RESUMO
To prevent the non-acceptable effects of agrochemicals on arable fields, Environmental Risk Assessment (ERA) aims to assess and protect against a wide range of risks due to stressors to non-target species. While exposure to stress is a key factor in ERA models, exposure values are difficult to obtain and rely on laboratory studies with often debatable relevance to field situations. To improve intake estimates, data from realistic field-based scenarios are needed. We developed calibration curves relating known seed numbers of up to 20 onion and carrot seeds consumed by wild-caught wood mice (Apodemus sylvaticus) to the seed DNA content in the feces. Based on these inferred quantitative relationships, a field trial was run to determine seed intake in a natural setting using realistic levels of seed spillage. Onion DNA was detected in the fecal samples of the wood mice caught in the field, which resembled a seed intake of up to 1 onion seed. No intake of carrot seeds was detected. This is the first-ever study to quantify seed intake in a realistic field scenario using a DNA-based analysis, showing that accurate seed intake estimates can be obtained. Our approach can help to improve risk assessment models through its minimally-invasive and accurate assessment of seed intake by ERA representative and non-target species, which would otherwise be undetectable with traditional methods. Our novel approach and its results are highly relevant to studies of food intake and diet composition for basic and applied research alike.
Assuntos
Mamíferos , Murinae , Animais , Camundongos , Fezes/química , DNA , Medição de Risco , SementesRESUMO
Autoantibodies neutralizing type I interferons (IFN-Is) can underlie infection severity. Here, we trace the development of these autoantibodies at high-resolution using longitudinal samples from 1,876 well-treated individuals living with HIV over a 35-year period. Similar to general populations, â¼1.9% of individuals acquired anti-IFN-I autoantibodies as they aged (median onset â¼63 years). Once detected, anti-IFN-I autoantibodies persisted lifelong, and titers increased over decades. Individuals developed distinct neutralizing and non-neutralizing autoantibody repertoires at discrete times that selectively targeted combinations of IFNα, IFNß, and IFNω. Emergence of neutralizing anti-IFNα autoantibodies correlated with reduced baseline IFN-stimulated gene levels and was associated with subsequent susceptibility to severe COVID-19 several years later. Retrospective measurements revealed enrichment of pre-existing autoreactivity against other autoantigens in individuals who later developed anti-IFN-I autoantibodies, and there was evidence for prior viral infections or increased IFN at the time of anti-IFN-I autoantibody triggering. These analyses suggest that age-related loss of self-tolerance prior to IFN-I immune-triggering poses a risk of developing lifelong functional IFN-I deficiency.
Assuntos
Anticorpos Neutralizantes , Autoanticorpos , COVID-19 , Interferon Tipo I , Humanos , Autoanticorpos/imunologia , Interferon Tipo I/imunologia , Pessoa de Meia-Idade , Masculino , Feminino , COVID-19/imunologia , Anticorpos Neutralizantes/imunologia , Adulto , Idoso , SARS-CoV-2/imunologia , Infecções por HIV/imunologia , Interferon-alfa/imunologia , Estudos RetrospectivosRESUMO
Human metapneumovirus (HMPV) is one of the leading causes of respiratory illness (RI), primarily in infants. Worldwide, two genetic lineages (A and B) of HMPV are circulating that are antigenically distinct and can each be further divided into genetic sublineages. Surveillance combined with large-scale whole-genome sequencing studies of HMPV are scarce but would help to identify viral evolutionary dynamics. Here, we analyzed 130 whole HMPV genome sequences obtained from samples collected from individuals hospitalized with RI and partial fusion (n = 144) and attachment (n = 123) protein gene sequences obtained from samples collected from patients with RI visiting general practitioners between 2005 and 2021 in the Netherlands. Phylogenetic analyses demonstrated that HMPV continued to group in the four sublineages described in 2004 (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected in the Netherlands after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between samples obtained from patients with RI being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. In the past, different names for the newly energing lineages have been proposed, demonstrating the need for a consistent naming convention. Here, criteria are proposed for the designation of new genetic lineages to aid in moving toward a systematic HMPV classification. IMPORTANCE Human metapneumovirus (HMPV) is one of the major causative agents of human respiratory tract infections. Monitoring of virus evolution could aid toward the development of new antiviral treatments or vaccine designs. Here, we studied HMPV evolution between 2005 and 2021, with viruses obtained from samples collected from hospitalized individuals and patients with respiratory infections consulting general practitioners. Phylogenetic analyses demonstrated that HMPV continued to group in the four previously described sublineages (A1, A2, B1, and B2). However, one sublineage (A1) was no longer detected after 2006, while the others continued to evolve. No differences were observed in dominant (sub)lineages between patients being hospitalized and those consulting general practitioners. In both populations, viruses of lineage A2 carrying a 180-nucleotide or 111-nucleotide duplication in the attachment protein gene became the most frequently detected genotypes. These data were used to propose criteria for the designation of new genetic lineages to aid toward a systematic HMPV classification.
