Recently, a new variant first detected in Manaus/Amazonas in the North Region of Brazil has become a concern worldwide. The named P.1 lineage is descendent of B.1.1.28 lineage and carries a set of mutations with important biological significance, mainly at region encoding spike protein (E484K, K417T and N501Y) (N.R. Faria, et al., unpub. data, https://virological.org/t/genomic-characterisation-of-an-emergent-sars-cov-2-lineage-in-manaus-preliminary-findings/586).

The P.1 lineage emerged with high frequency in a short period of time, spreading fast in North of Brazil, and in the recent weeks in other cities from Southeastern Brazil (F. Naveca et al., unpub. data, https://virological.org/t/sars-cov-2-reinfection-by-the-new-variant-of-concern-voc-p-1-in-amazonas-brazil/596). Here we described the first detected case of SARS-CoV-2 P.1 lineage in Rio Grande do Sul, the southernmost state in Brazil. The case occurred in Gramado city, a mountain town with German colonization that receives 6.5 million tourists every year. Gramado belongs to the 5th Regional Health Coordination (an administrative division) that comprises 49 municipalities with a 1,240,319 estimated population, also known as Caxias do Sul Region (https://cidades.ibge.gov.br/brasil/panorama).

An 88-years-old male patient (A) presented acute respiratory symptoms on Jan 29^th, 2021 and was hospitalized on Feb 3^rd after medical examination. At the same day, 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. Respiratory secretion was collected on Feb 1^st and RT-qPCR for SARS-CoV-2 detection was performed at Central Laboratory of Public Health from Rio Grande do Sul (LACEN-RS). The RNA was extracted from respiratory clinical sample through MagMAX Viral/Pathogen II Isolation kit on KingFischer Flex extractor (Thermo Fisher Scientific, Waltham, USA). The multiple detection of 3 target genes (E gene, RdRP gene and N gene) from pathogen was performed on Applied Biosystems 7500 Real-Time PCR System (Thermo Fisher Scientific, Waltham, USA), using Allplex SARS-CoV-2 Assay (Seegene Inc, Seoul, Republic of Korea). The SARS-CoV-2 RT-qPCR was positive (Ct 22) and the result released on Feb 2^nd, 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 trimmomatic¹ 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 algorithm² (Mean coverage: 2.158,216). Consensus fasta was obtained with SAMtools³.

The sequenced genome was assigned to P.1 lineage on Pangolin (github.com/cov-lineages/pangolin). Thus, we aligned the consensus fasta with other 156 P.1 genomes from worldwide available on GISAID⁴ as of Feb 16, 2021 with MAFFT⁵ under default parameters. The aligned multi-fasta was used to construct a maximum-likelihood tree in IQ-Tree v.2.1.2⁶ (GTR+G4+F -alrt 1000 -nt AUTO), annotated in the iTOL web-based tool⁷ 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, Rondônia, Roraima and São Paulo state, along with sequences found in Japan and Colombia (Figure 1).

Figure 1
Maximumlikelihood tree from the first SARSCoV P1 genome identified in Rio Grande do Sul Brazil contextualized on 156 P1 genomes from worldwide The aLRT support values are shown in key nodes Tip colors indicate the origin of samples Triangles are indicating the samples from three patients from Amazonas Brazil transferred to Rio Grande do Sul to receive hospital care The first SARSCoV P1 genome identified in Rio Grande do Sul it is highlighted in purple

Figure 1 - Maximum-likelihood tree from the first SARS-CoV P.1 genome identified in Rio Grande do Sul (Brazil) contextualized on 156 P.1 genomes from worldwide. The aLRT support values are shown in key nodes. Tip colors indicate the origin of samples. Triangles are indicating the samples from three patients from Amazonas (Brazil), transferred to Rio Grande do Sul to receive hospital care. The first SARS-CoV P.1 genome identified in Rio Grande do Sul it is highlighted in purple.

The patient (A) had no travel history, and the epidemiological investigation revealed a transmission chain as follows (linkages were omitted for privacy-preserving): he lived in a rural area, nearby the city, under self-isolation, and had daily contact with a person (B). This person had previous contact in a diner with an individual (C) on Jan 23^rd, a tourism worker who used to have close contact with tourists and was positive to COVID-19 with onset symptoms on Jan 21^st. (B) presented her first symptoms on Jan 26th and had a positive RT-qPCR test for SARS-CoV-2 on Jan 31^st, presenting mild disease; (C) to date remains hospitalized with severe disease symptoms. Colleagues who shared the same workplace to (C) were also diagnosed with COVID-19 and some were admitted to the ICU with a severe disease presentation. In this same period the hospitalizations have risen in Gramado and more than 3-fold in the neighboring city, Canela, also a touristic city.

Our findings indicate a local transmission of P.1 lineage occurring in Gramado, and revealed a worrying emergence of severe COVID-19 cases in this region. It calls for future use of genomic surveillance as a regular and permanent tool to identify the underlying events as in the present case, where, due to genomic surveillance, it was possible to detect an unsuspected variant of concern P.1 on a case of COVID-19 death in a patient practicing self-isolation, living in a remote area.

Our analysis highlights monitoring protocols for new variants must consider key social mobilization sites in the state. From all cities of Rio Grande do Sul state, Gramado and Canela are by far the most important from the point of view of social mobility, as they receive tourists from Brazil and abroad. The Capital and the border/port regions must also be constantly monitored in order to guarantee success in monitoring new circulating strains. The joint observation of epidemiological and laboratory surveillance findings can assist the community in COVID-19 control measures.

Data availability: Whole genome sequence from SARS-CoV-2 genome sequenced in this work it is available on GISAID database under accession ID EPI_ISL_983865.

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