PKR-mediated stress response enhances dengue and Zika virus replication.

IMPORTANCE: One of the fundamental features that make viruses intracellular parasites is the necessity to use cellular translational machinery. Hence, this is a crucial checkpoint for controlling infections. Here, we show that dengue and Zika viruses, responsible for nearly 400 million infections every year worldwide, explore such control for optimal replication. Using immunocompetent cells, we demonstrate that arrest of protein translations happens after sensing of dsRNA and that the information required to avoid this blocking is contained in viral 5'-UTR. Our work, therefore, suggests that the non-canonical translation described for these viruses is engaged when the intracellular stress response is activated.

Pirahy virus: Identification of a new and potential emerging arbovirus in South Brazil.

Genomic and epidemiological surveillance are paramount for the discovery of new viruses with the potential to cross species barriers. Here, we present a new member of the genus Alphavirus found in Trichoprosopon and Wyeomia mosquitoes, tentatively named Pirahy virus (PIRAV). PIRAV was isolated from mosquito pools collected in a rural area of Piraí do Sul, South Brazil. In vitro assays revealed that PIRAV replicates and causes cytopathic effects in vertebrate cell lines such as Vero E6, SH-SY5Y, BHK-21 and UMNSAH/DF-1. Genomic signature analysis supports these results showing a dinucleotide and codon usage balance compatible with several hosts. Phylogenetic analyses placed PIRAV basal to the Venezuelan equine encephalitis complex. Genome analyses, electron microscopy, and biological characterization show findings that may alert for the emergence of a new arbovirus in South America.

A multiplex PCR protocol for rapid differential identification of four families of trematodes with medical and veterinary importance transmitted by Biomphalaria Preston, 1910 snails.

Trematodes have complex life cycles with multiple hosts. Biomphalaria snails commonly act as the first intermediate hosts of several species that can affect human and animal health. The specific identification of larval trematodes found in snails is difficult and limited, since the taxonomy of these flukes is based on morphological traits of the adults found in vertebrates. Despite recent advances worldwide, studies aiming at the use of molecular tools for the identification of cercariae found in snails are scarce in the South America. In fact, most studies are focused on Schistosoma mansoni, with few efforts directed towards the identification of larvae of other parasites found in planorbids. When reported, these other parasites are identified as cercarial types, an artificial morphological system of classification. Therefore, alternative strategies for a correct, rapid and inexpensive identification of larval trematodes found in Biomphalaria are needed. This work aimed at developing a methodology capable of distinguishing four important families of trematodes (Clinostomidae, Echinostomatidae, Schistosomatidae and Strigeidae) commonly found infecting species of Biomphalaria. Using the rDNA sequences of 34 species as input for the online tool TipMT, we designed trematode family-specific primers targeting the ITS region optimized to be used in multiplex PCR. The panel of primers identified in this study was effective at the same PCR condition. The specificity of the primers was confirmed, and the PCR sensitivity ranged from 0.1 ng to 1 ag of the DNA of the parasite. This methodology was also effective for the detection of coinfection. Through a simple, fast, accurate, and inexpensive methodology, it is possible to properly identify the trematode families included in this study in a single PCR reaction. A family level identification provides important information about probable hosts, pattern of life cycle and possible impacts that the infection generates in a specific region, thus allowing the design of better control strategies, especially for those infections that have medical and veterinary importance.

Vasculature-associated fat macrophages readily adapt to inflammatory and metabolic challenges.

Tissue-resident macrophages are the most abundant immune cell population in healthy adipose tissue. Adipose tissue macrophages (ATMs) change during metabolic stress and are thought to contribute to metabolic syndrome. Here, we studied ATM subpopulations in steady state and in response to nutritional and infectious challenges. We found that tissue-resident macrophages from healthy epididymal white adipose tissue (eWAT) tightly associate with blood vessels, displaying very high endocytic capacity. We refer to these cells as vasculature-associated ATMs (VAMs). Chronic high-fat diet (HFD) results in the accumulation of a monocyte-derived CD11c+CD64+ double-positive (DP) macrophage eWAT population with a predominant anti-inflammatory/detoxifying gene profile, but reduced endocytic function. In contrast, fasting rapidly and reversibly leads to VAM depletion, while acute inflammatory stress induced by pathogens transiently depletes VAMs and simultaneously boosts DP macrophage accumulation. Our results indicate that ATM populations dynamically adapt to metabolic stress and inflammation, suggesting an important role for these cells in maintaining tissue homeostasis.

