In silico analysis of non-structural protein 12 sequences from SARS-COV-2 found in Manaus, Amazonas, Brazil, reveals mutations linked to higher transmissibility.

The disease coronavirus COVID-19 has been the cause of millions of deaths worldwide. Among the proteins of SARS-CoV-2, non-structural protein 12 (NSP12) plays a key role during COVID infection and is part of the RNA-dependent RNA polymerase complex. The monitoring of NSP12 polymorphisms is extremely important for the design of new antiviral drugs and monitoring of viral evolution. This study analyzed the NSP12 mutations detected in circulating SARS-CoV-2 during the years 2020 to 2022 in the population of the city of Manaus, Amazonas, Brazil. The most frequent mutations found were P323L and G671S. Reports in the literature indicate that these mutations are related to transmissibility efficiency, which may have contributed to the extremely high numbers of cases in this location. In addition, two mutations described here (E796D and R914K) are close and have RMSD that is similar to the mutations M794V and N911K, which have been described in the literature as influential on the performance of the NSP12 enzyme. These data demonstrate the need to monitor the emergence of new mutations in NSP12 in order to better understand their consequences for the treatments currently used and in the design of new drugs.

SARS-CoV-2 intra-host diversity, antibody response, and disease severity after reinfection by the variant of concern Gamma in Brazil.

The rapid spread of the SARS-CoV-2 Variant of Concern (VOC) Gamma in Amazonas during early 2021 fueled a second large COVID-19 epidemic wave and raised concern about the potential role of reinfections. Very few cases of reinfection associated with the VOC Gamma have been reported to date, and their potential impact on clinical, immunological, and virological parameters remains largely unexplored. Here we describe 25 cases of SARS-CoV-2 reinfection in Brazil. SARS-CoV-2 genomic analysis confirmed that individuals were primo-infected with distinct viral lineages between March and December 2020 (B.1.1, B.1.1.28, B.1.1.33, B.1.195, and P.2) and reinfected with the VOC Gamma between 3 to 12 months after primo-infection. We found a similar mean cycle threshold (Ct) value and limited intra-host viral diversity in both primo-infection and reinfection samples. Sera of 14 patients tested 10-75 days after reinfection displayed detectable neutralizing antibodies (NAb) titers against SARS-CoV-2 variants that circulated before (B.1.*), during (Gamma), and after (Delta and Omicron) the second epidemic wave in Brazil. All individuals had milder or no symptoms after reinfection, and none required hospitalization. These findings demonstrate that individuals reinfected with the VOC Gamma may display relatively high RNA viral loads at the upper respiratory tract after reinfection, thus contributing to onward viral transmissions. Despite this, our study points to a low overall risk of severe Gamma reinfections, supporting that the abrupt increase in hospital admissions and deaths observed in Amazonas and other Brazilian states during the Gamma wave was mostly driven by primary infections. Our findings also indicate that most individuals analyzed developed a high anti-SARS-CoV-2 NAb response after reinfection that may provide some protection against reinfection or disease by different SARS-CoV-2 variants.

Spread of Gamma (P.1) Sub-Lineages Carrying Spike Mutations Close to the Furin Cleavage Site and Deletions in the N-Terminal Domain Drives Ongoing Transmission of SARS-CoV-2 in Amazonas, Brazil.

