Viral metagenomics unveils MW (Malawi) polyomavirus infection in Brazilian pediatric patients with acute respiratory disease.

Viral metagenomics has been extensively applied for the identification of emerging or poorly characterized viruses. In this study, we applied metagenomics for the identification of viral infections among pediatric patients with acute respiratory disease, but who tested negative for SARS-CoV-2. Twelve pools composed of eight nasopharyngeal specimens were submitted to viral metagenomics. Surprisingly, in two of the pools, we identified reads belonging to the poorly characterized Malawi polyomavirus (MWPyV). Then, the samples composing the positive pools were individually tested using quantitative polymerase chain reaction for identification of the MWPyV index cases. MWPyV-positive samples were also submitted to respiratory virus panel testing due to the metagenomic identification of different clinically important viruses. Of note, MWPyV-positive samples tested also positive for respiratory syncytial virus types A and B. In this study, we retrieved two complete MWPyV genome sequences from the index samples that were submitted to phylogenetic inference to investigate their viral origin. Our study represents the first molecular and genomic characterization of MWPyV obtained from pediatric patients in South America. The detection of MWPyV in acutely infected infants suggests that this virus might participate (coparticipate) in cases of respiratory symptoms. Nevertheless, future studies based on testing of a larger number of clinical samples and MWPyV complete genomes appear to be necessary to elucidate if this emerging polyomavirus might be clinically important.

The role of pets in SARS-CoV-2 transmission: an exploratory analysis.

PURPOSE: There is evidence that humans can transmit SARS-CoV-2 to cats and dogs. However, there is no evidence that they can transmit it back to humans or play any role in SARS-CoV-2 transmission. Here, we present an exploratory analysis on that matter. METHODS: We conducted a case-control study with participants with flu-like symptoms seeking care at a primary healthcare unit to be tested for COVID-19. They were asked if they owned pet cats and/or dogs in their residences, and this variable was evaluated as exposure. RESULTS: The odds ratio of "having dogs and/or cats in the residence" was 1.29 (95% CI 1.08-1.54) of "having only dogs and no cats" was 1.26 (1.05-1.52), and "no dogs and only cats" was 1.29 (0.95-1.75). CONCLUSION: Having a cat/dog in the house can affect the risk of infection by SARS-CoV-2.

Molecular identification of the emerging Human Gemykibivirus-2 (HuGkV-2) among Brazilian blood donors.

Human gemykibivirus-2 (HuGkV-2) belonging to the Gemykibivirus genus (Genomoviridae family) is an emerging DNA virus which has been described as a component of the virome of a wide variety of samples including clinical ones. So far, the HuGkV-2 DNA prevalence in the human population as well as its clinical impact are completely unknown. The objective of this study was to investigate the HuGkV-2 DNA prevalence among Brazilian healthy blood donors from three different geographic regions. A total of 450 blood samples were screened for HuGkV-2 DNA (150 samples were from the Brazilian Amazon, 150 from Midwest Brazil and 150 from South Brazil). The overall HuGkV-2 DNA prevalence was 7.8 %. Considering the examined regions, the highest prevalence was observed in the Brazilian Amazon (city of Macapa, state of Amapa), 15.3 %, followed by the Midwest Brazil (city of Brasilia, Federal District) (6.0 %) and South Brazil (city of Santa Maria, Rio Grande do Sul State) (2.0 %). This study gives preliminary insights on the molecular prevalence of HuGkV-2 DNA among Brazilian blood donors, highlighting that the highest HuGkV-2 prevalence was recorded in the Brazilian Amazon. However, more studies regarding the prevalence, transmission routes and any possible clinical effects appear to be crucial in order to understand the impact of this emerging viral agent.

Environmental spreading of clinically relevant carbapenem-resistant gram-negative bacilli: the occurrence of blaKPC-or-NDM strains relates to local hospital activities.

