Viral metagenomics is increasingly being used for the identification of emerging and re-emerging viral pathogens in clinical samples with unknown etiology. The objective of this study was to shield light on the metavirome composition in clinical samples obtained from patients with clinical history compatible with an arboviral infection, but that presented inconclusive results when tested using RT-qPCR. The inconclusive amplification results might be an indication of the presence of an emerging arboviral agent that is inefficiently amplified by conventional PCR techniques. A total of eight serum samples with inconclusive amplification results for the routinely tested arboviruses—dengue (DENV), Zika (ZIKV), and Chikungunya (CHIKV) obtained during DENV and CHIKV outbreaks registered in the state of Alagoas, Northeast Brazil between July and August 2021—were submitted to metagenomic next-generation sequencing assay using NextSeq 2000 and bioinformatic pipeline for viral discovery. The performed bioinformatic analysis revealed the presence of two arboviruses: DENV type 2 (DENV-2) and CHIKV with a high genome coverage. Further, the metavirome of those samples revealed the presence of multiple commensal viruses apparently without clinical significance. The phylogenetic analysis demonstrated that the DENV-2 genome belonged to the Asian/American genotype and clustered with other Brazilian strains. The identified CHIKV genome was taxonomically assigned as ECSA genotype, which is circulating in Brazil. Together, our results reinforce the utility of metagenomics as a valuable tool for viral identification in samples with inconclusive arboviral amplification. Viral metagenomics is one of the most potent methods for the identification of emerging arboviruses.

Enterovirus D68 (EV-D68) is a leading cause of acute respiratory disease outbreaks, especially among children. EV-D68 infections can rapidly progress to severe clinical complications and potentially fatal outcomes. In Brazil, no diagnostic or genomic surveillance of this virus is currently performed. Between July and September 2023, cases of acute EV-D68 infection were identified among pediatric patients in several municipalities within the State of Alagoas, Northeast Brazil. Infections were confirmed by RT-qPCR using nasopharyngeal samples, and the complete EV-D68 genomes were sequenced and analyzed through phylogenetic inference. EV-D68 RNA was identified in four children aged 1–9 years from four geographically distinct municipalities in Alagoas. All infections were associated with lower respiratory tract symptoms, including dyspnea and wheezing; however, no fatalities were reported. Complete genomic sequencing revealed that the samples belonged to genotype B, subgenotype B3. This is the first study to report complete genomic data on EV-D68 infections from Brazil and South America. Enhanced genomic surveillance and focused EV-D68 diagnosis are critical to better understanding and managing the regional and national dissemination of this virus.