Biology subjects

Chotiwan, N.

Publications and source records attributed to Chotiwan, N..

9 recordsLinked to original sources

bioRxiv2025

Mechanism of neuroinvasion and interferon response in human choroid plexus organoids during Japanese encephalitis virus infection

Orthoflaviviruses are medically important arthropod-borne pathogens that cause a significant global disease burden. Some of these viruses can cause neurological infections, requiring a clear understanding of how they enter the central nervous system (CNS). The choroid plexus (ChP) is a cerebrospinal fluid (CSF)-producing tissue residing in the brain ventricles. The ChP forms a blood-CSF barrier, which acts as a crucial peripheral-CNS interface and a physical defense against neuroinvasive pathogens. While this tissue is increasingly recognized as a potential entry site for hematogenous pathogens, how orthoflaviviruses overcome the blood-CSF barrier and the corresponding intrinsic immune response in a human model remains critically undefined. This study aimed to investigate the potential of the ChP to serve as a neuroinvasive site for orthoflaviviruses. ChP organoids (ChPOs) derived from human inducible pluripotent stem cells were established and determined for susceptibility to a panel of orthoflaviviruses, namely Japanese encephalitis virus (JEV), Zika virus (ZIKV), and Dengue virus (DENV). These viruses exhibited differential susceptibility to ChPOs, with the pathogenic JEV strain being the most infective. Transcriptomic analysis of ChPOs confirmed a robust interferon (IFN) response to JEV infection. Functional studies further demonstrated that antiviral defenses are crucial for controlling virus infection, and that type I IFN responses are superior to the epithelial-restricted type III IFN responses. Critically, despite the productive release of JEV into the CSF-like fluid within the ChP lumen, selective barrier integrity was maintained, confirming a nondestructive transcellular neuroinvasion mechanism across the blood-CSF barrier. This study highlights a valuable in vitro platform and its potential for studying virus neuroinvasion at the blood-CSF barrier.

microbiology↗

bioRxiv2025

Natural salivary gland barrier curtails Zika virus transmission in Thai Aedes aegypti

BackgroundVector competence is a critical determinant of arbovirus transmission dynamics, yet most studies in Thailand have primarily examined Aedes aegypti susceptibility to infection and systemic viral dissemination rather than the mosquitos potential to transmit infectious virions. Given that transmission potential varies among mosquito populations, identifying population-specific transmission barriers is crucial for optimizing vector control strategies especially under budget and resource constrains. Methodology/Principal FindingsThis study assessed the Zika virus (ZIKV) transmission potential of three Ae. aegypti populations from Thailand: two field-derived populations from Chiang Mai (CSP) and Nakhon Sawan (NAK) and a long-established laboratory strain (DMSC). Following oral exposure to a locally circulating ZIKV strain, viral transmission potential was evaluated. CSP mosquitoes demonstrated the earliest and highest ZIKV prevalence in salivary glands at 7 days post-infectious blood meal (dpibm), with DMSC and NAK populations reaching comparable infection levels at later time points. Despite this, NAK mosquitoes exhibited a strong barrier, resulting in significantly lower transmission potential. Genetic crosses revealed that this phenotype is inherited as an autosomal dominant trait and is similarly effective against dengue virus serotype 2 (DENV2), underscoring the broad-spectrum potential of NAK Ae. aegypti for transmission suppression. Furthermore, male NAK mosquitoes exhibited superior mating competitiveness, reinforcing their viability as a tool for vector control by population replacement. Conclusions/SignificanceOur findings highlight significant variability in ZIKV vector competence among Thai Ae. aegypti populations, emphasizing the importance of direct transmission assessments in vector competence studies. The discovery of a naturally occurring, heritable salivary gland escape barrier presents an opportunity for vector control strategies through NAK-based population replacement approaches. With their strong mating capability and broad arbovirus-blocking ability, NAK mosquitoes provide a natural alternative to Wolbachia-based and genetically modified mosquito interventions. Author summaryAedes aegypti is the primary vector of arboviruses such as Zika virus (ZIKV) and dengue virus (DENV), but not all mosquitoes that become infected successfully transmit the virus. While most research in Thailand has focused on mosquito infection and virus dissemination, fewer studies have examined the ability of mosquitoes to transmit infectious virus through their saliva. We discovered a mosquito population from Nakhon Sawan (NAK) that exhibits a natural barrier to virus release from the salivary glands, leading to significant blocking of virus transmission in ZIKV-infected mosquitoes. This salivary gland escape barrier of NAK is a heritable autosomal dominant characteristic that also restricts DENV transmission. Given their high mating competitiveness, NAK mosquitoes present a promising candidate for population replacement strategies aimed at reducing arbovirus transmission. Our findings emphasize the importance of assessing transmission rates rather than relying solely on infection and dissemination rates. Furthermore, the discovery of a naturally occurring mosquito population with reduced transmission potential offers a practical and sustainable vector control approach. The implementation of NAK-based population replacement strategies could serve as an alternative to traditional control methods, such as insecticide application or genetically modified mosquito releases. Future research should focus on field trials to validate the efficacy of this approach in real-world settings and assess its long-term impact on virus transmission dynamics.

