Microglia do not restrict SARS-CoV-2 replication following infection of the central nervous system of K18-hACE2 transgenic mice

bioRxiv : the preprint server for biology

2021 Nov 17

Olivarria, GM;Cheng, Y;Furman, S;Pachow, C;Hohsfield, LA;Smith-Geater, C;Miramontes, R;Wu, J;Burns, MS;Tsourmas, KI;Stocksdale, J;Manlapaz, C;Yong, WH;Teijaro, J;Edwards, R;Green, KN;Thompson, LM;Lane, TE;
PMID: 34816260 | DOI: 10.1101/2021.11.15.468761

Unlike SARS-CoV-1 and MERS-CoV, infection with SARS-CoV-2, the viral pathogen responsible for COVID-19, is often associated with neurologic symptoms that range from mild to severe, yet increasing evidence argues the virus does not exhibit extensive neuroinvasive properties. We demonstrate SARS-CoV-2 can infect and replicate in human iPSC-derived neurons and that infection shows limited anti-viral and inflammatory responses but increased activation of EIF2 signaling following infection as determined by RNA sequencing. Intranasal infection of K18 human ACE2 transgenic mice (K18-hACE2) with SARS-CoV-2 resulted in lung pathology associated with viral replication and immune cell infiltration. In addition, ∼50% of infected mice exhibited CNS infection characterized by wide-spread viral replication in neurons accompanied by increased expression of chemokine ( Cxcl9, Cxcl10, Ccl2, Ccl5 and Ccl19 ) and cytokine ( Ifn-λ and Tnf-α ) transcripts associated with microgliosis and a neuroinflammatory response consisting primarily of monocytes/macrophages. Microglia depletion via administration of colony-stimulating factor 1 receptor inhibitor, PLX5622, in SARS-CoV-2 infected mice did not affect survival or viral replication but did result in dampened expression of proinflammatory cytokine/chemokine transcripts and a reduction in monocyte/macrophage infiltration. These results argue that microglia are dispensable in terms of controlling SARS-CoV-2 replication in in the K18-hACE2 model but do contribute to an inflammatory response through expression of pro-inflammatory genes. Collectively, these findings contribute to previous work demonstrating the ability of SARS-CoV-2 to infect neurons as well as emphasizing the potential use of the K18-hACE2 model to study immunological and neuropathological aspects related to SARS-CoV-2-induced neurologic disease.Understanding the immunological mechanisms contributing to both host defense and disease following viral infection of the CNS is of critical importance given the increasing number of viruses that are capable of infecting and replicating within the nervous system. With this in mind, the present study was undertaken to evaluate the role of microglia in aiding in host defense following experimental infection of the central nervous system (CNS) of K18-hACE2 with SARS-CoV-2, the causative agent of COVID-19. Neurologic symptoms that range in severity are common in COVID-19 patients and understanding immune responses that contribute to restricting neurologic disease can provide important insight into better understanding consequences associated with SARS-CoV-2 infection of the CNS.

Single-Cell RNA-seq Reveals Angiotensin-Converting Enzyme 2 and Transmembrane Serine Protease 2 Expression in TROP2+ Liver Progenitor Cells: Implications in Coronavirus Disease 2019-Associated Liver Dysfunction

Frontiers in medicine

2021 Apr 22

Seow, JJW;Pai, R;Mishra, A;Shepherdson, E;Lim, TKH;Goh, BKP;Chan, JKY;Chow, PKH;Ginhoux, F;DasGupta, R;Sharma, A;
PMID: 33968947 | DOI: 10.3389/fmed.2021.603374

