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

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.

Severe fever with thrombocytopenia syndrome virus genotype B in Thailand.

Severe fever with thrombocytopenia syndrome virus (SFTSV) has been reported in many countries in Southeast Asia, which expands the original geographic range of China, Korea, and Japan. Here, we report the complete genome sequences of two Thai SFTSV strains previously identified in patients with undifferentiated febrile illness in 2020. Phylogenetically, both clustered with SFTSV genotype B strains and were most closely related to those previously reported in central China (≥99.0% nucleotide sequence identity) in the L, M, and S gene segments. Nine amino acid residues encoded by one or more Thai SFTSV genomes differed from those found in global strains. Interestingly, the observed differences in numerous residues between the Thai strains suggest possible separate introductions of different variants into the region.

Norovirus GII.3[P25] in Patients and Produce, Chanthaburi Province, Thailand, 2022.

An increase in acute gastroenteritis occurred in Chanthaburi Province, Thailand, during December 2021‒January 2022. Of the norovirus genotypes we identified in hospitalized patients and produce from local markets, genotype GII.3[P25] accounted for one third. We found no traceable link between patients and produce but found evidence of potential viral intake.

Molecular surveillance of arboviruses circulation and co-infection during a large chikungunya virus outbreak in Thailand, October 2018 to February 2020.

A large national outbreak of chikungunya virus (CHIKV) was recently reported in Thailand. While dengue virus (DENV) infection tends to occur year-round with an upsurge in the rainy season, Zika virus (ZIKV) also circulates in the country. The overlap in the distribution of these viruses increased the probability of co-infections during the heightened CHIKV activity. By examining 1806 patient serum samples submitted for CHIKV diagnostics from October 2018-February 2020 (511 CHIKV-negatives and 1295 CHIKV-positives), we used real-time reverse transcription-polymerase chain reaction to identify DENV and ZIKV individually. A total of 29 ZIKV and 36 DENV single-infections were identified. Interestingly, 13 co-infection cases were observed, of which 8 were CHIKV/DENV, 3 were CHIKV/ZIKV, and 2 were DENV/ZIKV. There were six DENV genotypes (13 DENV-1 genotype I, 10 DENV-2 Asian I, 10 DENV-2 Cosmopolitan, 6 DENV-3 genotype I, 2 DENV-3 genotype III, and 5 DENV-4 genotype I). Additionally, ZIKV strains identified in this study either clustered with strains previously circulating in Thailand and Singapore, or with strains previously reported in China, French Polynesia, and the Americas. Our findings reveal the co-infection and genetic diversity patterns of mosquito-borne viruses circulating in Thailand.

Severe Fever with Thrombocytopenia Syndrome Virus Infection, Thailand, 2019-2020.

Infection with severe fever with thrombocytopenia syndrome (SFTS) virus, which can cause hemorrhagic febrile illness, is often transmitted by ticks. We identified 3 patients with SFTS in or near Bangkok, Thailand. Our results underscore a need for heightened awareness by clinicians of possible SFTS virus, even in urban centers.

Chikungunya virus infection: molecular biology, clinical characteristics, and epidemiology in Asian countries.

Chikungunya virus (CHIKV) is a re-emerging mosquito-borne human pathogen that causes chikungunya fever, which is typically accompanied by severe joint pain. In Asia, serological evidence indicated that CHIKV first emerged in 1954. From the 1950's to 2005, sporadic CHIKV infections were attributed to the Asian genotype. However, the massive outbreak of CHIKV in India and the Southwest Indian Ocean Islands in 2005 has since raised chikungunya as a worldwide public health concern. The virus is spreading globally, but mostly in tropical and subtropical regions, particularly in South and Southeast Asia. The emergence of the CHIKV East/Central/South African genotype-Indian Ocean lineage (ECSA-IOL) has caused large outbreaks in South and Southeast Asia affected more than a million people over a decade. Notably, the massive CHIKV outbreaks before 2016 and the more recent outbreak in Asia were driven by distinct ECSA lineages. The first significant CHIKV ECSA strains harbored the Aedes albopictus-adaptive mutation E1: A226V. More recently, another mass CHIKV ECSA outbreak in Asia started in India and spread beyond South and Southeast Asia to Kenya and Italy. This virus lacked the E1: A226V mutation but instead harbored two novel mutations (E1: K211E and E2: V264A) in an E1: 226A background, which enhanced its fitness in Aedes aegypti. The emergence of a novel ECSA strain may lead to a more widespread geographical distribution of CHIKV in the future. This review summarizes the current CHIKV situation in Asian countries and provides a general overview of the molecular virology, disease manifestation, diagnosis, prevalence, genotype distribution, evolutionary relationships, and epidemiology of CHIKV infection in Asian countries over the past 65 years. This knowledge is essential in guiding the epidemiological study, control, prevention of future CHIKV outbreaks, and the development of new vaccines and antivirals targeting CHIKV.

