Biology subjects

Anderson, T. J.

Publications and source records attributed to Anderson, T. J..

At least 19 recordsLinked to original sources

bioRxiv2026

Early-Life Wildfire Smoke Exposure Is Associated with Long-Term Systemic Immune Remodeling and Epigenetic Reprogramming

Wildfire smoke is an escalating global public health threat exposing millions of people, including children, to hazardous air pollution each year. Although wildfire smoke toxicants have been linked to a range of adverse health outcomes, including immune dysregulation, the long-term consequences of real-world pediatric wildfire smoke exposure on health and development remain largely unknown. To investigate the persistent effects of early-life exposure on immune health, here we leveraged a cohort of rhesus macaques that experienced nine consecutive days of hazardous wildfire smoke exposure in infancy during the 2020 Oregon Labor Day wildfires. By integrating ex vivo immune stimulations, multiplex cytokine profiling, single-cell transcriptomics, and genome-wide DNA methylation profiling, we identified persistent immunological consequences across molecular and functional levels. We found that a single severe postnatal exposure, in the first three months of life, was associated with persistent change in the innate immune response, including reduced pro-inflammatory cytokine response to a bacterial endotoxin, with subtle but consistent transcriptional changes in myeloid cells, particularly among males. Wildfire smoke exposure was also associated with changes in proportion of B and T/NK cells, and within the T/NK cell compartment, exposed animals exhibited an expansion of cytotoxic cells. Consistent with this, CD8+ T cells displayed extensive transcriptional remodeling and shifted toward more differentiated effector states, with the greatest differentiation observed in animals exposed at the youngest ages. Genome-wide DNA methylation profiling identified smoke-associated methylation changes consistent with acceleration of epigenetic aging, as well as persistent epigenetic alterations impacting genes involved in oxidative stress responses, innate immunity, T cell differentiation, and hematopoiesis. These findings demonstrate that a single severe wildfire smoke exposure during a critical developmental window is associated with extensive immune and epigenetic remodeling that persist years after exposure, providing new insight into the long-term biological consequences of early-life wildfire smoke exposure.

immunology↗

bioRxiv2026

Pooled amplicon sequencing for characterizing mutations in the praziquantel molecular target TRPMPZQ in schistosome populations from Western Kenya

Mass drug administration (MDA) using Praziquantel is central to efforts to eliminate Schistosomiasis. However, regions which respond poorly to MDA ("persistent hotspots") have been reported in many regions of Africa, including in Western Kenya. One possible explanation for persistent hotspots is that these areas contain PZQ resistant schistosome parasites. Recent studies have shown that Sm.TRPMPZQ gene is the molecular target for PZQ in schistosome parasites and that mutations in this gene can result in PZQ resistance. This study characterized mutations within Sm.TRPMPZQ in 23,420 miracidia collected from both hotspot and non-hotspot villages in Siaya County, western Kenya. We collected triplicate pools of 780.67 (SD {+/-} 183.47) miracidia from 135 people in five hotspot villages, where S. mansoni prevalence remains high despite over 5 annual treatments, and from 62 people from 5 non-hotspots villages where annual treatment resulted in reduction in prevalence. We extracted DNA from each miracidia pool, amplified 15 amplicons covering 1,695bp of the Sm.TRPMPZQtransmembrane domain and sequenced these to high read depth (21,110x) using a Miseq at KEMRI-CGHR. We identified five high confidence (frequency [≥] 0.01) Sm.TRPMPZQ variants. These included four synonymous changes and a non-synonymous variant (p.L1476I). p.L1476I is found at similar frequency in non-hotspot (0.040 {+/-} 0.006) and hotspot villages (0.044 {+/-} 0.0050) (Mann Whitney U=18, p= 0.31) and does not impact PZQ-response in Ca2+ reporter assays. Our studies show that resistance variants in Sm.TRPMPZQ are rare or non-existent in the locations studied and do not explain the existence of hotspots in this region.

genomics↗

bioRxiv2026

Direct measurement of in vitro response to Praziquantel in Schistosoma mansoni populations from Western Kenya

