The mosquito midgut functions as a key interface between pathogen and vector. However, studies of midgut physiology and virus infection dynamics are scarce, and in Culex tarsalis - an extremely efficient vector of West Nile virus (WNV) - nonexistent. We performed single-cell RNA sequencing on Cx. tarsalis midguts, defined multiple cell types, and determined whether specific cell types are more permissive to WNV infection. We identified 20 cell states comprising 8 distinct cell types, consistent with existing descriptions of Drosophila and Aedes aegypti midgut physiology. Most midgut cell populations were permissive to WNV infection. However, there were higher levels of WNV RNA (vRNA) in enteroendocrine cells, suggesting enhanced replication in this population. In contrast, proliferating intestinal stem cells (ISC) had the lowest levels of vRNA, a finding consistent with studies suggesting ISC proliferation in the midgut is involved in infection control. ISCs were also found to have a strong transcriptional response to WNV infection; genes involved in ribosome structure and biogenesis, and translation were significantly downregulated in WNV-infected ISC populations. Notably, we did not detect significant WNV-infection induced upregulation of canonical mosquito antiviral immune genes (e.g., AGO2 , R2D2 , etc.) at the whole-midgut level. Rather, we observed a significant positive correlation between immune gene expression levels and vRNA load in individual cells, suggesting that within midgut cells, high levels of vRNA may trigger antiviral responses. Our findings establish a Cx. tarsalis midgut cell atlas, and provide insight into midgut infection dynamics of WNV by characterizing cell-type specific enhancement/restriction of, and immune response to, infection at the single-cell level.

Rift Valley fever phlebovirus (RVFV) is a zoonotic mosquito-borne pathogen endemic to sub-Saharan Africa and the Arabian Peninsula which causes Rift Valley fever in ruminant livestock and humans. Co-infection with divergent viral strains can produce reassortment among the L, S, and M segments of the RVFV genome. Reassortment events can produce novel genotypes with altered virulence, transmission dynamics, and/or mosquito host range. This can have severe implications in areas where RVFV is endemic and convolutes our ability to anticipate transmission and circulation in novel geographic regions. Previously, we evaluated the frequency of RVFV reassortment in a susceptible ruminant host and observed low rates of reassortment (0–1.7%). Here, we tested the hypothesis that reassortment occurs predominantly in the mosquito using a highly permissive vector, Culex tarsalis. Cells derived from Cx. tarsalis or adult mosquitoes were co-infected with either two virulent (Kenya-128B-15 and SA01-1322) or a virulent and attenuated (Kenya-128B-15 and MP-12) strain of RVFV. Our results showed approximately 2% of virus genotypes isolated from co-infected Cx. tarsalis-derived cells were reassortant. Co-infected mosquitoes infected via infectious bloodmeal resulted in a higher percentage of reassortant virus (2–60%) isolated from midgut and salivary tissues at 14 days post-infection. The percentage of reassortant genotypes isolated from the midguts of mosquitoes co-infected with Kenya-128B-15 and SA01-1322 was similar to that of mosquitoes co-infected with Kenya-128B-15 and MP-12- strains (60 vs. 47%). However, only 2% of virus isolated from the salivary glands of Kenya-128B-15 and SA01-1322 co-infected mosquitoes represented reassortant genotypes. This was contrasted by 54% reassortment in the salivary glands of mosquitoes co-infected with Kenya-128B-15 and MP-12 strains. Furthermore, we observed preferential inclusion of genomic segments from the three parental strains among the reassorted viruses. Replication curves of select reassorted genotypes were significantly higher in Vero cells but not in Culex—derived cells. These data imply that mosquitoes play a crucial role in the reassortment of RVFV and potentially contribute to driving evolution of the virus.