Human norovirus GII.4 Hong Kong variant shares common ancestry with GII.4 Osaka and emerged in Thailand in 2016.

Human norovirus is a leading cause of non-bacterial acute gastroenteritis, which affects all age groups and are found globally. Infections are highly contagious and often occur as outbreaks. Periodic emergence of new strains are not uncommon and novel variants are named after the place of first reported nucleotide sequence. Here, we identified human norovirus GII.4 Hong Kong variant in stool samples from Thai patients presented with acute gastroenteritis. Comparison of amino acid residues deduced from the viral nucleotide sequence with those of historical and contemporary norovirus GII.4 strains revealed notable differences, which mapped to the defined antigenic sites of the viral major capsid protein. Time-scaled phylogenetic analysis suggests that GII.4 Hong Kong shared common ancestry with GII.4 Osaka first reported in 2007, and more importantly, did not evolve from the now-prevalent GII.4 Sydney lineage. As circulation of norovirus minor variants can lead to eventual widespread transmission in susceptible population, this study underscores the potential emergence of the GII.4 Hong Kong variant, which warrants vigilant molecular epidemiological surveillance.

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.

A functional interaction between GRP78 and Zika virus E protein.

Zika virus (ZIKV) is a mosquito-transmitted virus that has caused significant public health concerns around the world, partly because of an association with microcephaly in babies born to mothers who were infected with ZIKV during pregnancy. As a recently emerging virus, little is known as to how the virus interacts with the host cell machinery. A yeast-2-hybrid screen for proteins capable of interacting with the ZIKV E protein domain III, the domain responsible for receptor binding, identified 21 proteins, one of which was the predominantly ER resident chaperone protein GRP78. The interaction of GRP78 and ZIKV E was confirmed by co-immunoprecipitation and reciprocal co-immunoprecipitation, and indirect immunofluorescence staining showed intracellular and extracellular co-localization between GRP78 and ZIKV E. Antibodies directed against the N-terminus of GRP78 were able to inhibit ZIKV entry to host cells, resulting in significant reductions in the levels of ZIKV infection and viral production. Consistently, these reductions were also observed after down-regulation of GRP78 by siRNA. These results indicate that GRP78 can play a role mediating ZIKV binding, internalization and replication in cells. GRP78 is a main regulator of the unfolded protein response (UPR), and the study showed that expression of GRP78 was up-regulated, and the UPR was activated. Increases in CHOP expression, and activation of caspases 7 and 9 were also shown in response to ZIKV infection. Overall these results indicate that the interaction between GRP78 and ZIKV E protein plays an important role in ZIKV infection and replication, and may be a potential therapeutic target.

Evaluation of the antiviral activity of orlistat (tetrahydrolipstatin) against dengue virus, Japanese encephalitis virus, Zika virus and chikungunya virus.

