A protease-activatable luminescent biosensor and reporter cell line for authentic SARS-CoV-2 infection.

Efforts to define serological correlates of protection against COVID-19 have been hampered by the lack of a simple, scalable, standardised assay for SARS-CoV-2 infection and antibody neutralisation. Plaque assays remain the gold standard, but are impractical for high-throughput screening. In this study, we show that expression of viral proteases may be used to quantitate infected cells. Our assays exploit the cleavage of specific oligopeptide linkers, leading to the activation of cell-based optical biosensors. First, we characterise these biosensors using recombinant SARS-CoV-2 proteases. Next, we confirm their ability to detect viral protease expression during replication of authentic virus. Finally, we generate reporter cells stably expressing an optimised luciferase-based biosensor, enabling viral infection to be measured within 24 h in a 96- or 384-well plate format, including variants of concern. We have therefore developed a luminescent SARS-CoV-2 reporter cell line, and demonstrated its utility for the relative quantitation of infectious virus and titration of neutralising antibodies.

Evolutionary analysis of species-specific duplications in flatworm genomes.

Platyhelminthes, also known as flatworms, is a phylum of bilaterian invertebrates infamous for their parasitic representatives. The classes Cestoda, Monogenea, and Trematoda comprise parasitic helminths inhabiting multiple hosts, including fishes, humans, and livestock, and are responsible for considerable economic damage and burden on human health. As in other animals, the genomes of flatworms have a wide variety of paralogs, genes related via duplication, whose origins could be mapped throughout the evolution of the phylum. Through in-silico analysis, we studied inparalogs, i.e., species-specific duplications, focusing on their biological functions, expression changes, and evolutionary rate. These genes are thought to be key players in the adaptation process of species to each particular niche. Our results showed that genes related with specific functional terms, such as response to stress, transferase activity, oxidoreductase activity, and peptidases, are overrepresented among inparalogs. This trend is conserved among species from different classes, including free-living species. Available expression data from Schistosoma mansoni, a parasite from the trematode class, demonstrated high conservation of expression patterns between inparalogs, but with notable exceptions, which also display evidence of rapid evolution. We discuss how natural selection may operate to maintain these genes and the particular duplication models that fit better to the observations. Our work supports the critical role of gene duplication in the evolution of flatworms, representing the first study of inparalogs evolution at the genome-wide level in this group.

Screening of a Library of Recombinant Schistosoma mansoni Proteins With Sera From Murine and Human Controlled Infections Identifies Early Serological Markers.

BACKGROUND: Schistosomiasis is a major global health problem caused by blood-dwelling parasitic worms, which is currently tackled primarily by mass administration of the drug praziquantel. Appropriate drug treatment strategies are informed by diagnostics that establish the prevalence and intensity of infection, which, in regions of low transmission, should be highly sensitive. METHODS: To identify sensitive new serological markers of Schistosoma mansoni infections, we have compiled a recombinant protein library of parasite cell-surface and secreted proteins expressed in mammalian cells. RESULTS: Together with a time series of sera samples from volunteers experimentally infected with a defined number of male parasites, we probed this protein library to identify several markers that can detect primary infections with as low as 10 parasites and as early as 5 weeks postinfection. CONCLUSIONS: These new markers could be further explored as valuable tools to detect ongoing and previous S mansoni infections, including in endemic regions where transmission is low.

HIV-1 Integrates Widely throughout the Genome of the Human Blood Fluke Schistosoma mansoni.

Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate.

A novel member of the let-7 microRNA family is associated with developmental transitions in filarial nematode parasites.

