HER2-driven breast cancer suppression by the JNK signaling pathway.

The HER2+ subtype of human breast cancer is associated with the malignant transformation of luminal ductal cells of the mammary epithelium. The sequence analysis of tumor DNA identifies loss of function mutations and deletions of the MAP2K4 and MAP2K7 genes that encode direct activators of the JUN NH2-terminal kinase (JNK). We report that in vitro studies of human mammary epithelial cells with CRISPR-induced mutations in the MAPK and MAP2K components of the JNK pathway caused no change in growth in 2D culture, but these mutations promoted epithelial cell proliferation in 3D culture. Analysis of gene expression signatures in 3D culture demonstrated similar changes caused by HER2 activation and JNK pathway loss. The mechanism of signal transduction cross-talk may be mediated, in part, by JNK-suppressed expression of integrin α6ß4 that binds HER2 and amplifies HER2 signaling. These data suggest that HER2 activation and JNK pathway loss may synergize to promote breast cancer. To test this hypothesis, we performed in vivo studies using a mouse model of HER2+ breast cancer with Cre/loxP-mediated ablation of genes encoding JNK (Mapk8 and Mapk9) and the MAP2K (Map2k4 and Map2k7) that activate JNK in mammary epithelial cells. Kaplan-Meier analysis of tumor development demonstrated that JNK pathway deficiency promotes HER2+-driven breast cancer. Collectively, these data identify JNK pathway genes as potential suppressors for HER2+ breast cancer.

coiaf: Directly estimating complexity of infection with allele frequencies.

In malaria, individuals are often infected with different parasite strains. The complexity of infection (COI) is defined as the number of genetically distinct parasite strains in an individual. Changes in the mean COI in a population have been shown to be informative of changes in transmission intensity with a number of probabilistic likelihood and Bayesian models now developed to estimate the COI. However, rapid, direct measures based on heterozygosity or FwS do not properly represent the COI. In this work, we present two new methods that use easily calculated measures to directly estimate the COI from allele frequency data. Using a simulation framework, we show that our methods are computationally efficient and comparably accurate to current approaches in the literature. Through a sensitivity analysis, we characterize how the distribution of parasite densities, the assumed sequencing depth, and the number of sampled loci impact the bias and accuracy of our two methods. Using our developed methods, we further estimate the COI globally from Plasmodium falciparum sequencing data and compare the results against the literature. We show significant differences in the estimated COI globally between continents and a weak relationship between malaria prevalence and COI.

Temporal and spatial analysis of Plasmodium falciparum genomics reveals patterns of parasite connectivity in a low-transmission district in Southern Province, Zambia.

BACKGROUND: Understanding temporal and spatial dynamics of malaria transmission will help to inform effective interventions and strategies in regions approaching elimination. Parasite genomics are increasingly used to monitor epidemiologic trends, including assessing residual transmission across seasons and importation of malaria into these regions. METHODS: In a low and seasonal transmission setting of southern Zambia, a total of 441 Plasmodium falciparum samples collected from 8 neighbouring health centres between 2012 and 2018 were genotyped using molecular inversion probes (MIPs n = 1793) targeting a total of 1832 neutral and geographically informative SNPs distributed across the parasite genome. After filtering for quality and missingness, 302 samples and 1410 SNPs were retained and used for downstream population genomic analyses. RESULTS: The analyses revealed most (67%, n = 202) infections harboured one clone (monogenomic) with some variation at local level suggesting low, but heterogenous malaria transmission. Relatedness identity-by-descent (IBD) analysis revealed variable distribution of IBD segments across the genome and 6% of pairs were highly-related (IBD ≥ 0.25). Some of the highly-related parasite populations persisted across multiple seasons, suggesting that persistence of malaria in this low-transmission region is fueled by parasites "seeding" across the dry season. For recent years, clusters of clonal parasites were identified that were dissimilar to the general parasite population, suggesting parasite populations were increasingly fragmented at small spatial scales due to intensified control efforts. Clustering analysis using PCA and t-SNE showed a lack of substantial parasite population structure. CONCLUSION: Leveraging both genomic and epidemiological data provided comprehensive picture of fluctuations in parasite populations in this pre-elimination setting of southern Zambia over 7 years.