Many mosquito transmitted viruses of the genera Alphavirus and Flavivirus are human pathogens of significant concern, and there is currently no specific antiviral for any member of these two genera. This study sought to investigate the broad utility of orlistat (tetrahydrolipstatin) in reducing virus infection for several mosquito borne viruses including flaviviruses (dengue virus (DENV; nine isolates analyzed), Japanese encephalitis virus (JEV; one isolate analyzed) and Zika virus (ZIKV; 2 isolates analyzed)) as well as an alphavirus (chikungunya virus; CHIKV; 2 isolates analyzed). Three different treatment regimens were evaluated, namely pre-treatment (only), post-treatment (only) and pre- and post-treatment, and three factors were evaluated, namely level of infection, virus titer and genome copy number. Results showed that all three treatment modalities were able to significantly reduce virus titer for all viruses investigated, with the exception of three isolates of DENV in the pre-treatment only regimen. Pre- and post-treatment was more effective in reducing the level of infection and genome copy number of all viruses investigated than either pre-treatment or post-treatment alone. Collectively, these results suggest orlistat has potential as a broad-spectrum agent against multiple mosquito transmitted viruses.

Enhanced noninvasive imaging of oncology models using the NIS reporter gene and bioluminescence imaging.

Noninvasive bioluminescence imaging (BLI) of luciferase-expressing tumor cells has advanced pre-clinical evaluation of cancer therapies. Yet despite its successes, BLI is limited by poor spatial resolution and signal penetration, making it unusable for deep tissue or large animal imaging and preventing precise anatomical localization or signal quantification. To refine pre-clinical BLI methods and circumvent these limitations, we compared and ultimately combined BLI with tomographic, quantitative imaging of the sodium iodide symporter (NIS). To this end, we generated tumor cell lines expressing luciferase, NIS, or both reporters, and established tumor models in mice. BLI provided sensitive early detection of tumors and relatively easy monitoring of disease progression. However, spatial resolution was poor, and as the tumors grew, deep thoracic tumor signals were massked by overwhelming surface signals from superficial tumors. In contrast, NIS-expressing tumors were readily distinguished and precisely localized at all tissue depths by positron emission tomography (PET) or single photon emission computed tomography (SPECT) imaging. Furthermore, radiotracer uptake for each tumor could be quantitated noninvasively. Ultimately, combining BLI and NIS imaging represented a significant enhancement over traditional BLI, providing more information about tumor size and location. This combined imaging approach should facilitate comprehensive evaluation of tumor responses to given therapies.

Proteomic analysis of monkey kidney LLC-MK2 cells infected with a Thai strain Zika virus.

Zika virus (ZIKV) has been endemic in Southeast Asian countries for several years, but the presence of the virus has not been associated with significant outbreaks of infection unlike other countries around the world where the Asian lineage ZIKV was introduced recently. However, few studies have been undertaken using the endemic virus. The Thai isolate was shown to have a similar tissue tropism to an African isolate of ZIKV, albeit that the Thai isolate infected cells at a lower level as compared to the African isolate. To further understand the pathogenesis of the Thai isolate, a 2D-gel proteomic analysis was undertaken of ZIKV infected LLC-MK2 cells. Seven proteins (superoxide dismutase [Mn], peroxiredoxin 2, ATP synthase subunit alpha, annexin A5 and annexin A1, carnitine o-palmitoyltransferase 2 and cytoskeleton-associated protein 2) were identified as differentially regulated. Of four proteins selected for validation, three (superoxide dismutase [Mn], peroxiredoxin 2, ATP synthase subunit alpha, and annexin A1) were shown to be differentially regulated at both the transcriptional and translational levels. The proteins identified were primarily involved in energy production both directly, and indirectly through mediation of autophagy, as well as in the response to oxidative stress, possibly occurring as a consequence of increased energy production. This study provides further new information on the pathogenesis of ZIKV.

Dengue virus requires apoptosis linked gene-2-interacting protein X (ALIX) for viral propagation.

The endosomal sorting complexes required for transport (ESCRT) pathway accessory protein apoptosis linked gene-2-interacting protein X (ALIX) has been shown to be upregulated during dengue virus (DENV) replication. Yeast-two-hybrid screens have additionally shown that ALIX interacts with DENV NS3 protein, but evaluation of the interaction through a replicon assay failed to show a functional significance to the interaction. In this study the interaction between DENV NS3 and ALIX was investigated by co-immunoprecipitation, and functional significance assessed by investigation of DENV production in ALIX expression regulated cells. The results showed that ALIX both interacted and co-localized with DENV NS3 protein and that upregulation of ALIX resulted in a significantly increased viral titer, while either siRNA or CRISPR-Cas9 mediated down regulation of ALIX significantly reduced viral production, without affecting relative DENV genome levels. These results are consistent with ALIX playing a significant role in the DENV replication cycle either during late infection or at viral egress.