BACKGROUND: Aquatic matrices impacted by sewage may shelter carbapenem-resistant (CR) Gram-negative bacilli (GNB) harboring resistance genes of public health concern. In this study, sewage treatment plants (STPs) servicing well-defined catchment areas were surveyed for the presence of CR-GNB bearing carbapenemase genes (blaKPC or blaNDM). RESULTS: A total of 325 CR-GNB were recovered from raw (RS) and treated (TS) sewage samples as well as from water body spots upstream (UW) and downstream (DW) from STPs. Klebsiella-Enterobacter (KE) group amounted to 116 isolates (35.7%). CR-KE isolates were recovered from TS, DW (35.7%) and RS samples (44.2%) (p = 0.001); but not from UW samples. KE isolates represented 65.8% of all blaKPC or blaNDM positive strains. The frequency of blaKPC-or-NDM strains was positively associated with the occurrence of district hospitals located near STPs, as well as with the number of hospitalizations and of sewer connections serviced by the STPs. blaKPC-or-NDM strains were recovered from ST samples in 7 out of 14 STPs, including four tertiary-level STPs; and from 6 out of 13 DW spots whose RS samples also had blaKPC-or-NDM strains. CONCLUSIONS: Clinically relevant GNB bearing blaKPC-or-NDM resist sewage treatments and spread into environmental aquatic matrices mainly from STPs impacted by hospital activities.

Chikungunya virus seroprevalence in asymptomatic blood donors during an outbreak in the Federal District of Brazil.

INTRODUCTION: Chikungunya virus (CHIKV) is a mosquito-borne alphavirus belonging to the Togaviridae family. The symptomatic infection is characterised by acute febrile disease which generally results in severe arthralgia and myalgia, however, most of the CHIKV infections remain asymptomatic. CHIKV RNA detection in asymptomatic volunteers may be responsible for the transfusion transmission of this infection, especially during outbreaks. There is no information for CHIKV seroprevalence among blood donors from the Federal District of Brazil. AIM: In early 2019, the Federal District of Brazil experienced a CHIKV outbreak, and this study evaluates the anti-CHIKV IgM and IgG presence in a well characterised cohort of blood donors from this region. METHODOLOGY: Blood samples were collected from 450 volunteer blood donors during a CHIKV outbreak and tested for the presence of anti-CHIKV IgG and IgM antibodies using ELISA. RESULTS: The CHIKV seroprevalence was 0.89% (n = 4/450) and anti-CHIKV IgM prevalence was 1.11% (n = 5/450). CONCLUSION: The obtained results demonstrated that at least some of the blood donors have experienced CHIKV infection which can be related to a hypothetical risk of CHIKV transfusion transmission. More studies are necessary in order to examine the impact of CHIKV on blood transfusion.

Dengue RNA detection and seroprevalence in blood donors during an outbreak in the São Paulo State, Brazil, 2016.

Most dengue virus (DENV) infections remain asymptomatic. This increases the risk of DENV transfusion transmission (TT-DENV) during outbreaks. We evaluated DENV viremia in 8475 blood donations assembled in minipools for the presence of DENV RNA. The tested samples were obtained between February and May, 2016, during a large DENV outbreak in Ribeirão Preto city, northeast region of the São Paulo State, Brazil. The DENV RNA + samples were serotyped and screened for DENV NS1. We also tested a significant number of plasma samples (n = 372) to estimate the DENV seroprevalence among blood donors in the region. We detected three DENV RNA + samples in the tested blood donations (n = 3/8475, 0.04%). From these, two samples were further serotyped as DENV-1 and one sample as DENV-2. All DENV RNA positive samples were negative for anti-DENV IgG, indicating the presence of primary acute infection. Moreover, two of the DENV RNA + samples were also NS1 antigen positive (antigenemia). The anti-DENV IgG seroprevalence among blood donor population was 50.8% (n = 189/372). Our results are in accordance with the presence of DENV primary infection in blood donors which can lead to transfusion transmission of the infection to recipients. Measures to exclude such donors should be adopted to prevent TT-DENV.

Downregulation of histone methyltransferase EHMT2 in CD4+ T-cells may protect HTLV-1-infected individuals against HAM/TSP development.

