RESUMEN
The effects of neuroinvasion by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) become clinically relevant due to the numerous neurological symptoms observed in Corona Virus Disease 2019 (COVID-19) patients during infection and post-COVID syndrome or long COVID. This study reports the biofabrication of a 3D bioprinted neural-like tissue as a proof-of-concept platform for a more representative study of SARS-CoV-2 brain infection. Bioink is optimized regarding its biophysical properties and is mixed with murine neural cells to construct a 3D model of COVID-19 infection. Aiming to increase the specificity to murine cells, SARS-CoV-2 is mouse-adapted (MA-SARS-CoV-2) in vitro, in a protocol first reported here. MA-SARS-CoV-2 reveals mutations located at the Orf1a and Orf3a domains and is evolutionarily closer to the original Wuhan SARS-CoV-2 strain than SARS-CoV-2 used for adaptation. Remarkably, MA-SARS-CoV-2 shows high specificity to murine cells, which present distinct responses when cultured in 2D and 3D systems, regarding cell morphology, neuroinflammation, and virus titration. MA-SARS-CoV-2 represents a valuable tool in studies using animal models, and the 3D neural-like tissue serves as a powerful in vitro platform for modeling brain infection, contributing to the development of antivirals and new treatments for COVID-19.
Asunto(s)
COVID-19 , SARS-CoV-2 , Animales , Encéfalo , COVID-19/complicaciones , Humanos , Ratones , Neuronas , Síndrome Post Agudo de COVID-19RESUMEN
DNA methylation is one of the epigenetic modifications that configures gene transcription programs. This study describes the DNA methylation profile of HIV-infected individuals with distinct characteristics related to natural and artificial viremia control. Sheared DNA from circulating mononuclear cells was subjected to target enrichment bisulfite sequencing designed to cover CpG-rich genomic regions. Gene expression was assessed through RNA-seq. Hypermethylation in virologic responders was highly distributed closer to Transcription Start Sites (p-value = 0.03). Hyper and hypomethylation levels within TSS adjacencies varied according to disease progression status (Kruskal-Wallis, p < 0.001), and specific differentially methylated regions associated genes were identified for each group. The lower the promoter methylation, the higher the gene expression in subjects undergoing virologic failure (R = - 0.82, p = 0.00068). Among the inversely correlated genes, those supporting glycolysis and its related pathways were hypomethylated and up-regulated in virologic failures. Disease progression heterogeneity was associated with distinct DNA methylation patterns in terms of rates and distribution. Methylation was associated with the expression of genes sustaining intracellular glucose metabolism in subjects undergoing antiretroviral virologic failure. Our findings highlight that DNA methylation is associated with latency, disease progression, and fundamental cellular processes.
Asunto(s)
Metilación de ADN , Epigénesis Genética , Regulación de la Expresión Génica , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , Respuesta Virológica Sostenida , Latencia del Virus/genética , Adulto , Antirretrovirales/uso terapéutico , Estudios de Casos y Controles , Islas de CpG , Progresión de la Enfermedad , Femenino , Estudio de Asociación del Genoma Completo , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Infecciones por VIH/patología , Humanos , Masculino , Persona de Mediana Edad , Regiones Promotoras GenéticasRESUMEN
[This corrects the article DOI: 10.1016/j.eclinm.2021.100981.].
