Deep phylogenetic-based clustering analysis uncovers new and shared mutations in SARS-CoV-2 variants as a result of directional and convergent evolution (preprint)

Nearly two decades after the last epidemic caused by a severe acute respiratory syndrome coronavirus (SARS-CoV), newly emerged SARS-CoV-2 quickly spread in 2020 and precipitated an ongoing global public health crisis. Both the continuous accumulation of point mutations, owed to the naturally imposed genomic plasticity of SARS-CoV-2 evolutionary processes, as well as viral spread over time, allow this RNA virus to gain new genetic identities, spawn novel variants and enhance its potential for immune evasion. Here, through an in-depth phylogenetic clustering analysis of upwards of 200,000 whole-genome sequences, we reveal the presence of not previously reported and hitherto unidentified mutations and recombination breakpoints in Variants of Concern (VOC) and Variants of Interest (VOI) from Brazil, India (Beta, Eta and Kappa) and the USA (Beta, Eta and Lambda). Additionally, we identify sites with shared mutations under directional evolution in the SARS-CoV-2 Spike-encoding protein of VOC and VOI, tracing a heretofore-undescribed correlation with viral spread in South America, India and the USA. Our evidence-based analysis provides well-supported evidence of similar pathways of evolution for such mutations in all SARS-CoV-2 variants and sub-lineages. This raises two pivotal points: the co-circulation of variants and sub-lineages in close evolutionary environments, which sheds light onto their trajectories into convergent and directional evolution (i), and a linear perspective into the prospective vaccine efficacy against different SARS-CoV-2 strains (ii).

A suitable murine model for studying respiratory coronavirus infection and therapeutic countermeasures in BSL-2 laboratories (preprint)

Several animal models are being used to explore important features of COVID-19, nevertheless none of them recapitulates all aspects of the disease in humans. The continuous refinement and development of other options of in vivo models are opportune, especially ones that are carried out at BSL-2 (Biosafety Level 2) laboratories. In this study, we investigated the suitability of the intranasal infection with the murine betacoronavirus MHV-3 to recapitulate multiple aspects of the pathogenesis of COVID-19 in C57BL/6J mice. We demonstrate that MHV-3 replicated in lungs 1 day after inoculation and triggered respiratory inflammation and dysfunction. This MHV-model of infection was further applied to highlight the critical role of TNF in cytokine-mediated coronavirus pathogenesis. Blocking TNF signaling by pharmacological and genetic strategies greatly increased the survival time and reduces lung injury of MHV-3-infected mice. In vitro studies showed that TNF blockage decreased SARS-CoV-2 replication in human epithelial lung cells and resulted in the lower release of IL-6 and IL-8 cytokines beyond TNF itself. Taken together, our results demonstrate that this model of MHV infection in mice is a useful BSL-2 screening platform for evaluating pathogenesis for human coronaviruses infections, such as COVID-19.

Reverse Transcription-Loop-Mediated Isothermal Amplification (RT-LAMP) is an effective alternative for SARS-CoV-2 molecular detection in middle-income countries (preprint)

Molecular diagnosis of SARS-CoV-2 in developing countries is still a big challenge. The reference standard, RT-qPCR, recommended by WHO, is not widely available, difficulting early identification of cases. Furthermore, the transport logistic between the sample collection point and the laboratory facilities can alter the samples, producing false negative results. RT-LAMP is a cheaper, simpler molecular technique that can be an interesting alternative to be offered in hospital laboratories. We present the evaluation of a RT-LAMP for diagnosis of SARS-CoV-2 in two steps: the laboratory standardization and the clinical validation, comparing it with the standard RT-qPCR. In the standardization phase, limit of detection and robustness values were obtained using RNA from a Peruvian SARS-CoV-2 strain. It presented 100% agreement between triplicates (RT-LAMP agreement with all RT-qPCR reactions that presented Ct [≤] 30) and robustness (RT-LAMP successful reactions with 80% reaction volume and 50% primer concentration). 384 nasal and pharyngeal swabs collected from symptomatic patients and stored in the INS biobank were tested and we obtained 98.75%, 87.41%, 97.65% and 92.96% for specificity, sensitivity, positive predictive value and negative predictive values respectively. Then, 383 samples from symptomatic patients with less than 15 days of disease, were tested both with the RT-LAMP and with the RT-qPCR, obtaining e 98.8%, 88.1%, 97.7% y 93.3% of specificity, sensitivity, positive predictive value and negative predictive values respectively. The laboratory standardization and the clinical validation presented the same value by Kappa-Cohen index (0.88) indicating an almost perfect agreement between RT-LAMP and RT-qPCR for molecular detection of SARS-CoV-2. We conclude that this RT-LAMP protocol presented high diagnostic performance values and can be an effective alternative for COVID-19 molecular diagnosis in hospitals, contributing to early diagnosis and reducing the spread of virus transmission in the Peruvian population.