Approximately 5% of human T-cell leukemia virus type 1 (HTLV-1)-infected individuals will develop one of the HTLV-1-related diseases, such as HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) or adult T-cell leukemia. However, the mechanisms responsible for the appearance of symptoms have not been fully clarified. It is believed that viral factors, host genetic and epigenetic mechanisms are implicated in this process. Studies have shown the involvement of histone methyltransferases in retrovirus infection, but no study observed their expression in HTLV-1-infected patients. Among them, euchromatic histone-lysine N-methyltransferase (EHMT)-1 and EHMT-2 were related to retroviral latency in HIV-1 infection. We investigated whether histone methyltransferases EHMT1 and EHMT2 exert any influence on HAM/TSP development by assessing their expression levels in CD4+ T-cells from HTLV-1-infected patients. CD4+ T-cells were immunomagnetically isolated from peripheral blood mononuclear cells of HTLV-1-infected or non-infected individuals and the expression levels of EHMT1 and EHMT2 were determined by RT-qPCR. We observed that EHMT2 was negatively regulated in HTLV-1 asymptomatic carriers compared to non-infected individuals. No difference was observed for EHMT1. These results suggest that EHMT2 downregulation in CD4+ T-cells may be linked to a protection mechanism against the development of HAM/TSP.

Clinical relevance of telomerase polymorphism for breast cancer: A systematic review.

PURPOSE: To perform a systematic review to explore the clinical relevance of hTERT polymorphisms for breast cancer (BC). METHODS: Twenty-nine polymorphic regions were evaluated after comprehensive searching of 1236 articles, and selection of 9 publications (total of 12986 cases and 16758 controls). RESULTS: About the influence of hTERT variants in BC risk, 3 studies showed that the variant rs2736098 was associated with increasing risk. The variants rs10069690 and rs2853676 were also described as risk factors for BC. Only one variant rs2736100 presented as risk factor for BC. MNS16A genotype influenced the risk of BC in an Iranian, but not in the Greek and American populations. The associations of 5 hTERT variants with expression of hormone receptors were also evaluated in some studies. One study showed that the variant rs10069690 was associated to the estrogen receptor (ER)-negative and triple negative subtype, but other authors did not find the same results. In addition, the association of rs273618 with ER-/progesterone receptor (PR)+ cases, and rs10069690, rs2735940, rs4246742 and rs2736100 with both negative receptors were described. After data reanalyses, we found that the variant rs2735940 and rs2736100 were associated with ER-/PR- cases among patients with BC. Also, the variant rs2736100 was associated with ER+/PR+ cases and the variant rs2736118 was associated to ER+/PR+ and ER-/PR+ cases. CONCLUSIONS: The associations between hTERT variants and BC risk and outcomes could be useful since a polymorphism can be identified before the diagnosis, but the heterogeneity of data and analyses found in different studies lead to many controversies.

HLA-G 3'-untranslated region polymorphisms are associated with HTLV-1 infection, proviral load and HTLV-associated myelopathy/tropical spastic paraparesis development.

Most human T-lymphotropic virus type 1 (HTLV-1)-infected patients remain asymptomatic throughout life. The factors associated with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) development have not been fully elucidated; immunological and genetic factors may be involved. The association of 14 bp INS/DEL HLA-G polymorphism with HTLV-1 infection susceptibility has been reported previously. Here, other polymorphic sites at the HLA-G 3'-UTR (14-bp D/I, +3003C/T, +3010C/G, +3027A/C, +3035C/T, +3142C/G, +3187A/G and +3196C/G) were evaluated in 37 HTLV-1-infected individuals exhibiting HAM/TSP, 45 HTLV-1 asymptomatic carriers (HAC) and 153 uninfected individuals, followed up at University Hospital of the Faculty of Medicine of Ribeirão Preto, University of São Paulo, Brazil. It was observed that: (i) 14bpDI genotype is a risk factor for HTLV-1 infection, while the 14bpDD and +3142CC genotypes were associated with protection against infection; (ii) the +3142C allele and the +3003CT and +3142CC genotypes were associated with susceptibility, while 14bpII and +3003TT genotypes were associated with protection against HAM/TSP development; and (iii) the 14bpII, +3010CC, +3142GG and +3187AA genotypes were associated with lower HTLV-1 proviral load compared to respective counterpart genotypes. Findings that HLA-G has a well-recognized immunomodulatory role and that the genetic variability at HLA-G 3'-UTR may post-transcriptionally modify HLA-G production indicate a differential genetic susceptibility to: (i) the development of HTLV-1 infection, (ii) the magnitude of HTLV-1 proviral load and (iii) HAM/TSP development.