microbiology↗

bioRxiv2024

Cryo-electron tomography reveals coupled flavivirus replication, budding and maturation

Flaviviruses replicate their genomes in replication organelles (ROs) formed as bud-like invaginations on the endoplasmic reticulum (ER) membrane, which also functions as the site for virion assembly. While this localization is well established, it is not known to what extent viral membrane remodeling, genome replication, virion assembly, and maturation are coordinated. Here, we imaged tick-borne flavivirus replication in human cells using cryo-electron tomography. We find that the RO membrane bud is shaped by a combination of a curvature-establishing coat and the pressure from intraluminal template RNA. A protein complex at the RO base extends to an adjacent membrane, where immature virions bud. Naturally occurring furin site variants determine whether virions mature in the immediate vicinity of ROs. We further visualize replication in mouse brain tissue by cryo-electron tomography. Taken together, these findings reveal a close spatial coupling of flavivirus genome replication, budding, and maturation.

microbiology↗

bioRxiv2023

Differential intra-host infection kinetics in Aedes aegypti underlie superior transmissibility of African relative to Asian Zika virus

Despite numerous studies highlighting the higher transmissibility of the African Zika virus (ZIKV) lineage compared to the Asian lineage in mosquito vectors, little is known about how the viruses interact with different tissues during the early steps of mosquito infection. To address this gap, we aimed to characterize intra-host infection barriers by combining a tissue-level monitoring of infection using plaque assays and a novel quantitative analysis of single-cell level infection kinetics by in situ immunofluorescent staining. Our results revealed that, in Aedes aegypti, an African ZIKV strain exhibited a higher replication rate across various tissues than an Asian ZIKV strain. This difference was potentially due to a higher virus production in individual cells, faster spread within tissues, or a combination of both factors. Furthermore, we observed that higher bloodmeal titers resulted in a faster viral spread to neighboring cells suggesting that intra-host infection dynamics depend on inoculum size. We also identified a significant bottleneck during midgut infection establishment for both ZIKV lineages, with only a small percentage of the virus population successfully initiating infection. Finally, the in situ immunofluorescent staining technique enabled the examination of virus infection characteristics in different cell types and revealed heterogeneity in viral replication. Together, these findings demonstrate that differences in intra-host infection kinetics underlie differential transmissibility between African and Asian ZIKV lineages. This information could serve as a starting point to further investigate the underlying mechanisms and ultimately inform the development of alternative control strategies. ImportanceThe recent Zika virus (ZIKV) epidemic in the Americas highlights its potential public health threat. While the Asian ZIKV lineage has been identified as the main cause of the epidemic, the African lineage, which has been primarily confined to Africa, has shown evidence of higher transmissibility in Aedes mosquitoes. To gain a deeper understanding of this differential transmissibility, our study employed a combination of tissue-level infection kinetics and single-cell level infection kinetics using in situ immunofluorescent staining. We discovered that the African ZIKV lineage propagates more rapidly and spreads more efficiently within mosquito cells and tissues than its Asian counterpart. This information lays the groundwork for future exploration of the viral and host determinants driving these variations in propagation efficiency.

microbiology↗

bioRxiv2022

An MR-based brain template and atlas for optical projection tomography and light sheet fluorescence microscopy

