Evaluation of a rapid diagnostic test for measles IgM detection; accuracy and the reliability of visual reading using sera from the measles surveillance programme in Brazil, 2015.

Laboratory-based case confirmation is an integral part of measles surveillance programmes; however, logistical constraints can delay response. Use of RDTs during initial patient contact could enhance surveillance by real-time case confirmation and accelerating public health response. Here, we evaluate performance of a novel measles IgM RDT and assess accuracy of visual interpretation using a representative collection of 125 sera from the Brazilian measles surveillance programme. RDT results were interpreted visually by a panel of six independent observers, the consensus of three observers and by relative reflectance measurements using an ESEQuant Reader. Compared to the Siemens anti-measles IgM EIA, sensitivity and specificity of the RDT were 94.9% (74/78, 87.4-98.6%) and 95.7% (45/47, 85.5-99.5%) for consensus visual results, and 93.6% (73/78, 85.7-97.9%) and 95.7% (45/47, 85.5-99.5%), for ESEQuant measurement, respectively. Observer agreement, determined by comparison between individuals and visual consensus results, and between individuals and ESEQuant measurements, achieved average kappa scores of 0.97 and 0.93 respectively. The RDT has the sensitivity and specificity required of a field-based test for measles diagnosis, and high kappa scores indicate this can be accomplished accurately by visual interpretation alone. Detailed studies are needed to establish its role within the global measles control programme.

Two-Step In Vitro Model to Evaluate the Cellular Immune Response to SARS-CoV-2.

The cellular immune response plays an important role in COVID-19, caused by SARS-CoV-2. This feature makes use of in vitro models' useful tools to evaluate vaccines and biopharmaceutical effects. Here, we developed a two-step model to evaluate the cellular immune response after SARS-CoV-2 infection-induced or spike protein stimulation in peripheral blood mononuclear cells (PBMC) from both unexposed and COVID-19 (primo-infected) individuals (Step1). Moreover, the supernatants of these cultures were used to evaluate its effects on lung cell lines (A549) (Step2). When PBMC from the unexposed were infected by SARS-CoV-2, cytotoxic natural killer and nonclassical monocytes expressing inflammatory cytokines genes were raised. The supernatant of these cells can induce apoptosis of A549 cells (mock vs. Step2 [mean]: 6.4% × 17.7%). Meanwhile, PBMCs from primo-infected presented their memory CD4+ T cells activated with a high production of IFNG and antiviral genes. Supernatant from past COVID-19 subjects contributed to reduce apoptosis (mock vs. Step2 [ratio]: 7.2 × 1.4) and to elevate the antiviral activity (iNOS) of A549 cells (mock vs. Step2 [mean]: 31.5% × 55.7%). Our findings showed features of immune primary cells and lung cell lines response after SARS-CoV-2 or spike protein stimulation that can be used as an in vitro model to study the immunity effects after SARS-CoV-2 antigen exposure.

Investigation of Human IFITM3 Polymorphisms rs34481144A and rs12252C and Risk for Influenza A(H1N1)pdm09 Severity in a Brazilian Cohort.

Influenza is a major public health problem that causes acute respiratory infection in humans. Identification of host factors influencing in disease outcome is critical for recognition of individuals with increased risk. Investigations on the role of rs34481144A and rs12252C IFITM3 polymorphisms in influenza A(H1N1)pdm09 severity is not yet conclusively determined. This study aimed to evaluate such polymorphisms frequencies and IFITM3 levels in an infected Brazilian cohort of 314 influenza A(H1N1)pdm09 cases and its putative association with clinical, epidemiological and virological data. Individuals were clinically classified into mild, severe and fatal cases. IFITM3 polymorphisms were detected by specific Taqman probes in real time PCR reactions. IFITM3 levels were determined by quantitative real time PCR. Thus, the different clinical groups presented similar distribution of rs34481144 and rs12252 genotypes and allelic frequencies. There was no significant association between the polymorphisms with severity of disease by using distinct genetic models. Additionally, geographic distribution of mutants showed that rs34481144A allele was more predominant in Brazilian Southern region. In contrast, rs12252C allele presented similar frequencies in all regions. Individuals with the distinct rs34481144 and rs12252 genotypes showed similar levels of IFITM3 and viral load in their respiratory specimens. Furthermore, IFITM3 levels were comparable in the distinct clinical groups and were not correlated with influenza viral load in analyzed samples. Thereby, rs34481144A and rs12252C polymorphisms were not associated with severity or mortality of influenza A(H1N1)pdm09 infection nor with IFITM3 transcript levels and influenza viral load in upper respiratory tract samples in a Brazilian cohort.