Dengue viruses (DENVs) are the most prevalent arboviruses affecting humans. Four billion people are at risk of infection and this burden is rapidly increasing due to geographic expansion of the mosquito vector. Infection with any of the four serotypes of DENV can result in a self-limiting disease but debilitating febrile illness (DF), and some infections progress to severe disease with manifestations such as hemorrhage and shock. DENV infection drives the metabolic state of host cells for viral benefit and induces a host-immune response that has metabolic implications that link to disease. In this study, a dynamic metabolic response to DENV infection and disease was measured in 535 pediatric patient sera using liquid chromatography-mass spectrometry. The metabolome was interrogated to discover biochemical pathways and identify key metabolites perturbed in severe dengue disease. A biomarker panel of thirty-two perturbed metabolites was utilized to classify DF, and severe dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS) with high sensitivity and specificity equating to a balanced accuracy of 96.9%. Some metabolites that were structurally confirmed here belong to important biochemical pathways of omega-3 and omega-6 fatty acids, sphingolipids, purines, and tryptophan metabolism. A previously reported trend between serotonin and platelets in DHF patients has been expanded upon here to reveal a major depletion of serotonin, but not platelets, in DSS patients. This study differentiated and classified DF and DHF/DSS using a serum metabolic biomarker panel based on perturbed biochemical pathways that have potential implications for severe dengue disease. One sentence summary: Metabolic biomarkers distinguish dengue hemorrhagic fever and dengue shock syndrome from dengue fever and lend insight to severe disease pathology ### Competing Interest Statement The authors have declared no competing interest.

Science communication has historically been inequitable, with certain voices and perspectives holding the power and dominant ways of knowing being promoted over others. Recently, there has been a push toward inclusive science communication, which values diverse perspectives and ways of knowing in collaborative conversations to solve complex socioscientific issues. However, there is a lack of both trainings in inclusive science communication for undergraduate science, technology, engineering, and mathematics (STEM) students as well as established ways to evaluate the efficacy of these trainings. To address this need, we designed a new multifactorial survey based on the Theory of Planned Behavior to assess students' attitudes/norms, self-efficacy, behavioral intents, and behaviors in inclusive science communication, which we termed the Planned Behaviors in Inclusive Science Communication (PB-ISC) Scale. We utilized expert review, exploratory factor analysis, confirmatory factor analysis, cognitive interviews, and quantitative measures to gather evidence of validity supporting the proposed use of the final 4-factor, 26-item survey. This survey can be used as a tool by science communication educators and researchers to assess students' planned behavior in inclusive science communication in response to trainings or experiences in science communication or related topics like socioscientific issues, civic engagement, and citizen science.

Bluetongue virus (BTV) is a segmented, double-stranded RNA orbivirus listed by the World Organization for Animal Health and transmitted by Culicoides biting midges. Segmented viruses can reassort, which facilitates rapid and important genotypic changes. Our study evaluated reassortment in Culicoides sonorensis midges coinfected with different ratios of BTV-10 and BTV-17. Midges were fed blood containing BTV-10, BTV-17, or a combination of both serotypes at 90:10, 75:25, 50:50, 25:75, or 10:90 ratios. Midges were collected every other day and tested for infection using pan BTV and cox1 (housekeeping gene) qRT-PCR. A curve was fit to the ∆Ct values (pan BTV Ct—cox1 Ct) for each experimental group. On day 10, the midges were processed for BTV plaque isolation. Genotypes of the plaques were determined by next-generation sequencing. Pairwise comparison of ∆Ct curves demonstrated no differences in viral RNA levels between coinfected treatment groups. Plaque genotyping indicated that most plaques fully aligned with one of the parental strains; however, reassortants were detected, and in the 75:25 pool, most plaques were reassortant. Reassortant prevalence may be maximized upon the occurrence of reassortant genotypes that can outcompete the parental genotypes. BTV reassortment and resulting biological consequences are important elements to understanding orbivirus emergence and evolution.

Feline infectious peritonitis (FIP) is a systemic disease manifestation of feline coronavirus (FCoV) and is the most important cause of infectious disease-related deaths in domestic cats. FIP has a variable clinical manifestation but is most often characterized by widespread vasculitis with visceral involvement and/or neurological disease that is typically fatal in the absence of antiviral therapy. Using an aptamer-based proteomics assay, we analyzed the plasma protein profiles of cats who were naturally infected with FIP (n = 19) in comparison to the plasma protein profiles of cats who were clinically healthy and negative for FCoV (n = 17) and cats who were positive for the enteric form of FCoV (n = 9). We identified 442 proteins that were significantly differentiable; in total, 219 increased and 223 decreased in FIP plasma versus clinically healthy cat plasma. Pathway enrichment and associated analyses showed that differentiable proteins were related to immune system processes, including the innate immune response, cytokine signaling, and antigen presentation, as well as apoptosis and vascular integrity. The relevance of these findings is discussed in the context of previous studies. While these results have the potential to inform diagnostic, therapeutic, and preventative investigations, they represent only a first step, and will require further validation.