DSP30 enhances the immunosuppressive properties of mesenchymal stromal cells and protects their suppressive potential from lipopolysaccharide effects: A potential role of adenosine.

Multipotent mesenchymal stromal cells (MSC) are imbued with an immunosuppressive phenotype that extends to several immune system cells. In this study, we evaluated how distinct Toll-like receptor (TLR) agonists impact immunosuppressive properties of bone marrow (BM)-MSC and explored the potential mechanisms involved. We show that TLR4 stimulation by lipopolysaccharide (LPS) restricted the ability of MSC to suppress the proliferation of T lymphocytes, increasing the gene expression of interleukin (IL)-1ß and IL-6. In contrast, stimulation of TLR9 by DSP30 induced proliferation and the suppressive potential of BM-MSC, coinciding with reducing tumor necrosis factor (TNF)-α expression, increased expression of transforming growth factor (TGF)-ß1, increased percentages of BM-MSC double positive for the ectonucleotidases CD39+CD73+ and adenosine levels. Importantly, following simultaneous stimulation with LPS and DSP30, BM-MSC's ability to suppress T lymphocyte proliferation was comparable with that of non-stimulated BM-MSC levels. Moreover, stimulation of BM-MSC with LPS reduced significantly the gene expression levels, on co-cultured T lymphocyte, of IL-10 and interferon (IFN)γ, a cytokine with potential to enhance the immunosuppression mediated by MSC and ameliorate the clinical outcome of patients with graft-versus-host disease (GVHD). Altogether, our findings reiterate the harmful effects of LPS on MSC immunosuppression, besides indicating that DSP30 could provide a protective effect against LPS circulating in the blood of GVHD patients who receive BM-MSC infusions, ensuring a more predictable immunosuppressive effect. The novel effects and potential mechanisms following the stimulation of BM-MSC by DSP30 might impact their clinical use, by allowing the derivation of optimal "licensing" protocols for obtaining therapeutically efficient MSC.

Potential roles of microRNA-29a in the molecular pathophysiology of T-cell acute lymphoblastic leukemia.

Recent evidence has shown that deregulated expression of members of the microRNA-29 (miR-29) family may play a critical role in human cancer, including hematological malignancies. However, the roles of miR-29 in the molecular pathophysiology of T-cell acute lymphoblastic leukemia (T-ALL) has not been investigated. Here, we show that lower levels of miR-29a were significantly associated with higher blast counts in the bone marrow and with increased disease-free survival in T-ALL patients. Furthermore, miR-29a levels are extremely reduced in T-ALL cells compared to normal T cells. Microarray analysis following introduction of synthetic miR-29a mimics into Jurkat cells revealed the downregulation of several predicted targets (CDK6, PXDN, MCL1, PIK3R1, and CXXC6), including targets with roles in active and passive DNA demethylation (such as DNMT3a, DNMT3b, and members of the TET family and TDG). Restoring miR-29a levels in Jurkat and Molt-4 T-ALL cells led to the demethylation of many genes commonly methylated in T-ALL. Overall, our results suggest that reduced miR-29a levels may contribute to the altered epigenetic status of T-ALL, highlighting its relevance in the physiopathology of this disease.

RHCE and Kell genotyping and alloimmunization profile in patients with sickle cell disease in the Federal District of Brazil.