Sensitive, Highly Multiplexed Sequencing of Microhaplotypes From the Plasmodium falciparum Heterozygome.

BACKGROUND: Targeted next-generation sequencing offers the potential for consistent, deep coverage of information-rich genomic regions to characterize polyclonal Plasmodium falciparum infections. However, methods to identify and sequence these genomic regions are currently limited. METHODS: A bioinformatic pipeline and multiplex methods were developed to identify and simultaneously sequence 100 targets and applied to dried blood spot (DBS) controls and field isolates from Mozambique. For comparison, whole-genome sequencing data were generated for the same controls. RESULTS: Using publicly available genomes, 4465 high-diversity genomic regions suited for targeted sequencing were identified, representing the P. falciparum heterozygome. For this study, 93 microhaplotypes with high diversity (median expected heterozygosity = 0.7) were selected along with 7 drug resistance loci. The sequencing method achieved very high coverage (median 99%), specificity (99.8%), and sensitivity (90% for haplotypes with 5% within sample frequency in dried blood spots with 100 parasites/µL). In silico analyses revealed that microhaplotypes provided much higher resolution to discriminate related from unrelated polyclonal infections than biallelic single-nucleotide polymorphism barcodes. CONCLUSIONS: The bioinformatic and laboratory methods outlined here provide a flexible tool for efficient, low-cost, high-throughput interrogation of the P. falciparum genome, and can be tailored to simultaneously address multiple questions of interest in various epidemiological settings.

Decreased Susceptibility to Dihydrofolate Reductase Inhibitors Associated With Genetic Polymorphisms in Ugandan Plasmodium falciparum Isolates.

BACKGROUND: The Plasmodium falciparum dihydrofolate reductase (PfDHFR) inhibitors pyrimethamine and cycloguanil (the active metabolite of proguanil) have important roles in malaria chemoprevention, but drug resistance challenges their efficacies. A new compound, P218, was designed to overcome resistance, but drug-susceptibility data for P falciparum field isolates are limited. METHODS: We studied ex vivo PfDHFR inhibitor susceptibilities of 559 isolates from Tororo and Busia districts, Uganda, from 2016 to 2020, sequenced 383 isolates, and assessed associations between genotypes and drug-susceptibility phenotypes. RESULTS: Median half-maximal inhibitory concentrations (IC50s) were 42 100 nM for pyrimethamine, 1200 nM for cycloguanil, 13000 nM for proguanil, and 0.6 nM for P218. Among sequenced isolates, 3 PfDHFR mutations, 51I (100%), 59R (93.7%), and 108N (100%), were very common, as previously seen in Uganda, and another mutation, 164L (12.8%), had moderate prevalence. Increasing numbers of mutations were associated with decreasing susceptibility to pyrimethamine, cycloguanil, and P218, but not proguanil, which does not act directly against PfDHFR. Differences in P218 susceptibilities were modest, with median IC50s of 1.4 nM for parasites with mixed genotype at position 164 and 5.7 nM for pure quadruple mutant (51I/59R/108N/164L) parasites. CONCLUSIONS: Resistance-mediating PfDHFR mutations were common in Ugandan isolates, but P218 retained excellent activity against mutant parasites.

Susceptibilities of Ugandan Plasmodium falciparum Isolates to Proteasome Inhibitors.

The proteasome is a promising target for antimalarial chemotherapy. We assessed ex vivo susceptibilities of fresh Plasmodium falciparum isolates from eastern Uganda to seven proteasome inhibitors: two asparagine ethylenediamines, two macrocyclic peptides, and three peptide boronates; five had median IC50 values <100 nM. TDI8304, a macrocylic peptide lead compound with drug-like properties, had a median IC50 of 16 nM. Sequencing genes encoding the ß2 and ß5 catalytic proteasome subunits, the predicted targets of the inhibitors, and five additional proteasome subunits, identified two mutations in ß2 (I204T, S214F), three mutations in ß5 (V2I, A142S, D150E), and three mutations in other subunits. The ß2 S214F mutation was associated with decreased susceptibility to two peptide boronates, with IC50s of 181 nM and 2635 nM against mutant versus 62 nM and 477 nM against wild type parasites for MMV1579506 and MMV1794229, respectively, although significance could not be formally assessed due to the small number of mutant parasites with available data. The other ß2 and ß5 mutations and mutations in other subunits were not associated with susceptibility to tested compounds. Against culture-adapted Ugandan isolates, two asparagine ethylenediamines and the peptide proteasome inhibitors WLW-vinyl sulfone and WLL-vinyl sulfone (which were not studied ex vivo) demonstrated low nM activity, without decreased activity against ß2 S214F mutant parasites. Overall, proteasome inhibitors had potent activity against P. falciparum isolates circulating in Uganda, and genetic variation in proteasome targets was uncommon.

