Millions of Norway rats (Rattus norvegicus) inhabit New York City (NYC), presenting the potential for transmission of SARS-CoV-2 from humans to rats and other wildlife. We evaluated SARS-CoV-2 exposure among 79 rats captured from NYC during the fall of 2021. Results showed that 13 of 79 rats (16.5%) tested IgG or IgM positive, and partial genomes of SARS-CoV-2 were recovered from four rats that were qRT-PCR positive. Using a virus challenge study, we also showed that Alpha, Delta, and Omicron variants can cause robust infections in wild-type Sprague Dawley (SD) rats, including high level replications in the upper and lower respiratory tracts and induction of both innate and adaptive immune responses. Additionally, the Delta variant resulted in the highest infectivity. In summary, our results indicated that rats are susceptible to infection with Alpha, Delta, and Omicron variants, and rats in the NYC municipal sewer systems have been exposed to SARS-CoV-2. Our findings highlight the potential risk of secondary zoonotic transmission from urban rats and the need for further monitoring of SARS-CoV-2 in those populations.

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