The recent coronavirus disease 2019 (COVID-19) pandemic is caused by severe acute respiratory syndrome coronavirus 2. COVID-19 was first reported in China (December 2019) and is now prevalent across the globe. Entry of severe acute respiratory syndrome coronavirus 2 into mammalian cells requires the binding of viral Spike (S) proteins to the angiotensin-converting enzyme 2 receptor. Once entered, the S protein is primed by a specialized serine protease, transmembrane serine protease 2 in the host cell. Importantly, besides the respiratory symptoms that are consistent with other common respiratory virus infections when patients become viremic, a significant number of COVID-19 patients also develop liver comorbidities. We explored whether a specific target cell-type in the mammalian liver could be implicated in disease pathophysiology other than the general deleterious response to cytokine storms. Here, we used single-cell RNA-seq to survey the human liver and identified potentially implicated liver cell-type for viral ingress. We analyzed ~300,000 single cells across five different (i.e., human fetal, healthy, cirrhotic, tumor, and adjacent normal) liver tissue types. This study reports on the co-expression of angiotensin-converting enzyme 2 and transmembrane serine protease 2 in a TROP2+ liver progenitor population. Importantly, we detected enrichment of this cell population in the cirrhotic liver when compared with tumor tissue. These results indicated that in COVID-19-associated liver dysfunction and cell death, a viral infection of TROP2+ progenitors in the liver might significantly impair liver regeneration in patients with liver cirrhosis.

EXPRESS: Mechanisms of SARS-CoV-2 Induced Lung Vascular Disease: Potential Role of Complement

Pulmonary Circulation

2021 Apr 23

Stenmark, K;Frid, M;gerasimovskaya, e;zhang, h;McCarthy, M;Thurman, J;Morrison, T;
| DOI: 10.1177/20458940211015799

: The outbreak of COVID-19 disease, caused by SARS-CoV-2 beta-coronovirus, urges a focused search for the underlying mechanisms and treatment options. The lung is the major target organ of COVID-19, wherein the primary cause of mortality is hypoxic respiratory failure, resulting from acute respiratory disease syndrome (ARDS), with severe hypoxemia, often requiring assisted ventilation. While similar in some ways to ARDS secondary to other causes, lungs of some patients dying with COVID-19 exhibit distinct features of vascular involvement, including severe endothelial injury and cell death via apoptosis and/or pyroptosis, widespread capillary inflammation and thrombosis. Furthermore, the pulmonary pathology of COVID-19 is characterized by focal inflammatory cell infiltration, impeding alveolar gas exchange resulting in areas of local tissue hypoxia, consistent with potential amplification of COVID-19 pathogenicity by hypoxia. Vascular endothelial cells play essential roles in both innate and adaptive immune responses, and are considered to be âconditional innate immune cellsâ centrally participating in various inflammatory, immune pathologies. Activated endothelial cells produce cytokines/chemokines, dynamically recruit and activate inflammatory cells and platelets, and centrally participate in pro-thrombotic processes (thrombotic microangiopathies). Initial reports presented pathological findings of localized direct infection of vascular endothelial cells with SARS-CoV-2, yet emerging evidence does not support direct infection of endothelial or other vascular wall cell and thus widespread endothelial cell dysfunction and inflammation may be better explained as secondary responses to epithelial cell infection and inflammation. Endothelial cells are also actively engaged in a cross-talk with the complement system, the essential arm of innate immunity. Recent reports present evidence for complement deposition in SARS-CoV-2-damaged lung microcirculation, further strengthening the idea that, in severe cases of COVID-19, complement activation is an essential player, generating destructive hemorrhagic, capillariitis-like tissue damage, clotting, and hyper-inflammation. Thus, complement-targeted therapies are actively in development. This review is intended to explore in detail these ideas.