Optical projection tomography (OPT) and light sheet fluorescence microscopy (LSFM) are high-resolution optical imaging techniques operating in the mm-cm range, ideally suited for ex vivo 3D whole mouse brain imaging. Although these techniques exhibit high sensitivity and specificity for antibody-labeled targets, the provided anatomical information remains limited. To allow anatomical mapping of fluorescent signal in whole brain, we developed a novel magnetic resonance (MR) - based template with its associated tissue priors and atlas labels, specifically designed for brains subjected to tissue processing protocols required for 3D optical imaging. We investigated the effect of tissue pre-processing and clearing on brain size and morphology and developed optimized templates for BABB/Murrays clear (OCUM) and DBE/iDISCO (iOCUM) cleared brains. By creating optical-(i)OCUM fusion images using our mapping procedure, we localized dopamine transporter and translocator protein expression and tracer innervation from the eye to the lateral geniculate nucleus of thalamus and superior colliculus. These fusion images allowed for precise anatomical identification of fluorescent signal in discrete brain areas. As such, these templates enable applications in a broad range of research areas integrating optical 3D brain imaging by providing an MR template for cleared brains.

neuroscience↗

bioRxiv2022

Shifting a Cellular Metabolic Landscape Identifies a Refractory Environment for Flavivirus Replication

Host-targeted therapeutics to control viral infection are gaining prominence given the vulnerability of viral replication at select host-interaction points and the limited possibility of developing drug resistant mutants. Nevertheless, the chemical and biological impact of many host-targeted therapeutics on both the cell and virus has not been elucidated and remains a key complication. Previously, it has been demonstrated that inhibition of fatty acid metabolism has significant antiviral potential. Here, we use a multidisciplinary approach to demonstrate how inhibition of fatty acid biosynthesis creates a metabolically refractory environment that drives viral dependence on alternate metabolic pathways for survival. By profiling the global metabolic landscape following inhibition of fatty acid biosynthesis, we identified additional biochemical pathways that, when inhibited in combination with fatty acid biosynthesis, displayed increased antiviral potential. Our studies also demonstrated that there was a direct link between changes in cellular chemical composition and the ultrastructural membrane architecture induced by viral gene products. Utilizing inhibitors to change these metabolic environments significantly impacted early viral replication and disrupted the membrane architecture critical for the viral life cycle. Here, we have defined at a molecular level how shifting metabolic landscapes can be exploited to identify combinations of therapeutics that have a greater antiviral effect. Author SummaryDengue viruses are transmitted by Aedes aegypti mosquitoes which are prevalent in the tropical and subtropical regions of the world. These viruses cause over 350 million infections annually. There are no antivirals to combat infection and the only vaccine available is suboptimal. Since these viruses are obligate pathogens, they hijack lipid metabolic pathways in host cells to drive new lipid synthesis critically required for their replication. Mechanisms of how lipid synthesis impacts viral replication is unknown. These viruses also rearrange cellular membranes to form platforms for assembly of viral replication complexes. Here, for the first time, we show that virus-hijacking of de novo fatty acid biosynthesis pathways is required for the formation of membranous replication platforms and if inhibited disrupted synthesis of replicative form viral RNA. Importantly, these inhibitors drastically rearranged the metabolic landscape of the cell resulting in an activation of compensatory nucleotide synthesis pathways that allowed the virus to survive at a low level through the inhibition. However, if both pathways were inhibited in combination, infectious virus release was reduced to below detection limits. The study demonstrates how understanding the metabolic landscape altered by specific inhibitors can lead to the discovery of compensatory metabolic pathways and targets that in combination can enhance intervention efficacy.

microbiology↗

bioRxiv2022

Expression of fatty acid synthase genes and their role in development and arboviral infection of Aedes aegypti