Assuntos
Metapneumovirus , Infecções por Paramyxoviridae , Infecções Respiratórias , Lactente , Humanos , Metapneumovirus/genética , Infecções por Paramyxoviridae/epidemiologia , Filogenia , Variação Genética , Genótipo , NucleotídeosRESUMO
DNA-based approaches have greatly improved the applicability of dietary studies aimed at investigating ecological processes. These studies have provided direct insights into, otherwise difficult to measure, interactions between species and trophic levels, food web structure and ecosystem functioning. However, despite these advances, DNA-based methods have struggled to accurately quantify the whole breadth of diet constituents because of methodological biases, such as amplification bias and digestive processes. The present study is, to our knowledge, the first diet study to use droplet digital PCR to quantify diet constituents. We manipulated the diet of wild-caught wood mice (Apodemus sylvaticus) by feeding them with a known amount of small vegetable seeds (onion and carrot) and quantified the DNA traces of these diet constituents in faecal samples. The sensitivity of the technique combined with the control on the experimental design allowed mitigation of methodological bias. We were able to accurately determine DNA concentrations of small vegetable seeds in the diet of wood mice. Quantification of target DNA demonstrated significant differences in DNA content when one vs. five seeds were consumed. These differences remained significant when the age, sex and other diet constituents of the mice were altered. Different DNA markers, targeting different parts of the chloroplast, influenced onion DNA detectability. However, all onion and carrot markers showed higher DNA content for higher seed numbers. Overall, the sensitive DNA-based approach developed in this study allows for minimally invasive quantification of small diet constituents in faeces, which would otherwise be undetectable with traditional methods.
Assuntos
Dieta , Ecossistema , Animais , DNA/genética , Fezes , Mamíferos , CamundongosRESUMO
Human metapneumovirus (HMPV), a member of the Pneumoviridae family, is a causative agent of respiratory illness in young children, the elderly, and immunocompromised individuals. Globally, viruses belonging to two main genetic lineages circulate, A and B, which are further divided into four genetic sublineages (A1, A2, B1, B2). Classical genotyping of HMPV is based on the sequence of the fusion (F) and attachment (G) glycoprotein genes, which are under direct antibody-mediated immune pressure. Whole genome sequencing provides more information than sequencing of subgenomic fragments and is therefore a powerful tool for studying virus evolution and disease epidemiology and for identifying transmission events and nosocomial outbreaks. Here, we report a robust method to obtain whole genome sequences for HMPV using MinION Nanopore technology. This assay is able to generate HMPV whole genome sequences from clinical specimens with good coverage of the highly variable G gene and is equally sensitive for strains of all four genetic HMPV sublineages. This method can be used for studying HMPV genetics, epidemiology, and evolutionary dynamics.