RESUMEN
BACKGROUND: The absence of specific antivirals to treat COVID-19 leads to the repositioning of candidates' drugs. Nitazoxanide (NTZ) has a broad antiviral effect. METHODS: This was a randomized, double-blind pilot clinical trial comparing NTZ 600 mg BID versus Placebo for seven days among 50 individuals (25 each arm) with SARS-COV-2 RT-PCR+ (PCR) that were hospitalized with mild respiratory insufficiency from May 20th, 2020, to September 21st, 2020 (ClinicalTrials.gov NCT04348409). Clinical and virologic endpoints and inflammatory biomarkers were evaluated. A five-point scale for disease severity (SSD) was used. FINDINGS: Two patients died in the NTZ arm compared to 6 in the placebo arm (p = 0.564). NTZ was superior to placebo when considering SSD (p < 0001), the mean time for hospital discharge (6.6 vs. 14 days, p = 0.021), and negative PCR at day 21 (p = 0.035), whereas the placebo group presented more adverse events (p = 0.04). Among adverse events likely related to the study drug, 14 were detected in the NTZ group and 22 in placebo (p = 0.24). Among the 30 adverse events unlikely related, 21 occurred in the placebo group (p = 0.04). A decrease from baseline was higher in the NTZ group for d-Dimer (p = 0.001), US-RCP (p < 0.002), TNF (p < 0.038), IL-6 (p < 0.001), IL-8 (p = 0.014), HLA DR. on CD4+ T lymphocytes (p < 0.05), CD38 in CD4+ and CD8+ T (both p < 0.05), and CD38 and HLA-DR. on CD4+ (p < 0.01). INTERPRETATION: Compared to placebo in clinical and virologic outcomes and improvement of inflammatory outcomes, the superiority of NTZ warrants further investigation of this drug for moderate COVID-19 in larger clinical trials. A higher incidence of adverse events in the placebo arm might be attributed to COVID-19 related symptoms.
RESUMEN
DNA methylation is one of the epigenetic modifications that configures gene transcription programs. This study describes the DNA methylation profile of HIV-infected individuals with distinct characteristics related to natural and artificial viremia control. Sheared DNA from circulating mononuclear cells was subjected to target enrichment bisulfite sequencing designed to cover CpG-rich genomic regions. Gene expression was assessed through RNA-seq. Hypermethylation in virologic responders was highly distributed closer to Transcription Start Sites (p-value = 0.03). Hyper and hypomethylation levels within TSS adjacencies varied according to disease progression status (Kruskal–Wallis, p < 0.001), and specific differentially methylated regions associated genes were identified for each group. The lower the promoter methylation, the higher the gene expression in subjects undergoing virologic failure (R = − 0.82, p = 0.00068). Among the inversely correlated genes, those supporting glycolysis and its related pathways were hypomethylated and up-regulated in virologic failures. Disease progression heterogeneity was associated with distinct DNA methylation patterns in terms of rates and distribution. Methylation was associated with the expression of genes sustaining intracellular glucose metabolism in subjects undergoing antiretroviral virologic failure. Our findings highlight that DNA methylation is associated with latency, disease progression, and fundamental cellular processes.
RESUMEN
Two hundred forty million people are chronically infected with hepatitis B virus (HBV) worldwide. The rise of globalization has facilitated the emergence of novel HBV recombinants and genotypes. We evaluated HBV genotypes and recombinants, mutations associated with resistance to antivirals (AVs), progression of hepatic illness, and inefficient hepatitis B vaccination responses in chronically infected individuals in the city of São Paulo, Brazil. Forty-five full-length and 24 partial-length sequences were obtained. The genotype distribution was as follows: A (66.7%), D (15.9%), F (11.6%) and C (4.3%). We describe a new recombinant (D2/D3), confirmed through next-generation sequencing (NGS) and reconstruction of the quasispecies sequences in silico. Primary resistance and major vaccine escape mutations were not found. We did, however, find mutations in the S region that might may be related to HBV antigenicity changes, as well as Pre-S deletions. The precore/core mutations A1762T + G1764A (40.9%) were found mostly in genotypes A and D, and G1896A (29.55%) was more frequent in genotype D than in genotype A. The genotypic distribution reflects the history of Brazilian immigration. This is the first description of recombination between genotypes D2 and D3 in Brazil. It is also the first confirmation through NGS and reconstruction of the quasispecies in silico. However, little is known about the response to treatment of recombinants. This demonstrates the need for molecular epidemiology studies involving the analysis of full-length HBV sequences.