Endothelial cells are not productively infected by SARS-CoV-2

Clinical & translational immunology

2021 Oct 24

Schimmel, L;Chew, KY;Stocks, CJ;Yordanov, TE;Essebier, P;Kulasinghe, A;Monkman, J;Dos Santos Miggiolaro, AFR;Cooper, C;de Noronha, L;Schroder, K;Lagendijk, AK;Labzin, LI;Short, KR;Gordon, EJ;
PMID: 34721846 | DOI: 10.1002/cti2.1350

Thrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious.Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier.We show that primary human endothelial cells express very low levels of the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2, which blocks their capacity for productive viral infection, and limits their capacity to produce infectious virus. Accordingly, endothelial cells can only be infected when they overexpress ACE2, or are exposed to very high concentrations of SARS-CoV-2. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. Endothelial cells do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines.Taken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection may only occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells may still play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

Binding of SARS-CoV-2 to the avb6 Integrins May Promote Severe COVID in Patients with IPF

TP105. TP105 BASIC MECHANISMS OF LUNG INFECTIONS: FROM SARS-COV-2 TO INFLUENZA

2021 May 01

Joseph, C;Peacock, T;Calver, J;John, A;Organ, L;Fainberg, H;Porte, J;Mukhopadhyay, S;Barton, L;Stroberg, E;Duval, E;Copin, M;Poissy, J;Steinestel, K;Tatler, A;Barclay, W;Jenkins, G;
| DOI: 10.1164/ajrccm-conference.2021.203.1_MeetingAbstracts.A4170

RATIONALE: Patients with idiopathic pulmonary fibrosis (IPF) have worse outcomes following COVID-19. SARSCoV-2 (2019-nCoV) spike protein (S1) harbors an RGD motif in its receptor-binding domain (RBD). Although SARS-CoV-2 is to exploit human Angiotensin Converting Enzyme-2 (ACE2) receptors for cell entry. Single Cell RNA-seq showed that normal lung expresses low levels of ACE2 with very low expression (1.5%) in Alveolar type 2 epithelial cells. It is possible that SARS-CoV-2 needs a cellular co-receptor, which could include integrins, to promote alveolar cell internalization and pneumonitis.METHODS: Solid-phase binding assays were used to investigate S1 binding to ACE2 or αv containing integrins. Pseudovirus entry assays were used to measure the internalization of SARS-CoV-2 into Human embryonic kidney 293T cells expressing different combinations of potential receptors. RNAscope was used to visualize the co-localization of SARS-CoV-2, ACE2, and integrin mRNAs. Immunohistochemistry was used to evaluate the expression of αvβ6 integrins and ACE2 in lung tissue.RESULTS: Binding assays demonstrated that the RGD containing αvβ3 and αvβ6 integrins bound robustly to the SARS-CoV-2 S1 subunit of Spike protein and overexpression of the αvβ6 integrin modestly augments ACE2 mediated SARS-CoV-2 pseudoviral entry into epithelial cells. In COVID-19 damaged lung ACE2 levels are low but the αvβ6 integrin levels are increased in alveolar epithelium whereas both ACE2 and αvβ6 integrin are increased in lung sections from idiopathic pulmonary fibrosis compared with normal lung samples. CONCLUSION: The SARS-CoV-2 S1 subunit can bind αvβ6 integrins augmenting ACE2-dependent internalization of pseudovirus. In IPF patients, ACE2 levels and αvβ6 integrin levels are increased. Increased binding of the SARS-CoV-2 to ACE2 and the αvβ6 integrin within fibrotic lung may explain the increased risk of severe COVID in patients with IPF.

Determinants of SARS-CoV-2 entry and replication in airway mucosal tissue and susceptibility in smokers

Cell reports. Medicine

2021 Oct 19

Nakayama, T;Lee, IT;Jiang, S;Matter, MS;Yan, CH;Overdevest, JB;Wu, CT;Goltsev, Y;Shih, LC;Liao, CK;Zhu, B;Bai, Y;Lidsky, P;Xiao, Y;Zarabanda, D;Yang, A;Easwaran, M;Schürch, CM;Chu, P;Chen, H;Stalder, AK;McIlwain, DR;Borchard, NA;Gall, PA;Dholakia, SS;Le, W;Xu, L;Tai, CJ;Yeh, TH;Erickson-Direnzo, E;Duran, JM;Mertz, KD;Hwang, PH;Haslbauer, JD;Jackson, PK;Menter, T;Andino, R;Canoll, PD;DeConde, AS;Patel, ZM;Tzankov, A;Nolan, GP;Nayak, JV;
PMID: 34604819 | DOI: 10.1016/j.xcrm.2021.100421

