Philippines in 1953 and further transmitted to the countries in the region of South-East Asia and Western Pacific. Plasma leakages is the main pathophysiological hallmark that distinguishes DHF from DF. Severe plasma leakage can result in hypovolemic shock. Various factors are thought to impact disease presentation and severity. Virus virulence, preexisting dengue antibodies, immune dysregulation, lipid change and host genetic susceptibility are factors reported to be correlated with the development of DHF. However, the exact reasons and mechanisms that triggers DHF remains controversial. Currently, no specific drugs and licensed vaccines are available to treat dengue disease in any of its clinical presentations. CONCLUSION: This study concludes that antibody-dependent enhancement, cytokine dysregulation and variation of lipid profiles are correlated with DHF occurrence. Prompt diagnosis, appropriate treatment, active and continuous surveillance of cases and vectors are the essential determinants for dengue prevention and control.

The role of galectins in virus infection - A systemic literature review.

BACKGROUND: Galectins are ß-Galactose binding lectins expressed in numerous cells and play multiple roles in various physiological and cellular functions. However, few information is available regarding the role of galectins in virus infections. Here, we conducted a systemic literature review to analyze the role of galectins in human virus infection. METHODS: This study uses a systematic method to identify and select eligible articles according to the PRISMA guidelines. References were selected from PubMed, Web of Science and Google Scholar database covering publication dated from August 1995 to December 2018. RESULTS: Results indicate that galectins play multiple roles in regulation of virus infections. Galectin-1 (Gal-1), galectin-3 (Gal-3), galectin-8 (Gal-8), and galectin-9 (Gal-9) were found as the most predominant galectins reported to participate in virus infection. The regulatory function of galectins occurs by extracellularly binding to viral glycosylated envelope proteins, interacting with ligands or receptors on immune cells, or acting intracellularly with viral or cellular components in the cytoplasm. Several galectins express either positive or negative regulatory role, while some had dual regulatory capabilities on virus propagation based on the conditions and their localization. However, limited information about the endogenous function of galectins were found. Therefore, the endogenous effects of galectins in host-virus regulation remains valuable to investigate. CONCLUSIONS: This study offers information regarding the various roles galectins shown in viral infection and suggest that galectins can potentially be used as viral therapeutic targets or antagonists.

Importation of SARS-CoV-2 infection leads to major COVID-19 epidemic in Taiwan.

OBJECTIVE: COVID-19 has recently become a pandemic affecting many countries worldwide. This study aims to evaluate the current status of COVID-19 in Taiwan and analyze the source of infection. METHODS: National data regarding SARS-CoV-2 infection were obtained from Taiwan. CDC at the end of April 2020. These data were subjected to analysis of the current status and correlation between indigenous and imported COVID-19 cases. A phylogenetic tree was created to analyze the phylogeny of Taiwanese SARS-CoV-2 isolates. RESULTS: The first case of SARS-CoV-2 infection in Taiwan was detected on January 21, 2020. Epidemiological data indicate that by April 30, there were a total of 429 COVID-19 confirmed cases with the death rate of 1.3%. Most cases were identified as imported (79.9%; 343/429), with the majority originating from the United States of America (22.1%) and the United Kingdom (17.6%). Results from phylogenetic tree analyses indicate that the Taiwanese SARS-CoV-2 isolates were clustered with the SARS-CoV-2 isolates from other countries (bootstrap value 98%) and sub-clustered with bat SARS-like coronaviruses (bootstrap value 99%). CONCLUSION: This study suggests that the importation of SARS-CoV-2 infection was the primary risk-factor resulting in the COVID-19 epidemic in Taiwan.

The Examination of Viral Characteristics of HIV-1 CRF07_BC and Its Potential Interaction with Extracellular Galectin-3.

