Memorias do Instituto Oswaldo Cruz最新文献

Background: Arboviruses represent a potential threat to global public health due to their ability to infect various vertebrate hosts and vectors, as well as their adaptability to diverse ecosystems, allowing them to expand geographically across continents.

Objective: The present study aimed to describe the molecular epidemiology of encephalitic arboviruses, including Venezuelan equine encephalitis virus (VEEV), Eastern equine encephalitis virus (EEEV), Western equine encephalitis virus (WEEV), West Nile virus (WNV), Saint Louis encephalitis virus (SLEV), Toscana virus (TOSV), and Rift Valley fever virus (RVFV) in nervous tissue samples from domestic and wild animals from the Northern region of Brazil, between February 2023 and June 2024.

Methods: Samples negative for rabies virus were analysed by reverse transcription real-time polymerase chain reaction (RT-qPCR) targeting Alphavirus, Orthoflavivirus and Phlebovirus species.Positive samples were subjected to viral isolation in cell culture and whole-genome sequencing using next-generation sequencing.Phylogenetic and molecular clock analyses were performed to characterise viral lineages and evolutionary relationships.

Findings: Two samples tested positive for arboviruses by RT-qPCR: one SLEV sample from the state of Amazonas, which showed a low viral load, preventing virus isolation and sequencing, and one Madariaga virus (MADV) sample from the state of Pará, which could be isolated and sequenced.The isolated agent belongs to lineage III of EEEV, showing most similarity to strains from Guyana and Argentina.

Main conclusions: The present study detected two arboviruses in animals, suggesting its circulation in the study area.

Background: In light of the biotechnological potential demonstrated by Aspergillus species and, considering the great need for further research into the search for new sources of active molecules and the biodiversity of these microorganisms occurring in the Amazon region.

Objectives: This research aimed to investigate the biotechnological potential of the fungus Aspergillus japonicus Amazon Fungi Collection (CFAM) 0234, a fungal strain isolated from Amazonian soil and stored in the CFAM.

Methods: For this purpose, the Aspergillus species was investigated through comparative genomic analysis and antimicrobial activity assays.

Findings: Genome sequencing revealed a fragmented assembly (72.67 Mbp, N50 = 152 kbp) containing 106 biosynthetic clusters (BGCs), surpassing the reference strain CBS 114.51 (57 BGCs). Among the clusters identified, NRPS, PKS type I and hybrid NRPS-PKS systems stood out, including clusters exclusive to betalactones and isocyanides, potentially involved in the synthesis of β-lactam antibiotics and innovative metabolites. BiG-SCAPE analysis identified 63 BGC families unique to CFAM 0234, suggesting evolutionary adaptations to the competitive environment of the Amazon. Biological assays demonstrated selective antimicrobial activity of the ethyl acetate extract against Escherichia coli, Shigella sonnei and Sthapylococcus aureus (MRSA), with inhibition halos ranging from 8 mm to 6 mm in diameter, pathogens classified as priorities for research into new antibiotics. The correlation between predicted BGCs and antimicrobial activity reinforces the strain's biotechnological potential. Despite the fragmentation of the genome, the high completeness assessed by BUSCO (98.5%) confirms the quality of the assembly, while the detection of single nucleotide polymorphisms (SNPs) in regulatory regions and rearrangements close to BGCs suggests evolutionary pressure for metabolic diversification. The lack of correspondence with the minimum information about a biosynthetic gene cluster (MIBiG) bank and the limitations of crude extracts highlight the need for complementary techniques, such as long-read sequencing (Oxford Nanopore) and metabolomic analysis [liquid chromatography-mass espectrometry (LC-MS)], to link clusters to active metabolites.

Main conclusions: Aspergillus japonicus CFAM 0234 represents a promising microbial resource for bioprospecting in the Amazon, offering relevant genomic and chemical insights for the development of new antimicrobial agents. Future studies will focus on the purification of compounds and activation of silent BGCs, aiming at sustainable pharmaceutical applications.