Understanding viral tropism is an essential step toward reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, decreasing mortality from coronavirus disease 2019 (COVID-19) and limiting opportunities for mutant strains to arise. Currently, little is known about the extent to which distinct tissue sites in the human head and neck region and proximal respiratory tract selectively permit SARS-CoV-2 infection and replication. In this translational study, we discover key variabilities in expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential SARS-CoV-2 entry factors, among the mucosal tissues of the human proximal airways. We show that SARS-CoV-2 infection is present in all examined head and neck tissues, with a notable tropism for the nasal cavity and tracheal mucosa. Finally, we uncover an association between smoking and higher SARS-CoV-2 viral infection in the human proximal airway, which may explain the increased susceptibility of smokers to developing severe COVID-19. This is at least partially explained by differences in interferon (IFN)-β1 levels between smokers and non-smokers.

Comparison of the pathogenesis of SARS-CoV-2 infection in K18-hACE2 mice and Syrian golden hamster models

Disease models & mechanisms

2022 Oct 12

Jeong, H;Lee, YW;Park, IH;Noh, H;Kim, SH;Kim, J;Jeon, D;Jang, HJ;Oh, J;On, D;Uhm, C;Cho, K;Oh, H;Yoon, S;Seo, JS;Kim, JJ;Seok, SH;Lee, YJ;Hong, SM;An, SH;Kim, SY;Kim, YB;Hwang, JY;Lee, HJ;Kim, HB;Jeong, DG;Song, D;Song, M;Park, MS;Choi, KS;Park, JW;Seo, JY;Yun, JW;Shin, JS;Lee, HY;Nam, KT;Seong, JK;
PMID: 36222118 | DOI: 10.1242/dmm.049632

SARS-CoV-2, the etiological agent of COVID-19, causes life-threatening disease. This novel coronavirus enters host cells via the respiratory tract, promoting the formation of severe pulmonary lesions and systemic disease. Few animal models can simulate the clinical signs and pathology of COVID-19 patients. Diverse preclinical studies using K18-hACE2 mice and Syrian golden hamsters, which are highly permissive to SARS-CoV-2 in the respiratory tract, are emerging; however, the systemic pathogenesis and cellular tropism of these models remain obscure. We intranasally infected K18-hACE2 mice and Syrian golden hamsters with SARS-CoV-2, and compared the clinical features, pathogenesis, cellular tropism, and infiltrated immune-cell subsets. In K18-hACE2 mice, SARS-CoV-2 persistently replicated in alveolar cells and caused pulmonary and extra-pulmonary disease, resulting in fatal outcomes. Conversely, in Syrian golden hamsters, transient SARS-CoV-2 infection in bronchial cells caused reversible pulmonary disease, without mortality. Our findings provide comprehensive insights into the pathogenic spectrum of COVID-19 using pre-clinical models.

Rapid endotheliitis and vascular damage characterize SARS-CoV-2 infection in a human lung-on-chip model

EMBO reports

2021 Apr 28

Thacker, VV;Sharma, K;Dhar, N;Mancini, GF;Sordet-Dessimoz, J;McKinney, JD;
PMID: 33908688 | DOI: 10.15252/embr.202152744