Assuntos
Metapneumovirus , Nanoporos , Infecções por Paramyxoviridae , Infecções Respiratórias , Idoso , Criança , Pré-Escolar , Variação Genética , Humanos , Lactente , Metapneumovirus/genética , Filogenia , Tecnologia , Sequenciamento Completo do GenomaRESUMO
The HIV latent reservoir forms the major hurdle to an HIV cure. The discovery of CD32 as marker of this reservoir has aroused much interest, but subsequent reports have challenged this finding. Here, we observe a positive correlation between the percentages of CD32+ cells among CD4+ T cells of aviremic cART-treated, HIV-infected individuals and their HIV DNA loads in peripheral blood. Moreover, optimization of the CD32+CD4+ T cell purification protocol reveals prominent enrichment for HIV DNA (mean, 292-fold) in these cells. However, no enrichment for HIV RNA is observed in CD32+CD4+ cells, yielding significantly reduced HIV RNA/DNA ratios. Furthermore, HIV proviruses in CD32+CD4+ cells can be reactivated ex vivo to produce virus, strongly suggesting that these cells support HIV transcriptional latency. Our results underscore the importance of isolating pure, bona fide CD32+CD4+ T cells for future studies and indicate that CD32 remains a promising candidate marker of the HIV reservoir.
Assuntos
Linfócitos T CD4-Positivos/metabolismo , DNA Viral/genética , HIV-1/genética , Receptores de IgG/metabolismo , Latência Viral/genética , HumanosRESUMO
BACKGROUND: The latent HIV-1 reservoir in treated patients primarily consists of resting memory CD4+ T cells. Stimulating the T-cell receptor (TCR), which facilitates transition of resting into effector T cells, is the most effective strategy to purge these latently infected cells. Here we supply evidence that TCR-stimulated effector T cells still frequently harbor latent HIV-1. METHODS: Primary HIV-1 infected cells were used in a latency assay with or without dendritic cells (DCs) and reversion of HIV-1 latency was determined, in the presence or absence of specific pathway inhibitors. FINDINGS: Renewed TCR-stimulation or subsequent activation with latency reversing agents (LRAs) did not overcome latency. However, interaction of infected effector cells with DCs triggered further activation of latent HIV-1. When compared to TCR-stimulation only, CD4+ T cells from aviremic patients receiving TCRâ¯+â¯DC-stimulation reversed latency more frequently. Such a "one-two punch" strategy seems ideal for purging the reservoir. We determined that DC contact activates the PI3K-Akt-mTOR pathway in CD4+ T cells. INTERPRETATION: This insight could facilitate the development of a novel class of potent LRAs that purge latent HIV beyond levels reached by T-cell activation.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , HIV-1/fisiologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Latência Viral , Adulto , Idoso , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/metabolismo , Linfócitos T CD4-Positivos/virologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Infecções por HIV/tratamento farmacológico , Infecções por HIV/imunologia , HIV-1/efeitos dos fármacos , Humanos , Ativação Linfocitária/imunologia , Masculino , Pessoa de Meia-Idade , Modelos Biológicos , NF-kappa B/metabolismo , Fosforilação , Ligação Proteica , Proteínas Proto-Oncogênicas c-fos/química , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-jun/química , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Latência Viral/imunologiaRESUMO
After initiating antiretroviral therapy (ART), a rapid decline in HIV viral load is followed by a long period of undetectable viremia. Viral outgrowth assay suggests the reservoir continues to decline slowly. Here, we use full-length sequencing to longitudinally study the proviral landscape of four subjects on ART to investigate the selective pressures influencing the dynamics of the treatment-resistant HIV reservoir. We find intact and defective proviruses that contain genetic elements favoring efficient protein expression decrease over time. Moreover, proviruses that lack these genetic elements, yet contain strong donor splice sequences, increase relatively to other defective proviruses, especially among clones. Our work suggests that HIV expression occurs to a significant extent during ART and results in HIV clearance, but this is obscured by the expansion of proviral clones. Paradoxically, clonal expansion may also be enhanced by HIV expression that leads to splicing between HIV donor splice sites and downstream human exons.