Changing Prevalence of Potential Mediators of Aminoquinoline, Antifolate, and Artemisinin Resistance Across Uganda.

BACKGROUND: In Uganda, artemether-lumefantrine is recommended for malaria treatment and sulfadoxine-pyrimethamine for chemoprevention during pregnancy, but drug resistance may limit efficacies. METHODS: Genetic polymorphisms associated with sensitivities to key drugs were characterized in samples collected from 16 sites across Uganda in 2018 and 2019 by ligase detection reaction fluorescent microsphere, molecular inversion probe, dideoxy sequencing, and quantitative polymerase chain reaction assays. RESULTS: Considering transporter polymorphisms associated with resistance to aminoquinolines, the prevalence of Plasmodium falciparum chloroquine resistance transporter (PfCRT) 76T decreased, but varied markedly between sites (0-46% in 2018; 0-23% in 2019); additional PfCRT polymorphisms and plasmepsin-2/3 amplifications associated elsewhere with resistance to piperaquine were not seen. For P. falciparum multidrug resistance protein 1, in 2019 the 86Y mutation was absent at all sites, the 1246Y mutation had prevalence ≤20% at 14 of 16 sites, and gene amplification was not seen. Considering mutations associated with high-level sulfadoxine-pyrimethamine resistance, prevalences of P. falciparum dihydrofolate reductase 164L (up to 80%) and dihydropteroate synthase 581G (up to 67%) were high at multiple sites. Considering P. falciparum kelch protein propeller domain mutations associated with artemisinin delayed clearance, prevalence of the 469Y and 675V mutations has increased at multiple sites in northern Uganda (up to 23% and 41%, respectively). CONCLUSIONS: We demonstrate concerning spread of mutations that may limit efficacies of key antimalarial drugs.

Selection of Cytochrome b Mutants Is Rare among Plasmodium falciparum Patients Failing Treatment with Atovaquone-Proguanil in Cambodia.

Atovaquone-proguanil remains effective against multidrug-resistant Plasmodium falciparum in Southeast Asia, but resistance is mediated by a single point mutation in cytochrome b (cytb) that can arise during treatment. Among 14 atovaquone-proguanil treatment failures in a clinical trial in Cambodia, only one recrudescence harbored the cytb mutation Y268C. Deep sequencing did not detect the mutation at baseline or in the first 3 days of treatment, suggesting that it arose de novo Further sequencing across cytb similarly found no low-frequency cytb mutations that were up-selected from baseline to recrudescence. Copy number amplification in dihydroorotate dehydrogenase (DHODH) and cytb as markers of atovaquone tolerance was also absent. Cytb mutation played a minor role in atovaquone-proguanil treatment failures in an active comparator clinical trial.

Associations between Varied Susceptibilities to PfATP4 Inhibitors and Genotypes in Ugandan Plasmodium falciparum Isolates.

Among novel compounds under recent investigation as potential new antimalarial drugs are three independently developed inhibitors of the Plasmodium falciparum P-type ATPase (PfATP4): KAE609 (cipargamin), PA92, and SJ733. We assessed ex vivo susceptibilities to these compounds of 374 fresh P. falciparum isolates collected in Tororo and Busia districts, Uganda, from 2016 to 2019. Median IC50s were 65 nM for SJ733, 9.1 nM for PA92, and 0.5 nM for KAE609. Sequencing of pfatp4 for 218 of these isolates demonstrated many nonsynonymous single nucleotide polymorphisms; the most frequent mutations were G1128R (69% of isolates mixed or mutant), Q1081K/R (68%), G223S (25%), N1045K (16%), and D1116G/N/Y (16%). The G223S mutation was associated with decreased susceptibility to SJ733, PA92, and KAE609. The D1116G/N/Y mutations were associated with decreased susceptibility to SJ733, and the presence of mutations at both codons 223 and 1116 was associated with decreased susceptibility to PA92 and SJ733. In all of these cases, absolute differences in susceptibilities of wild-type (WT) and mutant parasites were modest. Analysis of clones separated from mixed field isolates consistently identified mutant clones as less susceptible than WT. Analysis of isolates from other sites demonstrated the presence of the G223S and D1116G/N/Y mutations across Uganda. Our results indicate that malaria parasites circulating in Uganda have a number of polymorphisms in PfATP4 and that modestly decreased susceptibility to PfATP4 inhibitors is associated with some mutations now present in Ugandan parasites.

