Since 2002, beta coronaviruses (CoV) have caused three zoonotic outbreaks, SARS-CoV in 2002–2003, MERS-CoV in 2012, and the newly emerged SARS-CoV-2 in late 2019. However, little is currently known about the biology of SARS-CoV-2. Here, using SARS-CoV-2 S protein pseudovirus system, we confirm that human angiotensin converting enzyme 2 (hACE2) is the receptor for SARS-CoV-2, find that SARS-CoV-2 enters 293/hACE2 cells mainly through endocytosis, that PIKfyve, TPC2, and cathepsin L are critical for entry, and that SARS-CoV-2 S protein is less stable than SARS-CoV S. Polyclonal anti-SARS S1 antibodies T62 inhibit entry of SARS-CoV S but not SARS-CoV-2 S pseudovirions. Further studies using recovered SARS and COVID-19 patients’ sera show limited cross-neutralization, suggesting that recovery from one infection might not protect against the other. Our results present potential targets for development of drugs and vaccines for SARS-CoV-2.

Studies have demonstrated that ~60%–80% of emerging infectious diseases (EIDs) in humans originated from wild life. Bats are natural reservoirs of a large variety of viruses, including many important zoonotic viruses that cause severe diseases in humans and domestic animals. However, the understanding of the viral population and the ecological diversity residing in bat populations is unclear, which complicates the determination of the origins of certain EIDs. Here, using bats as a typical wildlife reservoir model, virome analysis was conducted based on pharyngeal and anal swab samples of 4440 bat individuals of 40 major bat species throughout China. The purpose of this study was to survey the ecological and biological diversities of viruses residing in these bat species, to investigate the presence of potential bat-borne zoonotic viruses and to evaluate the impacts of these viruses on public health. The data obtained in this study revealed an overview of the viral community present in these bat samples. Many novel bat viruses were reported for the first time and some bat viruses closely related to known human or animal pathogens were identified. This genetic evidence provides new clues in the search for the origin or evolution pattern of certain viruses, such as coronaviruses and noroviruses. These data offer meaningful ecological information for predicting and tracing wildlife-originated EIDs.

Severe acute respiratory syndrome (SARS)–coronavirus (CoV) produces a devastating primary viral pneumonia with diffuse alveolar damage and a marked increase in circulating cytokines. One of the major cell types to be infected is the alveolar type II cell. However, the innate immune response of primary human alveolar epithelial cells infected with SARS-CoV has not been defined. Our objectives included developing a culture system permissive for SARS-CoV infection in primary human type II cells and defining their innate immune response. Culturing primary human alveolar type II cells at an air–liquid interface (A/L) improved their differentiation and greatly increased their susceptibility to infection, allowing us to define their primary interferon and chemokine responses. Viral antigens were detected in the cytoplasm of infected type II cells, electron micrographs demonstrated secretory vesicles filled with virions, virus RNA concentrations increased with time, and infectious virions were released by exocytosis from the apical surface of polarized type II cells. A marked increase was evident in the mRNA concentrations of interferon–β and interferon–λ (IL-29) and in a large number of proinflammatory cytokines and chemokines. A surprising finding involved the variability of expression of angiotensin-converting enzyme–2, the SARS-CoV receptor, in type II cells from different donors. In conclusion, the cultivation of alveolar type II cells at an air–liquid interface provides primary cultures in which to study the pulmonary innate immune responses to infection with SARS-CoV, and to explore possible therapeutic approaches to modulating these innate immune responses.

Little is known about the biology of the emerging human group c betacoronavirus, Middle East Respiratory Syndrome coronavirus (MERS-CoV). Because coronavirus spike glycoproteins (S) mediate virus entry, affect viral host range, and elicit neutralizing antibodies, analyzing the functions of MERS-CoV S protein is a high research priority. MERS-CoV S on lentivirus pseudovirions mediated entry into a variety of cell types including embryo cells from New World Eptesicus fuscus bats. Surprisingly, a polyclonal antibody to the S protein of MHV, a group a murine betacoronavirus, cross-reacted in immunoblots with the S2 domain of group c MERS-CoV spike protein. MERS pseudovirions released from 293T cells contained only uncleaved S, and pseudovirus entry was blocked by lysosomotropic reagents NH₄Cl and bafilomycin and inhibitors of cathepsin L. However, when MERS pseudovirions with uncleaved S protein were adsorbed at 4°C to Vero E6 cells, brief trypsin treatment at neutral pH triggered virus entry at the plasma membrane and syncytia formation. When 293T cells producing MERS pseudotypes co-expressed serine proteases TMPRSS-2 or -4, large syncytia formed at neutral pH, and the pseudovirions produced were non-infectious and deficient in S protein. These experiments show that if S protein on MERS pseudovirions is uncleaved, then viruses enter by endocytosis in a cathepsin L-dependent manner, but if MERS-CoV S is cleaved, either during virus maturation by serine proteases or on pseudovirions by trypsin in extracellular fluids, then viruses enter at the plasma membrane at neutral pH and cause massive syncytia formation even in cells that express little or no MERS-CoV receptor. Thus, whether MERS-CoV enters cells within endosomes or at the plasma membrane depends upon the host cell type and tissue, and is determined by the location of host proteases that cleave the viral spike glycoprotein and activate membrane fusion.

Coronaviruses have evolved diverse mechanisms to recognize different receptors for their cross-species transmission and host-range expansion. Mouse hepatitis coronavirus (MHV) uses the N-terminal domain (NTD) of its spike protein as its receptor-binding domain. Here we present the crystal structure of MHV NTD complexed with its receptor murine carcinoembryonic antigen-related cell adhesion molecule 1a (mCEACAM1a). Unexpectedly, MHV NTD contains a core structure that has the same β-sandwich fold as human galectins (S-lectins) and additional structural motifs that bind to the N-terminal Ig-like domain of mCEACAM1a. Despite its galectin fold, MHV NTD does not bind sugars, but instead binds mCEACAM1a through exclusive protein–protein interactions. Critical contacts at the interface have been confirmed by mutagenesis, providing a structural basis for viral and host specificities of coronavirus/CEACAM1 interactions. Sugar-binding assays reveal that galectin-like NTDs of some coronaviruses such as human coronavirus OC43 and bovine coronavirus bind sugars. Structural analysis and mutagenesis localize the sugar-binding site in coronavirus NTDs to be above the β-sandwich core. We propose that coronavirus NTDs originated from a host galectin and retained sugar-binding functions in some contemporary coronaviruses, but evolved new structural features in MHV for mCEACAM1a binding.