Dominance of P-glycoprotein 12 in phenotypic resistance conversion against ivermectin in Caenorhabditis elegans.

While diseases caused by nematodes remains a considerable drawback for the livestock, agriculture and public health, anthelmintics drug resistance has been observed over the past years and is a major concern for parasite control. Ivermectin, initially considered as a highly potent drug, currently presents a reduced anti-helminthic efficacy, which is influenced by expression of several ATP-binding cassette transporters (ABC), among them the P-glycoproteins (Pgps). Here we present some evidences of Pgps dominance during Ivermectin resistance/susceptibility using Pgps double silencing in C. elegans and the phylogenetic relationship of Pgps among nematodes, which strengthen the use of this model for study of drug resistance in nematodes. Firstly, we evaluated the quantitative gene expression of 12 out the 15 known Pgps from resistant and WT strains of C. elegans, we demonstrated the upregulation of Pgps 12 and 13 and downregulation of all remaining Pgps in ivermectin resistant strain. By using an RNAi loss-of-function approach we observed that Pgp 12 gene silencing reverts the resistance phenotype to ivermectin, while Pgp 4 gene silencing does not alter the resistance phenotype but induces a resistance in wild type strain. Interestingly, the dual silencing of Pgp 12 and Pgp 4 expression demonstrates the dominance of phenotype promoted by Pgp 12 silencing. Finally, in silico analysis reveals a close relationship between Pgps from C. elegans and several nematodes parasites. Taken together, our results indicate that Pgp 12 is crucial for the resistance to ivermectin and thus a good candidate for further studies aiming to develop specific inhibitors to this transporter, allowing the continuous use of ivermectin to control the burden on animal and human health inflicted by nematode parasites globally.

Predicting the proteins of Angomonas deanei, Strigomonas culicis and their respective endosymbionts reveals new aspects of the trypanosomatidae family.

Endosymbiont-bearing trypanosomatids have been considered excellent models for the study of cell evolution because the host protozoan co-evolves with an intracellular bacterium in a mutualistic relationship. Such protozoa inhabit a single invertebrate host during their entire life cycle and exhibit special characteristics that group them in a particular phylogenetic cluster of the Trypanosomatidae family, thus classified as monoxenics. In an effort to better understand such symbiotic association, we used DNA pyrosequencing and a reference-guided assembly to generate reads that predicted 16,960 and 12,162 open reading frames (ORFs) in two symbiont-bearing trypanosomatids, Angomonas deanei (previously named as Crithidia deanei) and Strigomonas culicis (first known as Blastocrithidia culicis), respectively. Identification of each ORF was based primarily on TriTrypDB using tblastn, and each ORF was confirmed by employing getorf from EMBOSS and Newbler 2.6 when necessary. The monoxenic organisms revealed conserved housekeeping functions when compared to other trypanosomatids, especially compared with Leishmania major. However, major differences were found in ORFs corresponding to the cytoskeleton, the kinetoplast, and the paraflagellar structure. The monoxenic organisms also contain a large number of genes for cytosolic calpain-like and surface gp63 metalloproteases and a reduced number of compartmentalized cysteine proteases in comparison to other TriTryp organisms, reflecting adaptations to the presence of the symbiont. The assembled bacterial endosymbiont sequences exhibit a high A+T content with a total of 787 and 769 ORFs for the Angomonas deanei and Strigomonas culicis endosymbionts, respectively, and indicate that these organisms hold a common ancestor related to the Alcaligenaceae family. Importantly, both symbionts contain enzymes that complement essential host cell biosynthetic pathways, such as those for amino acid, lipid and purine/pyrimidine metabolism. These findings increase our understanding of the intricate symbiotic relationship between the bacterium and the trypanosomatid host and provide clues to better understand eukaryotic cell evolution.

