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.

Potential use of high-resolution melting analyses for SARS-CoV-2 genomic surveillance

The pandemic caused by COVID-19 and the emergence of new variants of SARS-CoV-2 have generated clinical and epidemiological impacts on a global scale. The use of strategies for monitoring viral circulation and identifying mutations in genomic regions involved in host interaction are important measures to mitigate viral dissemination and reduce its likely complications on population health. In this context, the objective of this work was to explore the potential of high-resolution melting (HRM) analysis combined with one-step real-time reverse transcription PCR in a closed-tube system, as a fast and convenient method of screening for SARS-CoV-2 mutations with possible implications on host-pathogen interactions. The HRM analyses allowed the distinction of the Gamma, Zeta, Alpha, Delta, and Omicron variants against the predecessors (B.1.1.28, B.1.1.33) of occurrence in Brazil. It is concluded that the molecular tool standardized here has the potential to optimize the genomic surveillance of SARS-CoV-2, and could be adapted for genomic surveillance of other pathogens, due to its ability to detect, prior to sequencing, samples suggestive of new variants, selecting them more assertively and earlier for whole genome sequencing when compared to random screening.

Genomic Surveillance of SARS-CoV-2 in Healthcare Workers: A Critical Sentinel Group for Monitoring the SARS-CoV-2 Variant Shift.

SARS-CoV-2 genome surveillance is important for monitoring risk groups and health workers as well as data on new cases and mortality rate due to COVID-19. We characterized the circulation of SARS-CoV-2 variants from May 2021 to April 2022 in the state of Santa Catarina, southern Brazil, and evaluated the similarity between variants present in the population and healthcare workers (HCW). A total of 5291 sequenced genomes demonstrated the circulation of 55 strains and four variants of concern (Alpha, Delta, Gamma and Omicron-sublineages BA.1 and BA.2). The number of cases was relatively low in May 2021, but the number of deaths was higher with the Gamma variant. There was a significant increase in both numbers between December 2021 and February 2022, peaking in mid-January 2022, when the Omicron variant dominated. After May 2021, two distinct variant groups (Delta and Omicron) were observed, equally distributed among the five Santa Catarina mesoregions. Moreover, from November 2021 to February 2022, similar variant profiles between HCW and the general population were observed, and a quicker shift from Delta to Omicron in HCW than in the general population. This demonstrates the importance of HCW as a sentinel group for monitoring disease trends in the general population.

Emergence of Two Distinct SARS-CoV-2 Gamma Variants and the Rapid Spread of P.1-like-II SARS-CoV-2 during the Second Wave of COVID-19 in Santa Catarina, Southern Brazil.

The western mesoregion of the state of Santa Catarina (SC), Southern Brazil, was heavily affected as a whole by the COVID-19 pandemic in early 2021. This study aimed to evaluate the dynamics of the SARS-CoV-2 virus spreading patterns in the SC state from March 2020 to April 2021 using genomic surveillance. During this period, there were 23 distinct variants, including Beta and Gamma, among which the Gamma and related lineages were predominant in the second pandemic wave within SC. A regionalization of P.1-like-II in the Western SC region was observed, concomitant to the increase in cases, mortality, and the case fatality rate (CFR) index. This is the first evidence of the regionalization of the SARS-CoV-2 transmission in SC and it highlights the importance of tracking the variants, dispersion, and impact of SARS-CoV-2 on the public health systems.

Emergence of Two Distinct SARS-CoV-2 Gamma Variants and the Rapid Spread of P.1-like-II SARS-CoV-2 during the Second Wave of COVID-19 in Santa Catarina, Southern Brazil

The western mesoregion of the state of Santa Catarina (SC), Southern Brazil, was heavily affected as a whole by the COVID-19 pandemic in early 2021. This study aimed to evaluate the dynamics of the SARS-CoV-2 virus spreading patterns in the SC state from March 2020 to April 2021 using genomic surveillance. During this period, there were 23 distinct variants, including Beta and Gamma, among which the Gamma and related lineages were predominant in the second pandemic wave within SC. A regionalization of P.1-like-II in the Western SC region was observed, concomitant to the increase in cases, mortality, and the case fatality rate (CFR) index. This is the first evidence of the regionalization of the SARS-CoV-2 transmission in SC and it highlights the importance of tracking the variants, dispersion, and impact of SARS-CoV-2 on the public health systems.