Bluetongue virus (BTV) is a segmented, double-stranded RNA virus transmitted by Culicoides midges that infects ruminants. As global temperatures increase and geographical ranges of midges expand, there is increased potential for BTV outbreaks from incursions of novel serotypes into endemic regions. However, an understanding of the effect of temperature on reassortment is lacking. The objectives of this study were to compare how temperature affected Culicoides survival, virogenesis, and reassortment in Culicoides sonorensis coinfected with two BTV serotypes. Midges were fed blood meals containing BTV-10, BTV-17, or BTV serotype 10 and 17 and maintained at 20 °C, 25 °C, or 30 °C. Midge survival was assessed, and pools of midges were collected every other day to evaluate virogenesis of BTV via qRT-PCR. Additional pools of coinfected midges were collected for BTV plaque isolation. The genotypes of plaques were determined using next-generation sequencing. Warmer temperatures impacted traits related to vector competence in offsetting ways: BTV replicated faster in midges at warmer temperatures, but midges did not survive as long. Overall, plaques with BTV-17 genotype dominated, but BTV-10 was detected in some plaques, suggesting parental strain fitness may play a role in reassortment outcomes. Temperature adds an important dimension to host–pathogen interactions with implications for transmission and evolution.

Arthropod-borne virus (arbovirus) populations exist as mutant swarms that are maintained between arthropods and vertebrates. West Nile virus (WNV) population dynamics are host-dependent. In American crows, purifying selection is weak and population diversity is high compared to American robins, which have 100- to 1000-fold lower viremia. WNV passed in robins leads to fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows for higher genetic diversity within individual avian peripheral blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a molecularly barcoded WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes in each. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to the maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation.

Error-prone replication of RNA viruses generates the genetic diversity required for adaptation within rapidly changing environments. Thus, arthropod-borne virus (arbovirus) populations exist in nature as mutant swarms that are maintained between arthropods and vertebrates. Previous studies have demonstrated that West Nile virus (WNV) population dynamics are host dependent: In American crows, which experience extremely high viremia, purifying selection is weak and population diversity is high compared to American robins, which have 100 to 1000-fold lower viremia. WNV passed in robins experiences fitness gains, whereas that passed in crows does not. Therefore, we tested the hypothesis that high crow viremia allows higher genetic diversity within individual avian peripheral-blood mononuclear cells (PBMCs), reasoning that this could have produced the previously observed host-specific differences in genetic diversity and fitness. Specifically, we infected cells and birds with a novel, barcoded version of WNV and sequenced viral RNA from single cells to quantify the number of WNV barcodes that each contained. Our results demonstrate that the richness of WNV populations within crows far exceeds that in robins. Similarly, rare WNV variants were maintained by crows more frequently than by robins. Our results suggest that increased viremia in crows relative to robins leads to maintenance of defective genomes and less prevalent variants, presumably through complementation. Our findings further suggest that weaker purifying selection in highly susceptible crows is attributable to this higher viremia, polyinfections and complementation. These studies further document the role of particular, ecologically relevant hosts in shaping virus population structure.Author Summary:WNV mutational diversity in vertebrates is species-dependent. In crows, low frequency variants are common, and viral populations are more diverse. In robins, fewer mutations become permanent fixtures of the overall viral population. We infected crows, robins and a chicken cell line with a genetically marked (barcoded) WNV. Higher levels of virus led to multiple unique WNV genomes infecting individual cells, even when a genotype was present at low levels in the input viral stock. Our findings suggest that higher levels of circulating virus in natural hosts allow less fit viruses to survive in RNA virus populations through complementation by more fit viruses. This is significant as it allows less represented and less fit viruses to be maintained at low levels until they potentially emerge when virus environments change. Overall our data reveal new insights on the relationships between host susceptibility to high viremia and virus evolution.