Identification of strain-specific B-cell epitopes in Trypanosoma cruzi using genome-scale epitope prediction and high-throughput immunoscreening with peptide arrays.

BACKGROUND: The factors influencing variation in the clinical forms of Chagas disease have not been elucidated; however, it is likely that the genetics of both the host and the parasite are involved. Several studies have attempted to correlate the T. cruzi strains involved in infection with the clinical forms of the disease by using hemoculture and/or PCR-based genotyping of parasites from infected human tissues. However, both techniques have limitations that hamper the analysis of large numbers of samples. The goal of this work was to identify conserved and polymorphic linear B-cell epitopes of T. cruzi that could be used for serodiagnosis and serotyping of Chagas disease using ELISA. METHODOLOGY: By performing B-cell epitope prediction on proteins derived from pair of alleles of the hybrid CL Brener genome, we have identified conserved and polymorphic epitopes in the two CL Brener haplotypes. The rationale underlying this strategy is that, because CL Brener is a recent hybrid between the TcII and TcIII DTUs (discrete typing units), it is likely that polymorphic epitopes in pairs of alleles could also be polymorphic in the parental genotypes. We excluded sequences that are also present in the Leishmania major, L. infantum, L. braziliensis and T. brucei genomes to minimize the chance of cross-reactivity. A peptide array containing 150 peptides was covalently linked to a cellulose membrane, and the reactivity of the peptides was tested using sera from C57BL/6 mice chronically infected with the Colombiana (TcI) and CL Brener (TcVI) clones and Y (TcII) strain. FINDINGS AND CONCLUSIONS: A total of 36 peptides were considered reactive, and the cross-reactivity among the strains is in agreement with the evolutionary origin of the different T. cruzi DTUs. Four peptides were tested against a panel of chagasic patients using ELISA. A conserved peptide showed 95.8% sensitivity, 88.5% specificity, and 92.7% accuracy for the identification of T. cruzi in patients infected with different strains of the parasite. Therefore, this peptide, in association with other T. cruzi antigens, may improve Chagas disease serodiagnosis. Together, three polymorphic epitopes were able to discriminate between the three parasite strains used in this study and are thus potential targets for Chagas disease serotyping.

The MASP family of Trypanosoma cruzi: changes in gene expression and antigenic profile during the acute phase of experimental infection.

BACKGROUND: Trypanosoma cruzi is the etiological agent of Chagas disease, a debilitating illness that affects millions of people in the Americas. A major finding of the T. cruzi genome project was the discovery of a novel multigene family composed of approximately 1,300 genes that encode mucin-associated surface proteins (MASPs). The high level of polymorphism of the MASP family associated with its localization at the surface of infective forms of the parasite suggests that MASP participates in host-parasite interactions. We speculate that the large repertoire of MASP sequences may contribute to the ability of T. cruzi to infect several host cell types and/or participate in host immune evasion mechanisms. METHODS: By sequencing seven cDNA libraries, we analyzed the MASP expression profile in trypomastigotes derived from distinct host cells and after sequential passages in acutely infected mice. Additionally, to investigate the MASP antigenic profile, we performed B-cell epitope prediction on MASP proteins and designed a MASP-specific peptide array with 110 putative epitopes, which was screened with sera from acutely infected mice. FINDINGS AND CONCLUSIONS: We observed differential expression of a few MASP genes between trypomastigotes derived from epithelial and myoblast cell lines. The more pronounced MASP expression changes were observed between bloodstream and tissue-culture trypomastigotes and between bloodstream forms from sequential passages in acutely infected mice. Moreover, we demonstrated that different MASP members were expressed during the acute T. cruzi infection and constitute parasite antigens that are recognized by IgG and IgM antibodies. We also found that distinct MASP peptides could trigger different antibody responses and that the antibody level against a given peptide may vary after sequential passages in mice. We speculate that changes in the large repertoire of MASP antigenic peptides during an infection may contribute to the evasion of host immune responses during the acute phase of Chagas disease.