Large-scale treatment with praziquantel (PZQ) monotherapy is used to control schistosomiasis, leading to concerns about the emergence of PZQ-resistance. In Western Kenya, schistosome-infected patients frequently remain egg-positive following PZQ treatment, and several "hotspot" villages have been observed where transmission remains high, despite annual mass PZQ treatments. This project asks (i) whether PZQ-resistant parasites are found in Western Kenya and (ii) whether "hotspot" villages can be explained by a higher prevalence of PZQ-resistant parasites. We established a simple platform for directly assaying worm motility following in vitro PZQ-exposure in adult schistosomes isolated from a field setting. To do this, we established snail and hamster breeding colonies, and generated large populations of field-derived adult worms, by (i) harvesting S. mansoni eggs from multiple infected patients; (ii) infecting Biomphalaria spp snails with miracidia; (iii) infecting hamsters with released cercariae; (iv) perfusing adult worms from hamsters, and (iv) examining drug response following exposure to PZQ (1 {micro}g/ml for 1 day) in individual S. mansoni worms using an automated movement assay. We measured PZQ-response in 1,800 adult male parasites, representing an estimated 185 parasite genotypes. We identified a single worm that remained motile after PZQ-exposure among the 185 parasite genotypes surveyed (frequency = 0.54%; 95% CI 0.01 - 2.97%, exact binomial) consistent with PZQ-resistant worms being extremely rare or absent. Our direct phenotypic screening results suggests that (i) PZQ-resistance is not currently an obstacle for S. mansoni control in Western Kenya, and (ii) that other factors explain the existence of persistent hotspots.

microbiology↗

bioRxiv2026

A Multidimensional Analysis of the Bimodal Piperaquine Response in Plasmodium falciparum

Malaria remains a pressing global health challenge, with the continued emergence of resistance threatening the long-term efficacy of artemisinin-based combination therapies (ACTs). Piperaquine (PPQ), an important partner drug in artemisinin-based combination therapies exhibits a unique bimodal dose-response phenotype associated with reduced susceptibility, yet the biological mechanism underlying this phenotype remains unknown. This phenotype is strongly associated with mutations in pfcrt and copy number amplification of plasmepsin II/III (pm II/III). Given that plasmepsins play a central role in hemoglobin degradation within the blood stage parasite digestive vacuole, and that PPQ accumulates within this compartment and perturbs heme detoxification, this phenotype likely reflects alterations in fundamental biological processes alongside drug-specific effects. We used isogenic PPQ-resistant parasite clones differing only in pm II/III copy number to integrate phenotypes with metabolic changes, and transcriptional responses to ascertain the impact of genotype combinations on parasite response to PPQ. Across increasing PPQ concentrations, parasites with elevated pm II/III copy number exhibited distinct metabolic responses compared to single-copy parasites, specifically, an altered abundance of peptides derived from hemoglobin degradation, directly implicating a core biological pathway long associated with plasmepsin function. The combination of metabolic and transcriptional data with phenotypic measurements supports a model in which increased plasmepsin expression enhances the parasites capacity to sustain hemoglobin digestion and associated metabolic activity under high PPQ concentrations. This points to a mechanistic basis for continued parasite survival, indicating that changes in hemoglobin processing within the digestive vacuole contribute to the bimodal response to PPQ. Molecular dynamics simulations further support a direct interaction between PPQ and PM II/III, as a mechanism by which these proteins impact PPQ response dynamics through both modulation of hemoglobin digestion and protein-drug interactions within the digestive vacuole.

molecular biology↗

bioRxiv2026

Misleading inference of schistosome epidemiology from ribosomal internal transcribed spacer (ITS) and mitochondrial DNA

