Molecular evolution and structural analyses of the spike glycoprotein from Brazilian SARS-CoV-2 genomes: the impact of the fixation of selected mutations (preprint)

The COVID-19 pandemic caused by Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has reached by July 2021 almost 200 million cases and more than 4 million deaths worldwide since its beginning in late 2019, leading to enhanced concern in the scientific community and the general population. One of the most important pieces of this host-pathogen interaction is the spike protein, which binds to the human Angiotensin-converting enzyme 2 (hACE2) cell receptor, mediates the membrane fusion and is the major target of neutralizing antibodies against SARS-CoV-2. The multiple amino acid substitutions observed in this region, specially in the Receptor Binding Domain (RBD), mainly after almost one year of its emergence (late 2020), have enhanced the hACE2 binding affinity and led to several modifications in the mechanisms of SARS-CoV-2 pathogenesis, improving the viral fitness and/or promoting immune evasion, with potential impact in the vaccine development. In this way, the present work aimed to evaluate the effect of positively selected mutations fixed in the Brazilian SARS-CoV-2 lineages and to check for mutational evidence of coevolution. Additionally, we evaluated the impact of selected mutations identified in some of the VOC and VOI lineages (C.37, B.1.1.7, P.1, and P.2) of Brazilian samples on the structural stability of the spike protein, as well as their possible association with more aggressive infection profiles by estimating the binding affinity in the RBD-hACE2 complex. We identified 48 sites under selective pressure in Brazilian spike sequences, 17 of them with the strongest evidence by the HyPhy tests, including VOC related mutation sites 138, 142, 222, 262, 484, 681, and 845, among others. The coevolutionary analysis identified a number of 28 coevolving sites that were found not to be conditionally independent, such as the couple E484K - N501Y from P.1 and B.1.351 lineages. Finally, the molecular dynamics and free energy estimates showed the structural stabilizing effect and the higher impact of E484K for the improvement of the binding affinity between the spike RBD and the hACE2 in P.1 and P.2 lineages, as well as the stabilizing and destabilizing effects for the positively selected sites.

Predominance of the SARS-CoV-2 lineage P.1 and its sublineage P.1.2 in patients from the metropolitan region of Porto Alegre, Southern Brazil in March 2021: a phylogenomic analysis (preprint)

Almost a year after the COVID-19 pandemic had begun, The United Kingdom, South Africa, and Brazil became the epicenter of new lineages, the Variant of Concern (VOCs), B.1.1.7, B.1.351, and P.1, respectively. These VOCs are increasingly associated with enhanced transmissibility, immunity evasion, and mortality. The previous most prevalent lineages in the state of Rio Grande do South (Brazil), B.1.1.28 and B.1.1.33 were rapidly replaced by P.1 and P.2, two B.1.1.28-derived lineages harboring the E484K mutation. To perform a genomic characterization of SARS-CoV-2 samples from COVID-19 patients from the metropolitan region of Porto Alegre (Rio Grande do Sul, Southern Brazil), in this second pandemic wave, we sequenced viral samples from patients of this region to: (i) identify the prevalence of SARS-CoV-2 lineages in the region, the state and bordering countries/states, (ii) characterize the mutation spectra, and (iii) hypothesize possible viral dispersal routes by using phylogenetic and phylogeographic approaches. As results, we not only confirmed that 96.4% of the samples belonged to the P.1 lineage but also that approximately 20% of which could be assigned as the newer P.1.2 (a P.1 derived new sublineage harboring new signature substitutions recently described and present in other Brazilian states and foreign countries). Moreover, P.1 sequences from this study were allocated in several distinct branches (four clades and five clusters) of the P.1 phylogeny, suggesting multiple introductions of P.1 in Rio Grande do Sul still in 2020 and placing this state as a potential core of diffusion and emergence of P.1-derived clades. It is still uncertain if the emergence of P.1.2 and other P.1 clades are related to further virological, clinical, or epidemiological consequences. However, the clear signs of viral molecular diversification from recently introduced P.1 warrant further genomic surveillance.

