The outbreak of a novel corona Virus Disease 2019 in the city of Wuhan, China has resulted in more than 1.7 million laboratory confirmed cases all over the world. Recent studies showed that SARS-CoV-2 was likely originated from bats, but its intermediate hosts are still largely unknown. In this study, we assembled the complete genome of a coronavirus identified in 3 sick Malayan pangolins. The molecular and phylogenetic analyses showed that this pangolin coronavirus (pangolin-CoV-2020) is genetically related to the SARS-CoV-2 as well as a group of bat coronaviruses but do not support the SARS-CoV-2 emerged directly from the pangolin-CoV-2020. Our study suggests that pangolins are natural hosts of Betacoronaviruses. Large surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of coronaviruses in pangolins. In addition to conservation of wildlife, minimizing the exposures of humans to wildlife will be important to reduce the spillover risks of coronaviruses from wild animals to humans. Author summaryRecently, a novel coronavirus, SARS-CoV-2, caused a still ongoing pandemic. Epidemiological study suggested this virus was associated with a wet market in Wuhan, China. However, the exact source of this virus is still unknown. In this study, we attempted to assemble the complete genome of a coronavirus identified from two groups of sick PLOS PATHOGENSMalayan pangolins, which were likely to be smuggled for black market trade. The molecular and evolutionary analyses showed that this pangolin coronavirus we assembled was genetically associated with the SARS-CoV-2 but was not likely its precursor. This study suggested that pangolins are natural hosts of coronaviruses. Determining the spectrum of coronaviruses in pangolins can help understand the natural history of coronaviruses in wildlife and at the animal-human interface, and facilitate the prevention and control of coronavirus-associated emerging diseases.

Th17 cells have recently emerged as a major player in inflammatory and autoimmune diseases via the production of pro-inflammatory cytokines IL-17, IL-17F, and IL-22. The differentiation of Th17 cells and the associated cytokine production is directly controlled by ROR␥t. Here we show that ursolic acid (UA), a small molecule present in herbal medicine, selectively and effectively inhibits the function of ROR␥t, resulting in greatly decreased IL-17 expression in both developing and differentiated Th17 cells. In addition, treatment with UA ameliorated experimental autoimmune encephalomyelitis. The results thus suggest UA as a valuable drug candidate or leading compound for developing treatments of Th17-mediated inflammatory diseases and cancer. Th17 cells have been recently discovered as the third effector CD4ϩ T helper subset (1, 2). Th17 cells produce 4). Although Th17 cells play important roles in host defense against bacterial and fungal infections, they have been also linked to many immune-related diseases, including psoriasis, rheumatoid arthritis, multiple sclerosis, inflammatory bowel diseases, periodontal diseases, and asthma/airway inflammatory diseases (4, 5). Anti-IL-17 was recently shown to have good efficacy in treatment of multiple human diseases (6).In Th17 cells, the transcription of IL-17 and IL-17F is mediated by Th17-specific transcriptional regulators ROR␥t 6 and ROR␣, although the latter plays a less significant role in mice (7,8). Mice deficient in ROR␥ and those deficient in both ROR␥t and ROR␣ are defective in production of IL-17 and IL-17F and are resistant to experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis (7,8). Therefore, developing ROR inhibitors represents a promising therapeutic strategy in treatment of Th17-mediated diseases.In the current study, we screened a small chemical library and identified ursolic acid (UA), a natural carboxylic acid ubiquitously present in plants, as a strong and selective inhibitor for ROR␥t function. UA inhibited IL-17 production not only in developing Th17 cells but also in mature Th17 cells. Mice receiving UA were resistant to EAE, indicating that UA can be used for developing treatment of Th17-mediated diseases. EXPERIMENTAL PROCEDUREST Cell Analysis-Human and mouse T cell differentiation and retroviral transduction were performed and analyzed by intracellular staining or by quantitative real-time RT-PCR assays as described (8 -10). UA (dissolved in DMSO) or DMSO was added into the culture medium for inhibition assays.Luciferase Reporter Assays-The CNS2-Il17a and RORE reporter constructs were used for Dual-Luciferase reporter assays in EL4 and 293T cells, respectively, as reported (8, 11). The reporter activity was normalized against Renilla luciferase activity.Co-activator Binding Assays-The effect of UA on the interaction of coactivator peptides with ROR␥ was determined by terbium-mediated time-resolved fluorescence energy transfer assays using the LanthaScreen TR-FRET from Invitrogen. The experiments were conduc...

Summary T helper 17 (Th17) cells specifically transcribe the Il17 and Il17f genes, which are localized in the same chromosome region, but the underlying mechanism is unclear. Here, we report a cis element that we previously named conserved noncoding sequence 2 (CNS2), physically interacted with both Il17 and Il17f gene promoters and was sufficient for regulating their selective transcription in Th17 cells. Targeted deletion of CNS2 resulted in impaired retinoic acid-related orphan receptor gamma (RORγ)-driven IL-17 expression in vitro. CNS2-deficient T cells also produced substantially decreased amounts of IL-17F. These cytokine defects were associated with defective chromatin remodeling in the Il17-Il17f gene locus, possibly due to effects on CNS2-mediated recruitment of histone modifying enzymes p300 and JmjC domain-containing protein 3 (JMJD3). CNS2-deficient animals were also shown to be resistant to experimental autoimmune encephalomyelitis (EAE). Our results thus suggest that CNS2 is sufficient and necessary for Il17 and optimal Il17f gene transcription in Th17 cells.

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The outbreak of 2019-nCoV pneumonia in the city of Wuhan, China has resulted in more than 70,000 laboratory confirmed cases, and recent studies showed that 2019-nCoV (SARS-CoV-2) could be of bat origin but involve other potential intermediate hosts. In this study, we assembled the genomes of coronaviruses identified in sick pangolins. The molecular and phylogenetic analyses showed that pangolin Coronaviruses (pangolin-CoV) are genetically related to both the 2019-nCoV and bat Coronaviruses but do not support the 2019-nCoV arose directly from the pangolin-CoV. Our study also suggested that pangolin be natural host of Betacoronavirus, with a potential to infect humans. Large surveillance of coronaviruses in pangolins could improve our understanding of the spectrum of coronaviruses in pangolins. Conservation of wildlife and limits of the exposures of humans to wildlife will be important to minimize the spillover risks of coronaviruses from wild animals to humans. author/funder. All rights reserved. No reuse allowed without permission.