Many animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and could act as reservoirs; however, transmission in free-living animals has not been documented. White-tailed deer, the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 is circulating in deer, 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021 were assayed for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 (33.2%) deer samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, following the November 2020 peak of human cases in Iowa, and coinciding with the onset of winter and the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples collected over a 7-wk period. Whole genome sequencing of all 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%) and B.1.311 (n = 19; 20%) accounting for ∼75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple human-to-deer transmission events followed by subsequent deer-to-deer spread. These discoveries have important implications for the long-term persistence of the SARS-CoV-2 pandemic. Our findings highlight an urgent need for a robust and proactive “One Health” approach to obtain enhanced understanding of the ecology, molecular evolution, and dissemination of SARS-CoV-2. Significance The results provide strong evidence of extensive SARS-CoV-2 infection of white-tailed deer, a free-living wild animal species with widespread distribution across North, Central, and South America. The analysis shows infection of deer resulted from multiple spillovers from humans, followed by efficient deer-to-deer transmission. The discovery of widespread infection of white-tailed deer indicates their establishment as potential reservoir hosts for SARS-CoV-2, a finding with important implications for the ecology, long-term persistence, and evolution of the virus, including the potential for spillback to humans.

Individuals can express a range of disease phenotypes during infection, with important implications for epidemics. Tolerance, in particular, is a host response that minimizes the per-pathogen fitness costs of infection. Because tolerant hosts show milder clinical signs and higher survival, despite similar pathogen burdens, their potential for prolonged pathogen shedding may facilitate the spread of pathogens. To test this, we simulated outbreaks of mycoplasmal conjunctivitis in house finches, asking how the speed of transmission varied with tissue-specific and behavioural components of tolerance, milder conjunctivitis and anorexia for a given pathogen load, respectively. Because tissue-specific tolerance hinders pathogen deposition onto bird feeders, important transmission hubs, we predicted it would slow transmission. Because behavioural tolerance should increase interactions with bird feeders, we predicted it would speed transmission. Our findings supported these predictions, suggesting that variation in tolerance could help identify individuals most likely to transmit pathogens.

Lesion severity scales have been developed for a number of wildlife diseases causing external pathology. Perhaps the best known and most widely used scoring system has been developed for finch mycoplasmosis in which observers measure conjunctival pathology along a four-point scale of increasing severity. We developed novel techniques to characterize variation in host phenotype based on occupancy of multidimensional trait space (disease space). First, we used shape analysis to track distortions of the inner and outer eye rims, defined by 16 anatomical landmarks. Then, we used community analysis to evaluate pathology based on the presence or absence of a unique set of binary descriptors. We applied these techniques to experimental infection data to relate differences in conjunctival pathology to stage of infection. Specifically, by comparing specimens that received the same severity score at different time points in infection, we asked if shape or community analyses could distinguish between individuals in early infection versus those in recovery. We found that individual eyes followed predictable loops through disease space, tracking further from their origin with more severe pathology. Also, certain pathological descriptors were more likely to appear earlier versus later in infection. Our results indicated that leveraging differences in pathology captured in complex trait space could complement severity scores by better resolving the time course of infection from limited data points.

Despite thousands of individuals entering the illegal wildlife trade each year, assessments of pangolin populations are largely non-existent, even in areas with high exploitation and limited personnel and field equipment. Although pangolins have unique keratin-based scales, there is no universal scale-marking method for individuals despite some pangolin conservation programmes utilizing marking for reference and cataloguing. Each programme currently establishes and manages its own system, resulting in inconsistencies and limiting data sharing. To facilitate pangolin monitoring and research, we developed a standardized method for assigning individual identification numbers, which we call the Pangolin Universal Notching System. This system is neither resource nor training intensive, which could facilitate its adoption and implementation globally. Its application could help to address knowledge gaps in pangolin ageing, reproduction, survivorship, migration and local trafficking patterns, and could be used in combination with other tagging techniques for research on pangolin biology.

The potential infectivity of SARS-CoV-2 in animals raises a public health and economic concern, particularly the high susceptibility of white-tailed deer (WTD) to SARS-CoV-2. The disparity in the disease outcome between humans and WTD is very intriguing, as the latter are often asymptomatic, subclinical carriers of SARS-CoV-2. To date, no studies have evaluated the innate immune factors responsible for the contrasting SARS-CoV-2-associated disease outcomes in these mammalian species. A comparative transcriptomic analysis in primary respiratory epithelial cells of human (HRECs) and WTD (Deer-RECs) infected with SARS-CoV-2 was assessed throughout 48 hours post inoculation (hpi). Both HRECs and Deer-RECs were susceptible to SARS-COV-2, with significantly ( P < 0.001) lower virus replication in Deer-RECs. The number of differentially expressed genes (DEG) gradually increased in Deer-RECs but decreased in HRECs throughout the infection. The ingenuity pathway analysis of DEGs further identified that genes commonly altered during SARS-CoV-2 infection mainly belong to cytokine and chemokine response pathways mediated via IL-17 and NF-κB signaling pathways. Inhibition of the NF-κB signaling in the Deer-RECs pathway was predicted as early as 6 hpi. The findings from this study could explain the lack of clinical signs reported in WTD in response to SARS-CoV-2 infection as opposed to the severe clinical outcomes reported in humans. HIGHLIGHTS: White-tailed deer primary respiratory epithelial cells are susceptible to SARS- CoV-2 without causing hyper cytokine gene expression. Downregulation of IL-17 and NF-κB signaling pathways after SARS-CoV-2 infection could be key to the regulated cytokine response in deer cells. Deer innate immune system could play a critical role in early antiviral and tissue repair response following SARS-CoV-2 infection.