The nuclear, internal transcribed spacer (ITS) and mitochondrial cox1 markers are widely used to differentiate Schistosoma haematobium from its livestock counterparts, S. bovis and S. curassoni. Schistosoma isolated from humans with ITS and cox1 alleles from livestock parasites are typically inferred to be zoonotic infections and those with heterozygous ITS alleles (suggesting mixed species ancestry) are classified as recent hybrids. These classifications assume that the ITS and cox1 markers accurately reflect genome-wide ancestry. Here, we evaluated the reliability of this classification scheme by genotyping ITS and cox1 from 132 parasites isolated from human urine, and from 37 adult schistosomes collected from cattle at 14 Nigerian locations. We also genome sequenced each sample to empirically determine livestock schistosome ancestry. ITS/cox1 genotyping suggested extensive recent hybridization and zoonotic infection. Among parasites from humans, 10.1% carried both S. curassoni and S. haematobium ITS, consistent with F1 or early generation hybrids, 21% had livestock schistosome markers at both cox1 and ITS suggesting zoonotic infection, while 13.7% carried S. bovis cox1 alongside mixed S. curassoni and S. haematobium ITS, suggesting more complex ancestry. Genome sequencing revealed a very different picture. All parasites from humans formed a tight cluster regardless of ITS or cox1 genotype, while all worms from cattle were well differentiated. We found no schistosomes containing 50% livestock parasite ancestry consistent with F1s. Instead, we observed regionally varying levels of S. bovis introgression, with modest levels in southern Nigeria (mean = 4.9%) and low levels in northern Nigeria (mean = 0.06%). These results demonstrate that: (i) two-locus genotyping is uninformative for detecting zoonotic infection or recent hybridization between S. haematobium and livestock schistosomes and (ii) previous data generated using this approach requires reinterpretation. These findings reveal the limitations of widely-used approaches for documenting zoonotic infection and hybridization between S. haematobium and livestock schistosome species. Author SummaryMolecular markers provide useful tools for investigating epidemiology of closely related pathogens, but care is required when interpreting the data generated. This study critically examines the interpretation of two widely used molecular markers - the maternally-inherited mitochondrial cox1 locus and ribosomal internal transcribed spacer (ITS) - that are widely used for molecular epidemiology studies of schistosome parasites of humans and livestock. Data from these two markers have been used to infer recent hybridization between livestock and human parasites, and to identify livestock-to-human transmission. To examine the accuracy of epidemiological inferences from the cox1/ITS genotypes we collected parasites from both cattle and people in Nigeria. We genotyped each parasite using cox1 and ITS, as well as using whole-genome sequencing. Cox1/ITS genotyping suggested high rates of hybridization and livestock-to-human transmission. By contrast, whole-genome data show that parasites sampled from humans and cattle are distinct and correspond to independent species. The discrepant results demonstrate that genotyping just two markers provide insufficient resolution and can lead to erroneous conclusions about parasite epidemiology and poor management decisions. We suggest that future work should sample multiple markers from each parasite, using amplicon sequencing or other cost-effective approaches, to improve our understanding of schistosome epidemiology.

evolutionary biology↗

bioRxiv2025

TRPtracker: a community database for monitoring praziquantel sensitivity at TRPMPZQ variants

The anthelmintic praziquantel (PZQ) has been used for decades as the clinical therapy for schistosomiasis, and remains the only available drug. As a cheap and effective drug therapy for all human disease-causing Schistosoma species, usage of PZQ underpins mass drug administration strategies aimed at eliminating schistosomiasis as a public health problem by 2030. Concern over the potential emergence of resistance to PZQ is therefore warranted, as it would constitute a major threat to this approach. In terms of molecular adaptations conferring PZQ resistance, variation in the sequence and/or expression of the drug target is an obvious mechanism and should be a priority for surveillance efforts. The target of PZQ is a transient receptor potential ion channel, TRPMPZQ, which is established as a locus that regulates schistosome sensitivity to PZQ. Here, we describe the establishment of a community resource, TRPtracker, which coalesces data on TRPMPZQ natural variants together with measurements of individual variant sensitivity to PZQ. A compendium of laboratory-generated mutants in TRPMPZQ is also compiled in TRPtracker to map regions within TRPMPZQ critical for PZQ sensitivity. Aggregation of data from multiple research groups into TRPtracker permits rapid community-wide exchange of data, cataloguing which TRPMPZQ variants have been functionally profiled, where geographically these variants have been found, their frequency within populations and their potential impact on PZQ sensitivity.

microbiology↗

bioRxiv2025

Efficient amplicon panels for evaluating hybridization and genetic structure in the Schistosoma haematobium species group

Genetic markers for detecting hybridization and measuring population genetic parameters must be informative and cost-effective. Most population genetic studies within the Schistosoma haematobium species group rely on either a two-marker system consisting of the mitochondrial, cytochrome oxidase 1 (cox1) and the nuclear internal transcribed spacer (ITS) markers or, at the other extreme, millions of single nucleotide variants (SNVs) from whole genome/exome sequencing. cox1 and ITS studies contain minimal population genetic information but whole genome sequencing is cost-prohibitive. We examined [~]38 million previously published, whole genome SNVs genotyped in 162 S. haematobium and S. bovis sampled across Africa. We compared population genetic parameters from 4,000 panels of 10-100,000 randomly sampled SNVs to results from the whole genome dataset to test the resolution of reduced representation sequencing in schistosomes. We found that panels of 500 SNVs captured >99% of the population genetic information contained in the whole genome dataset by using Procrustes transformed principal component analyses and ancestry estimates (r{superscript 2} = 0.85). Additionally, the costs of genotyping parasites with an amplicon panel is two to three times less than whole genome sequencing. Our results show that moderately-sized amplicon panels targeting random SNVs provide an efficient approach to large scale, field-based schistosome surveillance.