BACKGROUND: Filarial nematodes are important pathogens in the tropics transmitted to humans via the bite of blood sucking arthropod vectors. The molecular mechanisms underpinning survival and differentiation of these parasites following transmission are poorly understood. microRNAs are small non-coding RNA molecules that regulate target mRNAs and we set out to investigate whether they play a role in the infection event. RESULTS: microRNAs differentially expressed during the early post-infective stages of Brugia pahangi L3 were identified by microarray analysis. One of these, bpa-miR-5364, was selected for further study as it is upregulated ~12-fold at 24 hours post-infection, is specific to clade III nematodes, and is a novel member of the let-7 family, which are known to have key developmental functions in the free-living nematode Caenorhabditis elegans. Predicted mRNA targets of bpa-miR-5364 were identified using bioinformatics and comparative genomics approaches that relied on the conservation of miR-5364 binding sites in the orthologous mRNAs of other filarial nematodes. Finally, we confirmed the interaction between bpa-miR-5364 and three of its predicted targets using a dual luciferase assay. CONCLUSIONS: These data provide new insight into the molecular mechanisms underpinning the transmission of third stage larvae of filarial nematodes from vector to mammal. This study is the first to identify parasitic nematode mRNAs that are verified targets of specific microRNAs and demonstrates that post-transcriptional control of gene expression via stage-specific expression of microRNAs may be important in the success of filarial infection.

The genome of the blood fluke Schistosoma mansoni.

Schistosoma mansoni is responsible for the neglected tropical disease schistosomiasis that affects 210 million people in 76 countries. Here we present analysis of the 363 megabase nuclear genome of the blood fluke. It encodes at least 11,809 genes, with an unusual intron size distribution, and new families of micro-exon genes that undergo frequent alternative splicing. As the first sequenced flatworm, and a representative of the Lophotrochozoa, it offers insights into early events in the evolution of the animals, including the development of a body pattern with bilateral symmetry, and the development of tissues into organs. Our analysis has been informed by the need to find new drug targets. The deficits in lipid metabolism that make schistosomes dependent on the host are revealed, and the identification of membrane receptors, ion channels and more than 300 proteases provide new insights into the biology of the life cycle and new targets. Bioinformatics approaches have identified metabolic chokepoints, and a chemogenomic screen has pinpointed schistosome proteins for which existing drugs may be active. The information generated provides an invaluable resource for the research community to develop much needed new control tools for the treatment and eradication of this important and neglected disease.

SchistoDB: an updated genome resource for the three key schistosomes of humans.

The new release of SchistoDB (http://SchistoDB.net) provides a rich resource of genomic data for key blood flukes (genus Schistosoma) which cause disease in hundreds of millions of people worldwide. SchistoDB integrates whole-genome sequence and annotation of three species of the genus and provides enhanced bioinformatics analyses and data-mining tools. A simple, yet comprehensive web interface provided through the Strategies Web Development Kit is available for the mining and visualization of the data. Genomic scale data can be queried based on BLAST searches, annotation keywords and gene ID searches, gene ontology terms, sequence motifs, protein characteristics and phylogenetic relationships. Search strategies can be saved within a user's profile for future retrieval and may also be shared with other researchers using a unique web address.

Progressive cross-reactivity in IgE responses: an explanation for the slow development of human immunity to schistosomiasis?

People in regions of Schistosoma mansoni endemicity slowly acquire immunity, but why this takes years to develop is still not clear. It has been associated with increases in parasite-specific IgE, induced, some investigators propose, to antigens exposed during the death of adult worms. These antigens include members of the tegumental-allergen-like protein family (TAL1 to TAL13). Previously, in a group of S. mansoni-infected Ugandan males, we showed that IgE responses to three TALs expressed in worms (TAL1, -3, and -5) became more prevalent with age. Now, in a subcohort we examined associations of these responses with resistance to reinfection and use the data to propose a mechanism for the slow development of immunity. IgE was measured 9 weeks posttreatment and at reinfection at 2 years (n = 144). An anti-TAL5 IgE (herein referred to as TAL5 IgE) response was associated with reduced reinfection even after adjusting for age using regression analysis (geometric mean odds ratio, 0.24; P = 0.016). TAL5 IgE responders were a subset of TAL3 IgE responders, themselves a subset of TAL1 responders. TAL3 IgE and TAL5 IgE were highly cross-reactive, with TAL3 the immunizing antigen and TAL5 the cross-reactive antigen. Transcriptional and translational studies show that TAL3 is most abundant in adult worms and that TAL5 is most abundant in infectious larvae. We propose that in chronic schistosomiasis, older individuals have repeatedly experienced IgE antigens exposed when adult worms die (e.g., TAL3) and that this leads to increasing cross-reactivity with antigens of invading larvae (e.g., TAL5). Progressive accumulation of worm/larvae cross-reactivity could explain the age-dependent immunity observed in areas of endemicity.