Atazanavir, Alone or in Combination with Ritonavir, Inhibits SARS-CoV-2 Replication and Proinflammatory Cytokine Production.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is already responsible for far more deaths than previous pathogenic coronaviruses (CoVs) from 2002 and 2012. The identification of clinically approved drugs to be repurposed to combat 2019 CoV disease (COVID-19) would allow the rapid implementation of potentially life-saving procedures. The major protease (Mpro) of SARS-CoV-2 is considered a promising target, based on previous results from related CoVs with lopinavir (LPV), an HIV protease inhibitor. However, limited evidence exists for other clinically approved antiretroviral protease inhibitors. Extensive use of atazanavir (ATV) as antiretroviral and previous evidence suggesting its bioavailability within the respiratory tract prompted us to study this molecule against SARS-CoV-2. Our results show that ATV docks in the active site of SARS-CoV-2 Mpro with greater strength than LPV, blocking Mpro activity. We confirmed that ATV inhibits SARS-CoV-2 replication, alone or in combination with ritonavir (RTV) in Vero cells and a human pulmonary epithelial cell line. ATV/RTV also impaired virus-induced enhancement of interleukin 6 (IL-6) and tumor necrosis factor alpha (TNF-α) levels. Together, our data strongly suggest that ATV and ATV/RTV should be considered among the candidate repurposed drugs undergoing clinical trials in the fight against COVID-19.

Human CCR5Δ32 (rs333) polymorphism has no influence on severity and mortality of influenza A(H1N1)pdm09 infection in Brazilian patients from the post pandemic period.

BACKGROUND: Influenza is an acute and highly contagious viral respiratory infection that causes significant morbidity and mortality. The identification of host genetic factors associated with susceptibility and severity of influenza virus infection is of paramount importance. Previous studies evaluating the potential involvement of the CCR5Δ32 polymorphism (rs333), a 32 base pair deletion in CC motif chemokine receptor 5 (CCR5) gene, in severity and mortality of influenza A(H1N1)pdm09 infected individuals have been reported, but their results are quite conflicting. OBJECTIVES: The aim of this study was the evaluation of the CCR5Δ32 frequency in individuals with mild, severe and fatal influenza A(H1N1)pdm09 infection and its putative association with clinical and epidemiologic data. PATIENTS/METHODS: A total of 432 individuals were included in this study and classified according to their clinical status, into the following groups: influenza like illness (ILI) (n = 153); severe acute respiratory infection (SARI) (n = 173) and fatal (n = 106) cases. The samples were collected in the post pandemic period, from 2012 to 2018. Individuals were further stratified according to their clinical and epidemiological data. The CCR5Δ32 variant was genotyped by PCR amplification and a subset of samples was further submitted to Sanger sequencing. RESULTS: The different clinical groups (ILI, SARI and fatal) presented similar distribution of wt/wt and wt/Δ32 genotypes and CCR5Δ32 allele frequencies. Genotype Δ32/Δ32 was not detected in our study. Additionally, no association between wt/wt and wt/Δ32 genotypes and dyspnea, a clinical factor for influenza complications was found. Similarly, no significant differences in the distribution of wt/wt and wt/Δ32 genotypes and CCR5Δ32 variant allele frequencies were observed in samples from the different Brazilian geographical regions. CONCLUSIONS: The CCR5Δ32 variant does not influence the susceptibility to influenza A(H1N1)pdm09 severe disease or mortality in individuals from Brazil.

Phosphatidyl Inositol 3 Kinase-Gamma Balances Antiviral and Inflammatory Responses During Influenza A H1N1 Infection: From Murine Model to Genetic Association in Patients.