Epstein-Barr Virus Genomes Reveal Population Structure and Type 1 Association with Endemic Burkitt Lymphoma.

Endemic Burkitt lymphoma (eBL), the most prevalent pediatric cancer in sub-Saharan Africa, is distinguished by its inclusion of Epstein-Barr virus (EBV). In order to better understand the impact of EBV variation in eBL tumorigenesis, we improved viral DNA enrichment methods and generated a total of 98 new EBV genomes from both eBL cases (n = 58) and healthy controls (n = 40) residing in the same geographic region in Kenya. Using our unbiased methods, we found that EBV type 1 was significantly more prevalent in eBL patients (74.5%) than in healthy children (47.5%) (odds ratio = 3.24, 95% confidence interval = 1.36 to 7.71, P = 0.007), as opposed to similar proportions in both groups. Controlling for EBV type, we also performed a genome-wide association study identifying six nonsynonymous variants in the genes EBNA1, EBNA2, BcLF1, and BARF1 that were enriched in eBL patients. In addition, viruses isolated from plasma of eBL patients were identical to their tumor counterparts consistent with circulating viral DNA originating from the tumor. We also detected three intertypic recombinants carrying type 1 EBNA2 and type 2 EBNA3 regions, as well as one novel genome with a 20-kb deletion, resulting in the loss of multiple lytic and virion genes. Comparing EBV types, viral genes displayed differential variation rates as type 1 appeared to be more divergent, while type 2 demonstrated novel substructures. Overall, our findings highlight the complexities of the EBV population structure and provide new insight into viral variation, potentially deepening our understanding of eBL oncogenesis.IMPORTANCE Improved viral enrichment methods conclusively demonstrate EBV type 1 to be more prevalent in eBL patients than in geographically matched healthy controls, which previously underrepresented the prevalence of EBV type 2. Genome-wide association analysis between cases and controls identifies six eBL-associated nonsynonymous variants in EBNA1, EBNA2, BcLF1, and BARF1 genes. Analysis of population structure reveals that EBV type 2 exists as two genomic subgroups and was more commonly found in female than in male eBL patients.

Describing the current status of Plasmodium falciparum population structure and drug resistance within mainland Tanzania using molecular inversion probes.

High-throughput Plasmodium genomic data is increasingly useful in assessing prevalence of clinically important mutations and malaria transmission patterns. Understanding parasite diversity is important for identification of specific human or parasite populations that can be targeted by control programmes, and to monitor the spread of mutations associated with drug resistance. An up-to-date understanding of regional parasite population dynamics is also critical to monitor the impact of control efforts. However, this data is largely absent from high-burden nations in Africa, and to date, no such analysis has been conducted for malaria parasites in Tanzania countrywide. To this end, over 1,000 P. falciparum clinical isolates were collected in 2017 from 13 sites in seven administrative regions across Tanzania, and parasites were genotyped at 1,800 variable positions genome-wide using molecular inversion probes. Population structure was detectable among Tanzanian P. falciparum parasites, approximately separating parasites from the northern and southern districts and identifying genetically admixed populations in the north. Isolates from nearby districts were more likely to be genetically related compared to parasites sampled from more distant districts. Known drug resistance mutations were seen at increased frequency in northern districts (including two infections carrying pfk13-R561H), and additional variants with undetermined significance for antimalarial resistance also varied by geography. Malaria Indicator Survey (2017) data corresponded with genetic findings, including average region-level complexity-of-infection and malaria prevalence estimates. The parasite populations identified here provide important information on extant spatial patterns of genetic diversity of Tanzanian parasites, to which future surveys of genetic relatedness can be compared.