Severe cases of SARS-CoV-2 infection are characterized by hypercoagulopathies and systemic endotheliitis of the lung microvasculature. The dynamics of vascular damage, and whether it is a direct consequence of endothelial infection or an indirect consequence of an immune cell-mediated cytokine storm remain unknown. Using a vascularized lung-on-chip model, we find that infection of alveolar epithelial cells leads to limited apical release of virions, consistent with reports of monoculture infection. However, viral RNA and proteins are rapidly detected in underlying endothelial cells, which are themselves refractory to apical infection in monocultures. Although endothelial infection is unproductive, it leads to the formation of cell clusters with low CD31 expression, a progressive loss of barrier integrity and a pro-coagulatory microenvironment. Viral RNA persists in individual cells generating an inflammatory response, which is transient in epithelial cells but persistent in endothelial cells and typified by IL-6 secretion even in the absence of immune cells. Inhibition of IL-6 signalling with tocilizumab reduces but does not prevent loss of barrier integrity. SARS-CoV-2-mediated endothelial cell damage thus occurs independently of cytokine storm.

Mouse-Adapted SARS-CoV-2 MA10 Strain Displays Differential Pulmonary Tropism and Accelerated Viral Replication, Neurodissemination, and Pulmonary Host Responses in K18-hACE2 Mice

mSphere

2023 Feb 21

Thieulent, CJ;Dittmar, W;Balasuriya, UBR;Crossland, NA;Wen, X;Richt, JA;Carossino, M;
PMID: 36728430 | DOI: 10.1128/msphere.00558-22

Several models were developed to study the pathogenicity of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) as well as the in vivo efficacy of vaccines and therapeutics. Since wild-type mice are naturally resistant to infection by ancestral SARS-CoV-2 strains, several transgenic mouse models expressing human angiotensin-converting enzyme 2 (hACE2) were developed. An alternative approach has been to develop mouse-adapted SARS-CoV-2 strains. Here, we compared the clinical progression, viral replication kinetics and dissemination, pulmonary tropism, and host innate immune response dynamics between the mouse-adapted MA10 strain and its parental strain (USA-WA1/2020) following intranasal inoculation of K18-hACE2 mice, a widely used model. Compared to its parental counterpart, the MA10 strain induced earlier clinical decline with significantly higher viral replication and earlier neurodissemination. Importantly, the MA10 strain also showed a wider tropism, with infection of bronchiolar epithelia. While both SARS-CoV-2 strains induced comparable pulmonary cytokine/chemokine responses, many proinflammatory and monocyte-recruitment chemokines, such as interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-α), IP-10/CXCL10, and MCP-1/CCL2, showed an earlier peak in MA10-infected mice. Furthermore, both strains induced a similar downregulation of murine Ace2, with only a transient downregulation of Tmprss2 and no alterations in hACE2 expression. Overall, these data demonstrate that in K18-hACE2 mice, the MA10 strain has a pulmonary tropism that more closely resembles SARS-CoV-2 tropism in humans (airways and pneumocytes) than its parental strain. Its rapid replication and neurodissemination and early host pulmonary responses can have a significant impact on the clinical outcomes of infection and are, therefore, critical features to consider for study designs using these strains and mouse model. IMPORTANCE The COVID-19 pandemic, caused by SARS-CoV-2, is still significantly impacting health care systems around the globe. Refined animal models are needed to study SARS-CoV-2 pathogenicity as well as efficacy of vaccines and therapeutics. In line with this, thorough evaluation of animal models and virus strains/variants are paramount for standardization and meaningful comparisons. Here, we demonstrated differences in replication dynamics between the Wuhan-like USA-WA1/2020 strain and the derivative mouse-adapted MA10 strain in K18-hACE2 mice. The MA10 strain showed accelerated viral replication and neurodissemination, differential pulmonary tropism, and earlier pulmonary innate immune responses. The observed differences allow us to better refine experimental designs when considering the use of the MA10 strain in the widely utilized K18-hACE2 murine model.