Influenza A virus (IAV) infection causes severe pulmonary disease characterized by intense leukocyte infiltration. Phosphoinositide-3 kinases (PI3Ks) are central signaling enzymes, involved in cell growth, survival, and migration. Class IB PI3K or phosphatidyl inositol 3 kinase-gamma (PI3Kγ), mainly expressed by leukocytes, is involved in cell migration during inflammation. Here, we investigated the contribution of PI3Kγ for the inflammatory and antiviral responses to IAV. PI3Kγ knockout (KO) mice were highly susceptible to lethality following infection with influenza A/WSN/33 H1N1. In the early time points of infection, infiltration of neutrophils was higher than WT mice whereas type-I and type-III IFN expression and p38 activation were reduced in PI3Kγ KO mice resulting in higher viral loads when compared with WT mice. Blockade of p38 in WT macrophages infected with IAV reduced levels of interferon-stimulated gene 15 protein to those induced in PI3Kγ KO macrophages, suggesting that p38 is downstream of antiviral responses mediated by PI3Kγ. PI3Kγ KO-derived fibroblasts or macrophages showed reduced type-I IFN transcription and altered pro-inflammatory cytokines suggesting a cell autonomous imbalance between inflammatory and antiviral responses. Seven days after IAV infection, there were reduced infiltration of natural killer cells and CD8+ T lymphocytes, increased concentration of inflammatory cytokines in bronchoalveolar fluid, reduced numbers of resolving macrophages, and IL-10 levels in PI3Kγ KO. This imbalanced environment in PI3Kγ KO-infected mice culminated in enhanced lung neutrophil infiltration, reactive oxygen species release, and lung damage that together with the increased viral loads, contributed to higher mortality in PI3Kγ KO mice compared with WT mice. In humans, we tested the genetic association of disease severity in influenza A/H1N1pdm09-infected patients with three potentially functional PIK3CG single-nucleotide polymorphisms (SNPs), rs1129293, rs17847825, and rs2230460. We observed that SNPs rs17847825 and rs2230460 (A and T alleles, respectively) were significantly associated with protection from severe disease using the recessive model in patients infected with influenza A(H1N1)pdm09. Altogether, our results suggest that PI3Kγ is crucial in balancing antiviral and inflammatory responses to IAV infection.

Susceptibility of Brazilian influenza A(H1N1)pdm09 viruses to neuraminidase inhibitors in the 2014-2016 seasons: Identification of strains bearing mutations associated with reduced inhibition profile.

Neuraminidase inhibitors (NAIs) are the main class of antivirals currently used for the treatment of influenza infections. As influenza viruses are constantly evolving, drug-resistance can emerge resulting in reduced effectiveness of treatment. This study evaluated the presence of molecular markers associated with NAI susceptibility in 724 influenza A(H1N1)pdm09 positive samples from Brazilian surveillance system from the 2014-2016 seasons, including 76 isolates tested for oseltamivir (OST) susceptibility and 23 isolates also tested for zanamivir, peramivir and laninamivir susceptibility. We identified the H275Y (n = 3) and I223K (n = 1) NA substitutions, associated with reduced inhibition (RI) by the NAIs. Noteworthy, no epidemiological links were identified among the patients infected with the mutant viruses. Phylogenetic analysis from NA and hemagglutinin genes showed that mutant viruses were not clustered. All mutant virus strains carried the permissive substitutions V241I and N369K, in addition to the N386K, which has been shown to destabilize the NA structure. Functional NA analysis of one virus containing the H275Y mutation confirmed its highly RI profile to OST and peramivir and demonstrated that it had decreased viral replication and NA thermostability compared to the wild type virus. The remaining tested isolates presented normal inhibition profile to the NAIs tested. In conclusion, the overall frequency of influenza A(H1N1)pdm09 viruses bearing mutations associated with NAI RI was 0.6%, similar to what has been observed in recent global studies.

Both osteopontin-c and osteopontin-b splicing isoforms exert pro-tumorigenic roles in prostate cancer cells.

BACKGROUND: Alternative splicing of the osteopontin (opn, spp1) gene generates three protein splicing isoforms (OPN-SI), designated as OPNa, OPNb, and OPNc, which have demonstrated specific roles in different tumor models. This work aims to investigate the roles of each OPN-SI in prostate cancer (PCa) progression by using in vivo and in vitro functional assays. METHODS: The expression levels of OPN-SI in prostate cell lines were analyzed by qRT-PCR. PC-3 was stably transfected with expression vectors containing OPNa, OPNb, and OPNc, as well as empty vector controls. PC-3 cells overexpressing each construct were analyzed for in vivo tumor growth and in relation to different aspects mimicking tumor progression, such as cell proliferation, migration, invasion, and soft agar colony formation. RESULTS: OPN-SI are overexpressed in PCa as compared to non-tumoral prostate cell lines. OPNc and OPNb overexpressing cells significantly activated enhanced xenograft tumor growth and PC-3 proliferation, migration, invasion, and soft agar colony formation, as well as the expression of MMP-2, MMP-9, and VEGF. These isoforms also support sustained proliferative survival. We found that both OPNc and OPNb pro-tumorigenic roles are mainly mediated through PI3K signaling. Inhibition of this pathway by using LY294002 specifically inhibited tumor progression features evoked by OPNc and OPNb overexpression. CONCLUSIONS: Our data provide evidence that both OPNc and OPNb splicing isoforms promote distinct aspects of PCa progression by inducing PI3K signaling. These data give support to strategies aiming to downregulate OPNc and OPNb expression as an approach to inhibit PCa progression.

Cholesterol depletion by methyl-beta-cyclodextrin enhances myoblast fusion and induces the formation of myotubes with disorganized nuclei.

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-beta-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric alpha-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and beta-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.