B-208 Implementation of Genomic Surveillance of Epidemic Viruses in a Clinical Laboratory in Brazil: Outcomes from Public-Private Collaborative Efforts

Background Genomic surveillance has been widely implemented in various countries during and after the COVID-19 pandemic. In this context, nanopore sequencing technology gained prominence, enabling the development of multiple protocols based on multiplex PCR product sequencing. In Brazil, private laboratories are responsible for a significant portion of laboratory testing, making them potential allies in genomic surveillance. In this context, we submitted a research project to a public funding agency in the Federal District of Brazil, aiming to sequence the genomes of epidemiologically relevant viruses. This work describes the main results obtained. Methods This study was approved by the appropriate ethics and research committee. Between 2023 and 2024, positive samples previously identified by real time PCR (qPCR) for Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), Monkeypox virus (MPXV), Chikungunya virus (CHIKV), Dengue virus (DENV) were selected. DNA and RNA were extracted using the Starlet or Maxwell extractor. RNA samples were subjected to cDNA synthesis using LunaScript RT SuperMix. Multiplex PCR was performed using specific primer pools for SARS-CoV-2 and CHIKV. The amplified products were quantified by fluorimetry, normalized, and sequencing libraries were prepared using the Sequencing chemistry V14 (SQK-LSK114, SQK-NBD114.24 or SQK-RBK114.96. Sequencing experiments were conducted on the MinION sequencer using R10 flowcells (FLO-MIN114) or Flongles R10 (FLO-FLG114). Raw signal data (.pod5) were processed using Dorado software with the super-accuracy protocol for basecalling and demultiplexing. The resulting reads (.fastq) were used for genome assembly with virus-specific pipelines in the Epi2Me software. The MPXV genome was sequenced directly from total extracted nucleic acid using a shotgun approach (without PCR amplification). The genotypes of DENV samples were determined by serotype-specific qPCR primers and probes. Results Between 2023 and 2024, a total of 163 SARS-CoV-2 genomes were sequenced, the most frequently identified variant in 2023 being XBB.1.5 (n = 43) and in 2024 BA.2 (n = 115). During the same period, 17 CHIKV genomes were obtained with an average coverage of 82,88% (range: 54,18% – 95,04%), all belonging to East/Central/South African genotype. Regarding MPXV, only one genome was successfully sequenced by shotgun approach resulting in 3,082,296 reads, from which 28,230 (0,9%) mapped in the MPXV genome, with 99,95% coverage and average depth of 110. The analysis classified this genome within clade IIb, consistent with previous reports in the region. For DENV, 21 positive samples were genotyped by RT-qPCR, identifying three samples of serotype 1, 17 of serotype 2, and one of serotype 4. All generated sequences were deposited in public databases, ensuring accessibility for further epidemiological studies. Conclusion The use of samples from routine diagnostic testing in private laboratories proved to be a viable and effective approach for genomic surveillance, contributing to the monitoring of epidemiologically relevant viral variants. This study generated nearly 200 viral sequences using the portable MinION sequencer, a low-cost and easily implementable technology. Furthermore, our results highlight the importance of collaborative projects between public research institutions and private laboratories, which can enhance genomic surveillance capabilities, expand data collection networks, and improve response strategies to emerging viral threats.