SARS-CoV-2 infects human pancreatic β cells and elicits β cell impairment

Cell Metabolism

2021 May 01

Wu, C;Lidsky, P;Xiao, Y;Lee, I;Cheng, R;Nakayama, T;Jiang, S;Demeter, J;Bevacqua, R;Chang, C;Whitener, R;Stalder, A;Zhu, B;Chen, H;Goltsev, Y;Tzankov, A;Nayak, J;Nolan, G;Matter, M;Andino, R;Jackson, P;
| DOI: 10.1016/j.cmet.2021.05.013

Emerging evidence points toward an intricate relationship between the pandemic of coronavirus disease 2019 (COVID-19) and diabetes. While preexisting diabetes is associated with severe COVID-19, it is unclear whether COVID-19 severity is a cause or consequence of diabetes. To mechanistically link COVID-19 to diabetes, we tested whether insulin-producing pancreatic β cells can be infected by SARS-CoV-2 and cause β cell depletion. We found that the SARS-CoV-2 receptor, ACE2, and related entry factors (TMPRSS2, NRP1, and TRFC) are expressed in β cells, with selectively high expression of NRP1. We discovered that SARS-CoV-2 infects human pancreatic β cells in patients who succumbed to COVID-19 and selectively infects human islet β cells in vitro. We demonstrated that SARS-CoV-2 infection attenuates pancreatic insulin levels and secretion and induces β cell apoptosis, each rescued by NRP1 inhibition. Phosphoproteomic pathway analysis of infected islets indicates apoptotic β cell signaling, similar to that observed in type 1 diabetes (T1D). In summary, our study shows SARS-CoV-2 can directly induce β cell killing.

SARS-CoV-2 infects the human kidney and drives fibrosis in kidney organoids

Cell Stem Cell

2021 Dec 01

Jansen, J;Reimer, K;Nagai, J;Varghese, F;Overheul, G;de Beer, M;Roverts, R;Daviran, D;Fermin, L;Willemsen, B;Beukenboom, M;Djudjaj, S;von Stillfried, S;van Eijk, L;Mastik, M;Bulthuis, M;Dunnen, W;van Goor, H;Hillebrands, J;Triana, S;Alexandrov, T;Timm, M;Tideman van den Berge, B;van den Broek, M;Nlandu, Q;Heijnert, J;Bindels, E;Hoogenboezem, R;Mooren, F;Kuppe, C;Miesen, P;Grünberg, K;Ijzermans, T;Steenbergen, E;Czogalla, J;Schreuder, M;Sommerdijk, N;Akiva, A;Boor, P;Puelles, V;Floege, J;Huber, T;van Rij, R;Costa, I;Schneider, R;Smeets, B;Kramann, R;
| DOI: 10.1016/j.stem.2021.12.010

Kidney failure is frequently observed during and after COVID-19, but it remains elusive whether this is a direct effect of the virus. Here, we report that SARS-CoV-2 directly infects kidney cells and is associated with increased tubule-interstitial kidney fibrosis in patient autopsy samples. To study direct effects of the virus on the kidney independent of systemic effects of COVID-19, we infected human induced pluripotent stem cell-derived kidney organoids with SARS-CoV-2. Single cell RNA-sequencing indicated injury and dedifferentiation of infected cells with activation of pro-fibrotic signaling pathways. Importantly, SARS-CoV-2 infection also led to increased collagen 1 protein expression in organoids. A SARS-CoV-2 protease inhibitor was able to ameliorate the infection of kidney cells by SARS-CoV-2. Our results suggest that SARS-CoV-2 can directly infect kidney cells and induce cell injury with subsequent fibrosis. These data could explain both acute kidney injury in COVID-19 patients and the development of chronic kidney disease in Long-COVID.