Background: The Mayaro virus (MAYV) is an alphavirus endemic to Central and South America, primarily transmitted by mosquitoes of the Haemagogus genus. Human infection causes "Mayaro fever," characterized by symptoms similar to dengue and chikungunya, including debilitating arthralgia. Despite its potential for urbanisation, many aspects of MAYV-host interactions, particularly the role of host microRNAs (miRNAs), remain poorly understood.

Objectives: This study aimed to investigate the expression profile of miRNAs in Vero cells infected with MAYV and to predict their potential biological targets and associated pathways.

Methods: Infection was performed using the MAYV strain (BeAr 20290), and small RNA libraries were prepared from infected and control cells. Initial experiments were conducted to evaluate viral replication, cell viability, and small RNA expression. Based on these parameters, the 24-h post-infection time point was selected for small RNA sequencing. Bioinformatic tools were used to identify differentially expressed miRNAs and predict their targets in Homo sapiens and the MAYV genome.

Findings: Among the 348 miRNAs identified, 46 were differentially expressed at 24 h (42 upregulated and four downregulated). Principal component analysis (PCA) indicated a clear separation between infected and control groups. In silico predictions of the targets of these miRNAs suggest potential associations with biological processes that may be relevant to virus-host interactions, such as immune response, programmed cell death pathways, viral replication, and persistence. Additionally, one miRNA detected in Vero cells was predicted to target a viral non-structural protein.

Main conclusions: Our findings indicate a potential dual role for host miRNAs during MAYV infection, involving both the modulation of host responses by the virus to enhance replication and a possible antiviral effect. While these interactions underscore the prospective relevance of miRNAs as biomarkers and therapeutic targets in arboviral infections, it is important to note that these conclusions are based solely on computational analyses. Therefore, they should be interpreted with caution until they are supported by further experimental validation.

Background: Plasmodium vivax rosetting is a cytoadhesion phenomenon associated with parasite virulence and clinical manifestations of malaria. However, the molecular mechanisms underlying this process remain poorly understood. Comparative transcriptomic analysis between isolates with different rosetting capacities may provide insights into the molecular base and clinical outcome of parasite populations with distinct rosetting characteristics.

Objectives: Our study aims to identify and describe the transcription profile of P. vivax isolates with high and low rosetting rates.

Methods: We used RNA-seq to compare the transcriptomes of 10 field P. vivax isolates from the Brazilian Amazon.

Findings: Among the 492 differentially expressed genes of P. vivax isolates with high rosetting (HR) versus low rosetting (LR) formation, 172 (34,96%) are annotated as genes conserved within Plasmodium and of unknown function. The expression profiles of the other 320 genes (65,04%) highlight the importance of integral membrane proteins and membrane-associated proteins with adhesive or adhesin-like properties, representing 10% of the transcribed genes (53 genes), such as Plasmodium Helical Interspersed Sub-telomeric (PHIST) proteins in rosetting phenotypes. Transcriptomic analyses revealed that approximately 4% (19 genes) of differentially expressed genes were kinases and 50% (248 genes) other proteins. Among cell surface proteins and integral/membrane-associated proteins, differentiated expression and positive regulation of representative 6-cysteine gene family were observed in HR formation group, which includes a tryptophan-rich protein (TRAG16), the 41K blood stage antigen precursor 41-3 protein, and merozoite surface protein 7-like (MSP7-like).

Main conclusions: These results contribute to understanding the molecular basis of P. vivax rosetting.

Background: Yellow fever virus (YFV) re-emerged among non-human primates (NHPs) in Rio Grande do Sul in early 2021, more than a decade after its last detection in the state. The spread of the virus was accompanied by increased mortality among NHPs.

Objectives: To conduct entomological surveillance and molecular detection of YFV and other Orthoflavivirus species in mosquito samples collected from affected and potentially receptive areas.

Methods: Mosquitoes were collected during epizootics using human landing catches, BG-Pro traps, and ovitraps. Virus detection was performed using reverse transcription real-time polymerase chain reaction (RT-qPCR) assays targeting YFV and pan-Orthoflavivirus sequences.

