Climate, demography, immunology, and virology combine to drive two decades of dengue virus dynamics in Cambodia.

The incidence of dengue virus disease has increased globally across the past half-century, with highest number of cases ever reported in 2019 and again in 2023. We analyzed climatological, epidemiological, and phylogenomic data to investigate drivers of two decades of dengue in Cambodia, an understudied endemic setting. Using epidemiological models fit to a 19-y dataset, we first demonstrate that climate-driven transmission alone is insufficient to explain three epidemics across the time series. We then use wavelet decomposition to highlight enhanced annual and multiannual synchronicity in dengue cycles between provinces in epidemic years, suggesting a role for climate in homogenizing dynamics across space and time. Assuming reported cases correspond to symptomatic secondary infections, we next use an age-structured catalytic model to estimate a declining force of infection for dengue through time, which elevates the mean age of reported cases in Cambodia. Reported cases in >70-y-old individuals in the 2019 epidemic are best explained when also allowing for waning multitypic immunity and repeat symptomatic infections in older patients. We support this work with phylogenetic analysis of 192 dengue virus (DENV) genomes that we sequenced between 2019 and 2022, which document emergence of DENV-2 Cosmopolitan Genotype-II into Cambodia. This lineage demonstrates phylogenetic homogeneity across wide geographic areas, consistent with invasion behavior and in contrast to high phylogenetic diversity exhibited by endemic DENV-1. Finally, we simulate an age-structured, mechanistic model of dengue dynamics to demonstrate how expansion of an antigenically distinct lineage that evades preexisting multitypic immunity effectively reproduces the older-age infections witnessed in our data.

Evaluation of cutaneous immune response in a controlled human in vivo model of mosquito bites.

Mosquito-borne viruses are a growing global threat. Initial viral inoculation occurs in the skin via the mosquito 'bite', eliciting immune responses that shape the establishment of infection and pathogenesis. Here we assess the cutaneous innate and adaptive immune responses to controlled Aedes aegypti feedings in humans living in Aedes-endemic areas. In this single-arm, cross-sectional interventional study (trial registration #NCT04350905), we enroll 30 healthy adult participants aged 18 to 45 years of age from Cambodia between October 2020 and January 2021. We perform 3-mm skin biopsies at baseline as well as 30 min, 4 h, and 48 h after a controlled feeding by uninfected Aedes aegypti mosquitos. The primary endpoints are measurement of changes in early and late innate responses in bitten vs unbitten skin by gene expression profiling, immunophenotyping, and cytokine profiling. The results reveal induction of neutrophil degranulation and recruitment of skin-resident dendritic cells and M2 macrophages. As the immune reaction progresses T cell priming and regulatory pathways are upregulated along with a shift to Th2-driven responses and CD8+ T cell activation. Stimulation of participants' bitten skin cells with Aedes aegypti salivary gland extract results in reduced pro-inflammatory cytokine production. These results identify key immune genes, cell types, and pathways in the human response to mosquito bites and can be leveraged to inform and develop novel therapeutics and vector-targeted vaccine candidates to interfere with vector-mediated disease.

Metagenomic Pathogen Sequencing in Resource-Scarce Settings: Lessons Learned and the Road Ahead.

Metagenomic next-generation sequencing (mNGS) is the process of sequencing all genetic material in a biological sample. The technique is growing in popularity with myriad applications including outbreak investigation, biosurveillance, and pathogen detection in clinical samples. However, mNGS programs are costly to build and maintain, and additional obstacles faced by low- and middle-income countries (LMICs) may further widen global inequities in mNGS capacity. Over the past two decades, several important infectious disease outbreaks have highlighted the importance of establishing widespread sequencing capacity to support rapid disease detection and containment at the source. Using lessons learned from the COVID-19 pandemic, LMICs can leverage current momentum to design and build sustainable mNGS programs, which would form part of a global surveillance network crucial to the elimination of infectious diseases.

Case Report: Cambodian National Malaria Surveillance Program Detection of Plasmodium knowlesi.