evolutionary biology↗

bioRxiv2025

Advantages of outcrossing in Plasmodium falciparum: insights from genetic crosses using fluorescent labelled parasites

Malaria parasites are obligately sexual hermaphrodite protozoans with gamete fusion occurring in the mosquito midgut, followed by meiosis and recombination. Malaria parasite populations show a spectrum of populations structures ranging from predominantly selfing to highly outcrossed. However, the fitness consequences of selfing and outcrossing for malaria parasites are poorly understood. This project was designed to investigate the dynamics of gamete fusion within the mosquito midgut and the relative fitness of selfed and outcrossed zygotes. We generated florescent-labelled clones of NF54 (mCherry), an African parasite, and NHP4026 (GFP), a Thai parasite, crossed these parasites, and scored genotypes of 8540 oocysts from 435 mosquitoes sampled from 7 to 14 days post infection. We observed decreasing proportions of outcrossed oocysts and increasing levels of inbreeding over the course of the infection in two independently replicated crosses. These results are consistent with the faster maturation of transmissible sporozoites derived from outcrossed compared with selfed oocysts. Our results suggest a substantial outcrossing advantage, perhaps because this allows for the removal of deleterious mutations accumulated during asexual parasite replication in the vertebrate host. We also found that selfed NF54 oocysts were larger than outcrossed or selfed NHP4026 oocysts, which may influence production of sporozoites and onward transmission. We conclude that fluorescent labelled parasites provide clear resolution of mating patterns, temporal dynamics and transmission potential of malaria parasites in mosquitoes. Importantly, faster maturation of outcrossed parasites can maximize levels of recombination in transmitted malaria parasite populations.

genetics↗

bioRxiv2025

Bioluminescent reporter influenza A viruses to track viral infections

Influenza A viruses (IAV) infect a wide range of mammal and bird species and are responsible for seasonal outbreaks and occasional pandemics. Studying IAV requires methods to detect the presence of the virus in infected cells or animal models. Recombinant IAV expressing fluorescent proteins have allowed monitoring viral infection in cultured cells and ex vivo in the organs of infected animals. However, fluorescent-expressing IAV are often attenuated and are not suited for the imaging of infected animals using in vivo imaging systems (IVIS). To overcome this limitation, we generated a recombinant A/California/04/2009 H1N1 (pH1N1) expressing nanoluciferase (Nluc) from the non-structural (NS) viral segment (pH1N1-Nluc) that replicates efficiently in vitro, with growth kinetics and plaque morphology comparable to wild-type pH1N1 (pH1N1-WT). We used this pH1N1-Nluc to demonstrate its ability to identify neutralizing antibodies and antivirals, with neutralization and inhibition results comparable to pH1N1-WT. In mice, pH1N1-Nluc was able to induce similar body weight loss and mortality, and viral titers comparable to pH1N1-WT, results that were recapitulated in a ferret model of IAV infection. Using IVIS, pH1N1-Nluc enabled non-invasive, real-time tracking of viral infection in vivo and ex vivo following infection of mice with viral titers comparable to pH1N1-WT. The flexibility of this approach was further demonstrated by the generation of a Nluc-expressing recombinant A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Altogether, our results demonstrate that Nluc-expressing recombinant IAV represent a valuable tool for in vitro and in vivo studies, including the identification of antivirals and/or neutralizing antibodies, and to assess protective efficacy of vaccines. IMPORTANCEDespite the availability of recombinant influenza A viruses (IAV) expressing fluorescent reporter genes to track viral infections in vitro and ex vivo, these viruses are often attenuated and do not represent the best option for imaging entire animals using in vivo imaging systems (IVIS). To solve this limitation, we generated recombinant influenza pandemic A/California/04/2009 H1N1 expressing nanoluciferase (pH1N1-Nluc) from the viral non-structural (NS) segment and demonstrate how expression of Nluc does not affect viral replication in vitro or viral pathogenesis in vivo. Importantly, we demonstrate the feasibility of detecting pH1N1-Nluc infection in vivo using IVIS. We also validate the flexibility of this approach by generating an influenza A/Puerto Rico/8/1934 H1N1 (PR8-Nluc). Our results support the feasibility of using these recombinant IAVs expressing Nluc from the NS segment for in vitro and in vivo studies, including the identification of neutralizing antibodies and/or antivirals, and to assess protective efficacy of vaccines.

