bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.452903; this version posted July 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Viral receptor profiles of masked palm civet revealed by single-cell

2 transcriptomics

3 Dongsheng Chen1,†, Zhihua Ou1, 2,†, Haoyu Wang1,3,†, Yanan Zhang1,4,†, Jiacheng Zhu1,3, Fuyu An5, Jinqian Xu5,

4 Xiangning Ding1,3, Peiwen Ding1,3, Lihua Luo1,3, Weiying Wu1, Qiuyu Qin1,6, Yanan Wei1,6, Wandong Zhao1,6,

5 Zhiyuan Lv1,6, Tianming Lan1, Meiling Li1, Wensheng Zhang7, Huan Liu1,*, Yan Hua5,*

6 1. BGI-Shenzhen, Shenzhen 518083, China

7 2. Shenzhen Key Laboratory of Unknown Pathogen Identification, BGI-Shenzhen, Shenzhen 518083, China.

8 3. College of Life Sciences, University of Chinese Academy of Sciences, Beijing 100049, China

9 4. Tsinghua-Berkeley Shenzhen Institute, Tsinghua University, Shenzhen, China

10 5. Guangdong Provincial Key Laboratory of Silviculture, Protection and Utilization, Guangdong Academy of

11 Forestry, Guangzhou 510520, China

12 6. School of Basic Medicine, Qingdao University, Qingdao 266071, China

13 7. School of Basic Medical Sciences, Binzhou Medical University, No. 346, Guanhai Road, Laishan District,

14 Yantai City, Shandong, China

15 † These authors contributed equally

16 * Correspondence should be addressed to Huan Liu ([email protected]) and Yan Hua

17 ([email protected])

18 Abstract 19 Civets are small mammals belonging to the family Viverridae. The masked palm 20 civets (Paguma larvata) served as an intermediate host in the bat-to-human 21 transmission of severe acute respiratory syndrome coronavirus (SARS-CoV) in 20031. 22 Because of their unique role in the SARS outbreak, civets were suspected as a 23 potential intermediate host of SARS-CoV-2, the etiological pathogen of the 24 COVID-19 pandemic. Besides their susceptibility to coronaviruses, civets can also be 25 infected by other viruses, such as canine distemper viruses2, parvoviruses3, influenza 26 viruses4, etc. Regarding the ecological and economical role of civets, it is vital to 27 evaluate the potential threats from different pathogens to these animals. Receptor 28 binding is a necessary step for virus entry into host cells. Understanding the 29 distribution of receptors of various viruses provides hints to their potential tissue bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.452903; this version posted July 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

30 tropisms. Herein, we characterized the cell atlas of five important organs (the frontal 31 lobe, lung, liver, spleen and kidney) of masked palm civets (Paguma larvata) and 32 described the expression profiles of receptor associated genes of 132 viruses from 25 33 families, including 16 viruses from 10 families reported before that can attack civets 34 and 116 viruses with little infection record. 35 36 Results 37 To build a comprehensive cell atlas of civet organs, we performed single-cell RNA 38 sequencing to five organs of an adult male masked palm civet. After pre-processing, a 39 total of 66,553 cells (Fig. 1a), including 6,593 cells from the frontal lobe, 13,009 cells 40 from the lung, 34,883 cells from the liver, 10,138 cells from the spleen and 1,930 cells 41 from the kidney were acquired. We conducted unsupervised clustering and annotated 42 resulted cell clusters based on canonical markers (Fig. 1b-f, Table S1). In the frontal 43 lobe, we identified 8 major cell types for 25 clusters, including astrocytes (AST), 44 excitatory neurons (EX), inhibitory neurons (IN), oligodendrocytes (OLG), OLG 45 progenitor cells (OPC), microglia (MG), smooth muscle cells (SMC), and endothelial 46 cells (END). In lung, 7 cell types were annotated for 30 clusters, including pulmonary 47 alveolar type I (AT1), pulmonary alveolar type II (AT2), macrophages (MAC), 48 epithelial cells (EC), ciliated cells (CC), fibroblasts (FIB) and END. 5 cell types were 49 characterized for 19 clusters in liver, including hepatic stellate cells (HSC), immune 50 cells (IC), liver sinusoidal endothelial cells (LSEC), cholangiocytes and hepatocytes. 51 In spleen, we annotated 21 clusters to 11 cell types, including END, FIB, neural cells 52 (NEU), and several immune cell types, which were naive T cells, T cells, T follicular 53 helper cells (Tfh), regulatory T cells (Treg), B cells, macrophages (MAC), dendritic 54 cells (DC) and natural killer cells (NK). In kidney, 11 clusters were characterized to 8 55 cell types, including proximal tubule cells (PCT), distal convoluted tubule cells 56 (DCT), podocytes (Podo), loop of Henle cells (LOH), collecting duct intercalated 57 cells (CD-IC), collecting duct principal cells (CD-PC), pericytes (PER) and END. To 58 share the single cell atlas of civet, we constructed an online platform, 59 http://120.79.46.200:81/Civet, allowing researchers freely exploring our data set and bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.452903; this version posted July 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