Despite recent success in reducing the regional incidence of Plasmodium falciparum malaria, cases of zoonotic malaria are on the rise in Southeast Asia. The Cambodian National Malaria Surveillance Program has previously relied on rapid diagnostic tests and blood smear microscopy with confirmatory polymerase chain reaction (PCR) testing in a subset of cases to further distinguish P. falciparum, P. malariae, P. ovale, and P. vivax species. Here, metagenomic next-generation sequencing identified P. knowlesi mono-infection in six Cambodian patients initially diagnosed with P. malariae by blood smear microscopy in February-May 2020. These findings of recent human infections with P. knowlesi in Cambodia led to the incorporation of P. knowlesi-specific PCR diagnostics to national malaria surveillance efforts.

Discovering disease-causing pathogens in resource-scarce Southeast Asia using a global metagenomic pathogen monitoring system.

SignificanceMetagenomic pathogen sequencing offers an unbiased approach to characterizing febrile illness. In resource-scarce settings with high biodiversity, it is critical to identify disease-causing pathogens in order to understand burden and to prioritize efforts for control. Here, metagenomic next-generation sequencing (mNGS) characterization of the pathogen landscape in Cambodia revealed diverse vector-borne and zoonotic pathogens irrespective of age and gender as risk factors. Identification of key pathogens led to changes in national program surveillance. This study is a "real world" example of the use of mNGS surveillance of febrile individuals, executed in-country, to identify outbreaks of vector-borne, zoonotic, and other emerging pathogens in a resource-scarce setting.

Development of Inapparent Dengue Associated With Increased Antibody Levels to Aedes aegypti Salivary Proteins: A Longitudinal Dengue Cohort in Cambodia.

BACKGROUND: We established the first prospective cohort to understand how infection with dengue virus is influenced by vector-specific determinants such as humoral immunity to Aedes aegypti salivary proteins. METHODS: Children aged 2-9 years were enrolled in the PAGODAS (Pediatric Assessment Group of Dengue and Aedes Saliva) cohort with informed consent by their guardians. Children were followed semi-annually for antibodies to dengue and to proteins in Ae. aegypti salivary gland homogenate using enzyme-linked immunosorbent assays and dengue-specific neutralization titers. Children presented with fever at any time for dengue testing. RESULTS: From 13 July to 30 August 2018, we enrolled 771 children. At baseline, 22% (173/770) had evidence of neutralizing antibodies to 1 or more dengue serotypes. By April 2020, 51 children had symptomatic dengue while 148 dengue-naive children had inapparent dengue defined by neutralization assays. In a multivariate model, individuals with higher antibodies to Ae. aegypti salivary proteins were 1.5 times more likely to have dengue infection (hazard ratio [HR], 1.47 [95% confidence interval {CI}, 1.05-2.06]; P = .02), particularly individuals with inapparent dengue (HR, 1.64 [95% CI, 1.12-2.41]; P = .01). CONCLUSIONS: High levels of seropositivity to Ae. aegypti salivary proteins are associated with future development of dengue infection, primarily inapparent, in dengue-naive Cambodian children. CLINICAL TRIALS REGISTRATION: NCT03534245.

IDseq-An open source cloud-based pipeline and analysis service for metagenomic pathogen detection and monitoring.

BACKGROUND: Metagenomic next-generation sequencing (mNGS) has enabled the rapid, unbiased detection and identification of microbes without pathogen-specific reagents, culturing, or a priori knowledge of the microbial landscape. mNGS data analysis requires a series of computationally intensive processing steps to accurately determine the microbial composition of a sample. Existing mNGS data analysis tools typically require bioinformatics expertise and access to local server-class hardware resources. For many research laboratories, this presents an obstacle, especially in resource-limited environments. FINDINGS: We present IDseq, an open source cloud-based metagenomics pipeline and service for global pathogen detection and monitoring (https://idseq.net). The IDseq Portal accepts raw mNGS data, performs host and quality filtration steps, then executes an assembly-based alignment pipeline, which results in the assignment of reads and contigs to taxonomic categories. The taxonomic relative abundances are reported and visualized in an easy-to-use web application to facilitate data interpretation and hypothesis generation. Furthermore, IDseq supports environmental background model generation and automatic internal spike-in control recognition, providing statistics that are critical for data interpretation. IDseq was designed with the specific intent of detecting novel pathogens. Here, we benchmark novel virus detection capability using both synthetically evolved viral sequences and real-world samples, including IDseq analysis of a nasopharyngeal swab sample acquired and processed locally in Cambodia from a tourist from Wuhan, China, infected with the recently emergent SARS-CoV-2. CONCLUSION: The IDseq Portal reduces the barrier to entry for mNGS data analysis and enables bench scientists, clinicians, and bioinformaticians to gain insight from mNGS datasets for both known and novel pathogens.