microbiology↗

bioRxiv2025

Malaria parasite population genomics during an elimination program in Eastern Myanmar

BackgroundWe investigated parasite population genomics during an intensive malaria elimination program in Kayin State (Myanmar), in which malaria posts were used for rapid detection and treatment of malaria cases, while mass drug administration (MDA) was used in villages with high submicroscopic reservoirs. MethodsWe collected 5014 dried blood spots from Plasmodium falciparum infected patients from 413 malaria posts, over 58-months (November 2015 - August 2020), and sequenced 2270 parasite genomes, each with geographic references (latitude and longitude). We used identity-by-descent (IBD) relationships to examine how control efforts impact parasite population structure. FindingsParasites were genetically depauperate: 1726 single-genotype infections comprised 166 unique genomes ([≥]90% IBD), while nine families ([≥]45% IBD) accounted for 62{middle dot}5% of parasites sampled. We observed localized, temporally stable transmission of unique parasite genotypes, identifying transmission chains. Parasite relatedness was positively correlated up to approximately 20 km revealing the scale of parasite subpopulations. Kelch13 diversity was stable from 2016-2019, but only one predominant clonal genotype (kelch13-R561H) remained in 2020. MDA resulted in parasite founder effects, providing genomic evidence for the efficacy of this malaria control tool. InterpretationOur genomic data show that parasite population size decreased over the study period, and we observed regional distribution of parasite genotypes, which can define operational units for parasite control. One parasite genotype (kelch13-R561H) from the north rose to high frequency in 2020, because transmission was halted elsewhere in the control area. Future surveillance will reveal whether this genotype spreads to neighboring regions. Genetic drift may have a stronger impact on parasite population structure than selection in low-transmission elimination settings. FundingNational Institutes of Health; Wellcome Trust; Global Fund to Fight against AIDS, Tuberculosis and Malaria; The Bill and Melinda Gates Foundation. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSThis study describes the genomic analysis of malaria parasites collected during a malaria elimination program targeting four townships (Myawaddy, Kawkareik, Hlaingbwe, and Hpapun) in Kayin State, eastern Myanmar. We searched PubMed on May 10, 2025, using the terms: "malaria", "transmission", "drug resistance", "southeast Asia" and "genetic surveillance" without any date or language restrictions. Our search yielded 58 publications, including reports on declined immunity to malaria and increased drug resistance in southeast Asia; genetic diversity and population structure shaped by mass drug administration, transmission intensity, human movement and mosquito ecology; and the spread of kelch13 and pfcrt mutations conferring dihydroartemimsinin-piperaquine resistance in east southeast Asia countries. Added value of this studyWe sequenced and analyzed 2270 Plasmodium falciparum whole genomes (each with geographic location) collected between November 2015 and August 2020 in Kayin State. We described fine-scale molecular epidemiology changes during intense malaria elimination interventions. We observed localized, temporally stable transmission of parasite genotypes, with different parasite families and kelch13 haplotypes predominating in different subregions over the 5-year period. We found kelch13 haplotypes with local or regional origin, but none originating from eastern southeast Asia. The genomic data indicated decreasing parasite population size, but we observed no selection towards drug-resistance parasites. In 2020, only one predominant lineage (kelch13-R561H) remained in our studying region, consistent with genetic drift in a pre-elimination setting. Implications of all the available evidenceThe parasite population was genetically depauperate, with a spatially localized and temporally stable distribution of parasite lineages, and declining population size. In this situation genetic drift may play a heightened role in parasite epidemiology. Consistent with this, parasites carrying kelch13-R561H rose to high frequency in 2020, because transmission was eliminated in all regions except the north of Kayin State, where parasites bearing this kelch13 genotype have predominated since 2017. Future surveillance will determine whether this parasite genotype is transmitted to neighboring regions, and thus related to changes in P. falciparum drug resistance dynamics on the Thailand Myanmar border. These data illustrate how genetic drift in small populations can result in instantaneous changes in the resistance status of parasite populations in near elimination settings.

genomics↗

bioRxiv2025

Genetic crosses reveal genomic loci responsible for virulence in Cryptosporidium parvum infection