A bioactive phlebovirus-like envelope protein in a hookworm endogenous virus.

Endogenous viral elements (EVEs), accounting for 15% of our genome, serve as a genetic reservoir from which new genes can emerge. Nematode EVEs are particularly diverse and informative of virus evolution. We identify Atlas virus-an intact retrovirus-like EVE in the human hookworm Ancylostoma ceylanicum, with an envelope protein genetically related to GN-GC glycoproteins from the family Phenuiviridae. A cryo-EM structure of Atlas GC reveals a class II viral membrane fusion protein fold not previously seen in retroviruses. Atlas GC has the structural hallmarks of an active fusogen. Atlas GC trimers insert into membranes with endosomal lipid compositions and low pH. When expressed on the plasma membrane, Atlas GC has cell-cell fusion activity. With its preserved biological activities, Atlas GC has the potential to acquire a cellular function. Our work reveals structural plasticity in reverse-transcribing RNA viruses.

A beginner's guide to manual curation of transposable elements.

BACKGROUND: In the study of transposable elements (TEs), the generation of a high confidence set of consensus sequences that represent the diversity of TEs found in a given genome is a key step in the path to investigate these fascinating genomic elements. Many algorithms and pipelines are available to automatically identify putative TE families present in a genome. Despite the availability of these valuable resources, producing a library of high-quality full-length TE consensus sequences largely remains a process of manual curation. This know-how is often passed on from mentor-to-mentee within research groups, making it difficult for those outside the field to access this highly specialised skill. RESULTS: Our manuscript attempts to fill this gap by providing a set of detailed computer protocols, software recommendations and video tutorials for those aiming to manually curate TEs. Detailed step-by-step protocols, aimed at the complete beginner, are presented in the Supplementary Methods. CONCLUSIONS: The proposed set of programs and tools presented here will make the process of manual curation achievable and amenable to all researchers and in special to those new to the field of TEs.

stepRNA: Identification of Dicer cleavage signatures and passenger strand lengths in small RNA sequences.

The enzyme Dicer is a component of many small RNA (sRNA) pathways involved in RNA processing for post-transcriptional regulation, anti-viral response and control of transposable elements. Cleavage of double-stranded RNA by Dicer produces a signature overhanging sequence at the 3' end of the sRNA sequence relative to a complementary passenger strand in a RNA duplex. There is a need for reliable tools to computationally search for Dicer cleavage signatures to help characterise families of sRNAs. This is increasingly important due to the rising popularity of sRNA sequencing, especially in non-model organisms. Here, we present stepRNA, a fast, local tool that identifies (i) overhang signatures strongly indicative of Dicer cleavage in RNA sequences, and (ii) the length of the passenger strand in sRNAs duplexes. We demonstrate the use of stepRNA with simulated and biological datasets to detect Dicer cleavage signatures in experimentally validated examples. Compared to currently available tools, stepRNA is more accurate, requires only sRNA sequence data rather than a reference genome, and provides information about other important features such as passenger strand length. stepRNA is freely available at https://github.com/Vicky-Hunt-Lab/stepRNA and is easily installable.

Correction: MiR-277/4989 regulate transcriptional landscape during juvenile to adult transition in the parasitic helminth Schistosoma mansoni.

[This corrects the article DOI: 10.1371/journal.pntd.0005559.].

Mapping Rora expression in resting and activated CD4+ T cells.

The transcription factor Rora has been shown to be important for the development of ILC2 and the regulation of ILC3, macrophages and Treg cells. Here we investigate the role of Rora across CD4+ T cells in general, but with an emphasis on Th2 cells, both in vitro as well as in the context of several in vivo type 2 infection models. We dissect the function of Rora using overexpression and a CD4-conditional Rora-knockout mouse, as well as a RORA-reporter mouse. We establish the importance of Rora in CD4+ T cells for controlling lung inflammation induced by Nippostrongylus brasiliensis infection, and have measured the effect on downstream genes using RNA-seq. Using a systematic stimulation screen of CD4+ T cells, coupled with RNA-seq, we identify upstream regulators of Rora, most importantly IL-33 and CCL7. Our data suggest that Rora is a negative regulator of the immune system, possibly through several downstream pathways, and is under control of the local microenvironment.