INTRODUCTION: Sickle cell disease (SCD) is the most important hemoglobinopathy worldwide. The treatment often requires phenotype-matched red blood cell (RBC) transfusions, but alloimmunization to non-ABO antigens may occur in a part of the SCD patients. The genotyping has been used for RBC antigen prediction, reducing the possibility of the alloimmunization. OBJECTIVE AND METHOD: In this study we performed the genotyping for the Kell and RHCE blood groups in samples from 77 phenotyped Brazilian SCD patients, whose alloimmunization profiles were also assessed. RESULTS: Discrepancies between genotyping and phenotyping for the RHCE and Kell blood groups systems were observed in 22.07% (17/77) of the SCD patients. We found C/c and E/e discrepancies in 11.68% and 9.09% of patients, respectively; one SCD patient (1.3%) presented a discrepancy in the Kell group. Two SCD patients with discrepancies between genotype and phenotype were alloimmunized. In total, twenty-eight patients (36.4%) developed alloantibodies, of which 55.17% were directed against antigens in the Rh system, 8.62% were directed against antigens in the Kell system and 36.20%, against other groups. Finally, the frequency of discrepancies is significantly higher in non-alloimmunized patients (30.61%), compared to alloimmunized patients (7.14%) (p = 0.0217). CONCLUSION: In part, the alloimmunization of the SCD patients may have been triggered by these discrepancies, indicating that the integration of serological and molecular tests in the immunohematology routine could help to increase the transfusion safety. However, the higher number of alloimmunized patients without discrepancies showed that reasons other than the discrepancies appear to have influenced more strongly the alloimmunization in the SCD patients in this study.

Merkel cell polyomavirus (MCPyV) DNA prevalence in Brazilian blood donors.

Merkel cell polyomavirus (MCPyV) is an oncogenic virus that has been etiologically linked to Merkel cell carcinoma. Low levels of MCPyV DNA have been detected in blood donors with unclear impact on transfusion. The prevalence of MCPyV DNA in Brazilian blood donors is unclear. Therefore, the objective of this study was to evaluate the MCPyV DNA prevalence among Brazilian blood donors. We examined the presence of MCPyV DNA by real-time PCR (qPCR) in a total of 450 serum samples obtained from blood donors from three Brazilian regions (North, Central-West and South). The overall estimated MCPyV DNA prevalence was 1.1% (CI = 95%, 0.16-2.06%). Divided by region, in North Brazil (city of Macapa, state of Amapá) and South Brazil (city of Santa Maria, state of Rio Grande do Sul), the MCPyV prevalence was the same: 1.33% (CI = 95%, range 0.0-3.14%). In Central-West Brazil (city of Brasilia), the MCPyV prevalence was 0.6% (CI = 95%, 0.0-1.96%). All MCPyV positive samples showed a high cycle threshold (median Ct = 35.5), most probably related to the low viral load. More studies are necessary to unveil the impact of this oncogenic virus on transfusion medicine and if such exists, especially in regards of its infectivity and transmission potential.

Seroprevalence of SARS-CoV-2 and Vaccination Coverage among Residents of a Lower-Middle-Class Population in the Federal District, Brazil.

Estimating seroprevalence and vaccination coverage against COVID-19 is crucial to the development of well-targeted public health policies at the local level. Here, we estimated seroprevalence and vaccination coverage in a lower-middle-class population in Brazil. We conducted an observational, cross-sectional, population-based survey from 24 September to 19 December 2021. CMIA tests were used to detect anti-SARS-CoV-2 IgG against the N-protein. The overall seroprevalence was 24.15% (177/733), and vaccination coverage was 91.40% (670/733); 72.09% (483/670) were fully vaccinated. Among vaccinated participants, seroprevalence was 24.77% (95% CI 21.50-28.04; 166/670), with a prevalence ratio (PR) of 1.03 (95% CI 0.98-1.08; p-value 0.131). Among participants who received an mRNA vaccine with S-based epitope (485), seroprevalence was 16.29% (95% CI 13.04-19.85; 79/485). Among unvaccinated participants, seroprevalence was 17.46% (95% CI 10.04-28.62; 11/63). Finally, in spite of the political climate and other possible causes for vaccine hesitancy, the positive Brazilian culture towards vaccination might have curbed hesitancy.

Human Pegivirus-1 Detection and Genotyping in Brazilian Patients with Fulminant Hepatitis.

