Biology subjects

Jupatanakul, N.

Publications and source records attributed to Jupatanakul, N..

12 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↗

bioRxiv2025

Dengue virus susceptibility in Aedes aegypti linked to natural cytochrome P450 variants

The mosquito Aedes aegypti is the primary vector for dengue virus (DENV), which infects millions of people annually. Variability in DENV susceptibility among wild Ae. aegypti populations is governed by genetic factors, but specific causal variants are unknown. Here, we identify a cytochrome P450-encoding gene (CYP4G15) whose variants drive differences in DENV susceptibility in a natural Ae. aegypti population. CYP4G15 is transiently upregulated in DENV-resistant midguts, while knockdown increases susceptibility, and transgenic overexpression enhances resistance. A naturally occurring 18-base-pair promoter deletion reduces CYP4G15 expression and confers higher DENV susceptibility. The unexpected role of a cytochrome P450 in DENV susceptibility challenges the long-standing focus on canonical immune pathways and opens new avenues for understanding antiviral defense and DENV transmission in mosquitoes.

microbiology↗

bioRxiv2025

Immunological surveillance using gSG6-P1 biomarker reveals spatio-temporal dynamics of Anopheles exposure and gaps in malaria risk assessment

Malaria risk assessment often relies on the entomological inoculation rate (EIR), which quantifies infectious bites per person over time. However, this approach does not account for human behavioral variability, limiting its effectiveness in accurately reflecting malaria risk at the population level. Recent studies have attempted to estimate mosquito-bite risk through the measurement of antibody responses to Anopheles salivary peptides. However, this approach has yet to be applied to evaluate malaria transmission risks along the Thailand-Cambodia border. This study aims to employ an immune biomarker for Anopheles salivary peptide (anti-gSG6-P1) to identify key risk factors associated with exposure to Anopheles mosquitoes in Sisaket Province, a region bordering Thailand and Cambodia. Blood samples from the same set of 184 participants were collected via finger prick during each season: rainy (August 2022), cool-dry (December 2022), and hot-dry (April 2023). Anti-gSG6-P1 antibody levels were quantified using enzyme-linked immunosorbent assay (ELISA). Factor Analysis of Mixed Data (FAMD) revealed that seasonality exerts the strongest influence on antibody levels. This pattern was likely driven by human activities, particularly the frequency of rubber tapping activity in the rubber plantation area where Anopheles species, especially Anopheles dirus, are present. While this study successfully identifies seasonality and human factors as critical influences on antibody responses, it also highlights gaps in understanding the kinetics of anti-salivary peptide responses to Anopheles bites, particularly in species with low salivary peptides sequence similarity to that of Anopheles gambiae. These findings emphasize the need for the development of new serological tools tailored to malaria vectors in the Greater Mekong Subregion to enhance malaria risk assessment and improve vector control strategies.

immunology↗

bioRxiv2024

Comprehensive intra-host infection kinetics reveals high arbo-flavivirus transmission potential by neglected vector species, Aedes scutellaris

