The B.1.1.33, known as a Brazilian lineage, has been detected in many countries. Brazil (84%), the USA (4%), Chile (3%), Canada (1%), and Argentina (1%) encompass most of the cases. Its earliest description date back to 2020–03-01, and the largest circulation in the world appears to have occurred between March 2020 and May 2021 (https://cov-lineages.org/lineage.html?lineage=B.1.1.33), the time interval in which our samples were collected.
The four sequenced samples belong to the B.1.1.33 lineage, which descends from lineage B.1. The latter is a large European lineage, whose origin, in turn, roughly corresponds to the Northern Italian outbreak early in 2020 (https://cov-lineages.org/lineage.html?lineage=B.1). On February 25, 2020, a Brazilian man, who traveled days before to Lombardy, Northern Italy, was confirmed as the first Brazilian case of COVID-19 . Since the LMM6378 sample was one of the first COVID-19 positive samples in our laboratory and our diagnostic service started in a pioneering way in the region in March of 2020, we highlight that B.1.1.33 was one of the precursor lineages in the spread of SARS-CoV-2 in RS.
Through the analysis described, it was possible to evaluate all the amino acid changes in FASTq reads even though these changes were not frequent enough to compose the consensus sequences of their respective samples. Some of the nonsynonymous mutations found in these consensus sequences, when compared to the reference sample, corroborated the data from the variant calling approach, thus making it possible to observe the gradual genome shift in these sequences. As seen in the S gene (Table 2), the amino acid changes that occurred with higher frequency were N63T in LMM6378 (42.3%) and LMM17042 (38%), and D484E in LMM14497 (46.4%). Since these changes had not enough frequency, mutations T63N and E484D were seen in the S gene translation alignment (Fig. 3).
B.1.1.33 is characterized by mutations in genes ORF1b (P314L), S (D614G), ORF6 (I33T), ORF8 (S84L), and N (R203K, G204R, I292T) (https://outbreak.info/situation-reports?pango=B.1.1.33). Since we analyzed only the S gene sequence, S:D614G was the signature mutation found in our four sequences. Finding this mutation in all sequences was expected since it is already a B.1.1.33 lineage characteristic mutation that has been described as important for viral spread and higher infection titers .
Another mutation, which is not the lineage signature but has been detected worldwide in different lineages, is S:H655Y, which was found in sequence LMM14497. According to Escalera et al. (2022) , SARS-CoV-2 variants harboring S:655Y show an enhanced spike protein cleavage and fusogenic ability in vitro, increasing transmissibility in vivo, and an enhanced phenotype in the human primary airway model. Worldwide, 2,648,195 sequences with the S:H655Y mutation have been detected since its identification in January 2020. In Brazil, 71,726 sequences were described, being 1328 in RS (data from March 28, 2022: https://outbreak.info/). Here, we would like to highlight that our sequence (LMM14497), collected on July 9, 2020, corresponds to the earliest sample detected containing this mutation in RS.
The other mutations identified in our sequences have considerably low detection frequencies worldwide and in Brazil. S:T63N, the only mutation that was found in two of the sequenced samples in this study (LMM6378 and LMM17042), has been previously detected in 101 samples worldwide, being 13 of them from Brazil, with almost all of them (12) coming from RS (data from March 28, 2022: https://outbreak.info/). Interestingly, sample LMM6378 corresponds to the earliest isolate reported to contain mutation S:T63N in RS, being also one of the first positive samples for COVID-19 in the region. The detection of this mutation started with an increase but ended up not being broadly widespread.
In the state of RS, the other mutations identified (S:N74K, E96D, E484D, N148K) were described only in our sequences since then. LMM14497 showed the mutations: S:N74K (detected in 152 sequences worldwide being 5 in Brazil), S:E96D (detected in 2,243 sequences worldwide being 24 in Brazil), and S:E484D (299 sequences worldwide being 6 in Brazil). LMM17042 presented the S:N148K that has been detected in 210 sequences worldwide, being two sequences in Brazil (data from March 28, 2022: https://outbreak.info/). Through the sequencing and analysis of mutations found in introductory sequences to the pandemic in the region, it is possible to trace the changes in the viral genome that occurred within the same lineage. Therefore, it is possible to infer how mutations may have interfered with the evolution and viral dissemination, and how some became characteristic of the lineage due to adaptive advantage when compared to others that were not able to maintain their circulation.
The viral isolation method was effective to isolate the four samples with the possibility of increasing the viral titter after a passage in VERO cells, being possible its detection through qPCR and its titration. Therefore, cell culture-based methods can be used to produce stock in laboratories contributing to the detection and identification of the studied virus. Although these samples are representative of the same lineage, their genomes are different. Therefore, the analysis of the profiles and signatures of mutational sequences was approached in an important way to better understand the studied sequences. Thus, it was possible to detect in our samples important mutations that were identified for the first time in the southernmost state of Brazil.
In conclusion, we highlight the importance of studies that track the genome profile of the early SARS-CoV-2 lineages being possible to evaluate mutations that became more frequent over time showing their effect on viral fitness and, consequently, the viral evolution. Also, these data point out the importance of constant evaluation and monitoring of SARS-CoV-2 in the population, supporting efforts that help to delineate strategies in the face of possible adversities such as the emergence of new variants, vaccine immune escape, and increase in virulence.