Zika virus in Thailand.

This review examines the historic reports of the presence of Zika virus (ZIKV) in Thailand, as well as collates such information as exists on the current situation in Thailand with regards to ZIKV. We suggest that considerable caution must be applied in interpreting early serological studies, but that ZIKV is presently circulating over much of Thailand, with increasing numbers of cases being reported.

Ubiquitin-Conjugating Enzyme E2 L3 is Downregulated by the Chikungunya Virus nsP2 Protease.

PURPOSE: Chikungunya virus (CHIKV) is a mosquito transmitted alphavirus that causes chikungunya fever in humans. The CHIKV non-structural protein 2 (nsP2) is a multifunctional protein that additionally modulates the host cell to dampen the innate immune response and inhibit other cellular processes. EXPERIMENTAL DESIGN: To further investigate the interactions of nsP2 with host cells, the protease domain of CHIKV nsP2 (nsP2-pro) is transfected into Hela cells, and differential protein expression is detected by 2D polyacrylamide gel electrophoresis. RESULTS: A total of 21 differentially regulated (six upregulated, 15 downregulated) spots are observed, of which five are identified by mass spectrometry. The downregulation of one of the identified proteins, ubiquitin-conjugating enzyme E2 L3 (UBE2L3) is confirmed by western blotting of both nsP2-pro transfection and CHIKV natural infection, and the downregulation of UBE2L3 is additionally shown to require an enzymatically active nsP2 protease domain. Transfection of full length UBE2L3 into HEK293T/17 cells prior to CHIKV infection reduce levels of infection and E protein expression but do not alter RNA genome levels. CONCLUSION: These results suggest that UBE2L3 is a cellular target of the CHIKV nsP2 protease, and this possibly mediates the pathogenesis of chikungunya fever.

Imported case of Middle East respiratory syndrome coronavirus (MERS-CoV) infection from Oman to Thailand, June 2015.

Thailand reported the first Middle East respiratory syndrome (MERS) case on 18 June 2015 (day 4) in an Omani patient with heart condition who was diagnosed with pneumonia on hospital admission on 15 June 2015 (day 1). Two false negative RT-PCR on upper respiratory tract samples on days 2 and 3 led to a 48-hour diagnosis delay and a decision to transfer the patient out of the negative pressure unit (NPU). Subsequent examination of sputum later on day 3 confirmed MERS coronavirus (MERS-CoV) infection. The patient was immediately moved back into the NPU and then transferred to Bamrasnaradura Infectious Disease Institute. Over 170 contacts were traced; 48 were quarantined and 122 self-monitored for symptoms. High-risk close contacts exhibiting no symptoms, and whose laboratory testing on the 12th day after exposure was negative, were released on the 14th day. The Omani Ministry of Health (MOH) was immediately notified using the International Health Regulation (IHR) mechanism. Outbreak investigation was conducted in Oman, and was both published on the World Health Organization (WHO) intranet and shared with Thailand's IHR focal point. The key to successful infection control, with no secondary transmission, were the collaborative efforts among hospitals, laboratories and MOHs of both countries.

Involvement of fatty acid synthase in dengue virus infection.

