Molecular epidemiology, clinical analysis, and genetic characterization of Zika virus infections in Thailand (2020–2023)

Abstract To investigate the clinical and molecular characteristics and evolution of the Zika virus (ZIKV) in Thailand from March 2020 to March 2023. In all, 751 serum samples from hospitalized patients in Bangkok and the surrounding areas were screened for ZIKV using real-time RT-PCR. Demographic data and clinical variables were evaluated. Phylogenetic and molecular clock analysis determined the genetic relationships among the ZIKV strains, emergence timing, and their molecular characteristics. Among the 90 confirmed ZIKV cases, there were no significant differences in infection prevalence when comparing age groups and sexes. Rash was strongly associated with ZIKV infection. Our ZIKV Thai isolates were categorized into two distinct clades: one was related to strains from Myanmar, Vietnam, Oceania, and various countries in the Americas, and the other was closely related to previously circulating strains in Thailand, one of which shared a close relation to a neurovirulent ZIKV strain from Cambodia. Moreover, ZIKV Thai strains could be further classified into multiple sub-clades, each exhibiting specific mutations suggesting the genetic diversity among the circulating strains of ZIKV in Thailand. Understanding ZIKV epidemiology and genetic diversity is crucial for tracking the virus's evolution and adapting prevention and control strategies

Large-scale outbreak of Chikungunya virus infection in Thailand, 2018-2019.

Between 2018 and 2019, the incidence of chikungunya was approximately 15,000 cases across 60 provinces in Thailand. Here, the clinical presentations in chikungunya, emergent pattern, and genomic diversity of the chikungunya virus (CHIKV) causing this massive outbreak were demonstrated. A total of 1,806 sera samples from suspected cases of chikungunya were collected from 13 provinces in Thailand, and samples were tested for the presence of CHIKV RNA, IgG, and IgM using real-time PCR, enzyme-linked immunoassay (ELISA), commercial immunoassay (rapid test). The phylogenetic tree of CHIKV whole-genome and CHIKV E1 were constructed using the maximum-likelihood method. CHIKV infection was confirmed in 547 (42.2%) male and 748 (57.8%) female patients by positive real-time PCR results and/or CHIKV IgM antibody titers. Unsurprisingly, CHIKV RNA was detected in >80% of confirmed cases between 1 and 5 days after symptom onset, whereas anti-CHIKV IgM was detectable in >90% of cases after day 6. Older age was clearly one of the risk factors for the development of arthralgia in infected patients. Although phylogenetic analysis revealed that the present CHIKV Thailand strain of 2018-2020 belongs to the East, Central, and Southern African (ECSA) genotype similar to the CHIKV strains that caused outbreaks during 2008-2009 and 2013, all present CHIKV Thailand strains were clustered within the recent CHIKV strain that caused an outbreak in South Asia. Interestingly, all present CHIKV Thailand strains possess two mutations, E1-K211E, and E2-V264A, in the background of E1-226A. These mutations are reported to be associated with virus-adapted Aedes aegypti. Taken together, it was likely that the present CHIKV outbreak in Thailand occurred as a result of the importation of the CHIKV strain from South Asia. Understanding with viral genetic diversity is essential for epidemiological study and may contribute to better disease management and preventive measures

Additional file 2: Figure S1. of Involvement of fatty acid synthase in dengue virus infection

Real-time PCR validation of siRNA mediated gene silencing of fatty acid synthase (FASN) gene. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with one of four siRNAs directed to FASN (FASN1 to FASN 4). On days 1 to 5 post transfection the level of FASN transcript was determined by real time PCR. Normalization expression data relative to actin is shown. Bars show mean +/−SD. (a) 1 day post transfection, (b) 2 days post transfection, (c) 3 days post transfection, (d) 4 days post transfection and (e) 5 days post transfection. Bars show mean +/−SD (*; p value <0.05). Figure S2. Assessment of cell viability after siRNA transfection. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with siRNAs directed to FASN (FASN1 and FASN 4). On day 2 post transfection cell viability was assessed by trypan blue staining and counting cells using a hemocytometer. Bars show mean +/−SD. Figure S3. Real-time PCR validation of siRNA mediated gene silencing of fatty acid synthase (FASN) gene. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with one of four siRNAs directed to FASN (FASN1 to FASN 4). On day 2 post transfection (a) the level of FASN transcript was determined by real time PCR and (b) amplification product was run on an agarose gel and products visualized after ethidium bromide staining. Normalization expression data relative to actin is shown. Bars show mean +/−SD (*; p value <0.05). Figure S4. Western analysis of FASN expression after siRNA treatment. HEK293T/17 cells were not treated (mock) or treated with a siRNA control (GFP) or treated with one of four siRNAs directed to FASN (FASN1 to FASN 4). On days 1 to 4 post transfection the level of FASN protein was determined by western blot analysis. Normalization expression data relative to actin is shown. Bars show mean +/−SD (* p value <0.05; ** p value <0.01). Figure S5. Determination of orlistat cytotoxicity to HEK293T/17 cells. HEK293T/17 cells were incubated with different concentrations of orlistat or not treated (−) for (a) 24 h or (b) 36 h followed by MTT cell viability assays. Data is derived from 8 replicates. Treatment with 5% DMSO was used as a positive control. Bars show mean +/−SD (*; p value <0.05). Figure S6. The morphology of HEK293T/17 cells after orlistat treatment. HEK293T/17 cells were incubated with different concentrations of orlistat or not treated (mock) for (a) 24 h or (b) 36 h followed by observation under an inverted microscope. Magnification × 20. Figure S7. Evaluation of virucidal activity of orlistat. Stock DENV-2 was incubated with orlistat at concentrations of 1, 10, 20, 50 μM for 1 h and then used in the standard infection protocol. At 24 h.p.i (a) flow cytometry was performed to determine the percentage of infection and (b) supernatants were used to determine the virus titers. No deficit was observed in either percentage cell infection or virus titer. (PDF 2701 kb