HIV-1 CRF07_BC is a B' and C subtype recombinant emerging virus and many of its viral characteristics remain unclear. Galectin-3 (Gal3) is a ß-galactose binding lectin that has been reported as a pattern recognition receptor (PRR) and is known to mediate adhesion between cells and microbes. This study aims to examine the viral characteristics of HIV-1 CRF07_BC virus and the role of extracellular galectin-3 in HIV-1 CRF07_BC infection. A total of 28 HIV-1+ injecting drug users (IDUs) were recruited and 24 (85.7%) were identified as HIV-1 CRF07_BC. Results indicate that significant higher serum galectin-3 was measured in CRF07_BC infected patients and CRF07_BC infection triggered significant galectin-3 expression (p < 0.01). Viral characteristics demonstrate that CRF07_BC virions display a higher level of envelope gp120 spikes. The virus infectivity assay demonstrated that co-treatment with galectin-3 significantly promoted CRF07_BC attachment and internalization (p < 0.01). A co-immunoprecipitation assay showed that pulldown galectin-3 co-precipitated both CD4 and gp120 proteins. Results from an enzyme-linked immunosorbent assay (ELISA) indicate that the galectin-3 promoting effect occurs through enhancement of the interaction between gp120 and CD4. This study suggests that CRF07_BC was predominant in HIV-1+ IDUs and CRF07_BC utilized extracellular galectin-3 to enhance its infectivity via stabilization of the gp120-CD4 interaction.

An epidemiological survey of the current status of Zika and the immune interaction between dengue and Zika infection in Southern Taiwan.

OBJECTIVES: This study was performed to examine the current status of Zika and the effects of pre-existing dengue immunity on Zika virus (ZIKV) infection in Southern Taiwan. METHODS: A phylogenetic tree was used to analyze the phylogeny of detected ZIKVs. Paired sera from dengue patients were collected for the determination of dengue and Zika infection. Plaque reduction neutralization tests (PRNT) and quantitative reverse transcription PCR (qRT-PCR) were used to determine the titers of neutralizing antibodies and viruses, respectively. An antibody-dependent enhancement (ADE) assay was used to evaluate the effect of anti-dengue antibodies on ZIKV infection. RESULTS: Epidemiological data indicated the continuous importation of ZIKV infection from neighboring Zika epidemic countries into Taiwan. A total of 78 dengue patients were enrolled and 21 paired serum samples were obtained. PRNT90 results for the 21 samples identified eight cases of primary dengue infection and 13 cases of secondary dengue infection; two samples were positive for ZIKV (MR766). Results from the ADE assay indicated that convalescent sera from primary and secondary dengue infection patients displayed significant ADE of the ZIKV infection when compared to healthy controls (p < 0.05). CONCLUSIONS: This study suggests that pre-existing dengue immunity facilitates ZIKV infection and that the continuous importation of ZIKV infection may pose a threat to indigenous Zika emergence in Southern Taiwan.

A clinical and epidemiological survey of the largest dengue outbreak in Southern Taiwan in 2015.

OBJECTIVES: This study examined the epidemiological, clinical, and immunological characteristics of the 2015 dengue outbreak in Taiwan. METHODS: Clinical data were collected from dengue fever (DF) and dengue hemorrhagic fever (DHF) patients. A phylogenetic tree was used to analyze the source of the outbreak strain. Paired plasma samples from DF/DHF patients were used for antibody-dependent enhancement (ADE) assay and cytokine multiplex biometric immunoassay to validate the immunological mechanism. RESULTS: This outbreak mainly occurred in two of the southern cities of Taiwan: Tainan (n=22 777; 52%) and Kaohsiung (n=19 784; 45%). A high DHF death rate was noted (34.6%). The case (DHF) and control (DF) study indicated that older age (>60 years), type II diabetes, and hypertension were risk factors correlated with the development of DHF (p< 0.0001). The phylogenetic tree results suggested that the outbreak-associated strain was dengue virus serotype 2 and cosmopolitan genotype, forming a stable cluster with the isolates from Thailand and Indonesia (bootstrap value of 99%). Cytokine analyses demonstrated that levels of interleukin (IL)-6, IL-4, IL-13, IL-1ß, interferon gamma (IFN-γ), and granulocyte-macrophage colony-stimulating factor (GM-CSF) were significantly higher in DHF patients compared to DF patients (p< 0.001). The ADE assay showed that diluted plasma containing preexisting dengue antibodies from DHF patients significantly enhanced dengue infection (p< 0.05). CONCLUSION: The results suggest that older age, type II diabetes, hypertension, immunological cytokine dysregulation, and preexisting dengue antibodies inducing ADE infection are correlated with dengue severity. This study also indicates that the largest dengue outbreak in Taiwan might have been a result of imported DF from dengue epidemic regions.