Fulminant hepatitis is a severe clinical disease characterized by a marked decline in liver function and encephalopathy. In a previous survey, using metagenomics in a group of 27 patients with this clinical condition, we observed an expressive quantity of reads of the Human pegivirus-1 (HPgV-1). Therefore, the objective of this study was to evaluate the frequency, molecular features, and HPgV-1 circulating genotypes in patients with fulminant hepatitis. After testing the collected plasma samples, we discovered twelve samples (44.4%) that were positive for HPgV-1 RNA (using both real-time and nested PCR). The positive samples presented a mean cycle threshold (Ct) of 28.5 (±7.3). Genotyping assignments revealed that all HPgV-1 positive samples belonged to the HPgV-1 genotype 2 (both subgenotypes 2A and 2B were identified). Although HPgV-1 is considered a commensal virus, little is known regarding its prevalence and genotypes in cases of fulminant hepatitis. More research is needed to understand whether HPgV-1 can be implicated in clinical disorders and infectious diseases.

Exploring Viral Metagenomics in Pediatric Patients with Acute Respiratory Infections: Unveiling Pathogens beyond SARS-CoV-2.

The emergence of SARS-CoV-2 and the subsequent pandemic have prompted extensive diagnostic and clinical efforts to mitigate viral spread. However, these strategies have largely overlooked the presence of other respiratory viruses. Acute respiratory diseases in pediatric patients can be caused by a diverse range of viral agents, and metagenomics represents a powerful tool for their characterization. This study aimed to investigate the viral abundance in pediatric patients with acute respiratory symptoms who tested negative for SARS-CoV-2 during the Omicron pandemic wave. To achieve this, viral metagenomics and next-generation sequencing were employed on 96 nasopharyngeal swab samples, which were organized into 12 pools, with each pool consisting of eight individual samples. Metagenomic analysis revealed that the most prevalent viruses associated with acute disease in pediatric patients were respiratory syncytial virus (detected in all pools) and enteroviruses, which are known to cause significant morbidity and mortality in children. Additionally, clinically significant viruses such as mumps orthorubulavirus, human metapneumovirus, influenza A, and a wide array of human herpesviruses (1, 3-7) were identified. These findings highlight the extensive potential of viral metagenomics in identifying viruses other than SARS-CoV-2 that contribute to acute infections in children. Consequently, this methodology should garner clinical attention in terms of differential diagnosis and the development of public policies to address such conditions in the global pediatric population.

SARS CoV-2 seroprevalence and diagnostic accuracy during a COVID-19 outbreak in a major penitentiary complex in Brazil, June to July 2020.

PURPOSE: This study aims to estimate the overall SARS-CoV-2 seroprevalence and evaluate the accuracy of an antibody rapid test compared to a reference serological assay during a COVID-19 outbreak in a prison complex housing over 13,000 prisoners in Brasília. DESIGN/METHODOLOGY/APPROACH: The authors obtained a randomized, stratified representative sample of each prison unit and conducted a repeated serosurvey among prisoners between June and July 2020, using a lateral-flow immunochromatographic assay (LFIA). Samples were also retested using a chemiluminescence enzyme immunoassay (CLIA) to compare SARS-CoV-2 seroprevalence and 21-days incidence, as well as to estimate the overall infection fatality rate (IFR) and determine the diagnostic accuracy of the LFIA test. FINDINGS: This study identified 485 eligible individuals and enrolled 460 participants. Baseline and 21-days follow-up seroprevalence were estimated at 52.0% (95% CI 44.9-59.0) and 56.7% (95% CI 48.2-65.3) with LFIA; and 80.7% (95% CI 74.1-87.3) and 81.1% (95% CI 74.4-87.8) with CLIA, with an overall IFR of 0.02%. There were 78.2% (95% CI 66.7-89.7) symptomatic individuals among the positive cases. Sensitivity and specificity of LFIA were estimated at 43.4% and 83.3% for IgM; 46.5% and 91.5% for IgG; and 59.1% and 77.3% for combined tests. ORIGINALITY/VALUE: The authors found high seroprevalence of anti-SARS-CoV-2 antibodies within the prison complex. The occurrence of asymptomatic infection highlights the importance of periodic mass testing in addition to case-finding of symptomatic individuals; however, the field performance of LFIA tests should be validated. This study recommends that vaccination strategies consider the inclusion of prisoners and prison staff in priority groups.