SARS-CoV-2 infection of the oral cavity and saliva

Nature medicine

2021 Mar 25

Huang, N;Pérez, P;Kato, T;Mikami, Y;Okuda, K;Gilmore, RC;Conde, CD;Gasmi, B;Stein, S;Beach, M;Pelayo, E;Maldonado, JO;Lafont, BA;Jang, SI;Nasir, N;Padilla, RJ;Murrah, VA;Maile, R;Lovell, W;Wallet, SM;Bowman, NM;Meinig, SL;Wolfgang, MC;Choudhury, SN;Novotny, M;Aevermann, BD;Scheuermann, RH;Cannon, G;Anderson, CW;Lee, RE;Marchesan, JT;Bush, M;Freire, M;Kimple, AJ;Herr, DL;Rabin, J;Grazioli, A;Das, S;French, BN;Pranzatelli, T;Chiorini, JA;Kleiner, DE;Pittaluga, S;Hewitt, SM;Burbelo, PD;Chertow, D;NIH COVID-19 Autopsy Consortium, ;HCA Oral and Craniofacial Biological Network, ;Frank, K;Lee, J;Boucher, RC;Teichmann, SA;Warner, BM;Byrd, KM;
PMID: 33767405 | DOI: 10.1038/s41591-021-01296-8

Despite signs of infection-including taste loss, dry mouth and mucosal lesions such as ulcerations, enanthema and macules-the involvement of the oral cavity in coronavirus disease 2019 (COVID-19) is poorly understood. To address this, we generated and analyzed two single-cell RNA sequencing datasets of the human minor salivary glands and gingiva (9 samples, 13,824 cells), identifying 50 cell clusters. Using integrated cell normalization and annotation, we classified 34 unique cell subpopulations between glands and gingiva. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) viral entry factors such as ACE2 and TMPRSS members were broadly enriched in epithelial cells of the glands and oral mucosae. Using orthogonal RNA and protein expression assessments, we confirmed SARS-CoV-2 infection in the glands and mucosae. Saliva from SARS-CoV-2-infected individuals harbored epithelial cells exhibiting ACE2 and TMPRSS expression and sustained SARS-CoV-2 infection. Acellular and cellular salivary fractions from asymptomatic individuals were found to transmit SARS-CoV-2 ex vivo. Matched nasopharyngeal and saliva samples displayed distinct viral shedding dynamics, and salivary viral burden correlated with COVID-19 symptoms, including taste loss. Upon recovery, this asymptomatic cohort exhibited sustained salivary IgG antibodies against SARS-CoV-2. Collectively, these data show that the oral cavity is an important site for SARS-CoV-2 infection and implicate saliva as a potential route of SARS-CoV-2 transmission.

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	Description
sense Example: Hs-LAG3-sense	Standard probes for RNA detection are in antisense. Sense probe is reverse complent to the corresponding antisense probe.
Intron# Example: Mm-Htt-intron2	Probe targets the indicated intron in the target gene, commonly used for pre-mRNA detection
Pool/Pan Example: Hs-CD3-pool (Hs-CD3D, Hs-CD3E, Hs-CD3G)	A mixture of multiple probe sets targeting multiple genes or transcripts
No-XSp Example: Hs-PDGFB-No-XMm	Does not cross detect with the species (Sp)
XSp Example: Rn-Pde9a-XMm	designed to cross detect with the species (Sp)
O# Example: Mm-Islr-O1	Alternative design targeting different regions of the same transcript or isoforms
CDS Example: Hs-SLC31A-CDS	Probe targets the protein-coding sequence only
EnEm	Probe targets exons n and m
En-Em	Probe targets region from exon n to exon m
Retired Nomenclature
tvn Example: Hs-LEPR-tv1	Designed to target transcript variant n
ORF Example: Hs-ACVRL1-ORF	Probe targets open reading frame
UTR Example: Hs-HTT-UTR-C3	Probe targets the untranslated region (non-protein-coding region) only
5UTR Example: Hs-GNRHR-5UTR	Probe targets the 5' untranslated region only
3UTR Example: Rn-Npy1r-3UTR	Probe targets the 3' untranslated region only
Pan Example: Pool	A mixture of multiple probe sets targeting multiple genes or transcripts

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