Search

The Covid-19 pandemic has created an unprecedented need for epidemiological monitoring using diverse strategies. We conducted a project combining prevalence, seroprevalence, and genomic surveillance approaches to describe the initial pandemic stages in Betim City, Brazil. We collected 3239 subjects in a population-based age-, sex- and neighbourhood-stratified, household, prospective; cross-sectional study divided into three surveys 21 days apart sampling the same geographical area. In the first survey, overall prevalence (participants positive in serological or molecular tests) reached 0.46% (90% CI 0.12% – 0.80%), followed by 2.69% (90% CI 1.88% – 3.49%) in the second survey and 6.67% (90% CI 5.42% - 7.92%) in the third. The underreporting reached 11, 19.6, and 20.4 times in each survey, respectively. We observed increased odds to test positive in females compared to males (OR 1.88 95% CI 1.25 – 2.82), while the single best predictor for positivity was ageusia/ anosmia (OR 8.12, 95% CI 4.72 – 13.98). Thirty-five SARS-CoV-2 genomes were sequenced, of which 18 were classified as lineage B.1.1.28, while 17 were B.1.1.33. Multiple independent viral introductions were observed. Integration of multiple epidemiological strategies was able to describe Covid-19 dispersion in the city adequately. Presented results have helped local government authorities to guide pandemic management.

Zika virus (ZIKV) emerged as a global health threat due to its association with severe outcomes in humans, including microcephaly and other neurological complications. ZIKV replication and induction of neuronal death are considered key factors for severe ZIKV-induced disease. Understanding the pathogenic mechanisms induced by ZIKV infection is crucial to identify potential therapeutic targets that may prevent or at least minimize the consequences in early phases of disease and adulthood. This chapter will discuss how ZIKV emerged in the past few years, will describe some aspects of the infection and, finally, will focus on the evidence of neuropathological mechanisms of the disease in humans and experimental models and its potential neuropsychiatric outcomes. The mechanisms explored are: (i) infection and virus replication, activation and apoptosis of neural progenitor cells, mature neurons and glial cells with concomitant induction of neuroinflammation; (ii) induction of neuronal excitotoxicity; and (iii) autophagy modulation during ZIKV infection.

Zika virus (ZIKV) infection of neurons leads to neurological complications and congenital malformations of the brain of neonates. To date, ZIKV mechanism of infection and pathogenesis is not entirely understood and different studies on gene regulation of ZIKV-infected cells have identified a dysregulation of inflammatory and stem cell maintenance pathways. MicroRNAs (miRNAs) are post-transcriptional regulators of cellular genes and they contribute to cell development in normal function and disease. Previous reports with integrative analyses of messenger RNAs (mRNAs) and miRNAs during ZIKV infection have not identified neurological pathway defects. We hypothesized that dysregulation of pathways involved in neurological functions will be identified by RNA profiling of ZIKV-infected fetal neurons. We therefore used microarrays to analyze gene expression levels following ZIKV infection of fetal murine neurons. We observed that the expression levels of transcription factors such as neural PAS domain protein 4 (Npas4) and of three members of the orphan nuclear receptor 4 (Nr4a) were severely decreased after viral infection. We confirmed that their downregulation was at both the mRNA level and at the protein level. The dysregulation of these transcription factors has been previously linked to aberrant neural functions and development. We next examined the miRNA expression profile in infected primary murine neurons by microarray and found that various miRNAs were dysregulated upon ZIKV infection. An integrative analysis of the differentially expressed miRNAs and mRNAs indicated that miR-7013-5p targets Nr4a3 gene. Using miRmimics, we corroborated that miR-7013-5p downregulates Nr4a3 mRNA and protein levels. Our data identify a profound dysregulation of neural transcription factors with an overexpression of miR-7013-5p that results in decreased Nr4a3 expression, likely a main contributor to ZIKV-induced neuronal dysfunction., Author summary Zika virus (ZIKV) is an emerging virus transmitted horizontally between humans through mosquito bites, and sexual intercourse generally inducing a mild disease. ZIKV is also transmitted vertically from mother-to-child producing congenital ZIKV syndrome (CZVS) in neonates. CZVS leads to severe microcephaly associated with neurological, ocular, musculoskeletal, genitourinary disorders and other disabilities. Although numerous studies have been performed on ZIKV infection of brain cells, we are still far from understanding how ZIKV infection leads to dysregulation of host genes, virus-induced cytopathicity and consequent pathology. Micro (mi)RNAs are small noncoding RNAs encoded and processed by the host cell. They regulate gene expression at the post-transcriptional level in a process called RNA interference (RNAi). Here, we evaluated the relationship between ZIKV infection and the level of mRNAs and miRNAs expressed in the cell. ZIKV infection of mouse embryo neurons downregulated several neural immediate-early genes (IEG). Moreover, we revealed that ZIKV infection led to aberrant regulation of several miRNAs, and identified one whose cognate target was a neural IEG. Our work identifies novel genes and miRNAs that are modulated upon ZIKV infection of fetal murine neurons, therefore linking neuronal dysfunction to transcription and the RNA interference pathway.