BackgroundDengue virus (DENV) and Zika virus (ZIKV) are primarily transmitted by Aedes mosquitoes. As most studies on vector competence have focused on Aedes aegypti and Aedes albopictus while neglecting other Aedes species, it is possible that the transmission risks might be underestimated. it is necessary to examine additional species that could potentially serve as competent vectors. This is particularly important considering the potential expansion of their geographical range due to climate change or species-specific vector reduction interventions. Methodology/Principal FindingsIn this study, we examined the infection kinetics and transmission potential of Aedes scutellaris from Thailand, comparing to Ae. aegypti and Ae. albopictus. Our findings demonstrated that Ae. scutellaris and Ae. albopictus had lower rates of midgut infection compared to Ae. aegypti due to smaller blood meal sizes during feeding. However, once the infection has established Ae. scutellaris exhibited efficient replication of ZIKV and DENV1-4 in the midguts, secondary organs, and salivary glands. Notably, Ae. scutellaris had a low salivary gland escape barrier, with comparable transmissibility as Ae. aegypti when inoculated with the same viral load. ConclusionThis study highlights the potential of Ae. scutellaris as a vector for DENV and ZIKV and emphasizes the importance of considering neglected mosquito species in arbovirus transmission and surveillance efforts. Author SummaryDengue and Zika are viral infections caused by arthropod-borne flaviviruses, and spread primarily through the bite of infected Aedes mosquitoes. Most research on DENV and ZIKV transmission has primarily focused on Aedes aegypti and Aedes albopictus while other Aedes species are overlooked, thus the epidemiology of the transmission might be underestimated. With climate change together with species-specific mosquito population reduction interventions these neglected Aedes species could become increasingly important in sustaining virus transmission. In this study, we examined Aedes scutellaris, a mosquito species that co-habitats with Ae. aegypti and Ae. albopictus, to assess its ability to transmit DENV and ZIKV using a combination of blood feeding and intrathoracic injection methods. Our findings show that although Ae. scutellaris had lower initial infection rates due to smaller blood meals, DENV and ZIKV were able to replicate and transmit at levels comparable to Ae. aegypti when exposed to similar virus loads. This highlights the need to study a broader range of species to improve virus control and outbreak prevention strategies.

microbiology↗

bioRxiv2023

Dirus Complex Species Identification PCR (DiCSIP) improves identification of Anopheles dirus complex from Greater Mekong Subregion

BackgroundThe Anopheles dirus complex plays a significant role as malaria vectors in the Greater Mekong Subregion (GMS), with varying degrees of vector competence among species. Accurate identification of sibling species in this complex is essential for understanding malaria transmission dynamics and deployment of effective vector control measures. This becomes increasingly crucial as the GMS advances towards malaria elimination while facing with the emergence of zoonotic simian malaria transmission. However, the original molecular identification assay, Dirus Allele Specific PCR (AS-PCR) targeting the ITS2 region have pronounced non-specific amplifications leading to ambiguous results and misidentification of the sibling species. This study investigates the underlying causes of these inconsistencies and develops new primers for accurate identification of species within the Anopheles dirus complex. Methodology/Principal findingsDespite several optimizations by reducing primer concentration, decreasing thermal cycling time, and increasing annealing temperature, the Dirus AS-PCR continued to produce inaccurate identifications, particularly for Anopheles dirus, Anopheles scanloni, and Anopheles nemophilous. Subsequently, in silico analyses pinpointed problematic primers with high GC content and multiple off-target binding sites. Through a series of in silico analyses and laboratory validation, a new set of primers for Dirus Complex Species Identification PCR (DiCSIP) has been developed. DiCSIP primers improve specificity, operational range, and sensitivity for accurate identification of five complex member species found in the GMS. Validation with laboratory and field An. dirus complex specimens demonstrated that DiCSIP could correctly identify all samples while the original Dirus AS-PCR misidentify An. dirus as other species when used with different thermocyclers. Conclusion/SignificanceThe DiCSIP assay offers a significant improvement in An. dirus complex identification, addressing challenges in specificity and efficiency of the previous ITS2-based assay. This new primer set provides a valuable tool for accurate entomological surveys, supporting effective vector control strategies to reduce transmission, and prevent the re-introduction of malaria in the GMS. Author SummarySeveral species of Anopheles mosquitoes belong to species complexes due to their indistinguishable morphology from closely related sibling species. However, members of the same species complex may exhibit varied vectorial capability, i.e. the ability to spread human pathogens, in this case malaria parasites, ranging from dominant vector to non-vector. The Dirus AS-PCR molecular assay to identify species within the An. dirus complex, a significant malaria vector species in the GMS, can produce inconsistent PCR results leading to misidentification. Furthermore, our analysis of An. dirus ITS2 sequences in the NCBI database indicated misidentification between these sibling species suggesting the need for a new set of primers to improve reproducibility and sensitivity in identifying members of the An. dirus complex. This study presents thorough analyses of the existing primers that cause difficulties in correct amplification and a development of a new set of primers for Dirus Complex Species Identification PCR (DiCSIP) assay. The DiCSIP assay offers several advantages over the original Dirus AS-PCR. It provides a higher specificity, sensitivity and wider operational range allowing for the use of the DNAzol direct reagent to process mosquito samples without the need for DNA extraction, saving both time and cost in sample processing. Our study provides a valuable molecular tool for entomological surveys, which is crucial for effective vector control measures in the GMS.