Mutation hotspots, geographical and temporal distribution of SARS-CoV-2 lineages in Brazil, February 2020-2021: insights and limitations from uneven sequencing efforts (preprint)

The COVID-19 pandemic has already reached approximately 110 million people and it is associated with 2.5 million deaths worldwide. Brazil is the third worst-hit country, with approximately 10.2 million cases and 250 thousand deaths. International efforts have been established to share information about SARS-CoV-2 epidemiology and evolution. However, sequencing facilities and research investments are very heterogeneous across different regions and countries. The understanding of the SARS-CoV-2 evolution plays a significant role in the development of effective strategies for public health and disease management. We aimed to analyze the available and high-quality genome sequences from Brazil between February 2020 and February 2021 to identify mutation hotspots, geographical and temporal distribution of SARS-CoV-2 lineages by using phylogenetics and phylodynamics analyses. We describe heterogeneous and episodic sequencing efforts, the progression of the different lineages along time, evaluating mutational spectra and frequency oscillations derived from the prevalence of novel and specific lineages across different Brazilian regions. We found at least seven major (1-7) and two minor clades (4.2 and 5.3) related to the six most prevalent lineages in the country and described its spatial distribution and dynamics. The emergence and recent frequency shift of lineages (P.1 and P.2) containing mutations of concern in the spike protein ( e. g ., E484K, N501Y) draws attention due to their association with immune evasion and enhanced receptor binding affinity. Improvements in genomic surveillance are of paramount importance and should be extended in Brazil to better inform policy makers and enable evidence-based decisions to fight the COVID-19 pandemic.

E484K as an innovative phylogenetic event for viral evolution: Genomic analysis of the E484K spike mutation in SARS-CoV-2 lineages from Brazil (preprint)

The COVID-19 pandemic caused by SARS-CoV-2 has affected millions of people since its beginning in 2019. The propagation of new lineages and the discovery of key mechanisms adopted by the virus to overlap the immune system are central topics for the entire public health policies, research and disease management. Since the second semester of 2020, the mutation E484K has been progressively found in the Brazilian territory, composing different lineages over time. It brought multiple concerns related to the risk of reinfection and the effectiveness of new preventive and treatment strategies due to the possibility of escaping from neutralizing antibodies. To better characterize the current scenario we performed genomic and phylogenetic analyses of the E484K mutated genomes sequenced from Brazilian samples in 2020. From October, 2020, more than 40% of the sequenced genomes present the E484K mutation, which was identified in three different lineages (P1, P2 and B.1.1.33) in four Brazilian regions. We also evaluated the presence of E484K associated mutations and identified selective pressures acting on the spike protein, leading us to some insights about adaptive and purifying selection driving the virus evolution.

Genomic Epidemiology of SARS-CoV-2 in Esteio, Rio Grande do Sul, Brazil (preprint)

Brazil is the third country most affected by Covid-19 pandemic. In spite of this, viral evolution in municipality resolution is poorly understood in Brazil and it is crucial to understand the epidemiology of viral spread. We identified four main circulating lineages in Esteio (Southern Brazil) and their relationship with global, national and regional lineages using phylogenetics and phylodynamics inferences from 21 SARS-CoV-2 genome sequences. We provided a comprehensive view of viral mutations from a time- and age-representative sampling from May to October 2020, in Esteio (RS, Brazil), highlighting two frequent mutations in Spike glycoprotein (D614G and V1176F), an emergent mutation (E484K) in Spike Receptor Binding Domain (RBD) characteristic of the South African lineage B.1.351, and the adjacent replacement of 2 amino acids in Nucleocapsid phosphoprotein (R203K and G204R). A significant viral diversity was evidenced with the identification of 80 different SNPs. The E484K replacement was found in two genomes (9.5%) from samples obtained in mid-October, which is to our best knowledge the earliest description of E484K harboring SARS-CoV-2 in South Brazil. This mutation identified in a small municipality from the RS state demonstrates that it was probably widely distributed in the Brazilian territory, but went unnoticed so far by the lack of genomic surveillance in Brazil. The introduction of E484K mutants shows temporal correlation with later increases in new cases in our state. Importantly, since it has been associated with immune evasion and enhanced interaction with hACE-2, lineages containing this substitution must be the subject of intense surveillance. Our date demonstrates multiple introductions of the most prevalent lineages (B.1.1.33 and B.1.1.248) and the major role of community transmission in viral spreading and the establishment of Brazilian lineages. This represents an important contribution to the epidemiology of SARS-CoV-2.

Population-based prevalence surveys during the COVID-19 pandemic: a systematic review (preprint)

Population-based prevalence surveys of COVID-19 contribute to establish the burden and epidemiology of infection, the role of asymptomatic and mild infections in transmission, and allow more precise decisions about reopen policies. We performed a systematic review to evaluate qualitative aspects of these studies, their reliability, and biases. The available data described 37 surveys from 19 countries, mostly from Europe and America and using antibody testing. They reached highly heterogeneous sample sizes and prevalence estimates. Disproportional prevalence was observed in minority communities. Important risk of bias was detected in four domains: sample size, data analysis with sufficient coverage, measurements in standard way, and response rate. The correspondence analysis showed few consistent patterns for high risk of bias. Intermediate risk of bias was related to American and European studies, blood samples and prevalence >1%. Low risk of bias was related to Asian studies, RT-PCR tests and prevalence <1%.