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host- Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a Δ bpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the Δ bpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase Δ cgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, Δ bpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.

Absent in melanoma 2 (AIM2) is a sensor of cytosolic dsDNA and is responsible for the activation of inflammatory and host immune responses to DNA viruses and intracellular bacteria. AIM2 is a member of the hematopoietic interferon-inducible nuclear proteins with a 200 amino-acid repeat (HIN200) family, containing a pyrin domain (PYD) at the N-terminus. Several studies have demonstrated that AIM2 is responsible for host defense against intracellular bacteria such as Francisella tularensis, Listeria monocytogenes and Mycobacerium tuberculosis. However, the role of AIM2 in host defenses against Brucella is poorly understood. In this study, we have shown that AIM2 senses Brucella DNA in dendritic cells to induce pyroptosis and regulates type I IFN. Confocal microscopy of infected cells revealed co-localization between Brucella DNA and endogenous AIM2. Dendritic cells from AIM2 KO mice infected with B. abortus showed impaired secretion of IL-1β as well as compromised caspase-1 cleavage. AIM2 KO mice displayed increased susceptibility to B. abortus infection in comparison to wild-type mice, and this susceptibility was associated with defective IL-1β production together with reduced IFN-γ responses. In summary, the increased bacterial burden observed in vivo in AIM2 KO animals confirmed that AIM2 is essential for an effective innate immune response against Brucella infection.

Immunity against Brucella abortus depends on the recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors (PRRs). Signaling pathways triggered by Brucella DNA involves TLR9, AIM2 and possibly STING and MAVS. Herein, we review the advances in B. abortus DNA sensing by host innate immune receptors and the progress in this field.

STING (stimulator of interferon genes) is a cytosolic sensor for cyclic dinucleotides and also an adaptor molecule for intracellular DNA receptors. Although STING has important functions in the host defense against pathogens and in autoimmune diseases, its physiological relevance in intestinal homeostasis is largely unknown. In this study, we show that STING-/- mice presented defective protective mechanisms of intestinal mucosa, including decreased number of goblet cells, diminished mucus production, and lower levels of secretory IgA, when compared with wild-type (WT) mice. Fecal content and microbiota DNA could activate STING, indicating a role of this molecule in gut. Microbiota composition was altered in STING-/- mice toward a more inflammatory profile, evidencing a reduction in the Allobacolum and Bifidobacterium groups along with increase in Disulfovibrio bacteria. Absence of STING lead to decrease in induced intraepithelial lymphocytes (IEL) and to increase in group 1 innate lymphoid cell (ILC1) as well as ILC3 frequencies and decrease in ILC2 in the colon. Development and function of Foxp3+ and LAP+ regulatory T cells were also compromised in STING-/- mice. Moreover, these mice were highly susceptible to dextran sodium sulfate-induced colitis, T-cell-induced colitis, and enteric Salmonella typhimurium infection when compared with WT animals. Therefore, our results identify an important role of STING in maintaining gut homeostasis and also a protective effect in controlling gut inflammation.