Intra-household contacts (HCs) of leprosy patients are at increased risk of infection by Mycobacterium leprae and about ∼5-10% will develop active disease. A prognostic tool to identify HCs with the greatest risk of progressing to active disease would enhance early leprosy diagnosis and optimize prophylactic intervention. Previous metabolomics studies suggest that host lipid mediators derived from ω-3 and ω-6 polyunsaturated fatty acids (PUFAs) are potential biomarkers for leprosy. In this study, we investigated retrospective sera of leprosy HCs by liquid chromatography-mass spectrometry and enzyme-linked immunoassay to determine whether circulating levels of ω-3 and ω-6 PUFA metabolites were altered in HCs that developed leprosy (HCDL) in comparison to those that did not (HCNDL). Sera were collected from HCs at the time of index case diagnosis and before clinical signs/symptoms of leprosy. Our findings showed that HCDL sera exhibited a distinct metabolic profile in comparison to HCDNL. Specifically, arachidonic acid, leukotriene B4, 11-hydroxyeicosatetraenoic acid, prostaglandin D2, and lipoxin A4 were elevated in HCDL. In contrast, prostaglandin E2 levels were reduced in HCDL. The ω-3 PUFAs, docosahexaenoic acid, eicosapentaenoic acid, and the docosahexaenoic acid-derived resolvin D1 and maresin-1 were also elevated in HCDL individuals compared to HCNDL. Principal component analyses provided further evidence that lipid mediators could serve as an early biomarker for progression to active leprosy. A logistic model identified resolvin D1 and D2, and prostaglandin D2 as having the greatest potential for early detection of HCs that will manifest leprosy.

The World Health Organization (WHO) emphasizes that tuberculosis (TB) in children and adolescents is often overlooked by healthcare providers and difficult to diagnose. As childhood TB cases rise, finding a diagnostic high in sensitivity and specificity is critical. In this study 91 urine samples from children aged 1-10 years were analyzed for tuberculostearic acid (TBSA) by gas chromatography/mass spectrometry (GC/MS) and capture ELISA (C-ELISA). In C-ELISA the CS35/A194-01 antibody performed very poorly with both curve-based and model-based cutoffs. The area under the ROC curve (AUC) of the CS35 OD450 values was only 0.60. Replacing the capture antibody with BJ76 gave a better performance in both sensitivity and specificity (AUC = 0.95). When these samples were analyzed by GC/MS, 41 classified as 'probable/possible' for TB were distinctly TBSA positive with ten samples having <3 ng/mL LAM. However, from the 50 samples with 'unlikely' TB classification, 36 were negative but 7 had >3 ng/mL and were designated as LAM positive. This experimental assay assessment study signifies that i) the antibody pair CS35/A194-01 that has been successful for adult active TB diagnosis is not adequate when LAM level is low as in pediatric TB; ii) no one mAb appears to recognize all TB-specific LAM epitopes.

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

We provide a pipeline for data preprocessing, biomarker selection, and classification of liquid chromatography-mass spectrometry (LCMS) serum samples to generate a prospective diagnostic test for Lyme disease. We utilize tools of machine learning (ML), e.g., sparse support vector machines (SSVM), iterative feature removal (IFR), and k-fold feature ranking to select several biomarkers and build a discriminant model for Lyme disease. We report a 98.13% test balanced success rate (BSR) of our model based on a sequestered test set of LCMS serum samples. The methodology employed is general and can be readily adapted to other LCMS, or metabolomics, data sets.

Multiple drug discovery initiatives for tuberculosis are currently ongoing to identify and develop new potent drugs with novel targets in order to shorten treatment duration. One of the drug classes with a new mode of action is DprE1 inhibitors targeting an essential process in cell wall synthesis of Mycobacterium tuberculosis. In this investigation, three DprE1 inhibitors currently in clinical trials, TBA-7371, PBTZ169, and OPC-167832, were evaluated side-by-side as single agents in the C3HeB/FeJ mouse model presenting with caseous necrotic pulmonary lesions upon tuberculosis infection. The goal was to confirm the efficacy of the DprE1 inhibitors in a mouse tuberculosis model with advanced pulmonary pathology and perform comprehensive analysis of plasma, lung, and lesion-centric drug levels to establish pharmacokinetic-pharmacodynamic (PK-PD) parameters predicting efficacy at the site of infection. Results showed significant efficacy for all three DprE1 inhibitors in the C3HeB/FeJ mouse model after 2 months of treatment. Superior efficacy was observed for OPC-167832 even at low-dose levels, which can be attributed to its low MIC, favorable distribution, and sustained retention above the MIC throughout the dosing interval in caseous necrotic lesions, where the majority of bacteria reside in C3HeB/FeJ mice. These results support further progression of the three drug candidates through clinical development for tuberculosis treatment.

