The global spread of new SARS-CoV-2 variants of concern underscore an urgent need of simple deployed molecular tools that can differentiate these lineages. Several tools and protocols have been shared since the beginning of the COVID-19 pandemic, but they need to be timely adapted to cope with SARS-CoV-2 evolution. Although whole-genome sequencing (WGS) of the virus genetic material has been widely used, it still presents practical difficulties such as high cost, shortage of available reagents in the global market, need of a specialized laboratorial infrastructure and well-trained staff. These limitations result in SARS-CoV-2 surveillance blackouts across several countries. Here we propose a rapid and accessible protocol based on Sanger sequencing of a single PCR fragment that is able to identify and discriminate all SARS-CoV-2 variants of concern (VOCs) identified so far, according to each characteristic mutational profile at the Spike-RBD region (K417N/T, E484K, N501Y, A570D). Twelve COVID-19 samples from Brazilian patients were evaluated for both WGS and Sanger sequencing: three P.2, two P.1, six B.1.1 and one B.1.1.117 lineage. All results from the Sanger sequencing method perfectly matched the mutational profile of VOCs and non-VOCs RBD's characterized by WGS. In summary, this approach allows a much broader network of laboratories to perform molecular surveillance of SARS-CoV-2 VOCs and report results within a shorter time frame, which is of utmost importance in the context of rapid public health decisions in a fast evolving worldwide pandemic.

The coronavirus disease 2019 (COVID-19) pandemic unfolded due to the widespread severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission reinforced the urgent need for affordable molecular diagnostic alternative methods for massive testing screening. We present the clinical validation of a pH-dependent colorimetric reverse transcription loop-mediated isothermal amplification (RT-LAMP) for SARS-CoV-2 detection. The method revealed a limit of detection of 19.3 ± 2.7 viral genomic copies/μL when using RNA extracted samples obtained from nasopharyngeal swabs collected in guanidine-containing viral transport medium. Typical RT-LAMP reactions were performed at 65°C for 30 min. When compared to reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR), up to cycle-threshold (Ct) value 32, RT-LAMP presented 98% [95% confidence interval (CI) = 95.3–99.5%] sensitivity and 100% (95% CI = 94.5–100%) specificity for SARS-CoV-2 RNA detection targeting E and N genes. No cross-reactivity was detected when testing other non–SARS-CoV virus, confirming high specificity. The test is compatible with primary RNA extraction–free samples. We also demonstrated that colorimetric RT-LAMP can detect SARS-CoV-2 variants of concern and variants of interest, such as variants occurring in Brazil named gamma (P.1), zeta (P.2), delta (B.1.617.2), B.1.1.374, and B.1.1.371. The method meets point-of-care requirements and can be deployed in the field for high-throughput COVID-19 testing campaigns, especially in countries where COVID-19 testing efforts are far from ideal to tackle the pandemics. Although RT-qPCR is considered the gold standard for SARS-CoV-2 RNA detection, it requires expensive equipment, infrastructure, and highly trained personnel. In contrast, RT-LAMP emerges as an affordable, inexpensive, and simple alternative for SARS-CoV-2 molecular detection that can be applied to massive COVID-19 testing campaigns and save lives.

Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has infected almost 200 million people worldwide by July 2021 and the pandemic has been characterized by infection waves of viral lineages showing distinct fitness profiles. The simultaneous infection of a single individual by two distinct SARS-CoV-2 lineages may impact COVID-19 disease progression and provides a window of opportunity for viral recombination and the emergence of new lineages with differential phenotype. Several hundred SARS-CoV-2 lineages are currently well phylogenetically defined, but two main factors have precluded major coinfection/codetection and recombination analysis thus far: (i) the low diversity of SARS-CoV-2 lineages during the first year of the pandemic, which limited the identification of lineage defining mutations necessary to distinguish coinfecting/recombining viral lineages; and the (ii) limited availability of raw sequencing data where abundance and distribution of intrasample/intrahost variability can be accessed. Here, we assembled a large sequencing dataset from Brazilian samples covering a period of 18 May 2020 to 30 April 2021 and probed it for unexpected patterns of high intrasample/intrahost variability. This approach enabled us to detect nine cases of SARS-CoV-2 coinfection with well characterized lineage-defining mutations, representing 0.61 % of all samples investigated. In addition, we matched these SARS-CoV-2 coinfections with spatio-temporal epidemiological data confirming its plausibility with the cocirculating lineages at the timeframe investigated. Our data suggests that coinfection with distinct SARS-CoV-2 lineages is a rare phenomenon, although it is certainly a lower bound estimate considering the difficulty to detect coinfections with very similar SARS-CoV-2 lineages and the low number of samples sequenced from the total number of infections.

The nuclear receptor binding SET domain protein 1 (NSD1) is recurrently mutated in human cancers including acute leukemia. We show that NSD1 knockdown alters erythroid clonogenic growth of human CD34 + hematopoietic cells. Ablation of Nsd1 in the hematopoietic system of mice induces a transplantable erythroleukemia. In vitro differentiation of Nsd1 −/− erythroblasts is majorly impaired despite abundant expression of GATA1, the transcriptional master regulator of erythropoiesis, and associated with an impaired activation of GATA1induced targets. Retroviral expression of wildtype NSD1, but not a catalytically-inactive NSD1 N1918Q SET-domain mutant induces terminal maturation of Nsd1 −/− erythroblasts. Despite similar GATA1 protein levels, exogenous NSD1 but not NSD N1918Q significantly increases the occupancy of GATA1 at target genes and their expression. Notably, exogenous NSD1 reduces the association of GATA1 with the co-repressor SKI, and knockdown of SKI induces differentiation of Nsd1 −/− erythroblasts. Collectively, we identify the NSD1 methyltransferase as a regulator of GATA1-controlled erythroid differentiation and leukemogenesis.