BACKGROUND: The mosquito transmitted Dengue virus (DENV) remains a significant public health problem in many tropical and subtropical countries. Increasing evidence has suggested that during the infection process cellular lipids play important roles at several stages of the replication cycle. This study sought to characterize the changes in lipid metabolism gene expression and investigated the role of one enzyme, fatty acid synthase, in DENV infection. METHODS: Transcriptional profiles of genes associated with lipid metabolism were evaluated by real-time PCR after infection of different cell lines (HepG2 and HEK293T/17) and with different DENVs (laboratory adapted and low passage). Expression profiles of genes were evaluated by western blotting. A critical lipid metabolism protein, fatty acid synthase was down-regulated through siRNA and inhibited with orlistat and the effect on DENV infection determined by flow cytometry, plaque assay, western blotting and confocal microscopy. RESULTS: The results showed alterations of gene transcription and expression were seen in genes variously associated with lipogenesis, lipolysis and fatty acid ß-oxidation during DENV infection. Interference of fatty acid synthase with either siRNA or orlistat had marked effects on virus production, with orlistat having an EC50 value of 10.07 µM at 24 h post infection. However, non-structural protein expression was largely unaffected. CONCLUSIONS: While drug treatment reduced virus titer by up to 3Log10, no significant effect on DENV non-structural protein expression was observed, suggesting that fatty acid synthase acts through an effect on virion formation.

Activity of andrographolide against dengue virus.

Dengue is the most prevalent arthropod-transmitted viral illness of humans, with an estimated 100 million symptomatic infections occurring each year and more than 2.5 billion people living at risk of infection. There are no approved antiviral agents against dengue virus, and there is only limited introduction of a dengue vaccine in some countries. Andrographolide is derived from Andrographis paniculata, a medicinal plant traditionally used to treat a number of conditions including infections. The antiviral activity of andrographolide against dengue virus (DENV) serotype 2 was evaluated in two cell lines (HepG2 and HeLa) while the activity against DENV 4 was evaluated in one cell line (HepG2). Results showed that andrographolide had significant anti-DENV activity in both cell lines, reducing both the levels of cellular infection and virus output, with 50% effective concentrations (EC50) for DENV 2 of 21.304 µM and 22.739 µM for HepG2 and HeLa respectively. Time of addition studies showed that the activity of andrographolide was confined to a post-infection stage. These results suggest that andrographolide has the potential for further development as an anti-viral agent for dengue virus infection.

Cell-type specific variation in the induction of ER stress and downstream events in chikungunya virus infection.

Over the last decade infections with the mosquito transmitted chikungunya virus (CHIKV) have become a major worldwide concern, and considerable efforts have been made in understanding the interaction of this virus with the host cell machinery. Studies have documented the induction of the unfolded protein response (UPR), as well as the induction of apoptosis and autophagy in response to CHIKV infection. This study comparatively analysed these three processes in two cell lines, Hela and HepG2. Infection of Hela cells was characterized by activation of the PERK/eIF2α branch of the UPR, the induction of autophagy and early apoptosis, while infection of HepG2 cells was characterized by activation of the IRE/XBP1 branch of the UPR, limited or no activation of autophagy and comparatively later apoptosis. These results show that the specific cell context is an important mediator of the host cell response to CHIKV infection.

Retrospective screening of acute undifferentiated fever serum samples with universal flavivirus primers.

INTRODUCTION: Fever is a common symptom of many tropical diseases and in many cases the etiologic agent remains unidentified as a consequence of either the etiologic agent not being part of routine diagnostic screening or as a consequence of false negatives on standard diagnostic tests. METHODOLOGY: This study screened a well characterized panel of 274 serum samples collected on day of admission from adult patients with acute undifferentiated fever admitted to a hospital in Nakhon Ratchasima, Thailand by RT-PCR using pan-flavivirus degenerate primers. RESULTS: Subsequent clinical diagnosis was achieved for 38 of the patients, and included 19 cases of dengue fever. RT-PCR screening identified seven positive samples (2.5%) which were revealed by sequence analysis to be dengue virus 1 (2 cases), dengue virus 2 (2 cases) and dengue virus 3 (3 cases). Only 5 out of 19 (26%) serum samples from patients subsequently diagnosed with dengue were positive, but 2 samples which clinically remained undiagnosed were shown to be positive for dengue virus. Sequence analysis suggested that the dengue virus 3 cases occurred as a result of importation of a strain of dengue from India or China. No other flaviviruses were identified. CONCLUSIONS: No evidence was found of other flaviviruses besides dengue circulating in this population. Despite improved diagnostic tests, cases of dengue are still evading correct diagnosis.