Findings: A total of 1,210 mosquitoes, representing 26 taxa, were collected across 17 municipalities. Psorophora ferox was the most abundant species, followed by Culex (Culex) spp., accounting for 27% and 12% of the specimens, respectively. Haemagogus leucocelaenus, the primary YFV vector in the region, was also among the most frequently captured species, representing 7%. In total, 203 mosquito pools were assembled by species, location, and date of collection. RT-qPCR analysis did not detect YFV or other Orthoflavivirus RNA in any of the samples.

Main conclusions: Although mosquitoes were collected during a period of active YFV circulation, the absence of virus detection suggests that arboviral circulation in vector populations may occur at low frequencies, even during outbreaks.

Background: Triatoma dimidiata is a widely distributed vector of Trypanosoma cruzi in Mesoamerica, but its epidemiological role in most regions of Panamá remains poorly understood.

Objectives: To investigate the presence, infection status, and feeding behaviour of T. dimidiata populations in peridomestic areas of Palmira Arriba, western Panamá.

Methods: Entomological surveys were conducted in five peridomestic sites of a rural highland community. Thirty-seven triatomines (13 adults and 24 nymphs) were collected from wooden piles and construction materials in contact with the ground. DNA from 30 specimens was analysed by polymerase chain reaction (PCR) for T. cruzi detection, genotyping [discrete typing unit (DTU) and haplotype identification], and blood meal source determination through cytochrome b amplification.

Findings: Twenty-one insects (70.0%) were positive for T. cruzi. Sixteen infections (76.2%) belonged to DTU I (TcI), including 13 TcIDOM and 14 TcIa genotypes, both linked to domestic and sylvatic cycles. Blood meal analysis revealed one mammalian and two avian feedings, indicating opportunistic behaviour.

Main conclusions: This study provides the first molecular confirmation of T. cruzi infection in T. dimidiata from Palmira Arriba. The combination of high infection prevalence, multiple developmental stages, and recent feeding suggests active local transmission favoured by humid and cool ecological conditions. Expanded surveillance and integrative One Health approaches are needed to elucidate transmission dynamics in highland rural Panamá.

Background: Egg detection still has a role in schistosomiasis control, as a screening strategy or to provide a reference standard for the assessment of the accuracy of other diagnostic tools. The Helmintex method is highly sensitive but laborious, and several improvements of it, including automated egg detection, are currently under development.

Objective: We conducted a preliminary evaluation of Schistosoma mansoni eggs' autofluorescence as a distinctive marker amid very complex fecal sediments.

Methods: Eggs from mouse livers and human feces were examined under a fluorescence microscope.

Findings: More intense green fluorescence (greater for miracidia than for eggshell) was consistently detected using a B-2A filter (FITC, 420-495 nm).

Main conclusions: These findings may help to improve diagnostic methods, especially with automated egg detection systems. Besides access to safe water and adequate sanitation, as well as health education and the treatment of infected individuals, laboratory diagnosis is a key measure that can help eliminate schistosomiasis as a public health problem.

Background: Plasmodium vivax rosetting is a cytoadhesion phenomenon associated with parasite virulence and clinical manifestations of malaria. However, the molecular mechanisms underlying this process remain poorly understood. Comparative transcriptomic analysis between isolates with different rosetting capacities may provide insights into the molecular base and clinical outcome of parasite populations with distinct rosetting characteristics.

Objectives: Our study aims to identify and describe the transcription profile of P. vivax isolates with high and low rosetting rates.

Methods: We used RNA-seq to compare the transcriptomes of 10 field P. vivax isolates from the Brazilian Amazon.