Rapid metagenomic characterization of a case of imported COVID-19 in Cambodia.

Rapid production and publication of pathogen genome sequences during emerging disease outbreaks provide crucial public health information. In resource-limited settings, especially near an outbreak epicenter, conventional deep sequencing or bioinformatics are often challenging. Here we successfully used metagenomic next generation sequencing on an iSeq100 Illumina platform paired with an open-source bioinformatics pipeline to quickly characterize Cambodia's first case of COVID-2019.

Structure/functional aspects of the human riboflavin transporter-3 (SLC52A3): role of the predicted glycosylation and substrate-interacting sites.

The human riboflavin (RF) transporter-3 (hRFVT-3; product of the SLC52A3 gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF. We addressed these issues using appropriate biochemical/molecular approaches, a protein-docking model, and established intestinal/renal epithelial cells. Our results showed that the hRFVT-3 protein is glycosylated and that glycosylation is important for its function. Mutating the predicted N-glycosylation sites at Asn94 and Asn168 led to a significant decrease in RF uptake; it also led to a marked intracellular (in the endoplasmic reticulum, ER) retention of the mutated proteins as shown by live-cell confocal imaging studies. The protein-docking model used in this study has identified a number of putative substrate-interacting sites: Ser16, Ile20, Trp24, Phe142, Thr314, and Asn315 Mutating these potential interacting sites was indeed found to lead to a significant inhibition in RF uptake and to intracellular (ER) retention of the mutated proteins (except for the Phe142 mutant). These results demonstrate that the hRFVT-3 protein is glycosylated and this glycosylation is important for its function and cell surface expression. This study also identified a number of residues in the hRFVT-3 polypeptide that are important for its function/cell surface expression.

Mechanisms Involved in the Development of the Chronic Gastrointestinal Syndrome in Nonhuman Primates after Total-Body Irradiation with Bone Marrow Shielding.

In this study, nonhuman primates (NHPs) exposed to lethal doses of total body irradiation (TBI) within the gastrointestinal (GI) acute radiation syndrome range, sparing ∼5% of bone marrow (TBI-BM5), were used to evaluate the mechanisms involved in development of the chronic GI syndrome. TBI increased mucosal permeability in the jejunum (12-14 Gy) and proximal colon (13-14 Gy). TBI-BM5 also impaired mucosal barrier function at doses ranging from 10-12.5 Gy in both small intestine and colon. Timed necropsies of NHPs at 6-180 days after 10 Gy TBI-BM5 showed that changes in small intestine preceded those in the colon. Chronic GI syndrome in NHPs is characterized by continued weight loss and intermittent GI syndrome symptoms. There was a long-lasting decrease in jejunal glucose absorption coincident with reduced expression of the sodium-linked glucose transporter. The small intestine and colon showed a modest upregulation of several different pro-inflammatory mediators such as NOS-2. The persistent inflammation in the post-TBI-BM5 period was associated with a long-lasting impairment of mucosal restitution and a reduced expression of intestinal and serum levels of alkaline phosphatase (ALP). Mucosal healing in the postirradiation period is dependent on sparing of stem cell crypts and maturation of crypt cells into appropriate phenotypes. At 30 days after 10 Gy TBI-BM5, there was a significant downregulation in the gene and protein expression of the stem cell marker Lgr5 but no change in the gene expression of enterocyte or enteroendocrine lineage markers. These data indicate that even a threshold dose of 10 Gy TBI-BM5 induces a persistent impairment of both mucosal barrier function and restitution in the GI tract and that ALP may serve as a biomarker for these events. These findings have important therapeutic implications for the design of medical countermeasures.

Interleukin-13 Receptor α1-Dependent Responses in the Intestine Are Critical to Parasite Clearance.