Unusual SARS-CoV-2 intrahost diversity reveals lineage superinfection.

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.

Identification of a novel SARS-CoV-2 P.1 sub-lineage in Brazil provides new insights about the mechanisms of emergence of variants of concern.

One of the most remarkable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) features is the significant number of mutations they acquired. However, the specific factors that drove the emergence of such variants since the second half of 2020 are not fully resolved. In this study, we describe a new SARS-CoV-2 P.1 sub-lineage circulating in Brazil, denoted here as Gamma-like-II, that as well as the previously described lineage Gamma-like-I shares several lineage-defining mutations with the VOC Gamma. Reconstructions of ancestor sequences support that most lineage-defining mutations of the Spike (S) protein, including those at the receptor-binding domain (RBD), accumulated at the first P.1 ancestor. In contrast, mutations outside the S protein were mostly fixed at subsequent steps. Our evolutionary analyses estimate that P.1-ancestral strains carrying RBD mutations of concern probably circulated cryptically in the Amazonas for several months before the emergence of the VOC Gamma. Unlike the VOC Gamma, the other P.1 sub-lineages displayed a much more restricted dissemination and accounted for a low fraction (<2 per cent) of SARS-CoV-2 infections in Brazil in 2021. The stepwise diversification of lineage P.1 through multiple inter-host transmissions is consistent with the hypothesis that partial immunity acquired from natural SARS-CoV-2 infections in heavily affected regions might have been a major driving force behind the natural selection of some VOCs. The lag time between the emergence of the P.1 ancestor and the expansion of the VOC Gamma and the divergent epidemic trajectories of P.1 sub-lineages support a complex interplay between the emergence of mutations of concern and viral spread in Brazil.

Identification of a novel SARS-CoV-2 P.1 sub-lineage in Brazil provides new insights about the mechanisms of emergence of variants of concern

One of the most remarkable severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOC) features is the significant number of mutations they acquired. However, the specific factors that drove the emergence of such variants since the second half of 2020 are not fully resolved. In this study, we describe a new SARS-CoV-2 P.1 sub-lineage circulating in Brazil, denoted here as Gamma-like-II, that as well as the previously described lineage Gamma-like-I shares several lineage-defining mutations with the VOC Gamma. Reconstructions of ancestor sequences support that most lineage-defining mutations of the Spike (S) protein, including those at the receptor-binding domain (RBD), accumulated at the first P.1 ancestor. In contrast, mutations outside the S protein were mostly fixed at subsequent steps. Our evolutionary analyses estimate that P.1-ancestral strains carrying RBD mutations of concern probably circulated cryptically in the Amazonas for several months before the emergence of the VOC Gamma. Unlike the VOC Gamma, the other P.1 sub-lineages displayed a much more restricted dissemination and accounted for a low fraction (<2 per cent) of SARS-CoV-2 infections in Brazil in 2021. The stepwise diversification of lineage P.1 through multiple inter-host transmissions is consistent with the hypothesis that partial immunity acquired from natural SARS-CoV-2 infections in heavily affected regions might have been a major driving force behind the natural selection of some VOCs. The lag time between the emergence of the P.1 ancestor and the expansion of the VOC Gamma and the divergent epidemic trajectories of P.1 sub-lineages support a complex interplay between the emergence of mutations of concern and viral spread in Brazil.

The ongoing evolution of variants of concern and interest of SARS-CoV-2 in Brazil revealed by convergent indels in the amino (N)-terminal domain of the spike protein.