Mesenchymal stem cells promote the sustained expression of CD69 on activated T lymphocytes: roles of canonical and non-canonical NF-κB signalling.

Mesenchymal stem cells (MSCs) are known to induce the conversion of activated T cells into regulatory T cells in vitro. The marker CD69 is a target of canonical nuclear factor kappa-B (NF-κB) signalling and is transiently expressed upon activation; however, stable CD69 expression defines cells with immunoregulatory properties. Given its enormous therapeutic potential, we explored the molecular mechanisms underlying the induction of regulatory cells by MSCs. Peripheral blood CD3(+) T cells were activated and cultured in the presence or absence of MSCs. CD4(+) cell mRNA expression was then characterized by microarray analysis. The drug BAY11-7082 (BAY) and a siRNA against v-rel reticuloendotheliosis viral oncogene homolog B (RELB) were used to explore the differential roles of canonical and non-canonical NF-κB signalling, respectively. Flow cytometry and real-time PCR were used for analyses. Genes with immunoregulatory functions, CD69 and non-canonical NF-κB subunits (RELB and NFKB2) were all expressed at higher levels in lymphocytes co-cultured with MSCs. The frequency of CD69(+) cells among lymphocytes cultured alone progressively decreased after activation. In contrast, the frequency of CD69(+) cells increased significantly following activation in lymphocytes co-cultured with MSCs. Inhibition of canonical NF-κB signalling by BAY immediately following activation blocked the induction of CD69; however, inhibition of canonical NF-κB signalling on the third day further induced the expression of CD69. Furthermore, late expression of CD69 was inhibited by RELB siRNA. These results indicate that the canonical NF-κB pathway controls the early expression of CD69 after activation; however, in an immunoregulatory context, late and sustained CD69 expression is promoted by the non-canonical pathway and is inhibited by canonical NF-κB signalling.

Upregulation of hsa-miR-125b in HTLV-1 asymptomatic carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis patients.

The retrovirus human T lymphotropic virus type 1 (HTLV-1) promotes spastic paraparesis, adult T cell leukaemia and other diseases. Recently, some human microRNAs (miRNAs) have been described as important factors in host-virus interactions. This study compared miRNA expression in control individuals, asymptomatic HTLV-1 carriers and HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis patients. The proviral load and Tax protein expression were measured in order to characterize the patients. hsa-miR-125b expression was significantly higher in patients than in controls (p = 0.0285) or in the HAM group (p = 0.0312). Therefore, our findings suggest that miR-125b expression can be used to elucidate the mechanisms of viral replication and pathogenic processes.

Viral Metagenomics for the Identification of Emerging Infections in Clinical Samples with Inconclusive Dengue, Zika, and Chikungunya Viral Amplification.

Viral metagenomics is increasingly being used for the identification of emerging and re-emerging viral pathogens in clinical samples with unknown etiology. The objective of this study was to shield light on the metavirome composition in clinical samples obtained from patients with clinical history compatible with an arboviral infection, but that presented inconclusive results when tested using RT-qPCR. The inconclusive amplification results might be an indication of the presence of an emerging arboviral agent that is inefficiently amplified by conventional PCR techniques. A total of eight serum samples with inconclusive amplification results for the routinely tested arboviruses-dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) obtained during DENV and CHIKV outbreaks registered in the state of Alagoas, Northeast Brazil between July and August 2021-were submitted to metagenomic next-generation sequencing assay using NextSeq 2000 and bioinformatic pipeline for viral discovery. The performed bioinformatic analysis revealed the presence of two arboviruses: DENV type 2 (DENV-2) and CHIKV with a high genome coverage. Further, the metavirome of those samples revealed the presence of multiple commensal viruses apparently without clinical significance. The phylogenetic analysis demonstrated that the DENV-2 genome belonged to the Asian/American genotype and clustered with other Brazilian strains. The identified CHIKV genome was taxonomically assigned as ECSA genotype, which is circulating in Brazil. Together, our results reinforce the utility of metagenomics as a valuable tool for viral identification in samples with inconclusive arboviral amplification. Viral metagenomics is one of the most potent methods for the identification of emerging arboviruses.