60 analysis results. 61 62 Among the viruses in analysis, we placed special focus on six virus families that 63 prone to cause inter-species transmissions associated with severe diseases, including 64 Coronaviridae, Filoviridae, Orthomyxoviridae, Paramyxoviridae, Parvoviridae and 65 Rhabdoviridae, though no civet infections by filoviruses have yet been reported. For 66 Coronaviridae, ACE2, the common receptor of SARS-CoV, SARS-CoV-2 and human 67 coronavirus NL63, was expressed by a small fraction of cells in the frontal lobe, lung, 68 liver, spleen and kidney, with the highest expression level in loop of Henle cells and 69 proximal tubule cells of the kidney. AXL, the common receptor of SARS-CoV-2, 70 ebolavirus and marburg marburgvirus, showed the same expression pattern as ACE2. 71 Importantly, NRP1, another receptor for SARS-CoV-2, was widely detected in all five 72 organs and expressed in a much higher level than ACE2. ANPEP, a common receptor 73 for animal and human multiple coronaviruses, was only expressed by a small fraction 74 of hepatocytes. For Filoviridae, multiple receptors (ITCH, NPC1 and MERTK) of 75 Ebola viruses and Marburg viruses displayed high expression in the five organs, 76 though the expression levels varied among cell types. For Orthomyxoviridae, three 77 receptor-associated genes for avian influenza A virus (UVRAG, ANXA5 and EGFR) 78 and one receptor-associated gene for bat influenza A virus H18N11 (CD74) showed 79 high expression patterns in all five organs, especially in the lung and spleen. For 80 Paramyxoviridae, SLAMF1 and NECTIN4 were both receptors of canine 81 morbilliviruses but SLAMF1 was detected in a small number of cells across the five 82 organs while NECTIN4 was only highly expressed by a few lung cells. EFNB2 is a 83 receptor of Henipaviruses, which was detected in all five organs with high 84 expressions in the lung, spleen and kidney. For Parvoviridae, TFRC, which is a 85 common receptor of parvoviruses, was widely expressed in the five organs with a 86 significant enrichment in the endothelial cells of the frontal lobe. For Rhabdoviridae, 87 the receptors of lyssaviruses, GRM2 and NCAM1, were detected in all five organs 88 with the highest expression in the frontal lobe. The receptors of vesicular stomatitis 89 virus, UVRAG and LDLR, were identified in five organs with lung cells showing the bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.452903; this version posted July 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