Our study sought to determine whether urine lipoarabinomannan (LAM) could be validated in a sample cohort that consisted mainly of HIV uninfected individuals that presented with tuberculosis symptoms. We evaluated two tests developed in our laboratory, and used them on clinical samples from Lima, Peru where incidence of HIV is low. ELISA analysis was performed on 160 samples (from 140 adult culture-confirmed TB cases and 20 symptomatic TB-negative child controls) using 100 μL of urine after pretreatment with Proteinase K. Two different mouse monoclonal antibodies-CS35 and CHCS9-08 were used individually for capture of urine LAM. Among cases, optical density (OD 450 ) values had a positive association with higher bacillary loads. The 20 controls had negative values (below the limit of detection). The assay correctly identified all samples (97–100% accuracy confidence interval). For an alternate validation of the ELISA results, we analyzed all 160 urine samples using an antibody independent chemoanalytical approach. Samples were called positive only when LAM surrogates—tuberculostearic acid (TBSA) and d -arabinose ( d -ara)—were found to be present in similar amounts. All TB cases, including the 40 with a negative sputum smear had LAM in detectable quantities in urine. None of the controls had detectable amounts of LAM. Our study shows that urinary LAM detection is feasible in HIV uninfected, smear negative TB patients.

The World Health Organization (WHO) calls for the development of a rapid, biomarker-based, non-sputum test capable of detecting all forms of tuberculosis (TB) at the point-of-care to enable immediate treatment initiation. Lipoarabinomannan (LAM) is the only WHO-endorsed TB biomarker that can be detected in urine, an easily collected sample matrix. For obtaining optimal sensitivity, we and others have shown that some form of sample pretreatment is necessary to remove background from patient urine samples. A number of systems are paper-based often destined for resource limited settings. Our current work presents incorporation of one such sample pretreatment, proteinase K (ProK) immobilized on paper (IPK) and test its performance in comparison to standard proteinase K (SPK) treatment that involves addition and deactivation at high temperature prior to performing a capture ELISA. Herein, a simple and economical method was developed for using ProK immobilized strips to pretreat urine samples. Simplification and cost reduction of the proposed pretreatment strip were achieved by using Whatman no.1 paper and by minimizing the concentration of ProK (an expensive but necessary reagent) used to pretreat the clinical samples prior to ELISA. To test the applicability of IPK, capture ELISA was carried out on either LAM-spiked urine or the clinical samples after pretreatment with ProK at 400 μg/mL for 30 minutes at room temperature. The optimal conditions and stability of the IPK were tested and validation was performed on a set of 25 previously analyzed archived clinical urine samples with known TB and HIV status. The results of IPK and SPK treated samples were in agreement showing that the urine LAM test currently under development has the potential to reach adult and pediatric patients regardless of HIV status or site of infection, and to facilitate global TB control to improve assay performance and ultimately treatment outcomes.

It is critical for science, technology, engineering, and mathematics (STEM) students to develop competencies in science communication, including science writing. However, it can be difficult for instructors and departments to assess the quality of their students' science writing. Many published science writing rubrics are specific to certain genres like lab reports. We thus developed a Universal Science Writing Rubric (USWR) that is usable regardless of the genre or audience of science writing. This tool enables students, instructors, and departments to assess science writing written to lay or scientific audiences, focusing on important rhetorical concerns like science content and interpretation rather than simply surface features like grammar. We demonstrate the use of our USWR on various life science lab reports, scientific review articles, grant proposals, and news articles, showing that the USWR is sensitive enough to highlight statistically significant differences between groups of student writing samples and valid enough to produce results that echo published and anecdotal observations of STEM student science writing skills. Thus, the USWR is a useful tool for assessment of STEM student science writing that is widely applicable in the classroom and laboratory.

