key: cord-0808287-icnqrpyg authors: Li, Xiaobing; Xiao, Kangpeng; Chen, Xiaoyuan; Liang, Xianghui; Zhang, Xu; Zhang, Zhipeng; Zhai, Junqiong; Wang, Ruichen; Zhou, Niu; Chen, Zu-Jin; Su, Renwei; Zhou, Fuqing; Holmes, Edward C.; Irwin, David M.; Chen, Rui-Ai; He, Qian; Wu, Ya-Jiang; Wang, Chen; Du, Xue-Qing; Peng, Shi-Ming; Xie, Wei-Jun; Shan, Fen; Li, Wan-Ping; Dai, Jun-Wei; Shen, Xuejuan; Feng, Yaoyu; Xiao, Lihua; Chen, Wu; Shen, Yongyi title: Pathogenicity, tissue tropism and potential vertical transmission of SARSr-CoV-2 in Malayan pangolins date: 2020-06-22 journal: bioRxiv DOI: 10.1101/2020.06.22.164442 sha: 9db801028ebecca7028844ca0ff596fb4f19e122 doc_id: 808287 cord_uid: icnqrpyg SARS-CoV-2 is having severe impact on public health at a global scale. Malayan pangolin SARS-CoV-2-related coronavirus (SARSr-CoV-2) is closely related to SARS-CoV-2. We show that CT scans of virus-positive pangolins reveal bilateral ground-glass opacities in lungs in similar manner to COVID-19 patients. The virus infected multiple organs in pangolins, with the lungs being the major target. Histological expression showed that ACE2 and TMPRSS2 are co-expressed with viral RNA. Transcriptome analysis revealed an inadequate interferon response, with different dysregulated chemokines and cytokines responses in pregnant and non-pregnant adults and fetuses. Viral RNA and protein were detected in three fetuses providing evidence for vertical virus transmission. In sum, our study identifies the biological framework of SARSr-CoV-2 in pangolins, revealing striking similarities to COVID-19 in humans. The ongoing pandemic of COVID-19 caused the coronavirus SARS-CoV-2 is severely effecting global health (1-3). After SARS-CoV and MERS-CoV, SARS-CoV-2 is the third coronavirus to cause severe respiratory illness in humans identified in the past two decades, although with a markedly lower infection fatality ratio (4) . SARS-CoV-2 belongs to the genus Betacoronavirus, sharing 79.5% overall genome nucleotide sequence identity with SARS-CoV and 96.2% with the bat-derived SARS-related coronavirus RaTG13 to which it is most closely related at the level of the whole genome (5) . A better understanding of the pathobiology of SARS-CoV-2 is critical for its control and the development of therapeutics. Several animal models are being developed to assist in better understanding of the pathogenicity of SARS-CoV-2. These include the development of transduced or transgenic mouse models that express human ACE2 and can be infected by SARS-CoV-2 (6) (7) (8) , and macaques that can be infected and recapitulate moderate disease (9, 10) . We previously isolated a SARS-CoV-2-related coronavirus (SARSr-CoV-2) in Malayan pangolins (Manis javanica) that is closely related to SARS-CoV-2, especially in the receptor-binding domain (RBD) of the spike protein (11, 12) . Understanding the biology of SARSr-CoV-2 in pangolins will be important for understanding key aspects of coronavirus disease. Herein, we studied 28 Malayan pangolins that were confiscated in Guangdong Province between March-August 2019 and initially sent to Guangzhou wildlife rescue center. Notably, seven of these animals were pregnant. We used the viral S (spike) gene to identify infection with the pangolin-derived SARSr-CoV-2, and found that 15 animals, including six pregnant females, naturally infected by the virus. Notably, the S genes amplified from these individuals exhibited no changes in the consensus sequence. Four samples were chosen for deep sequencing, enabling the de novo assembly of four near complete coronavirus genomes ( Figure S1 ). Analyses of these genomes revealed that they exhibited 99.6-99.9% genetic identity with the pangolin SARSr-CoV-2 reference genome (EPI_ISL_410721) and hence are indicative of a localized outbreak due to a single source of infection ( Figure S2 ). COVID-19 is characterized by a range of symptoms, but in most cases include fever, cough, dyspnoea, and myalgia (13) . Bilateral opacities on x-ray or patchy shadows and ground glass opacities visible in CT scans are the most common features of severe COVID-19 cases (1, 14) . Malayan pangolins that were positive for pangolin SARSr-CoV-2 exhibited respiratory symptoms such as cough and shortness of breath (11) . Blood gas tests revealed elevated levels of PCO 2 (58.2, and 60.25 mmHg), HCO 3 (29.9, and 34.6 mmol/L), and TCO 2 (31.5 mmol/L) in the infected animals (Table S1) Figure S4 ). These features are consistent with breathing difficulties. Alveoli were filled with desquamated epithelial cells and some macrophages with hemosiderin pigments ( Figure 2C ). To ensure that these lesions were associated with SARSr-CoV-2, we used immunohistochemistry to detect and localize the nucleocapsid (N protein) of the virus. Several alveolar macrophages and pneumocytes were found to be positive ( Figure 2E ). qRT-PCR and western blot analysis further supported the presence of SARSr-CoV-2 infection in the lungs ( Figure 2E , Figure 3A -B). These findings are similar to those of SARS-CoV-2 in humans and are compatible with a similar pathological mechanism, mainly by invading alveolar epithelial cells which results in respiratory symptoms (13) . Although respiratory symptoms dominate the clinical presentation of COVID-19, SARS-CoV-2 is also associated with multiple organ dysfunction syndrome (13) . Necropsy of virus-positive pangolins similarly revealed multiple organ injury ( Figure S1 ). We therefore used qRT-PCR to examine the tissue tropism of SARSr-CoV-2. Viral RNA was mainly detected in the lungs, although other organs such as