The Amazonas was one of the most heavily affected Brazilian states by the COVID-19 epidemic. Despite a large number of infected people, particularly during the second wave associated with the spread of the Variant of Concern (VOC) Gamma (lineage P.1), SARS-CoV-2 continues to circulate in the Amazonas. To understand how SARS-CoV-2 persisted in a human population with a high immunity barrier, we generated 1,188 SARS-CoV-2 whole-genome sequences from individuals diagnosed in the Amazonas state from 1st January to 6th July 2021, of which 38 were vaccine breakthrough infections. Our study reveals a sharp increase in the relative prevalence of Gamma plus (P.1+) variants, designated Pango Lineages P.1.3 to P.1.6, harboring two types of additional Spike changes: deletions in the N-terminal (NTD) domain (particularly Δ144 or Δ141-144) associated with resistance to anti-NTD neutralizing antibodies or mutations at the S1/S2 junction (N679K or P681H) that probably enhance the binding affinity to the furin cleavage site, as suggested by our molecular dynamics simulations. As lineages P.1.4 (S:N679K) and P.1.6 (S:P681H) expanded (Re > 1) from March to July 2021, the lineage P.1 declined (Re < 1) and the median Ct value of SARS-CoV-2 positive cases in Amazonas significantly decreases. Still, we did not find an increased incidence of P.1+ variants among breakthrough cases of fully vaccinated patients (71%) in comparison to unvaccinated individuals (93%). This evidence supports that the ongoing endemic transmission of SARS-CoV-2 in the Amazonas is driven by the spread of new local Gamma/P.1 sublineages that are more transmissible, although not more efficient to evade vaccine-elicited immunity than the parental VOC. Finally, as SARS-CoV-2 continues to spread in human populations with a declining density of susceptible hosts, the risk of selecting more infectious variants or antibody evasion mutations is expected to increase. IMPORTANCE The continuous evolution of SARS-CoV-2 is an expected phenomenon that will continue to happen due to the high number of cases worldwide. The present study analyzed how a Variant of Concern (VOC) could still circulate in a population hardly affected by two COVID-19 waves and with vaccination in progress. Our results showed that the answer behind that was a new generation of Gamma-like viruses, which emerged locally carrying mutations that made it more transmissible and more capable of spreading, partially evading prior immunity triggered by natural infections or vaccines. With thousands of new cases daily, the current pandemics scenario suggests that SARS-CoV-2 will continue to evolve and efforts to reduce the number of infected subjects, including global equitable access to COVID-19 vaccines, are mandatory. Thus, until the end of pandemics, the SARS-CoV-2 genomic surveillance will be an essential tool to better understand the drivers of the viral evolutionary process.

Structural analysis of SARS-Cov-2 nonstructural protein 1 polymorphisms found in the Brazilian Amazon.

The coronavirus disease COVID-19 has been the cause of millions of deaths worldwide. Among the SARS-CoV-2 proteins, the non-structural protein 1 (NSP1) has great importance during the virus infection process and is present in both alpha and beta-CoVs. Therefore, monitoring of NSP1 polymorphisms is crucial in order to understand their role during infection and virus-induced pathogenicity. Herein, we analyzed how mutations detected in the circulating SARS-CoV-2 in the population of the city of Manaus, Amazonas state, Brazil could modify the tertiary structure of the NSP1 protein. Three mutations were detected in the SARS-CoV-2 NSP1 gene: deletion of the amino acids KSF from positions 141 to 143 (delKSF), SARS-CoV-2, lineage B.1.195; and two substitutions, R29H and R43C, SARS-CoV-2 lineage B.1.1.28 and B.1.1.33, respectively. The delKSF was found in 47 samples, whereas R29H and R43C were found in two samples, one for each mutation. The NSP1 structures carrying the mutations R43C and R29H on the N-terminal portion (e.g. residues 10 to 127) showed minor backbone divergence compared to the Wuhan model. However, the NSP1 C-terminal region (residues 145 to 180) was severely affected in the delKSF and R29H mutants. The intermediate variable region (residues 144 to 148) leads to changes in the C-terminal region, particularly in the delKSF structure. New investigations must be carried out to analyze how these changes affect NSP1 activity during the infection. Our results reinforce the need for continuous genomic surveillance of SARS-CoV-2 to better understand virus evolution and assess the potential impact of the viral mutations on the approved vaccines and future therapies.

COVID-19 in Amazonas, Brazil, was driven by the persistence of endemic lineages and P.1 emergence.

The northern state of Amazonas is among the regions in Brazil most heavily affected by the COVID-19 epidemic and has experienced two exponentially growing waves, in early and late 2020. Through a genomic epidemiology study based on 250 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes from different Amazonas municipalities sampled between March 2020 and January 2021, we reveal that the first exponential growth phase was driven mostly by the dissemination of lineage B.1.195, which was gradually replaced by lineage B.1.1.28 between May and June 2020. The second wave coincides with the emergence of the variant of concern (VOC) P.1, which evolved from a local B.1.1.28 clade in late November 2020 and replaced the parental lineage in <2 months. Our findings support the conclusion that successive lineage replacements in Amazonas were driven by a complex combination of variable levels of social distancing measures and the emergence of a more transmissible VOC P.1 virus. These data provide insights to understanding the mechanisms underlying the COVID-19 epidemic waves and the risk of dissemination of SARS-CoV-2 VOC P.1 in Brazil and, potentially, worldwide.

COVID-19 epidemic in the Brazilian state of Amazonas was driven by long-term persistence of endemic SARS-CoV-2 lineages and the recent emergence of the new variant of concern P.1.