Mutations at both the receptor-binding domain (RBD) and the amino (N)-terminal domain (NTD) of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Spike (S) glycoprotein can alter its antigenicity and promote immune escape. We identified that SARS-CoV-2 lineages circulating in Brazil with mutations of concern in the RBD independently acquired convergent deletions and insertions in the NTD of the S protein, which altered the NTD antigenic-supersite and other predicted epitopes at this region. Importantly, we detected the community transmission of different P.1 lineages bearing NTD indels ∆69-70 (which can impact several SARS-CoV-2 diagnostic protocols), ∆144 and ins214ANRN, and a new VOI N.10 derived from the B.1.1.33 lineage carrying three NTD deletions (∆141-144, ∆211, and ∆256-258). These findings support that the ongoing widespread transmission of SARS-CoV-2 in Brazil generates new viral lineages that might be more resistant to antibody neutralization than parental variants of concern.

A Potential SARS-CoV-2 Variant of Interest (VOI) Harboring Mutation E484K in the Spike Protein Was Identified within Lineage B.1.1.33 Circulating in Brazil.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) epidemic in Brazil was dominated by two lineages designated as B.1.1.28 and B.1.1.33. The two SARS-CoV-2 variants harboring mutations at the receptor-binding domain of the Spike (S) protein, designated as lineages P.1 and P.2, evolved from lineage B.1.1.28 and are rapidly spreading in Brazil. Lineage P.1 is considered a Variant of Concern (VOC) because of the presence of multiple mutations in the S protein (including K417T, E484K, N501Y), while lineage P.2 only harbors mutation S:E484K and is considered a Variant of Interest (VOI). On the other hand, epidemiologically relevant B.1.1.33 deriving lineages have not been described so far. Here we report the identification of a new SARS-CoV-2 VOI within lineage B.1.1.33 that also harbors mutation S:E484K and was detected in Brazil between November 2020 and February 2021. This VOI displayed four non-synonymous lineage-defining mutations (NSP3:A1711V, NSP6:F36L, S:E484K, and NS7b:E33A) and was designated as lineage N.9. The VOI N.9 probably emerged in August 2020 and has spread across different Brazilian states from the Southeast, South, North, and Northeast regions.

Evolutionary Dynamics and Dissemination Pattern of the SARS-CoV-2 Lineage B.1.1.33 During the Early Pandemic Phase in Brazil.

A previous study demonstrates that most of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) Brazilian strains fell in three local clades that were introduced from Europe around late February 2020. Here we investigated in more detail the origin of the major and most widely disseminated SARS-CoV-2 Brazilian lineage B.1.1.33. We recovered 190 whole viral genomes collected from 13 Brazilian states from February 29 to April 31, 2020 and combined them with other B.1.1 genomes collected globally. Our genomic survey confirms that lineage B.1.1.33 is responsible for a variable fraction of the community viral transmissions in Brazilian states, ranging from 2% of all SARS-CoV-2 genomes from Pernambuco to 80% of those from Rio de Janeiro. We detected a moderate prevalence (5-18%) of lineage B.1.1.33 in some South American countries and a very low prevalence (<1%) in North America, Europe, and Oceania. Our study reveals that lineage B.1.1.33 evolved from an ancestral clade, here designated B.1.1.33-like, that carries one of the two B.1.1.33 synapomorphic mutations. The B.1.1.33-like lineage may have been introduced from Europe or arose in Brazil in early February 2020 and a few weeks later gave origin to the lineage B.1.1.33. These SARS-CoV-2 lineages probably circulated during February 2020 and reached all Brazilian regions and multiple countries around the world by mid-March, before the implementation of air travel restrictions in Brazil. Our phylodynamic analysis also indicates that public health interventions were partially effective to control the expansion of lineage B.1.1.33 in Rio de Janeiro because its median effective reproductive number (R e ) was drastically reduced by about 66% during March 2020, but failed to bring it to below one. Continuous genomic surveillance of lineage B.1.1.33 might provide valuable information about epidemic dynamics and the effectiveness of public health interventions in some Brazilian states.