Thioredoxin and glutathione systems differ in parasitic and free-living platyhelminths.

BACKGROUND: The thioredoxin and/or glutathione pathways occur in all organisms. They provide electrons for deoxyribonucleotide synthesis, function as antioxidant defenses, in detoxification, Fe/S biogenesis and participate in a variety of cellular processes. In contrast to their mammalian hosts, platyhelminth (flatworm) parasites studied so far, lack conventional thioredoxin and glutathione systems. Instead, they possess a linked thioredoxin-glutathione system with the selenocysteine-containing enzyme thioredoxin glutathione reductase (TGR) as the single redox hub that controls the overall redox homeostasis. TGR has been recently validated as a drug target for schistosomiasis and new drug leads targeting TGR have recently been identified for these platyhelminth infections that affect more than 200 million people and for which a single drug is currently available. Little is known regarding the genomic structure of flatworm TGRs, the expression of TGR variants and whether the absence of conventional thioredoxin and glutathione systems is a signature of the entire platyhelminth phylum. RESULTS: We examine platyhelminth genomes and transcriptomes and find that all platyhelminth parasites (from classes Cestoda and Trematoda) conform to a biochemical scenario involving, exclusively, a selenium-dependent linked thioredoxin-glutathione system having TGR as a central redox hub. In contrast, the free-living platyhelminth Schmidtea mediterranea (Class Turbellaria) possesses conventional and linked thioredoxin and glutathione systems. We identify TGR variants in Schistosoma spp. derived from a single gene, and demonstrate their expression. We also provide experimental evidence that alternative initiation of transcription and alternative transcript processing contribute to the generation of TGR variants in platyhelminth parasites. CONCLUSIONS: Our results indicate that thioredoxin and glutathione pathways differ in parasitic and free-living flatworms and that canonical enzymes were specifically lost in the parasitic lineage. Platyhelminth parasites possess a unique and simplified redox system for diverse essential processes, and thus TGR is an excellent drug target for platyhelminth infections. Inhibition of the central redox wire hub would lead to overall disruption of redox homeostasis and disable DNA synthesis.

TASOR is a pseudo-PARP that directs HUSH complex assembly and epigenetic transposon control.

The HUSH complex represses retroviruses, transposons and genes to maintain the integrity of vertebrate genomes. HUSH regulates deposition of the epigenetic mark H3K9me3, but how its three core subunits - TASOR, MPP8 and Periphilin - contribute to assembly and targeting of the complex remains unknown. Here, we define the biochemical basis of HUSH assembly and find that its modular architecture resembles the yeast RNA-induced transcriptional silencing complex. TASOR, the central HUSH subunit, associates with RNA processing components. TASOR is required for H3K9me3 deposition over LINE-1 repeats and repetitive exons in transcribed genes. In the context of previous studies, this suggests that an RNA intermediate is important for HUSH activity. We dissect the TASOR and MPP8 domains necessary for transgene repression. Structure-function analyses reveal TASOR bears a catalytically-inactive PARP domain necessary for targeted H3K9me3 deposition. We conclude that TASOR is a multifunctional pseudo-PARP that directs HUSH assembly and epigenetic regulation of repetitive genomic targets.

Antagonism of PP2A is an independent and conserved function of HIV-1 Vif and causes cell cycle arrest.

The seminal description of the cellular restriction factor APOBEC3G and its antagonism by HIV-1 Vif has underpinned two decades of research on the host-virus interaction. We recently reported that HIV-1 Vif is also able to degrade the PPP2R5 family of regulatory subunits of key cellular phosphatase PP2A (PPP2R5A-E; Greenwood et al., 2016; Naamati et al., 2019). We now identify amino acid polymorphisms at positions 31 and 128 of HIV-1 Vif which selectively regulate the degradation of PPP2R5 family proteins. These residues covary across HIV-1 viruses in vivo, favouring depletion of PPP2R5A-E. Through analysis of point mutants and naturally occurring Vif variants, we further show that degradation of PPP2R5 family subunits is both necessary and sufficient for Vif-dependent G2/M cell cycle arrest. Antagonism of PP2A by HIV-1 Vif is therefore independent of APOBEC3 family proteins, and regulates cell cycle progression in HIV-infected cells.