Dentre as 27 Unidades Federativas brasileiras, o estado do Amazonas foi uma das mais afetadas pela presente pandemia do novo coronavírus, com a ocorrência de uma segunda onda ao final de 2020 na qual um grande número de casos graves levou a um colapso no sistema de saúde. Este pre-print, resultado da análise do genoma completo de 250 amostras do SARS-CoV-2 coletadas no Amazonas entre março de 2020 e janeiro de 2021, descreve a dinâmica de sucessões de linhagens dominantes no estado. Mais recentemente, ele foi publicado na revista Nature Medicine, após editoração e a revisão por pesquisadores independentes. A publicação apresenta dados que embasam as hipóteses de que a linhagem responsável pela maioria dos casos no primeiro momento da pandemia (entre março e maio de 2020) foi a B.1.195, de que esta foi suplantada pela B.1.1.28, que tornou-se a linhagem dominante no estado entre maio e dezembro de 2020, e de que em dezembro o surgimento de uma variante da B.1.1.28, denominada P.1 e dotada de maior transmissibilidade, foi responsável pela nova ascensão no número de casos e mortes. Desta forma, a dinâmica local de surgimento de novas genéticas virais foi uma importante força-motriz para a forma com a qual a pandemia avançou sobre o estado do Amazonas, influenciada diretamente pela circulação da população e sua relação com o espalhamento do vírus, que culminou com a substituição da B.1.1.28 pela variante de preocupação P.1 em um processo que acredita-se ter durado apenas dois meses.

Three SARS-CoV-2 reinfection cases by the new Variant of Concern (VOC) P.1/501Y.V3

Neste relatório (pre-print), são apresentados três casos de reinfecção causados pela Variante de Preocupação (VOC) P.1, também conhecida como "cepa de Manaus". As três pacientes eram mulheres adultas, e tiveram a primeira infecção durante a primeira onda da pandemia na primeira metade de 2020. Nos três casos, a linhagem detectada no primeiro diagnóstico molecular era diferente da encontrada posteriormente, evidência da reinfecção. Dois dos casos de reinfecção tiveram apresentação de sintomas leves, enquanto o terceiro foi assintomático, apesar de a quantidade de material genético viral detectado sugerir cargas virais elevadas. As evidências aqui apresentadas sugerem que a imunidade após infecção primária por linhagens anteriores à circulação daquelas contendo a mutação E484K não impede uma nova infecção pela variante P.1, e nem mesmo que pessoas reinfectadas por esta variante espalhem o vírus, embora seja possível que tenha protegido estas três pacientes do desenvolvimento de sintomas graves.

Genomic and phylogenetic characterisation of an imported case of SARS-CoV-2 in Amazonas State, Brazil.

A new coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] is currently causing a life-threatening pandemic. In this study, we report the complete genome sequencing and genetic characterisation of a SARS-CoV-2 detected in Manaus, Amazonas, Brazil, and the protocol we designed to generate high-quality SARS-CoV-2 full genome data. The isolate was obtained from an asymptomatic carrier returning from Madrid, Spain. Nucleotide sequence analysis showed a total of nine mutations in comparison with the original human case in Wuhan, China, and support this case as belonging to the recently proposed lineage A.2. Phylogeographic analysis further confirmed the likely European origin of this case. To our knowledge, this is the first SARS-CoV-2 genome obtained from the North Brazilian Region. We believe that the information generated in this study may contribute to the ongoing efforts toward the SARS-CoV-2 emergence.

Genomic and phylogenetic characterisation of an imported case of SARS-CoV-2 in Amazonas State, Brazil

A new coronavirus [severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)] is currently causing a life-threatening pandemic. In this study, we report the complete genome sequencing and genetic characterisation of a SARS-CoV-2 detected in Manaus, Amazonas, Brazil, and the protocol we designed to generate high-quality SARS-CoV-2 full genome data. The isolate was obtained from an asymptomatic carrier returning from Madrid, Spain. Nucleotide sequence analysis showed a total of nine mutations in comparison with the original human case in Wuhan, China, and support this case as belonging to the recently proposed lineage A.2. Phylogeographic analysis further confirmed the likely European origin of this case. To our knowledge, this is the first SARS-CoV-2 genome obtained from the North Brazilian Region. We believe that the information generated in this study may contribute to the ongoing efforts toward the SARS-CoV-2 emergence.