90 highest expressions (Fig. S1). 91 92 Besides the above six families, we also categorized the receptor distributions of other 93 viruses capable of infecting civets. F11R, the receptor for viruses of Caliciviridae and 94 Reoviridae, was only detected in the civet lung (AT1 and AT2) and liver (liver 95 sinusoidal endothelial cells and hepatocytes). Another two receptors of reoviruses, 96 RTN4R and ITGB1, were both widely expressed in all five organs. CXCR4, a receptor 97 of feline immunodeficiency virus (Retroviridae), was widely detected in the frontal 98 lobe, lung, liver and spleen at low levels. TLR8 and TLR7, the receptors of severe 99 fever with thrombocytopenia syndrome virus (Bunyavirales, Phenuiviridae), were 100 both found in the lung and spleen but TLR8 was also distributed in the frontal lobe 101 and liver. The receptor of West Nile virus (Flaviviridae), ITGAV, showed expression 102 in all five organs (Fig. S2). 103 104 The receptor expressions of other viruses that have not been reported to infect civets 105 were also identified, including Adenoviridae, Arenaviridae, Flaviviridae, 106 Hepadnaviridae, Herpesviridae, etc. Multiple receptors for Adenoviridae (WWP2, 107 CXADR, ITGB5 and ITGAV), Herpesviridae (ITGB1, CR1, ITCH, ITGB8, ITGAV and 108 IDE), Picornaviridae (ITGAV, CXADR and ITGB8) and Reoviridae (ITGB1, HSPA8 109 and ITGAV) were abundantly expressed by cells of the five organs. Expressions of the 110 other receptors tended to be lower than the above-mentioned ones or concentrated in

111 certain organs (Fig. S1, Fig. S2).

112 The receptor expression profiles could help us better clarify or predict the 113 pathological outcomes caused by viral infection in civets. For example, the canine 114 parvoviruses were reported to cause diarrhea and deaths in civets and the viral DNA 115 can be detected in the brain, liver, heart, spleen and small intestine3, which is 116 consistent with the wide distribution of its receptor TFRC in the civet organs. 117 Lyssaviruses usually cause fatal encephalitic diseases in a wide range of mammals 118 with civet infections reported in Africa and Asia5,6. Our results showed an obvious bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.452903; this version posted July 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

119 enrichment of a related receptor, NCAM1, in multiple cell types of the civet frontal 120 lobe, which may contribute to the neurovirulence of these viruses. The receptor of 121 SARS-CoV-2, ACE2, was only expressed at moderate levels in the kidney and flow 122 cytometric experiment showed undetectable binding between the civet ACE2 ortholog 123 and the viral receptor-binding domain. However, two alternative receptors, NRP1 and 124 AXL, were both highly expressed in the lung and spleen, indicating potential 125 susceptibility of civets to SARS-CoV-2, although in vivo infection remains unclear.

126 Discussion 127 Taken together, we have built a comprehensive multi-organ cell atlas of masked palm 128 civet and described the distribution of various viral receptors in these tissues, 129 providing preliminary evidence of the potential tissue tropism of these viruses in 130 civets. The results could enhance our knowledge of the biological background of 131 civets and their susceptibility to various pathogens, which may facilitate the control 132 and prevention of enzootic and zoonotic viruses.

133

134 Acknowledgement 135 This work was supported by China National GeneBank (CNGB).

136

137 AUTHOR CONTRIBUTIONS

138 Y.H., H. L., D.C. and Z. O. conceived and designed the project. J.Z., F.A., J.X., were 139 responsible for sample collection and dissection. W.W. participated in single-nucleus 140 library construction and sequencing. H.W. performed single cell analysis. Y.Z., H.W., 141 Z. O., D.C., X.D., P.D., L.L., Q.Q., Y.W., W.D., Z.L., T.L., M.L., W.Z., participated in 142 data interpretation, data visualization and manuscript writing. Y.H., H. L. revised the 143 manuscript. 144 145 Conflict of Interest: The authors declare no competing interests.

146

147 Reference bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.452903; this version posted July 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

148 1. Guan, Y. et al. Isolation and characterization of viruses related to the SARS coronavirus from

149 animals in Southern China. Science (80-. ). 302, 276–278 (2003).