molecular biology↗

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↗

bioRxiv2021

New insights into antimalarial chemopreventive activity of antifolates

Antifolates targeting dihydrofolate reductase (DHFR) are antimalarial compounds that have long been used for malaria treatment and chemoprevention (inhibition of infection from mosquitoes to humans). Despite their extensive applications, the thorough understanding of antifolate activity against hepatic malaria parasites, especially resistant parasites, have yet to be achieved. Using a transgenic P. berghei harboring quadruple mutant dhfr from P. falciparum (Pb::Pfdhfr-4M), we demonstrate that quadruple mutations on Pfdhfr confer complete chemoprevention resistance to pyrimethamine, the previous generation of antifolate, but not a new class of antifolate designed to overcome the resistance such as P218. Detailed investigation to pin-point stage-specific chemoprevention further demonstrated that it is unnecessary for the drug to be present throughout hepatic development. The drug is most potent against the developmental stages from early hepatic trophozoite to late hepatic trophozoite, but is not effective at inhibiting sporozoite and early hepatic stage development from sporozoite to early trophozoite. Our data shows that P218 also inhibited the late hepatic stage development, from trophozoite to mature schizonts to a lesser extent. With a single dose of 15 mg/kg, P218 prevented infection from up to 25,000 pyrimentamine-resistant sporozoites, a number equal to thousands of infectious mosquito bites. Additionally, the hepatic stage of malaria parasite is much more susceptible to antifolates than the asexual blood stage. This study provides important insights into the activity of antifolates, as a chemopreventive therapeutic which could lead to a more efficient and cost effective treatment regime.

microbiology↗

bioRxiv2020

Transgenic pyrimethamine resistant Plasmodium berghei as a model for in vivo anti-DHFR drug testing

Inhibitors for Plasmodium falciparum dihydrofolate reductase (DHFR) form an important class of antimalarial drugs widely used for malaria treatment, but have been compromised by development of resistance to the drugs. Mutations in DHFR are the main contributing factors to the resistance. Although new, rationally designed antifolates active against resistant P. falciparum, such as P218, have been developed, the activity against the quadruple mutant P. falciparum (V1/S) has only been demonstrated in vitro, and in vivo activity has only been shown in SCID mice. A convenient in vivo model for antifolate testing is desirable. In this study, the endogenous P. berghei dihydrofolate reductase-thymidylate synthase (Pbdhfr-ts) gene was successfully replaced by quadruple dhfr-ts mutant gene from P. falciparum (N51I+C59R+S108N+I164L). The transgenic parasite gained resistance to pyrimethamine but not to other class of antimalarial drugs. While 30 mg/kg of pyrimethamine could not inhibit the transgenic parasite, P218 could inhibit the transgenic parasite with the ED50 of 0.11{+/-}0.02 mg/kg, a level similar to the P. falciparum in SCID mice model. These results demonstrated the validity of our model and showed that P218 was very potent against quadruple Pfdhfr-ts mutant parasite, in vivo.

microbiology↗

bioRxiv2020

Transgenic pyrimethamine-resistant P. falciparum reveals transmission blocking potency of P218, a novel antifolate

