An 88-years-old male patient presented acute respiratory symptoms on Jan 29th, 2021 and was hospitalized at Arcanjo São Miguel Hospital, Gramado city, on Feb 3rd after medical examination. At the same day of hospitalization, the patient was admitted at the intensive care unit with fever of 39ºC, no leg movements, chest pain, besides flu-like symptoms/Acute Respiratory Syndrome (sore throat, dyspnea, oxygen desaturation). Respiratory secretion was collected on Feb 1st, the RT-qPCR for COVID-19 was positive (Ct 22) and the patient died on Feb 10th, nine days after hospitalization.
As part of SARS-CoV-2 genomic surveillance in Rio Grande do Sul State, the collected sample, along with samples from different cities, were sequenced aiming to obtain the current scenario of SARS-CoV-2 genomic diversity in this region. Whole genome library preparation of SARS-CoV-2 was performed using QIAseq SARS-CoV-2 Primer Panel paired with QIAseq FX DNA Library UDI Kit, according to the manufacturer instructions. The sequencing was performed on an Illumina MiSeq machine using MiSeq Reagent Kit v3 (600-cycle). Raw FASTQ files from genome sequencing were firstly trimmed to remove adapter sequences and low-quality reads using trimmomatic  the read data quality assessed in fastQC (www.bioinformatics.babraham.ac.uk/projects/fastqc/) and then mapped against the reference genome Wuhan-Hu-1 (GenBank Accession MN908947.3) using the BWA-MEM algorithm (Mean coverage: 2.158,216). Consensus fasta was obtained with SAMtools , and analyzed with Pangolin (github.com/cov-lineages/pangolin) to determine SARS-CoV-2 lineage.
The sequenced genome was assigned to P.1 lineage on Pangolin. Thus, we aligned the consensus fasta with other 156 P.1 genomes from worldwide available on GISAID  as of February 16, 2021 with MAFFT  under default parameters. The aligned multi-fasta was used to construct a maximum-likelihood tree in IQ-Tree v.2.1.2  (GTR + G4 + F -alrt 1000 -nt AUTO), annotated in the iTOL web-based tool  and rooted on Wuhan-Hu-1 reference genome. The maximum-likelihood phylogenetic analyses revealed that the sequence from Gramado, Rio Grande do Sul, is branched in a monophyletic clade that comprises 25 genomes including sequences from Amazonas, Rondonia, Roraima and São Paulo state, along with sequences found in Japan and Colombia (Fig. 1). Interestingly, the sequences originated from three patients transferred from Manaus to be hospitalized in Rio Grande do Sul in the same period (February 2021), fell on different branches of the P.1 phylogeny, suggesting distinct transmission chains.