Antimalarial Drug Resistance Profiling of Plasmodium falciparum Infections in Ghana Using Molecular Inversion Probes and Next-Generation Sequencing.

A key drawback to monitoring the emergence and spread of antimalarial drug resistance in sub-Saharan Africa is early detection and containment. Next-generation sequencing methods offer the resolution, sensitivity, and scale required to fill this gap by surveilling for molecular markers of drug resistance. We performed targeted sequencing using molecular inversion probes to interrogate five Plasmodium falciparum genes (pfcrt, pfmdr1, pfdhps, pfdhfr, and pfk13) implicated in chloroquine, sulfadoxine-pyrimethamine (SP), and artemisinin resistance in two sites in Ghana. A total of 803 dried blood spots from children aged between 6 months and 14 years presenting with uncomplicated P. falciparum malaria at the Begoro District Hospital in Begoro and the Ewim Polyclinic in Cape Coast, Ghana, from 2014 to 2017 were prepared on filter paper. Thirteen years after the removal of drug pressure, chloroquine-sensitive parasite strains with pfcrt K76 have increased nearly to fixation in Begoro, in the forest area (prevalence = 95%), but at a lower rate in Cape Coast, in the coastal region (prevalence = 71%, Z = -3.5, P < 0.001). In addition, pfmdr1 184F-bearing parasites are under strong selection. The pfdhfr/pfdhps quadruple genotype ( IRNG K), associated with SP resistance, is near saturation. Our study identified at a 2 to 10% prevalence pfdhps 581G, which is a sulfadoxine resistance marker that correlates with the failure of SP prophylaxis in pregnancy and which has not been observed in Ghana. The differences in the reexpansion of chloroquine-sensitive strains observed at the two study sites, the stronger SP resistance, and the high prevalence of pfmdr1 184F should be further monitored to inform malaria control strategies in Ghana.

Falciparum malaria from coastal Tanzania and Zanzibar remains highly connected despite effective control efforts on the archipelago.

BACKGROUND: Tanzania's Zanzibar archipelago has made significant gains in malaria control over the last decade and is a target for malaria elimination. Despite consistent implementation of effective tools since 2002, elimination has not been achieved. Importation of parasites from outside of the archipelago is thought to be an important cause of malaria's persistence, but this paradigm has not been studied using modern genetic tools. METHODS: Whole-genome sequencing (WGS) was used to investigate the impact of importation, employing population genetic analyses of Plasmodium falciparum isolates from both the archipelago and mainland Tanzania. Ancestry, levels of genetic diversity and differentiation, patterns of relatedness, and patterns of selection between these two populations were assessed by leveraging recent advances in deconvolution of genomes from polyclonal malaria infections. RESULTS: Significant decreases in the effective population sizes were inferred in both populations that coincide with a period of decreasing malaria transmission in Tanzania. Identity by descent analysis showed that parasites in the two populations shared long segments of their genomes, on the order of 5 cM, suggesting shared ancestry within the last 10 generations. Even with limited sampling, two of isolates between the mainland and Zanzibar were identified that are related at the expected level of half-siblings, consistent with recent importation. CONCLUSIONS: These findings suggest that importation plays an important role for malaria incidence on Zanzibar and demonstrate the value of genomic approaches for identifying corridors of parasite movement to the island.

The changing landscape of Plasmodium falciparum drug resistance in the Democratic Republic of Congo.

BACKGROUND: Drug resistant malaria is a growing concern in the Democratic Republic of the Congo (DRC), where previous studies indicate that parasites resistant to sulfadoxine/pyrimethamine or chloroquine are spatially clustered. This study explores longitudinal changes in spatial patterns to understand how resistant malaria may be spreading within the DRC, using samples from nation-wide population-representative surveys. METHODS: We selected 552 children with PCR-detectable Plasmodium falciparum infection and identified known variants in the pfdhps and pfcrt genes associated with resistance. We compared the proportion of mutant parasites in 2013 to those previously reported from adults in 2007, and identified risk factors for carrying a resistant allele using multivariate mixed-effects modeling. Finally, we fit a spatial-temporal model to the observed data, providing smooth allele frequency estimates over space and time. RESULTS: The proportion of co-occurring pfdhps K540E/A581G mutations increased by 16% between 2007 and 2013. The spatial-temporal model suggests that the spatial range of the pfdhps double mutants expanded over time, while the prevalence and range of pfcrt mutations remained steady. CONCLUSIONS: This study uses population-representative samples to describe the changing landscape of SP resistance within the DRC, and the persistence of chloroquine resistance. Vigilant molecular surveillance is critical for controlling the spread of resistance.

