Detection and characterization of novel luchacoviruses, genus Alphacoronavirus, in meso-carnivores in the northeastern United States

Small to mid-sized carnivores, known as meso-carnivores, comprise a group of highly diverse mammals, many of which can easily adapt to anthropogenically disturbed environments. Due to commonly inhabiting urban landscapes, high population densities, and wide home ranges, wild meso-carnivores may get exposed to or spread pathogens to other species. To date, several coronaviruses (family Coronaviridae ) have been detected and characterized in domesticated and farmed meso-carnivores, but knowledge of the role of meso-carnivores in the epidemiology of coronaviruses (CoVs) remains limited. To increase our understanding of CoVs circulating in wild meso-carnivores, we screened 300 samples, including feces and tissues, from 9 species of free-ranging wild meso-carnivores in the northeastern United States collected between 2016 and 2022. Using a pan-CoV nested PCR approach, we detected CoVs in fecal samples of animals in three species: fisher ( Pekania pennanti ), bobcat ( Lynx rufus ) and red fox ( Vulpes vulpes ). Next generation sequencing revealed that all the detected viruses belonged to the Luchacovirus subgenus within the Alphacoronavirus genus, previously reported only in rodents and lagomorphs (i.e., rabbits and pikas). Genetic comparison of the 3’-end of the genome (∼12,000 bp) revealed that although the viruses detected group with, and have a genetic organization similar to other luchacoviruses, they are genetically distinct to luchacoviruses from rodents and lagomorphs. Genetic characterization of the spike (S) protein revealed that these luchacoviruses do not have an identifiable S1/S2 cleavage site but do have highly variable structural loops within S1 that comprise protease cleavage motifs or very similar cleavage motifs to those identified in certain pathogenic CoVs. This study highlights the importance of characterizing the S protein from CoVs in various wild animal species to target specific regions for further experimental analysis and epidemiologic monitoring. Importance Spillback of SARS-CoV-2 into free-ranging animal species has been well documented, but the repertoire of other coronaviruses that could serve as sources for recombination in many hosts is unknown. The cryptic circulation of possible animal-associated variant SARS-CoV-2 lineages in urban sewage surveillance, and canine alphacoronavirus infections in humans, highlight a critical need to survey animal populations in the US. In general, no large-scale pathogen discovery work has been conducted in North America; studies on other continents have largely focused on bats. While bats are a well-known reservoir for coronavirus emergence, the potential risks for other mammals are very poorly understood. Assessing the potential of US wildlife species as pathogen reservoirs is vastly under-explored and is essential to prepare for future emerging threats. Our focus here was on α and β CoV discovery from the Northeast USA as a potential “hotspot” area with high density of humans and urban wildlife. This study identifies novel alphacoronaviruses circulating in this area and explores their potential zoonotic risk based on Spike gene sequence. ### Competing Interest Statement The authors have declared no competing interest.