Findings: Among the 492 differentially expressed genes of P. vivax isolates with high rosetting (HR) versus low rosetting (LR) formation, 172 (34,96%) are annotated as genes conserved within Plasmodium and of unknown function. The expression profiles of the other 320 genes (65,04%) highlight the importance of integral membrane proteins and membrane-associated proteins with adhesive or adhesin-like properties, representing 10% of the transcribed genes (53 genes), such as Plasmodium Helical Interspersed Sub-telomeric (PHIST) proteins in rosetting phenotypes. Transcriptomic analyses revealed that approximately 4% (19 genes) of differentially expressed genes were kinases and 50% (248 genes) other proteins. Among cell surface proteins and integral/membrane-associated proteins, differentiated expression and positive regulation of representative 6-cysteine gene family were observed in HR formation group, which includes a tryptophan-rich protein (TRAG16), the 41K blood stage antigen precursor 41-3 protein, and merozoite surface protein 7-like (MSP7-like).

Main conclusions: These results contribute to understanding the molecular basis of P. vivax rosetting.

Background: Paracoccidioidomycosis (PCM) is a systemic infection that is endemic to Latin America, caused by thermodimorphic fungi from the Paracoccidioides genus. These fungi are facultative intracellular parasites of macrophages. LC3-associated phagocytosis (LAP), a non-canonical form of autophagy, plays a critical role in the response of these phagocytes to similar pathogens.

Objectives: In this study, we investigated the role of LAP in the macrophage responses to Paracoccidioides spp.

Methods: We detected LAP in macrophages infected with Paracoccidioides spp by immunofluorescence microscopy with antibodies to LC3. Piceatannol and diphenyleneiodonium chloride (DPI), respectively Syk and nicotinamide adenine dinucleotide phosphate oxidase (NADPH) inhibitors, were used to understand the role their pathways played. To determine the function of LAP, we targeted ATG5, a key autophagy gene, by RNA interference.

Findings: We observed LC3 recruitment to phagosomes containing Paracoccidioides spp. in RAW264.7 and J774.16 cell lines and in bone marrow-derived macrophages. ATG5 RNA interference reduced the antifungal activity of J774.16 cells, highlighting the importance of LC3 recruitment for effective fungal control. Interestingly, pharmacological inhibition of Syk kinase and NADPH oxidase pathways, essential for LAP against Aspergillus fumigatus and Candida albicans, did not impair LAP against P. brasiliensis.

Main conclusions: This suggests distinct triggering mechanisms, possibly due to differences in the fungal cell surface composition. These findings suggest that LAP plays a significant role in the host defense against Paracoccidioides spp. and may represent a promising target for host-directed PCM therapies.

Background: The life cycle of the parasitic protozoan Trypanosoma cruzi, the etiological agent of Chagas disease (CD), includes two well-recognised insect-dwelling stages: the replicative non-infective epimastigotes and the non-replicative infective metacyclic trypomastigotes. Nonetheless, the existence of multiple intermediate forms has been reported. Since nutrient restriction is considered one of the main factors driving metacyclogenesis and is very frequent due to the long-term starvation periods that the insect vectors commonly undergo, we have studied the transcriptomic effects of nutrient restriction on long-lasting epimastigote cultures. We previously reported that in these conditions, we observed a long stationary phase characterised by an RNA content per cell three times smaller than the epimastigote's and a distinctive transcriptomic profile. Remarkably, our study identified gene expression changes that distincty characterise transitional parasite forms enriched by nutrient restriction.

Objectives: In this work we focused on pathogenic genes to further characterise the transcriptomic dynamics accompanying the nutrient restriction within the insect-dwelling parasite stage.

Methods: The alterations of morphology, growth rate and complement resistance of parasite population on long-lasting epimastigote cultures as well as the transcriptomic dynamics was studied.

Findings: We found a gene expression early rise of surface proteins (such as trans-sialidase and GP63) and even a rise of TcTASV and δ-amastin, which is not accompanied by increased expression of metacyclic transcript markers. In addition, we found increased expression of genes coding for proteins involved in two other processes activated during the differentiation of epimastigotes to the infective form of the parasite: autophagy (Atg4, Atg7, Atg8.2) and complement resistance (TcCRP and T-DAF).

Main conclusions: Altogether, these results, plus our previous identification of transcriptomic markers for transitional parasites, further support earlier proposals of a specific parasite stage that morphologically resembles epimastigotes but exhibits distinctive biological characteristics, including key features related to infectivity.