Transcriptome of the parasitic flatworm Schistosoma mansoni during intra-mammalian development.

Schistosomes are parasitic blood flukes that survive for many years within the mammalian host vasculature. How the parasites establish a chronic infection in the hostile bloodstream environment, whilst evading the host immune response is poorly understood. The parasite develops morphologically and grows as it migrates to its preferred vascular niche, avoiding or repairing damage from the host immune system. In this study, we investigated temporal changes in gene expression during the intra-mammalian development of Schistosoma mansoni. RNA-seq data were analysed from parasites developing in the lung through to egg-laying mature adult worms, providing a comprehensive picture of in vivo intra-mammalian development. Remarkably, genes involved in signalling pathways, developmental control, and adaptation to oxidative stress were up-regulated in the lung stage. The data also suggested a potential role in immune evasion for a previously uncharacterised gene. This study not only provides a large and comprehensive data resource for the research community, but also reveals new directions for further characterising host-parasite interactions that could ultimately lead to new control strategies for this neglected tropical disease pathogen.

Peptides Derived of Kunitz-Type Serine Protease Inhibitor as Potential Vaccine Against Experimental Schistosomiasis.

Schistosomiasis is a significant public health problem in sub-Saharan Africa, China, Southeast Asia, and regions of South and Central America affecting about 189 million people. Kunitz-type serine protease inhibitors have been identified as important players in the interaction of other flatworm parasites with their mammalian hosts. They are involved in host blood coagulation, fibrinolysis, inflammation, and ion channel blocking, all of them critical biological processes, which make them interesting targets to develop a vaccine. Here, we evaluate the protective efficacy of chemically synthesized T- and B-cell peptide epitopes derived from a kunitz protein from Schistosoma mansoni. Putative kunitz-type protease inhibitor proteins were identified in the S. mansoni genome, and their expression was analyzed by RNA-seq. Gene expression analyses showed that the kunitz protein Smp_147730 (Syn. Smp_311670) was dramatically and significantly up-regulated in schistosomula and adult worms when compared to the invading cercariae. T- and B-cell epitopes were predicted using bioinformatics tools, chemically synthesized, and formulated in the Adjuvant Adaptation (ADAD) vaccination system. BALB/c mice were vaccinated and challenged with S. mansoni cercariae. Kunitz peptides were highly protective in vaccinated BALB/c mice showing significant reductions in recovery of adult females (89-91%) and in the numbers of eggs trapped in the livers (77-81%) and guts (57-77%) of mice. Moreover, liver lesions were significantly reduced in vaccinated mice (64-65%) compared to infected control mice. The vaccination regime was well-tolerated with both peptides. We propose the use of these peptides, alone or in combination, as reliable candidates for vaccination against schistosomiasis.

MiR-277/4989 regulate transcriptional landscape during juvenile to adult transition in the parasitic helminth Schistosoma mansoni.

Schistosomes are parasitic helminths that cause schistosomiasis, a disease affecting circa 200 million people, primarily in underprivileged regions of the world. Schistosoma mansoni is the most experimentally tractable schistosome species due to its ease of propagation in the laboratory and the high quality of its genome assembly and annotation. Although there is growing interest in microRNAs (miRNAs) in trematodes, little is known about the role these molecules play in the context of developmental processes. We use the completely unaware "miRNA-blind" bioinformatics tool Sylamer to analyse the 3'-UTRs of transcripts differentially expressed between the juvenile and adult stages. We show that the miR-277/4989 family target sequence is the only one significantly enriched in the transition from juvenile to adult worms. Further, we describe a novel miRNA, sma-miR-4989 showing that its proximal genomic location to sma-miR-277 suggests that they form a miRNA cluster, and we propose hairpin folds for both miRNAs compatible with the miRNA pathway. In addition, we found that expression of sma-miR-277/4989 miRNAs are up-regulated in adults while their predicted targets are characterised by significant down-regulation in paired adult worms but remain largely undisturbed in immature "virgin" females. Finally, we show that sma-miR-4989 is expressed in tegumental cells located proximal to the oesophagus gland and also distributed throughout the male worms' body. Our results indicate that sma-miR-277/4989 might play a dominant role in post-transcriptional regulation during development of juvenile worms and suggest an important role in the sexual development of female schistosomes.