Sara Mesquita Costa

SARS-CoV-2 high transmission and genomic mutations result in the emergence of new variants that impact COVID-19 vaccine efficacy and virus transmission by evading the host immune system. Wastewater-based epidemiology is an effective approach to monitor SARS-CoV-2 variants circulation in the population but is a challenge due to the presence of reaction inhibitors and the low concentrations of SARS-CoV-2 in this environment. Here, we aim to improve SARS-CoV-2 variant detection in wastewater by employing nested PCR followed by next-generation sequencing (NGS) of small amplicons of the S gene. Eight SARS-CoV-2 wastewater samples from Alegria Wastewater Treatment Plant, in Rio de Janeiro, Brazil, were collected monthly from February to September 2021. Samples were submitted to virus concentration, RNA extraction and nested PCR followed by NGS. The small amplicons were used to prepare libraries for sequencing without the need to perform any fragmentation step. We identified and calculated the frequencies of 29 mutations matching the Alpha, Beta, Gamma, Delta, Omicron, and P.2 variants. Omicron matching-mutations were detected before the lineage was classified as a variant of concern. SARS-CoV-2 wastewater sequences clustered with SARS-CoV-2 variants detected in clinical samples that circulated in 2021 in Rio de Janeiro. We show that sequencing of selected small amplicons of SARS-CoV-2 S gene allows the identification of SARS-CoV-2 variants matching mutations and their frequencies’ calculation. This approach may be expanded using customizing primers for additional genomic regions, in order to differentiate current variants. Approaches that allow us to learn how variants emerge and how they relate to clinical outcomes are crucial for our understanding of the dynamics of virus variants circulation, providing valuable data for public health management.

SARS-CoV-2 high transmission and genomic mutations result in the emergence of new variants that impact COVID-19 vaccine efficacy and virus transmission by evading the host immune system. Wastewater-based epidemiology is an effective approach to monitor SARS-CoV-2 variants circulation in the population but is a challenge due to the presence of reaction inhibitors and the low concentrations of SARS-CoV-2 in this environment. Here, we aimed to improve SARS-CoV-2 variant detection in wastewater by employing nested PCR followed by next-generation sequencing (NGS) of small amplicons of the S gene. Eight SARS-CoV-2 wastewater samples from Alegria Wastewater Treatment Plant, in Rio de Janeiro, Brazil, were collected monthly from February to September 2021. Samples were submitted to virus concentration, RNA extraction and nested PCR followed by NGS. The small amplicons were used to prepare libraries for sequencing without the need to perform any fragmentation step. We identified and calculated the frequencies of 29 mutations matching the Alpha, Beta, Gamma, Delta, Omicron and P.2 variants. Omicron matching-mutations were detected before the lineage was classified as a variant of concern. SARS-CoV-2 wastewater sequences clustered with SARS-CoV-2 variants detected in clinical samples that circulated in 2021 in Rio de Janeiro. We show that sequencing of selected small amplicons of SARS-CoV-2 S gene allows the identification of SARS-CoV-2 variants matching-mutations and their frequencies calculation. This approach may be expanded using customizing primers for additional genomic regions, in order to differentiate current variants. Approaches that allow us to learn how variants emerge and how they relate to clinical outcomes are crucial for our understanding of the dynamics of virus variants circulation, providing valuable data for public health management.

The spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the COVID-19 outbreak, posed a primary concern of public health worldwide. The most common changes in SARS-CoV-2 are single nucleotide substitutions, also reported insertions and deletions. This work investigates the presence of SARS-CoV-2 ORF7a deletions identified in COVID-19-positive individuals. Sequencing of SARS-CoV-2 complete genomes showed three different ORF7a size deletions (190-nt, 339-nt and 365-nt). Deletions were confirmed through Sanger sequencing. The ORF7a∆190 was detected in a group of five relatives with mild symptoms of COVID-19, and the ORF7a∆339 and ORF7a∆365 in a couple of co-workers. These deletions did not affect subgenomic RNAs (sgRNA) production downstream of ORF7a. Still, fragments associated with sgRNA of genes upstream of ORF7a showed a decrease in size when corresponding to samples with deletions. In silico analysis suggests that the deletions impair protein proper function; however, isolated viruses with partial deletion of ORF7a can replicate in culture cells similarly to wild-type viruses at 24 hpi, but with less infectious particles after 48 hpi. These findings on deleted ORF7a accessory protein gene, contribute to understanding SARS-CoV-2 phenotypes such as replication, immune evasion and evolutionary fitness as well insights into the role of SARS-CoV-2_ORF7a in the mechanism of virus-host interactions.