150 2. Techangamsuwan, S. et al. Pathologic and Molecular Virologic Characterization of a Canine

151 Distemper Outbreak in Farmed Civets. Vet. Pathol. 52, 724–731 (2015).

152 3. Mendenhall, I. H. et al. Evidence of canine parvovirus transmission to a civet cat (Paradoxurus

153 musangus) in Singapore. One Heal. 2, 122–125 (2016).

154 4. Roberton, S. I. et al. Avian influenza H5N1 in viverrids: Implications for wildlife health and

155 conservation. Proc. R. Soc. B Biol. Sci. 273, 1729–1732 (2006).

156 5. Sabeta, C. T. et al. Rabies in the African civet: An incidental host for lyssaviruses? Viruses 12,

157 (2020).

158 6. Matsumoto, T. et al. Novel sylvatic rabies virus variant in endangered golden palm civet, Sri

159 Lanka. Emerg. Infect. Dis. 17, 2346–2349 (2011).

160

161

162 Figure legends 163 Fig. 1. Single cell atlas of the frontal lobe, lung, liver, spleen and kidney of civet. a 164 Workflow of this study. b tSNE plot of the frontal lobe cells. Colors represent 165 different cell types. Feature plots indicate the expression of cell markers with red 166 color indicating high expression patterns. c tSNE plot of lung cells and feature plots 167 of cell markers. d tSNE plot of liver cells and feature plots of cell markers. e tSNE 168 plot of spleen cells and feature plots of cell markers. f tSNE plot of kidney cells and 169 feature plots of cell markers. 170 171 Supplementary materials 172 Supplementary Figures 173 Fig. S1. Distribution of viral receptors for Coronaviridae, Filoviridae, 174 Orthomyxoviridae, Paramyxoviridae, Parvoviridae and Rhabdoviridae in the 175 frontal lobe, lung, liver, spleen and kidney of civet. Bubble plot shows the viral 176 receptor expressions in different organs. Dot size represents the percentage of cells 177 expressing the corresponding receptor. Color saturation indicates the average scaled bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.452903; this version posted July 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

178 expression level. Viruses that are capable of infecting civets are in red text. 179 180 Fig. S2. Distribution of other viral receptors in civet organs. Bubble plot shows 181 the expressions of viral receptors in different organs. Viruses that are capable of 182 infecting civets are in red text. 183 184 Supplementary Tables 185 Table S1. Marker genes for cell types of frontal lobe, lung, liver, spleen and kidney 186 Table S2. Expression of viral receptor genes in civet 187 Table S3. DEG of cell types in frontal lobe, lung, liver, spleen and kidney 188 Table S4. GO term of cell type DEGs in frontal lobe, lung, liver, spleen and kidney Fig. 1 a bioRxiv preprint doi: https://doi.org/10.1101/2021.07.20.452903; this version posted July 21, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.Virus Receptor Profiling

b SLC1A2 SNAP25 GAD1 MBP VCAN

Excitatory Neurons Astrocytes Oligodendrocytes

Oligodendrocyte progenitor cells Smooth muscle cells Endothelial cells Inhibitory Neurons Microglia

tSNE 2 tSNE 1 c CYP4B1 CD36 CALCRL MRC1 CCDC39

Macrophages Epithelial cells pulmonary alveolar type II Fibroblasts Ciliated cells Endothelial cells

pulmonary alveolar type I

tSNE 2 tSNE 1 d RELN PTPRC KDR KRT8 ABCD3

Hepatic stellate cells

Immune cells Hepatocytes Liver sinusoidal endothelial cells

Cholangiocytes

tSNE 2 tSNE 1

e ICOS NRP1 BLNK LCP2 ANXA1

T cells Endothelial cells Naive T cells Neural cells Macrophages Regulatory T cells T follicular helper cells B cells Fibroblasts Dendritic cells

Natural killer cells

tSNE 2 tSNE 1 f PDZK1 SLC12A3 NPHS2 SLC12A1 ATP6V1G3

Proximal tubule cells Endothelial cells

Podocytes Collecting duct intercalated cells Collecting duct principal cells Loop of Henle cells Pericytes

Distal convoluted tubule cells

tSNE 2 tSNE 1