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researchsquare; 2021.


Background: COVID-19 has a broad spectrum of clinical manifestations, from asymptomatic to mild or moderate symptoms, reaching the most severe forms and death. The mechanisms underlying the SARS-CoV-2 infection and its clinical evolution are still unclear. Once SARS-CoV-2 infects individuals, host factors are activated by the presence of the virus inside the cells, such as the inflammasome system. The search of risk factors for COVID-19 is of relevance for clinical management. In this study, we investigated the impact of 11 single-base polymorphisms (SNPs) in the NLRP3, CARD8, AIM2, CASP-1, IFI16, and IL-1β inflammasome genes in SARS-CoV-2 infected individuals with distinct disease outcomes. Methods Patients were divided into two groups: (1) inpatients, with severe/critical disease (Hospitalized group, n=451), and (2) convalescent volunteers with prior SARS-CoV-2 infection and a history of asymptomatic to mild symptoms (Mild group, n=43). Patients hospitalized were followed up at a Hospital Center for COVID-19 Pandemic – National Institute of Infectology (INI)/FIOCRUZ, Rio de Janeiro, Brazil, from June 2020 to March 2021. The Mild group was recruited at Oswaldo Cruz Institute (IOC)/FIOCRUZ, Rio de Janeiro, Brazil, in 2020. Genotyping of the SNPs was determined by Real-Time PCR. Protection and risk estimations were performed by unconditional logistic regression models. Results Among the genotyped SNPs, significant differences in the NLRP3 rs1539019 and rs10754558 frequencies were observed between the groups. The C/C genotype (OR adj =6.31; P adj =0.026) or allele C (OR adj =1.05; P adj =0.002) in rs1539019 polymorphism were associated with the risk for hospitalization, while the C/G genotype (OR adj =0.16; P adj =0.016) or carrier-G (OR adj =0.2; P adj =0.028) in rs10754558 polymorphism were associated with protection for hospitalization. Regarding the NLRP3 genetic variants, the A-C-G-C-G haplotype (OR adj =0.14; P adj = 0.030) was associated with protection for hospitalization. No significant association was observed for the other polymorphisms. Conclusions As of our knowledge, this is the first study demonstrating the association of inflammasome NLRP3 variants with risk and/or protection for hospitalization in COVID-19. Studies linking the NLRP3 inflammasome and SARS-CoV-2 infection are still scarce due to the recent emergence of this pathogen. Our results contribute to the discussion of the impact of inflammasomes in the clinical evolution of COVID-19.

researchsquare; 2020.


The coronaviruses (CoVs) called the attention of the world for causing outbreaks of severe acute respiratory syndrome (SARS-CoV), in Asia in 2002-03, and respiratory disease in the Middle East (MERS-CoV), in 2012. In December 2019, yet again a new coronavirus (SARS-CoV-2) first identified in Wuhan, China, was associated with a severe respiratory infection, known today as COVID-19. This new virus is highly transmissible, and quickly spread throughout China and 30 additional countries. As result, the World Health Organization (WHO) elevated the status of the COVID-19 outbreak from emergency of international concern to pandemic on March 11, 2020. The impact of COVID-19 on public health and economy fueled a worldwide race to approve therapeutic and prophylactic agents, but so far, there are no specific antiviral drugs or vaccines available. In current scenario, the development of in vitro systems for viral mass production and for testing antiviral and vaccine candidates proves to be an urgent matter. Research groups around the world are strongly focused on this, and the susceptibility of different cell lines to SARS-CoV-2 infection has already been demonstrated by molecular techniques. However, data on the biology of SARS-CoV-2 at the ultrastructural level in these in vitro models is still scarce. In this study, we documented, by transmission electron microscopy and real-time RT-PCR, the infection of Vero-E6 cells with SARS-CoV-2 samples isolated from Brazilian patients. The infected cells presented cytopathic effects and SARS-CoV-2 particles were observed attached to the cell surface and inside cytoplasmic vesicles. The entry of the virus into cells occurred through the endocytic pathway or by fusion of the viral envelope with the cell membrane. Assembled nucleocapsids were verified inside rough endoplasmic reticulum cisterns (RER). Viral maturation seemed to occur by budding of viral particles from the RER into smooth membrane vesicles. Therefore, the susceptibility of Vero-E6 cells to SARS-CoV-2 infection and the viral pathway inside the cells were demonstrated by ultrastructural analysis.

COVID-19 , Severe Acute Respiratory Syndrome , Respiratory Tract Diseases , Respiratory Tract Infections
biorxiv; 2020.
Preprint in English | bioRxiv | ID: ppzbmed-10.1101.2020.04.30.069039


Genomic surveillance has become a useful tool for better understanding virus pathogenicity, origin and spread. Obtaining accurately assembled, complete viral genomes directly from clinical samples is still a challenging. Here, we describe three protocols using a unique primer set designed to recover long reads of SARS-CoV-2 directly from total RNA extracted from clinical samples. This protocol is useful, accessible and adaptable to laboratories with varying resources and access to distinct sequencing methods: Nanopore, Illumina and/or Sanger.