Assuntos
Terapia Antirretroviral de Alta Atividade , Antivirais/uso terapêutico , Infecções por HIV/tratamento farmacológico , HIV-1/efeitos dos fármacos , Adulto , Feminino , Infecções por HIV/virologia , HIV-1/genética , HIV-1/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , Mutação , Filogenia , Provírus/classificação , Provírus/efeitos dos fármacos , Provírus/genética , Carga Viral/efeitos dos fármacos , Viremia/prevenção & controle , Latência Viral/efeitos dos fármacosRESUMO
Betacoronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV), are important pathogens causing potentially lethal infections in humans and animals. Coronavirus RNA synthesis is thought to be associated with replication organelles (ROs) consisting of modified endoplasmic reticulum (ER) membranes. These are transformed into double-membrane vesicles (DMVs) containing viral double-stranded RNA and into other membranous elements such as convoluted membranes, together forming a reticulovesicular network. Previous evidence suggested that the nonstructural proteins (nsp's) 3, 4, and 6 of the severe acute respiratory syndrome coronavirus (SARS-CoV), which contain transmembrane domains, would all be required for DMV formation. We have now expressed MERS-CoV replicase self-cleaving polyprotein fragments encompassing nsp3-4 or nsp3-6, as well as coexpressed nsp3 and nsp4 of either MERS-CoV or SARS-CoV, to characterize the membrane structures induced. Using electron tomography, we demonstrate that for both MERS-CoV and SARS-CoV coexpression of nsp3 and nsp4 is required and sufficient to induce DMVs. Coexpression of MERS-CoV nsp3 and nsp4 either as individual proteins or as a self-cleaving nsp3-4 precursor resulted in very similar DMVs, and in both setups we observed proliferation of zippered ER that appeared to wrap into nascent DMVs. Moreover, when inactivating nsp3-4 polyprotein cleavage by mutagenesis, we established that cleavage of the nsp3/nsp4 junction is essential for MERS-CoV DMV formation. Addition of the third MERS-CoV transmembrane protein, nsp6, did not noticeably affect DMV formation. These findings provide important insight into the biogenesis of coronavirus DMVs, establish strong similarities with other nidoviruses (specifically, the arteriviruses), and highlight possible general principles in viral DMV formation.IMPORTANCE The RNA replication of positive stranded RNA viruses of eukaryotes is thought to take place at cytoplasmic membranous replication organelles (ROs). Double-membrane vesicles are a prominent type of viral ROs. They are induced by coronaviruses, such as SARS-CoV and MERS-CoV, as well as by a number of other important pathogens, yet little is known about their biogenesis. In this study, we explored the viral protein requirements for the formation of MERS-CoV- and SARS-CoV-induced DMVs and established that coexpression of two of the three transmembrane subunits of the coronavirus replicase polyprotein, nonstructural proteins (nsp's) 3 and 4, is required and sufficient to induce DMV formation. Moreover, release of nsp3 and nsp4 from the polyprotein by proteolytic maturation is essential for this process. These findings provide a strong basis for further research on the biogenesis and functionality of coronavirus ROs and may point to more general principles of viral DMV formation.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , RNA Viral/metabolismo , Proteínas não Estruturais Virais/genética , Replicação Viral/genética , Linhagem Celular , Vesículas Citoplasmáticas/ultraestrutura , Vesículas Citoplasmáticas/virologia , Tomografia com Microscopia Eletrônica , Retículo Endoplasmático/virologia , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/metabolismo , Biogênese de Organelas , Proteólise , Proteínas não Estruturais Virais/metabolismoRESUMO
Crassignathadanaugirangensis sp. n. (Araneae: Symphytognathidae) was discovered during a tropical ecology field course held at the Danau Girang Field Centre in Sabah, Malaysia. A taxonomic description and accompanying ecological study were completed as course activities. To assess the ecology of this species, which belongs to the ground-web-building spider community, three habitat types were surveyed: riparian forest, recently inundated riverine forest, and oil palm plantation. Crassignathadanaugirangensis sp. n. is the most abundant ground-web-building spider species in riparian forest; it is rare or absent from the recently inundated forest and was not found in a nearby oil palm plantation. The availability of this taxonomic description may help facilitate the accumulation of data about this species and the role of inundated riverine forest in shaping invertebrate communities.