The innate immune system is essential for the detection and elimination of Bacterial pathogens. Upon inflammasome activation, caspase-1 cleaves pro-IL-1β and pro-IL-18 to their mature forms IL-1β and IL-18, respectively, and the cell undergoes inflammatory death termed pyroptosis. Here, we reviewed recent findings demonstrating that Brucella abortus ligands activate NLRP3 and AIM2 inflammasomes which lead to control of infection. This protective effect is due to the inflammatory response caused by IL-1β and IL-18 rather than cell death. Brucella DNA is sensed by AIM2 and bacteria-induced mitochondrial reactiveoxygen species is detected by NLRP3. However, deregulation of pro-inflammatory cytokine production can lead to immunopathology. Nervous system invasion by bacteria of the genus Brucella results in an inflammatory disorder termedneurobrucellosis. Herein, we discuss the mechanism of caspase-1 activation and IL-1β secretion in glial cells infected with B. abortus.Our results demonstrate that the ASC inflammasome is indispensable for inducing the activation of caspase-1 and secretion of IL-1β upon infection of astrocytes and microglia with Brucella. Moreover, our results demonstrate that secretion of IL-1β by Brucella-infected glial cells depends on NLRP3 and AIM2 and leads to neurobrucellosis. Further, the inhibition of the host cell inflammasome as an immune evasion strategy has been described for bacterial pathogens. We discuss here that the bacterial type IV secretion system VirB is required for inflammasome activation in host cells during infection. Taken together, our results indicate that Brucella is sensed by ASC inflammasomes mainly NLRP3 andAIM2 that collectively orchestrate a robust caspase-1 activation and pro-inflammatory response. Fil: Marin, Fernanda M.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasil Fil: Franco, Miriam M. Costa. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasil Fil: Gomez, Marco Tulio R.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasil Fil: Miraglia, Maria Cruz. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Giambartolomei, Guillermo Hernan. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Inmunología, Genética y Metabolismo. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Inmunología, Genética y Metabolismo; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Hospital de Clínicas General San Martín; Argentina Fil: Oliveira, Sergio C.. Universidade Federal de Minas Gerais. Instituto de Ciências Biológicas; Brasil

Innate immunity serves as the first line of defense against infectious agents such as intracellular bacteria. The innate immune platform includes Toll-like receptors (TLRs), retinoid acid-inducible gene-I-like receptors and other cytosolic nucleic acid sensors, nucleotide-binding and oligomerization domain-like receptors, adaptors, kinases and other signaling molecules that are required to elicit effective responses against different pathogens. Our research group has been using the Gram-negative bacteria Brucella abortus as a model of pathogen. We have demonstrated that B. abortus triggers MAPK and NF-κB signaling pathways in macrophages in a MyD88 and IRAK-4-dependent manner. Furthermore, we claimed that so far TLR9 is the most important single TLR during Brucella infection. The identification of host receptors that recognize pathogen-derived nucleic acids has revealed an essential role for nucleic acid sensing in the triggering of immunity to intracellular pathogens. Besides TLRs, herein we describe recent advances in NOD1, NOD2, and type I IFN receptors in innate immune pathways during B. abortus infection.

CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico O Zika vírus (ZIKV) é um arbovírus pertencente à família Flaviviridae e sua sintomatologia está associada a sinais clínicos como febre aguda, dor de cabeça, mialgia, exantema maculopapular, dor de garganta e edema nos membros inferiores. Também são observadas complicações neurológicas associadas à infecção por esse vírus, como a Síndrome de Guillain-Barré (SGB) e a microcefalia em recém-nascidos, além de anomalias fetais, no qual é definido como Síndrome Congênita do Zika (SCZ). Mas as consequências da infecção na fase adulta ainda são investigadas, principalmente no contexto de co-circulação de outros flavivírus, que podem acarretar no fenômeno de ADE (aumento da infecção dependente de anticorpos). Por isso, o objetivo desse trabalho foi, através da análise de transcriptoma, avaliar a expressão de genes diferenciais na prole nascida de mães infectadas por ZIKV, na presença ou ausência de anticorpos anti-flavivírus. Para tal, camundongo fêmeas grávidas foram inoculadas com 1x106 UFP/animal de ZIKV por via intraperitoneal no dia embrionário 5,5, na presença ou ausência de 4G2. Após 12 semanas do nascimento da prole, os filhotes foram eutanasiados e o córtex cerebral coletado para análise do transcriptoma. Foram sequenciados 6 amostras de cada grupo por meio do equipamento HiSeq 2500 (Illumina, San Diego, CA, USA). A análise de transcriptoma seguiu os seguintes passos: controle de qualidade, alinhamento, sumarização, quantificação, normalização, análise de expressão diferencial e análise de enriquecimento de genes sets. Os grupos animais foram comparados entre si, sendo eles PBS, ZIKV e 4G2+ZIKV. Foram observados poucos genes diferencialmente expressos quando comparado PBS e ZIKV, sendo a maioria suprimida e estão relacionados principalmente ao ciclo celular e a morfogênese. Em contrapartida, quando comparado ZIKV e 4G2+ZIKV, foi observados uma quantidade grande de genes sendo ativados, referentes à resposta imunológica e produção de citocinas e mediadores inflamatórios, os quais alteraram vias importantes na tentativa do organismo hospedeiro em conter a infecção. Também foi observada a repressão de processos celulares e morfológicos, que corroboram com a patogenia da ZIKV, principalmente no desenvolvimento do sistema nervoso. As alterações neurológicas foram mais bem observadas na análise de splicing alternativo que revelou transcritos provindos de genes distintos àqueles diferencialmente expressos e que alteram principalmente o desenvolvimento neurológico no grupo com a presença de 4G2. Dessa forma, a análise do transcriptoma elucida a gravidade de co-infecções no contexto de ZIKV, ao observar alterações genéticas e metabólicas mais acentuadas na presença de 4G2, que contribuem para um fenótipo mais grave e complicações na fase adulta do indivíduo. Zika virus (ZIKV) is an arbovirus that belongs to the Flaviviridae family and its symptoms are associated with clinical signs as acute fever, headache, myalgia, maculopapular rash, sore throat and edema in the lower limbs. Neurological complications associated with this virus infection are also observed, as Guillain-Barré Syndrome (GBS) and microcephaly in newborns, further fetal anomalies, in which is defined as Congenital Zika Syndrome (CZS). But the consequences of infection in adulthood are still being investigated, especially in the context of co-circulation of other flaviviruses, which can cause tthe phenomenon of ADE (Antibody-Dependent Enhancement). Therefore, the objective of this work was, through transcriptome analysis, to evaluate the expression of differential genes in offspring born from mothers infected with ZIKV, in the presence or absence of anti-flavivirus antibodies. For this, pregnant female mice were inoculated with 1x106 PFU/animal of ZIKV intraperitoneally on embryonic day 5.5, in the presence or absence of 4G2. After 12 weeks of offspring birth, the puppies were euthanized and the cerebral cortex was collected for transcriptome analysis. Six samples from each group were sequenced using the HiSeq 2500 equipment (Illumina, San Diego, CA, USA). The transcriptome analysis followed the steps: quality control, alignment, summarization, quantification, normalization, differential expression analysis and analysis of set gene enrichment. The animal groups were compared to each other, which are PBS, ZIKV and 4G2+ZIKV. Few differentially expressed genes were observed when comparing PBS and ZIKV, most of which were suppressed and are mainly related to cell cycle and morphogenesis. On the other hand, when comparing ZIKV and 4G2+ZIKV, a large amount of genes activated was observed, referring to the immune response and production of cytokines and inflammatory mediators, which altered important pathways in the host organism's attempt to contain the infection. The repression of cellular and morphological processes was also observed, which corroborate the pathogenesis of ZIKV, especially in the development of the nervous system. Neurological alterations were better observed in the analysis of alternative splicing, which revealed transcripts originating from genes different from those differentially expressed, which mainly change neurological development in the group with the presence of 4G2. Therefore, the transcriptome analysis elucidates the severity of co-infections in the context of ZIKV, by observing more accentuated genetic and metabolic alterations in the presence of 4G2, which contribute to a more severe phenotype and complications in the individual's adult phase.