Untapped potential of wastewater for animal and potentially zoonotic virus surveillance: Pilot study to detect non-human animal viruses in urban settings

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INTRODUCTION: Wastewater surveillance has become an essential tool for monitoring viral outbreaks and surveillance of human viruses. While PCR-based methods are most frequently used, more advanced techniques, such as shotgun metagenomics in combination with viral capture methods, have been developed. These capture methods significantly improve the ability to detect nearly all (known) viruses at once in complex samples, including wastewater. In this study, we focus on tracking animal specific and zoonotic viruses in city wastewater using metagenomics combined with hybrid-capture approach. METHODS: We collected 6 wastewater samples from Leuven and Brussels, situated in the center of Belgium. Automated wastewater samplers collected 50 mL samples every 10 min resulting in a 24 h composite influent wastewater. All samples were processed using the TWIST comprehensive research panel capture, designed to target over 3,000 human and animal viruses species and 15,000 strains. Sequencing was performed on the AVITI sequencing platform, targeting an average of ten million reads per sample. The sequencing data were analyzed using the EsViritu tool. RESULTS: Over 2294 viral genomes or segments were recovered from wastewater samples. Of these, 168 were associated with non-human vertebrate animals, including cats, dogs, pigeons, and rats, spanning 51 virus species. We identified near-complete genomes of clinically relevant animal viruses, such as pigeon circovirus, chicken anemia virus, feline bocaparvovirus 2, canine minute virus, rat coronavirus, canine parvovirus, and porcine circovirus. Additionally, we noted the presence of viruses with known cross-species transmission potential, including porcine torovirus, rosavirus, hepatitis E virus, rat hepatitis virus, and cardiovirus. CONCLUSION: The results demonstrate the ability to track a wide range of animal viruses in urban wastewater, potentially forming an early warning system for zoonotic diseases, ultimately being a useful tool for One Health based public health approaches.

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