Nematode infection upregulates interleukin-4 (IL-4) and IL-13 and induces STAT6-dependent changes in gut function that promote worm clearance. IL-4 and IL-13 activate the type 2 IL-4 receptor (IL-4R), which contains the IL-13Rα1 and IL-4Rα chains. We used mice deficient in IL-13Rα1 (IL-13Rα1(-/-)) to examine the contribution of IL-13 acting at the type 2 IL-4R to immune and functional responses to primary (Hb1) and secondary (Hb2) infections with the gastrointestinal nematode parasite Heligmosomoides bakeri There were differences between strains in the IL-4 and IL-13 expression responses to Hb1 but not Hb2 infection. Following Hb2 infection, deficient mice had impaired worm expulsion and higher worm fecundity despite normal production of Th2-derived cytokines. The upregulation of IL-25 and IL-13Rα2 in Hb1- and Hb2-infected wild-type (WT) mice was absent in IL-13Rα1(-/-)mice. Goblet cell numbers and resistin-like molecule beta (RELM-ß) expression were attenuated significantly in IL-13Rα1(-/-)mice following Hb2 infections. IL-13Rα1 contributes to the development of alternatively activated macrophages, but the type 1 IL-4R is also important. Hb1 infection had no effects on smooth muscle function or epithelial permeability in either strain, while the enhanced mucosal permeability and changes in smooth muscle function and morphology observed in response to Hb2 infection in WT mice were absent in IL-13Rα1(-/-)mice. Notably, the contribution of claudin-2, which has been linked to IL-13, does not mediate the increased mucosal permeability following Hb2 infection. These results show that activation of IL-13Rα1 is critical for key aspects of the immune and functional responses to Hb2 infection that facilitate expulsion.

Enteric nematodes and the path to up-regulation of type 2 cytokines IL-4 and IL-13.

Protective immunity against enteric parasitic nematodes is dependent on IL-4, IL-13 activation of their exclusive transcription factor STAT6. The precise pathways by which enteric parasitic nematodes are recognized by the host is unclear, but elimination of this important interaction in developed nations is thought to contribute to the dysregulated immune responses that are a characteristic of autoimmune diseases. Nematode-derived products are involved in evading host defenses to promote their life cycle leading to modulation of host immune responses. Host protective immunity has adapted to enteric parasitic nematode infection by elaboration of mucins, increasing intraluminal fluid to control access to the surface epithelium, increasing cell turnover to maintain an effective barrier to their invasion, initiating immune responses through activation of resident immune cells, and recruitment of additional immune cells to release immune mediators that help orchestrate these responses. Both the immune and functional outcomes depend largely on IL-4/IL-13 signaling through STAT6, with a dominant role for IL-13 working through the type 2 IL-4 receptor (IL-4R). The recent observation that enteric nematode infection prevents the onset of a number of experimental models of IBD, diabetes, and several extraintestinal autoimmune diseases including multiple sclerosis has generated considerable interest in the identification of worm/egg products involved in the generation and maintenance of Th2 cytokines that may mediate the beneficial effects of nematode infection in autoimmune and inflammatory pathologies.

Role of macrophages in the altered epithelial function during a type 2 immune response induced by enteric nematode infection.

Parasitic enteric nematodes induce a type 2 immune response characterized by increased production of Th2 cytokines, IL-4 and IL-13, and recruitment of alternatively activated macrophages (M2) to the site of infection. Nematode infection is associated with changes in epithelial permeability and inhibition of sodium-linked glucose absorption, but the role of M2 in these effects is unknown. Clodronate-containing liposomes were administered prior to and during nematode infection to deplete macrophages and prevent the development of M2 in response to infection with Nippostrongylus brasiliensis. The inhibition of epithelial glucose absorption that is associated with nematode infection involved a macrophage-dependent reduction in SGLT1 activity, with no change in receptor expression, and a macrophage-independent down-regulation of GLUT2 expression. The reduced transport of glucose into the enterocyte is compensated partially by an up-regulation of the constitutive GLUT1 transporter consistent with stress-induced activation of HIF-1α. Thus, nematode infection results in a "lean" epithelial phenotype that features decreased SGLT1 activity, decreased expression of GLUT2 and an emergent dependence on GLUT1 for glucose uptake into the enterocyte. Macrophages do not play a role in enteric nematode infection-induced changes in epithelial barrier function. There is a greater contribution, however, of paracellular absorption of glucose to supply the energy demands of host resistance. These data provide further evidence of the ability of macrophages to alter glucose metabolism of neighboring cells.

SerpinB2 is critical to Th2 immunity against enteric nematode infection.