Antimalarial drug which target more than one life stage of the parasite are valuable tools in the fight against malaria. Previous generation of antifolate drugs are able to inhibit replicative stages of drug-sensitive, but not resistant parasites in humans, and mosquitoes. The lack of reliable gametocyte-producing, antifolate resistant P. falciparum hindrance the development of new antifolate compounds against mosquito stages. We used CRISPR-Cas9 technology to develop transgenic gametocyte producing P. falciparum with quadruple mutations in dhfr gene, using NF54 as a parental strain. The transgenic parasites gained pyrimethamine resistance while maintaining the gametocyte producing activity. In contrast to pyrimethamine that cannot inhibit exflagellation of the quadruple dhfr mutant parasite, the novel antifolate P218 showed a good potency for exflagellation inhibition (exflagellation IC50 10.74 {+/-} 4.22 nM). The exflagellation IC50 was 5.3 times lower than erythrocytic IC50 suggesting that the human to mosquito transmission poses as a strong barrier to prevent P218 resistant parasite among population. This study demonstrates that P218 can be considered as a highly potent tool to prevent the spread of antifolate resistant parasites. Graphical Abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY Research Highlights- Transgenic gametocyte producing pyrimethamine resistant P. falciparum was generated. - P218 asexual stage IC50 in NF54-4mutPfdhfr was 56.94 {+/-} 15.69 nM. - P218 exflagellation IC50 in NF54-4mutPfdhfr was 10.74 {+/-} 4.22 nM. - P218 exflagellation IC50 in NF54-4mutPfdhfr is 5.3 times lower than erythrocytic IC50. - P218 is an invaluable tool for malaria treatment and transmission control.

microbiology↗

bioRxiv2020

High transmissibility and fetal pathogenicity of recent Zika virus strains from the African lineage

The global emergence of Zika virus (ZIKV) in the last decade revealed the unprecedented ability for a mosquito-borne virus to cause congenital birth defects such as microcephaly. A puzzling aspect of ZIKV emergence is that all human outbreaks and birth defects to date have been exclusively associated with the Asian ZIKV lineage, despite a growing body of laboratory evidence pointing towards higher transmissibility and pathogenicity of the African ZIKV lineage. Whether this apparent paradox reflects the use of relatively old African ZIKV strains in most laboratory studies is unclear. Here, we experimentally compared the transmissibility and pathogenicity of seven low-passage ZIKV strains representing the recently circulating viral genetic diversity. We found that recent African ZIKV strains largely outperformed their Asian counterparts in mosquito transmission kinetics experiments, which translated into a markedly higher epidemic potential in outbreak computer simulations. In addition, African ZIKV strains were significantly more lethal than Asian ZIKV strains in immunocompromised adult mice. Finally, prenatal infection of immunocompetent mouse embryos with an African ZIKV strain resulted in embryonic death whereas it caused microcephaly with Asian ZIKV strains. Together, our results demonstrate the high epidemic potential and pathogenicity of recent ZIKV strains from Africa. Importantly, they also imply that the African ZIKV lineage could more easily go unnoticed by public health surveillance systems than the Asian ZIKV lineage due to its propensity to cause fetal loss rather than birth defects.

microbiology↗

bioRxiv2020

Serratia marcescens secretes proteases and chitinases with larvicidal activity against Anopheles dirus

Vector control, the most efficient tool to reduce mosquito-borne disease transmission, has been compromised by the rise of insecticide resistance. Recent studies suggest the potential of mosquito-associated microbiota as a source for new biocontrol agents or new insecticidal chemotypes. In this study, we identified a strain of Serratia marcescens that has larvicidal activity against Anopheles dirus, an important malaria vector in Southeast Asia. This bacterium secretes heat-labile larvicidal macromolecules when cultured under static condition at 25{degrees}C but not 37{degrees}C. Two major protein bands of approximately 55 kDa and 110 kDa were present in spent medium cultured at 25{degrees}C but not at 37{degrees}C. The Liquid Chromatography-Mass Spectrometry (LC-MS) analyses of these two protein bands identified several proteases and chitinases that were previously reported for insecticidal properties against agricultural insect pests. The treatment with protease and chitinase inhibitors led to a reduction in larvicidal activity, confirming that these two groups of enzymes are responsible for the macromolecules toxicity. Taken together, our results suggest a potential use of these enzymes in the development of larvicidal agents against Anopheles mosquitoes.

microbiology↗