Drug-Resistance and Population Structure of Plasmodium falciparum Across the Democratic Republic of Congo Using High-Throughput Molecular Inversion Probes.

A better understanding of the drivers of the spread of malaria parasites and drug resistance across space and time is needed. These drivers can be elucidated using genetic tools. Here, a novel molecular inversion probe (MIP) panel targeting all major drug-resistance mutations and a set of microsatellites was used to genotype Plasmodium falciparum infections of 552 children from the 2013-2014 Demographic and Health Survey conducted in the Democratic Republic of the Congo (DRC). Microsatellite-based analysis of population structure suggests that parasites within the DRC form a homogeneous population. In contrast, sulfadoxine-resistance markers in dihydropteroate synthase show marked spatial structure with ongoing spread of double and triple mutants compared with 2007. These findings suggest that parasites in the DRC remain panmictic despite rapidly spreading antimalarial-resistance mutations. Moreover, highly multiplexed targeted sequencing using MIPs emerges as a cost-effective method for elucidating pathogen genetics in complex infections in large cohorts.

Downregulation of the Host Gene jigr1 by miR-92 Is Essential for Neuroblast Self-Renewal in Drosophila.

Intragenic microRNAs (miRNAs), located mostly in the introns of protein-coding genes, are often co-expressed with their host mRNAs. However, their functional interaction in development is largely unknown. Here we show that in Drosophila, miR-92a and miR-92b are embedded in the intron and 3'UTR of jigr1, respectively, and co-expressed with some jigr1 isoforms. miR-92a and miR-92b are highly expressed in neuroblasts of larval brain where Jigr1 expression is low. Genetic deletion of both miR-92a and miR-92b demonstrates an essential cell-autonomous role for these miRNAs in maintaining neuroblast self-renewal through inhibiting premature differentiation. We also show that miR-92a and miR-92b directly target jigr1 in vivo and that some phenotypes due to the absence of these miRNAs are partially rescued by reducing the level of jigr1. These results reveal a novel function of the miR-92 family in Drosophila neuroblasts and provide another example that local negative feedback regulation of host genes by intragenic miRNAs is essential for animal development.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania, and continued local transmission. To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo District on the coastal mainland from 2016-2018. Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Our data support importation as a main source of genetic diversity and contribution to the parasite population on Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive for malaria reemergence due to susceptible hosts and competent vectors.

Strong isolation by distance and evidence of population microstructure reflect ongoing Plasmodium falciparum transmission in Zanzibar.

Background: The Zanzibar archipelago of Tanzania has become a low-transmission area for Plasmodium falciparum. Despite being considered an area of pre-elimination for years, achieving elimination has been difficult, likely due to a combination of imported infections from mainland Tanzania and continued local transmission. Methods: To shed light on these sources of transmission, we applied highly multiplexed genotyping utilizing molecular inversion probes to characterize the genetic relatedness of 282 P. falciparum isolates collected across Zanzibar and in Bagamoyo district on the coastal mainland from 2016 to 2018. Results: Overall, parasite populations on the coastal mainland and Zanzibar archipelago remain highly related. However, parasite isolates from Zanzibar exhibit population microstructure due to the rapid decay of parasite relatedness over very short distances. This, along with highly related pairs within shehias, suggests ongoing low-level local transmission. We also identified highly related parasites across shehias that reflect human mobility on the main island of Unguja and identified a cluster of highly related parasites, suggestive of an outbreak, in the Micheweni district on Pemba island. Parasites in asymptomatic infections demonstrated higher complexity of infection than those in symptomatic infections, but have similar core genomes. Conclusions: Our data support importation as a main source of genetic diversity and contribution to the parasite population in Zanzibar, but they also show local outbreak clusters where targeted interventions are essential to block local transmission. These results highlight the need for preventive measures against imported malaria and enhanced control measures in areas that remain receptive to malaria reemergence due to susceptible hosts and competent vectors. Funding: This research was funded by the National Institutes of Health, grants R01AI121558, R01AI137395, R01AI155730, F30AI143172, and K24AI134990. Funding was also contributed from the Swedish Research Council, Erling-Persson Family Foundation, and the Yang Fund. RV acknowledges funding from the MRC Centre for Global Infectious Disease Analysis (reference MR/R015600/1), jointly funded by the UK Medical Research Council (MRC) and the UK Foreign, Commonwealth & Development Office (FCDO), under the MRC/FCDO Concordat agreement and is also part of the EDCTP2 program supported by the European Union. RV also acknowledges funding by Community Jameel.

