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.

One of the most remarkable 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%) 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.

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SummaryThe rapid spread of the SARS-CoV-2 Variant of Concern (VOC) Gamma during late 2020 and early 2021 in Brazilian settings with high seroprevalence raised some concern about the potential role of reinfections in driving the epidemic. Very few cases of reinfection associated with the VOC Gamma, however, have been reported. Here we describe 25 cases of SARS-CoV-2 reinfection confirmed by real-time RT-PCR twice within months apart in Brazil. SARS-CoV-2 genomic analysis confirmed that individuals were primo-infected between March and December 2020 with distinct viral lineages, including B.1.1, B.1.1.28, B.1.1.33, B.1.195 and P.2, and then reinfected with the VOC Gamma between 3 to 12 months after primo-infection. The overall mean cycle threshold (Ct) value of the first (25.7) and second (24.5) episodes were roughly similar for the whole group and 14 individuals displayed mean Ct values < 25.0 at reinfection. Sera of 14 patients tested by plaque reduction neutralization test after reinfection displayed detectable neutralizing antibodies against Gamma and other SARS-CoV-2 variants (B.1.33, B.1.1.28 and Delta). All individuals have milder or no symptoms after reinfection and none required hospitalization. The present study demonstrates that the VOC Gamma was associated with reinfections during the second Brazilian epidemic wave in 2021 and raised concern about the potential infectiousness of reinfected subjects. Although individuals here analyzed failed to mount a long-term sterilizing immunity, they developed a high anti-Gamma neutralizing antibody response after reinfection that may provide some protection against severe disease.

The 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 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 characterized but two main factors have precluded major coinfection/codetection 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 viral lineages; and the ii) limited availability of raw sequencing data where abundance and distribution of intrasample/intrahost variability can be accessed. Here, we have put together 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. It enabled us to detect nine cases of SARS-CoV-2 coinfection with well characterized lineage-defining mutations. In addition, we matched these SARS-CoV-2 coinfections with spatio-temporal epidemiological data confirming their plausibility with the co-circulating lineages at the timeframe investigated. These coinfections represent around 0.61% of all samples investigated. Although our data suggests that coinfection with distinct SARS-CoV-2 lineages is a rare phenomenon, it is likely an underestimation and coinfection rates warrants further investigation.