The relationship between parasite genotype and pathogenesis is largely unknown for Cryptosporidium, a leading cause of diarrheal disease in children. An array of parasites with similar genomes produces varied disease outcomes in different hosts. Here, we isolate and characterize Cryptosporidium parvum strains that show marked differences in virulence and persistence in mice. Taking advantage of the sexual lifecycle of this eukaryotic pathogen, we use genetic crosses to discover the underlying chromosomal loci. Whole-genome sequencing and bulk segregant analysis of infection selected progeny mapped three loci on chromosomes 2, 6, and 7 associated with the ability to colonize and persist in mice and the positions of drug resistance genes. The chromosome 6 locus encodes the hyper-polymorphic surface glycoprotein GP60. Reverse genetic studies in both parental strains demonstrate that GP60 controls parasite burden and virulence, but not persistence, and reveal the dominance of the less virulent allele, suggesting it restricts virulence.

genetics↗

bioRxiv2025

A Live Attenuated Vaccine Candidate against Emerging Highly Pathogenic Cattle-Origin 2.3.4.4b H5N1 Viruses

Influenza viruses present a significant public health risk, causing substantial illness and death in humans each year. Seasonal flu vaccines must be updated regularly, and their effectiveness often decreases due to mismatches with circulating strains. Furthermore, inactivated vaccines do not provide protection against shifted influenza viruses that have the potential to cause a pandemic. The highly pathogenic avian influenza H5N1 clade 2.3.4.4b is prevalent among wild birds worldwide and is causing a multi-state outbreak affecting poultry and dairy cows in the United States (US) since March 2024. In this study, we have generated a NS1 deficient mutant of a low pathogenic version of the cattle-origin human influenza A/Texas/37/2024 H5N1, namely LPhTXdNS1, and validated its safety, immunogenicity, and protection efficacy in a prime vaccination regimen against wild-type (WT) A/Texas/37/2024 H5N1. The attenuation of LPhTXdNS1 in vitro was confirmed by its reduced replication in cultured cells and inability to control IFN{beta} promoter activation. In C57BL/6J mice, LPhTXdNS1 has reduced viral replication and pathogenicity compared to WT A/Texas/37/2024 H5N1. Notably, LPhTXdNS1 vaccinated mice exhibited high immunogenicity that reach its peak at weeks 3 and 4 post-immunization, leading to robust protection against subsequent lethal challenge with WT A/Texas/37/2024 H5N1. Altogether, we demonstrate that a single dose vaccination with LPhTXdNS1 is safe and able to induce protective immune responses against H5N1. Both safety profile and protection immunity suggest that LPhTXdNS1 holds promise as a potential solution to address the urgent need for an effective vaccine in the event of a pandemic for the treatment of infected animals and humans. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=143 HEIGHT=200 SRC="FIGDIR/small/646033v2_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@1ca0d0borg.highwire.dtl.DTLVardef@1e5232org.highwire.dtl.DTLVardef@a6694corg.highwire.dtl.DTLVardef@14b0832_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

A Human H5N1 Influenza Virus Expressing Bioluminescence for Evaluating Viral Infection and Identifying Therapeutic Interventions

A multistate outbreak of highly pathogenic avian influenza virus (HPAIV) H5N1 in dairy cows was first reported on March 25, 2024, in the United States (US), marking the first discovery of HPAIV H5N1 in cattle. Soon after, a dairy worker on an affected dairy farm became the first human case linked directly to this outbreak. Studies with influenza A virus (IAV) require secondary methods to detect the virus in infected cells or animal models of infection. We modified the non-structural (NS) genome segment of the human A/Texas/37/2024 (HPhTX) H5N1 virus to create a recombinant virus expressing nanoluciferase (HPhTX NSs-Nluc), enabling the tracking of virus in cultured cells and mice via in vitro, ex vivo, and in vivo imaging systems (IVIS). In vitro, HPhTX NSs-Nluc showed growth and plaque characteristics similar to its wild-type (WT) counterpart. In vivo, HPhTX NSs-Nluc allowed tracking viral infection in the entire animals and in the organs of infected animals using in vivo and ex vivo IVIS, respectively. Importantly, the morbidity, mortality, and replication titers of HPhTX NSs-Nluc were comparable to those of the WT HPhTX. In vitro, HPhTX NSs-Nluc was inhibited by Baloxavir acid (BXA) to levels observed with WT HPhTX. We also demonstrate the feasibility of using HPhTX NSs-Nluc to evaluate the antiviral activity of BXA in vivo. Our findings support that HPhTX NSs-Nluc represents an excellent tool for tracking viral infections, including the identification of prophylactics or therapeutics for the treatment of the HPAIV H5N1 responsible of the outbreak in dairy cows.

microbiology↗

bioRxiv2024

Measuring Growth, Resistance and Recovery after Artemisinin Treatment of Plasmodium falciparum in a semi-high-throughput Assay