IFIH1 (MDA5) is required for innate immune detection of intron-containing RNA expressed from the HIV-1 provirus.

Antiretroviral therapy (ART) suppresses HIV-1 viremia and prevents progression to AIDS. Nonetheless, chronic inflammation is a common problem for people living with HIV-1 on ART. One possible cause of inflammation is ongoing transcription from HIV-1 proviruses, whether or not the sequences are competent for replication. Previous work has shown that intron-containing RNA expressed from the HIV-1 provirus in primary human blood cells, including CD4+ T cells, macrophages, and dendritic cells, activates type 1 interferon. This activation required HIV-1 rev and was blocked by the XPO1 (CRM1)-inhibitor leptomycin. To identify the innate immune receptor required for detection of intron-containing RNA expressed from the HIV-1 provirus, a loss-of-function screen was performed with shRNA-expressing lentivectors targeting twenty-one candidate genes in human monocyte derived dendritic cells. Among the candidate genes tested, only knockdown of XPO1 (CRM1), IFIH1 (MDA5), or MAVS prevented activation of the IFN-stimulated gene ISG15. The importance of IFIH1 protein was demonstrated by rescue of the knockdown with non-targetable IFIH1 coding sequence. Inhibition of HIV-1-induced ISG15 by the IFIH1-specific Nipah virus V protein, and by IFIH1-transdominant inhibitory CARD-deletion or phosphomimetic point mutations, indicates that IFIH1 filament formation, dephosphorylation, and association with MAVS, are all required for innate immune activation in response to HIV-1 transduction. Since both IFIH1 and DDX58 (RIG-I) signal via MAVS, the specificity of HIV-1 RNA detection by IFIH1 was demonstrated by the fact that DDX58 knockdown had no effect on activation. RNA-Seq showed that IFIH1-knockdown in dendritic cells globally disrupted the induction of IFN-stimulated genes. Finally, specific enrichment of unspliced HIV-1 RNA by IFIH1 was revealed by formaldehyde crosslinking immunoprecipitation (f-CLIP). These results demonstrate that IFIH1 is required for innate immune activation by intron-containing RNA from the HIV-1 provirus, and potentially contributes to chronic inflammation in people living with HIV-1.

Plasmodium falciparum gametocyte carriage in longitudinally monitored incident infections is associated with duration of infection and human host factors.

Malaria transmission depends on the presence of Plasmodium gametocytes that are the only parasite life stage that can infect mosquitoes. Gametocyte production varies between infections and over the course of infections. Infection duration is highly important for gametocyte production but poorly quantified. Between 2017 and 2019 an all-age cohort of individuals from Tororo, eastern Uganda was followed by continuous passive and routine assessments. We longitudinally monitored 104 incident infections from 98 individuals who were sampled once every 28 days and on any day of symptoms. Among infections that lasted ≥ 3 months, gametocyte appearance was near-universal with 96% of infections having detectable gametocytes prior to clearance. However, most infections were of much shorter duration; 55.7% of asymptomatic infections were detected only once. When considering all asymptomatic infections, regardless of their duration, only 36.3% had detectable gametocytes on at least one time-point prior to parasite clearance. Infections in individuals with sickle-cell trait (HbAS) were more likely to have gametocytes detected (Hazard Rate (HR) = 2.68, 95% CI 1.12, 6.38; p = 0.0231) and had gametocytes detected at higher densities (Density Ratio (DR) = 9.19, 95% CI 2.79, 30.23; p = 0.0002) compared to infections in wildtype (HbAA) individuals. Our findings suggest that a large proportion of incident infections is too short in duration and of too low density to contribute to onward transmission.