liver, intestine, heart, kidney, spleen and muscle of some individuals also had detectable levels of virus RNA ( Figure 3A , Table S2 ). Western blotting further supported the presence of viral infection in these tissues ( Figure 3B ). The spike protein of SARS-CoV-2 binds to ACE2 receptors for entry into cells (5, 16) , while TMPRSS2 mediates spike protein activation and facilitates viral entry (16, 17) . Generally, virus-positive pangolins had much higher expression levels of ACE2 and TMPRSS2 in all tissues than virus-negative individuals ( Figure 3C and D). In humans, ACE2 is abundant in the epithelia of the lung, kidney, and small intestine (18) . In pangolins, lung, liver, and spleen had the highest levels of ACE2 and TMPRSS expression compared to other tissues examined. SARSr-CoV-2 RNA was also detected in the tissues expressing the highest levels of ACE2 and TMPRSS, supporting the idea that cells that express both ACE2 and TMPRSS2 are most susceptible to coronaviruses (19). However, SARSr-CoV-2 was also detected in intestine and kidney, tissues that do not have high expression of these receptor genes in the pangolins. As COVID-19 becomes a global pandemic, the potential risk of vertical transmission of SARS-CoV-2 has become a topic of concern (20, 21). The ACE2 receptor is widely expressed in the placenta during pregnancy ( Table S2 ). Hence, these results demonstrate it is possible that the vertical transmission of SARSr-CoV-2 can occur in utero. We next compared the transcriptional response of lung, spleen, muscle and production. The fetal immune system is unique and immature (28). Fetuses had no detectable virus RNA in lungs, and DEGs related to immune pathways were not enriched in lungs ( Figure 4D ). In the spleen and intestine, where virus RNA was detected in fetuses, down-regulated genes were related to immune pathways ( Figure 4D and Figure S6 ). In sum, we show that Malayan pangolins infected with SARSr-CoV-2 exhibit a strikingly similar pathogenicity to human COVID-19 patients. For example, the host response of pangolins to SARSr-CoV-2, reflected in a low level ISG response, is similar to that seen in human COVID-19. Although the SARS-CoV-2-related pangolin virus has a number of important genetic differences to SARS-CoV-2, we were able to show that SARSr-CoV-2 was present in pangolin fetuses highlights the potential for in utero virus transmission. A Table S2 . Table S4 . Protein was extracted with radio-immuno precipitation assay (RIPA) buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS) and a protease inhibitor cocktail. 10mg of total protein was separated on SDS-PAGE and then transferred to PVDF membranes using the Bio-Rad Trans-Blot Lungs from six Malayan pangolins, including four that were positive for SARSr-CoV-2, were fixed in 10% buffered formalin. Tissues were then washed to remove formalin, dehydrated in ascending grades of ethanol, cleared with chloroform, and embedded with molten paraffin wax in a template. The tissue blocks were sectioned with a microtome, and sections were transferred onto grease-free glass slides, deparaffinized, and rehydrated through descending grades of ethanol and distilled water. Sections were stained with a hematoxylin and eosin staining kit (Baso Diagnostics Inc., Wuhan Servicebio Technology Co., Ltd. (Table S3 ). The raw sequencing data sets were submitted to NCBI Gene Expression Omnibus (GEO) server (accession number PRJNA640246). Adaptor and low-quality sequences were trimmed using fastp (v0.19.7) (38) . Table S1 . Blood gas analysis and routine blood tests of pangolins. A novel coronavirus from patients with pneumonia in China A new coronavirus associated with human respiratory disease in China A novel coronavirus outbreak of global health concern Estimates of the severity of coronavirus disease 2019: a model-based analysis A pneumonia outbreak associated with a new coronavirus of probable bat origin Pathogenesis of SARS-CoV-2 in transgenic mice expressing human angiotensin-converting enzyme 2 A mouse model of SARS-CoV-2 infection and pathogenesis Generation of a Broadly Useful Model for COVID-19 Respiratory disease in rhesus macaques inoculated with SARS-CoV-2 Comparative pathogenesis of COVID-19, MERS, and SARS in a nonhuman primate model Isolation of SARS-CoV-2-related coronavirus from Malayan pangolins Identifying SARS-CoV-2 related coronaviruses in Malayan pangolins Clinical features of patients infected with 2019 novel coronavirus in Wuhan Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: a descriptive study COVID-19) pneumonia in 62 patients in Wuhan, China SARS-CoV-2 cell entry depends on ACE2 and TMPRSS2 and Is blocked by a clinically proven protease inhibitor Evidence that TMPRSS2 activates the severe acute respiratory syndrome coronavirus spike protein for membrane fusion and reduces viral control by the humoral immune response Tissue distribution of ACE2 protein, the functional receptor for SARS coronavirus. A first step in understanding SARS pathogenesis The Immunobiology of SARS* fastp: an ultra-fast all-in-one FASTQ preprocessor Fast and accurate long-read alignment with Burrows-Wheeler transform MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph Graph-based genome alignment and genotyping with HISAT2 and HISAT-genotype featureCounts: an efficient general purpose program for assigning sequence reads to genomic features Differential expression analysis for sequence count data He, clusterProfiler: an R package for comparing biological themes among gene clusters ggplot2: elegant graphics for data analysis The authors declare no competing interests.