BackgroundArtemisinin partial resistance (ART-R) has spread throughout Southeast Asia and mutations in pfKelch13, the molecular marker of resistance, are widely reported in East Africa. Effective in vitro assays and robust phenotypes are crucial for monitoring populations for the emergence and spread of resistance. The recently developed extended Recovery Ring-stage Survival Assay used a qPCR-based readout to reduce the labor intensiveness for in vitro phenotyping of ART-R and improved correlation with the clinical phenotype of ART-R. Here, we extend and refine this assay to include measurements of parasite growth and recovery after drug exposure. Clinical isolates and progeny from two genetic crosses were used to optimize and validate the reliability of a straight-from-blood, SYBR Green-based qPCR protocol in a 96-well plate format to accurately measure phenotypes for Growth, Resistance, and Recovery. ResultsThe assay determined growth between 6 h and 96 h, resistance at 120 h, and recovery from 120 h and 192 h. Growth can be accurately captured by qPCR and is shown by reproduction of previous growth phenotypes from HB3 x Dd2. Resistance measured at 120 h continually shows the most consistent phenotype for ring stage susceptibility. Recovery identifies an additional response to drug than parasites that are determined sensitive by Fold Change at 120 h. Comparison of progeny phenotypes for Growth vs Resistance showed a minor but significant correlation, whereas Growth vs Recovery and Resistance vs Recovery showed no significant correlation. Additionally, dried blood spot (DBS) samples matched Fold Change measured from liquid samples demonstrating Resistance can be easily quantified using either storage method. ConclusionsThe qPCR-based methodology provides the throughput needed to quickly measure large numbers of parasites for multiple relevant phenotypes. Growth can reveal fitness defects and illuminate relationships between proliferation rates and drug response. Recovery serves as a complementary phenotype to resistance that quantifies the ability of sensitive parasites to tolerate drug exposure. All three phenotypes offer a comprehensive assessment of parasite-drug interaction each with independent genetic determinants of main effect and overlapping secondary effects that should be further. By adapting our method to include DBS, readouts can be easily extended to ex vivo surveillance applications.

microbiology↗

bioRxiv2024

Transcriptional phenotype of the anti-parasitic benzodiazepine meclonazepam on the blood fluke Schistosoma mansoni

There are limited control measures for the disease schistosomiasis, despite the fact that infection with parasitic blood flukes affects hundreds of millions of people worldwide. The current treatment, praziquantel, has been in use since the 1980s and there is a concern that drug resistance may emerge with continued monotherapy. Given the need for additional antischistosomal drugs, we have re-visited an old lead, meclonazepam. In comparison to praziquantel, there has been relatively little work on its antiparasitic mechanism. Recent findings indicate that praziquantel and meclonazepam act through distinct receptors, making benzodiazepines a promising chemical series for further exploration. Previous work has profiled the transcriptional changes evoked by praziquantel treatment. Here, we examine in detail schistosome phenotypes evoked by in vitro and in vivo meclonazepam treatment. These data confirm that meclonazepam causes extensive tegument damage and directly kills parasites, as measured by pro-apoptotic caspase activation. In vivo meclonazepam exposure results in differential expression of many genes that are divergent in parasitic flatworms, as well as several gene products implicated in blood feeding and regulation of hemostasis in other parasites. Many of these transcripts are also differentially expressed with praziquantel exposure, which may reflect a common schistosome response to the two drugs. However, despite these similarities in drug response, praziquantel-resistant parasites retain susceptibility to meclonazepams schistocidal effects. These data provide new insight into the mechanism of antischistosomal benzodiazepines, resolving similarities and differences with the current frontline therapy, praziquantel.

pharmacology and toxicology↗

bioRxiv2024

Abundant genetic variation is retained in many laboratory schistosome populations