SerpinB2, a member of the serine protease inhibitor family, is expressed by macrophages and is significantly upregulated by inflammation. Recent studies implicated a role for SerpinB2 in the control of Th1 and Th2 immune responses, but the mechanisms of these effects are unknown. In this study, we used mice deficient in SerpinB2 (SerpinB2(-/-)) to investigate its role in the host response to the enteric nematode, Heligmosomoides bakeri. Nematode infection induced a STAT6-dependent increase in intestinal SerpinB2 expression. The H. bakeri-induced upregulation of IL-4 and IL-13 expression was attenuated in SerpinB2(-/-) mice coincident with an impaired worm clearance. In addition, lack of SerpinB2 in mice resulted in a loss of the H. bakeri-induced smooth muscle hypercontractility and a significant delay in infection-induced increase in mucosal permeability. Th2 immunity is generally linked to a CCL2-mediated increase in the infiltration of macrophages that develop into the alternatively activated phenotype (M2). In H. bakeri-infected SerpinB2(-/-) mice, there was an impaired infiltration and alternative activation of macrophages accompanied by a decrease in the intestinal CCL2 expression. Studies in macrophages isolated from SerpinB2(-/-) mice showed a reduced CCL2 expression, but normal M2 development, in response to stimulation of Th2 cytokines. These data demonstrate that the immune regulation of SerpinB2 expression plays a critical role in the development of Th2-mediated protective immunity against nematode infection by a mechanism involving CCL2 production and macrophage infiltration.

Parasitic nematode-induced modulation of body weight and associated metabolic dysfunction in mouse models of obesity.

Obesity is associated with a chronic low-grade inflammation characterized by increased levels of proinflammatory cytokines that are implicated in disrupted metabolic homeostasis. Parasitic nematode infection induces a polarized Th2 cytokine response and has been explored to treat autoimmune diseases. We investigated the effects of nematode infection against obesity and the associated metabolic dysfunction. Infection of RIP2-Opa1KO mice or C57BL/6 mice fed a high-fat diet (HFD) with Nippostrongylus brasiliensis decreased weight gain and was associated with improved glucose metabolism. Infection of obese mice fed the HFD reduced body weight and adipose tissue mass, ameliorated hepatic steatosis associated with a decreased expression of key lipogenic enzymes/mediators, and improved glucose metabolism, accompanied by changes in the profile of metabolic hormones. The infection resulted in a phenotypic change in adipose tissue macrophages that was characterized by upregulation of alternative activation markers. Interleukin-13 (IL-13) activation of the STAT6 signaling pathway was required for the infection-induced attenuation of steatosis but not for improved glucose metabolism, whereas weight loss was attributed to both IL-13/STAT6-dependent and -independent mechanisms. Parasitic nematode infection has both preventive and therapeutic effects against the development of obesity and associated features of metabolic dysfunction in mice.

IL-33-induced alterations in murine intestinal function and cytokine responses are MyD88, STAT6, and IL-13 dependent.

IL-33 is a recently identified cytokine member of the IL-1 family. The biological activities of IL-33 are associated with promotion of Th2 and inhibition of Th1/Th17 immune responses. Exogenous IL-33 induces a typical "type 2" immune response in the gastrointestinal tract, yet the underlying mechanisms remain to be fully elucidated. In addition, the role of IL-33 in the regulation of gastrointestinal function is not known. The present study investigated IL-33-dependent intestinal immunity and function in mice. Exogenous IL-33 induced a polarized type 2 cytokine response in the intestine that was entirely MyD88 dependent but STAT6 and IL-13 independent. Mice injected with recombinant IL-33 exhibited intestinal smooth muscle hypercontractility, decreased epithelial responses to acetylcholine and glucose, and increased mucosal permeability. IL-33 effects on intestinal epithelial function were STAT6 dependent, and both IL-4 and IL-13 appeared to play a role. The effects on smooth muscle function, however, were attributable to both STAT6-dependent and -independent mechanisms. In addition, IL-13 induction of insulin-like growth factor-1 was implicated in IL-33-induced smooth muscle hypertrophy. Finally, alternative activation of macrophages induced by IL-33 revealed a novel pathway that is IL-4, IL-13, and STAT6 independent. Thus manipulating IL-33 or related signaling pathways represents a potential therapeutic strategy for treating inflammatory diseases associated with dysregulated intestinal function.