COVID-19 monitoring with sparse sampling of sewered and non-sewered wastewater in urban and rural communities.

Equitable SARS-CoV-2 surveillance in low-resource communities lacking centralized sewers is critical as wastewater-based epidemiology (WBE) progresses. However, large-scale studies on SARS-CoV-2 detection in wastewater from low-and middle-income countries is limited because of economic and technical reasons. In this study, wastewater samples were collected twice a month from 186 urban and rural subdistricts in nine provinces of Thailand mostly having decentralized and non-sewered sanitation infrastructure and analyzed for SARS-CoV-2 RNA variants using allele-specific RT-qPCR. Wastewater SARS-CoV-2 RNA concentration was used to estimate the real-time incidence and time-varying effective reproduction number (Re). Results showed an increase in SARS-CoV-2 RNA concentrations in wastewater from urban and rural areas 14-20 days earlier than infected individuals were officially reported. It also showed that community/food markets were "hot spots" for infected people. This approach offers an opportunity for early detection of transmission surges, allowing preparedness and potentially mitigating significant outbreaks at both spatial and temporal scales.

Implications of SARS-CoV-2 Mutations for Genomic RNA Structure and Host microRNA Targeting.

The SARS-CoV-2 virus is a recently-emerged zoonotic pathogen already well adapted to transmission and replication in humans. Although the mutation rate is limited, recently introduced mutations in SARS-CoV-2 have the potential to alter viral fitness. In addition to amino acid changes, mutations could affect RNA secondary structure critical to viral life cycle, or interfere with sequences targeted by host miRNAs. We have analysed subsets of genomes from SARS-CoV-2 isolates from around the globe and show that several mutations introduce changes in Watson-Crick pairing, with resultant changes in predicted secondary structure. Filtering to targets matching miRNAs expressed in SARS-CoV-2-permissive host cells, we identified ten separate target sequences in the SARS-CoV-2 genome; three of these targets have been lost through conserved mutations. A genomic site targeted by the highly abundant miR-197-5p, overexpressed in patients with cardiovascular disease, is lost by a conserved mutation. Our results are compatible with a model that SARS-CoV-2 replication within the human host is constrained by host miRNA defences. The impact of these and further mutations on secondary structures, miRNA targets or potential splice sites offers a new context in which to view future SARS-CoV-2 evolution, and a potential platform for engineering conditional attenuation to vaccine development, as well as providing a better understanding of viral tropism and pathogenesis.