Schistosomes are obligately sexual blood flukes that can be maintained in the laboratory using freshwater snails as intermediate and rodents as definitive hosts. The genetic composition of laboratory schistosome populations is poorly understood: whether genetic variation has been purged due to serial inbreeding or retained is unclear. We sequenced 19 - 24 parasites from each of five laboratory Schistosoma mansoni populations and compared their genomes with published exome data from four S. mansoni field populations. We found abundant genomic variation (0.897 - 1.22 million variants) within laboratory populations: these retained on average 49% ({pi} = 3.27e-04 - 8.94e-04) of the nucleotide diversity observed in the four field parasite populations ({pi} = 1.08e-03 - 2.2e-03). However, the pattern of variation was very different in laboratory and field populations. Tajimas D was positive in all laboratory populations except SmBRE, indicative of recent population bottlenecks, but negative in all field populations. Current effective population size estimates of laboratory populations were lower (2 - 258) compared to field populations (3,174 - infinity). The distance between markers at which linkage disequilibrium (LD) decayed to 0.5 was longer in laboratory populations (59 bp - 180 kb) compared to field populations (9 bp - 9.5 kb). SmBRE was the least variable; this parasite also shows low fitness across the lifecycle, consistent with inbreeding depression. The abundant genetic variation present in most laboratory schistosome populations has several important implications: (i) measurement of parasite phenotypes, such as drug resistance, using laboratory parasite populations will determine average values and underestimate trait variation; (ii) genome-wide association studies (GWAS) can be conducted in laboratory schistosome populations by measuring phenotypes and genotypes of individual worms; (iii) genetic drift may lead to divergence in schistosome populations maintained in different laboratories. We conclude that the abundant genetic variation retained within many laboratory schistosome populations can provide valuable, untapped opportunities for schistosome research.

genomics↗

bioRxiv2024

Limited Impact of Schistosome Infection on Biomphalaria glabrata Snail Microbiomes

BackgroundThe microbiome of disease vectors can be a key determinant of their ability to transmit parasites. Conversely, parasite infection may modify vector microbiomes. We are exploring the interactions between the Biomphalaria glabrata snail microbiome and the blood fluke Schistosoma mansoni, responsible for an estimated 200,000 human deaths each year. Snail hosts vary in their susceptibility to schistosome parasites, and the underlying mechanisms driving this variation are not fully understood. We have previously shown that the snail hemolymph (i.e., blood) and organs harbor a diverse microbiome. Here we investigate the impact of schistosome infection on snail microbiomes, hypothesizing that invading schistosomes can alter the snail microbiomes in both composition and abundance over the course of infection, as developing schistosome parasites are in close contact with the host tissues. ResultWe generated cohorts of uninfected and S. mansoni infected snails. We collected snail hemolymph and hepatopancreas (i.e., liver) at 8 timepoints during the pre-patent and patent periods of schistosome infection. We quantified bacterial density using qPCR and profiled the microbiome composition of all samples by sequencing the V4 region of the 16S rRNA. Schistosome infection had surprisingly no effect on bacterial density and limited effect on the microbiome composition, affecting mainly the hemolymph during the pre-patent period (at day 7 and 21). Organ and hemolymph microbiomes were relatively stable overtime for both infected and uninfected snail cohorts. The sample type (hemolymph, hepatopancreas) was the major driver of the differences observed in microbiome composition. ConclusionsThe limited impact of schistosome infection on the host snail microbiomes might be explained by the long-term interaction of the two partners and the fact that parasite fitness is closely dependent on host fitness. Further investigations into the interactions between snails, their microbiomes, and schistosome parasites are essential for developing strategies to disrupt the parasite lifecycle and, consequently, schistosomiasis transmission.

microbiology↗

bioRxiv2024

An estimate of the longitudinal pace of aging from a single brain scan predicts dementia conversion, morbidity, and mortality

To understand how aging affects functional decline and increases disease risk, it is necessary to develop accurate and reliable measures of how fast a person is aging. Epigenetic clocks measure aging but require DNA methylation data, which many studies lack. Using data from the Dunedin Study, we introduce an accurate and reliable measure for the rate of longitudinal aging derived from cross-sectional brain MRI: the Dunedin Pace of Aging Calculated from NeuroImaging or DunedinPACNI. Exporting this measure to the Alzheimers Disease Neuroimaging Initiative and UK Biobank datasets revealed that faster DunedinPACNI predicted participants cognitive impairment, accelerated brain atrophy, and conversion to diagnosed dementia. Underscoring close links between longitudinal aging of the body and brain, faster DunedinPACNI also predicted physical frailty, poor health, future chronic diseases, and mortality in older adults. Furthermore, DunedinPACNI followed an established socioeconomic health gradient with people of lower socioeconomic status showing faster DunedinPACNI. Associations between DunedinPACNI and cognitive impairment were replicated in BrainLat, a sample of Latin American patients with dementia. When compared to brain age gap, an existing MRI aging biomarker, DunedinPACNI was similarly or more strongly related to clinical outcomes. DunedinPACNI is a "next generation" MRI measure that will be made publicly available to the research community to help accelerate aging research and evaluate the effectiveness of dementia prevention and anti-aging strategies.

neuroscience↗