BackgroundFatty acids are the building blocks of complex lipids essential for living organisms. In mosquitoes, fatty acids are involved in cell membrane production, energy conservation and expenditure, innate immunity, development, and reproduction. Fatty acids are synthesized by a multifunctional enzyme complex called fatty acid synthase (FAS). Several paralogues of FAS were found in the Aedes aegypti (Ae. aegypti) mosquito. However, the molecular characteristics and the expression of some of these paralogues have not been investigated. MethodsGenome assemblies of Ae. aegypti were analyzed and orthologues of human FAS were identified. Phylogenetic analysis and in silico molecular characterization were performed to identify the functional domains of the Ae. aegypti FAS (AaFAS). Quantitative analysis and loss-of-function experiments were performed to determine the significance of different AaFAS transcripts in various stages of development, expression following different diets and the impact of AaFAS on dengue virus, serotype 2 (DENV2) infection and transmission. ResultsWe identified seven putative FAS genes in the Ae. aegypti genome assembly, based on nucleotide similarity to the FAS proteins (tBLASTn) of humans, other mosquitoes and invertebrates. Bioinformatics and molecular analyses suggested that only five of the FAS genes produce mRNA and therefore represent complete gene models. Expression levels of AaFAS varied among developmental stages and between male and female Ae. aegypti. Quantitative analyses revealed that expression of AaFAS1, the putative orthologue of the human FAS, was highest in adult females. Transient knockdown (KD) of AaFAS1 did not induce a complete compensation by other AaFAS genes but limited DENV2 infection of Aag2 cells in culture and the midgut of the mosquito. ConclusionAaFAS1 is the predominant AaFAS in the adult mosquitoes. It has the highest amino acid similarity to human FAS and contains all enzymatic domains typical of human FAS. AaFAS1 also facilitated DENV2 replication in both cell culture and in mosquito midguts. Our data suggest that AaFAS1 may play a role in transmission of dengue viruses, and could represent a target for intervention strategies.

microbiology↗

bioRxiv2021

Acyl-CoA Thioesterases; a rheostat that controls activated fatty acids modulates dengue virus serotype 2 replication

During infection with dengue viruses (DENVs), the lipid landscape within host cells is significantly altered to assemble membrane platforms that support viral replication and particle assembly. Fatty acyl-CoAs are key intermediates in the biosynthesis of complex lipids that form these membranes. They also function as key signaling lipids in the cell. Here, we carried out loss of function studies on acyl-CoA thioesterases (ACOTs), a family of enzymes that hydrolyze fatty acyl-CoAs to free fatty acids and coenzyme A, to understand their influence on the lifecycle of DENVs. Loss of function of the type I ACOTs 1 (cytoplasmic) and 2 (mitochondrial) together significantly increased DENV serotype 2 (DENV2) viral replication and infectious particle release. However, isolated knockdown of mitochondrial ACOT2 significantly decreased DENV2 protein translation, genome replication, and infectious virus release. Furthermore, loss of ACOT7 function, a mitochondrial type II ACOT, similarly suppressed DENV2. As ACOT1 and ACOT2 are splice variants, these data suggest that location (cytosol and mitochondria, respectively) rather than function of these proteins may account for the differences in DENV2 infection phenotype. Additionally, loss of mitochondrial ACOT2 and ACOT7 expression also altered the expression of several ACOTs located in multiple organelle compartments within the cell highlighting a complex relationship between ACOTs in the DENV2 virus lifecycle.

microbiology↗

bioRxiv2021

Whole-brain fluorescence-MRI coregistration for precise anatomical mapping of virus infection

Viral tropism within the brain and the role(s) of vertebrate immune response to neurotropic flaviviruses infection is largely understudied. We combined multimodal imaging (cm-nm scale) with single nuclei RNA- sequencing to study Langat virus in wildtype and interferon alpha/beta receptor knockout (Ifnar-/-) mice to visualize viral pathogenesis and define molecular mechanisms. Whole brain viral infection was imaged by Optical Projection Tomography coregistered to ex vivo MRI. Infection was limited to grey matter of sensory systems in wildtype mice, but extended into white matter, meninges and choroid plexus in Ifnar-/-mice. Cells in wildtype displayed strong type I and II IFN responses, likely due to Ifnb expressing astrocytes, infiltration of macrophages and Ifng-expressing CD8+NK cells, whereas in Ifnar-/-, the absence of this response contributed to a shift in cellular tropism towards non-activated resident microglia. Multimodal imaging-transcriptomics exemplifies a powerful way to characterize mechanisms of viral pathogenesis and tropism.

microbiology↗