Background. Post-treatment Lyme disease symptoms/syndrome (PTLDS) occurs in approximately 10% of patients with Lyme disease following antibiotic treatment. Biomarkers or specific clinical symptoms to identify patients with PTLDS do not currently exist and the PTLDS classification is based on the report of persistent, subjective symptoms for >= 6 months following antibiotic treatment for Lyme disease. Methods. Untargeted liquid chromatography-mass spectrometry metabolomics was used to determine longitudinal metabolic responses and biosignatures in PTLDS and clinically cured non-PTLDS Lyme patients. Evaluation of biosignatures included (1) defining altered classes of metabolites, (2) elastic net regularization to define metabolites that most strongly defined PTLDS and non-PTLDS patients at different time points, (3) changes in the longitudinal abundance of metabolites, and (4) linear discriminant analysis to evaluate robustness in a second patient cohort. Results. This study determined that observable metabolic differences exist between PTLDS and non-PTLDS patients at multiple time points. The metabolites with differential abundance included those from glycerophospholipid, bile acid, and acylcarnitine metabolism. Distinct longitudinal patterns of metabolite abundance indicated a greater metabolic variability in PTLDS versus non-PTLDS patients. Small numbers of metabolites (6 to 40) could be used to define PTLDS versus non-PTLDS patients at defined time points, and the findings were validated in a second cohort of PTLDS and non-PTLDS patients. Conclusions. These data provide evidence that an objective metabolite-based measurement can distinguish patients with PTLDS and help understand the underlying biochemistry of PTLDS.

Competition between viruses and Wolbachia for host lipids is a proposed mechanism of Wolbachia -mediated virus blocking in insects. Yet, the metabolomic interaction between virus and symbiont within the mosquito has not been clearly defined. We compare the lipid profiles of Aedes aegypti mosquitoes bearing mono- or dual-infections of the Wolbachia w Mel strain and dengue virus serotype 3 (DENV3). We found metabolic signatures of infection-induced intracellular events but little evidence to support direct competition between Wolbachia and virus for host lipids. Lipid profiles of dual-infected mosquitoes resemble those of DENV3 mono-infected mosquitoes, suggesting virus-driven modulation dominates over that of Wolbachia . Interestingly, knockdown of key metabolic enzymes suggests cardiolipins are host factors for DENV3 and Wolbachia replication. These findings define the Wolbachia -DENV3 metabolic interaction as indirectly antagonistic, rather than directly competitive, and reveal new research avenues with respect to mosquito × virus interactions at the molecular level.

Identification of biomarkers for latent Mycobacterium tuberculosis infection and risk of progression to tuberculosis (TB) disease are needed to better identify individuals to target for preventive therapy, predict disease risk, and potentially predict preventive therapy efficacy. Our group developed multiple reaction monitoring mass spectrometry (MRM-MS) assays that detected M. tuberculosis peptides in serum extracellular vesicles from TB patients. We subsequently optimized this MRM-MS assay to selectively identify 40 M. tuberculosis peptides from 19 proteins that most commonly copurify with serum vesicles of patients with TB. Here, we used this technology to evaluate if M. tuberculosis peptides can also be detected in individuals with latent TB infection (LTBI). Serum extracellular vesicles from 74 individuals presumed to have latent M. tuberculosis infection (LTBI) based on close contact with a household member with TB or a recent tuberculin skin test (TST) conversion were included in this study. Twenty-nine samples from individuals with no evidence of TB infection by TST and no known exposure to TB were used as controls to establish a threshold to account for nonspecific/background signal. We identified at least one of the 40 M. tuberculosis peptides in 70 (95%) individuals with LTBI. A single peptide from the glutamine synthetase (GlnA1) enzyme was identified in 61/74 (82%) individuals with LTBI, suggesting peptides from M. tuberculosis proteins involved in nitrogen metabolism might be candidates for pathogen-specific biomarkers for detection of LTBI. The detection of M. tuberculosis peptides in serum extracellular vesicles from persons with LTBI represents a potential advance in the diagnosis of LTBI.