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Farmacorresistencia Viral/genética , Virus de la Hepatitis B/genética , Hepatitis B Crónica/epidemiología , Virus Reordenados/genética , Recombinación Genética , Adenina/análogos & derivados , Adenina/uso terapéutico , Adulto , Anciano , Antivirales/uso terapéutico , Brasil/epidemiología , Femenino , Genotipo , Virus de la Hepatitis B/clasificación , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/transmisión , Hepatitis B Crónica/virología , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Lamivudine/uso terapéutico , Masculino , Persona de Mediana Edad , Epidemiología Molecular , Mutación , Organofosfonatos/uso terapéutico , Filogenia , Virus Reordenados/clasificación , Virus Reordenados/efectos de los fármacos , Virus Reordenados/aislamiento & purificación , Estudios RetrospectivosRESUMEN
BACKGROUND: Continuous long-term treatment is recommended to reduce the hepatitis B virus (HBV) viral load. However, as a consequence, resistance mutations can emerge and be transmitted to other individuals. The polymerase (POL) gene overlaps the surface (S) gene. Thus, during treatment, mutations in the POL gene may lead to changes in hepatitis B surface antigen (HBsAg). The purpose of this study was to evaluate the frequency of lamivudine and vaccine escape mutations in HBsAg-positive blood donors from the city of Santos and in untreated HBV mono-infected patients from the city of São Paulo, Brazil. METHODS: HBV DNA was extracted from 80 serum samples, of which 61 were from volunteer blood donors and 19 were from untreated HBV patients. A fragment of the POL/S genes containing 593 base pairs was amplified using nested PCR. Thirty four were PCR-positive and sequencing was performed using an ABI Prism 3130 Genetic Analyzer. Alignments and mutation mapping were performed using BioEdit software. RESULTS: HBV DNA from 21 blood donors and 13 untreated patient samples were characterized using nucleotide sequencing PCR products from the POL/S genes. We were able to detect one sample with the resistance mutation to lamivudine rtM204V + rtL180M (2.94%), which was found in a volunteer blood donor that has never used antiviral drugs. The other samples showed only compensatory mutations, such as rtL80F (5.88%), rtL80V (2.94%), rtL82V + rtV207L (2.94%), rtT128P (5.88%), rtT128N/S (2.94%) and rtS219A (5.88%). We found modifications in the S gene in 14 of the 34 samples (41.16%). The mutations detected were as follows: sM133L + sI195T (2.94%), sI195M (2.94%), sP120T (2.94%), sY100S/F (2.94%), sY100C (17.64%), sI/T126P + sQ129P (2.94%), sM198I + sF183C (2.94%) and sS210R (5.88%). CONCLUSIONS: Our results suggest the transmission of lamivudine-resistant forms. Thus, the evaluation of HBV-infected subjects for lamivudine resistance would improve treatment regime. Moreover, the mutations in the S gene may impair HBsAg antigenicity and contribute to HBsAg failure detection and vaccine escape.
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Antivirales/farmacología , Farmacorresistencia Viral , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Hepatitis B Crónica/virología , Lamivudine/farmacología , Adulto , Brasil , ADN Viral/química , ADN Viral/genética , ADN Viral/aislamiento & purificación , Errores Diagnósticos , Reacciones Falso Negativas , Femenino , Antígenos de Superficie de la Hepatitis B/sangre , Vacunas contra Hepatitis B/inmunología , Hepatitis B Crónica/diagnóstico , Hepatitis B Crónica/tratamiento farmacológico , Humanos , Evasión Inmune , Masculino , Mutación Missense , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADNRESUMEN
Recent studies suggest that GB virus C/hepatitis G virus (GBV-C/HGV) infection in HIV-positive individuals is associated with a slower progression to AIDS, leading to a lower HIV viral load and higher counts of CD4(+) T cells, although many studies have failed to demonstrate these beneficial effects. We developed a Real-Time PCR (TaqMan RT qPCR) to quantify the viral load of GBV-C/HGV in 102 HIV-1-infected patients, who were also evaluated for the presence of anti-E2. The prevalence of GBV-C/HGV infection was 21% among infected patients and the mean plasma viral load was 3.62 ± 0.64 log(10) copies/ml. Despite the high prevalence, there was no statistical difference when we compared the mean viral load (p≤0.46) and the average count of CD4(+) (p≤0.29) and CD8(+) (p≤0.64) among patients infected by GBV-C/HGV and HIV and patients infected only by HIV. This fact can be explained by the number of patients included in the study. Nevertheless, compared to other studies, we observed a discrete number of patients with undetectable HIV load and lower median viral load in the group presenting GBV-C/HGV RNA. Our study suggests that there may be an impact on HIV viral load in GBV-C/HGV-coinfected patients. However, further studies are needed to elucidate the molecular and cellular mechanisms involved in this viral interaction, previously reported in other studies, with the aim of contributing to the development of new targets for drugs against HIV.