While coronaviruses were first identified nearly 60 years ago, they received notoriety in 2003 when one of their members was identified as the etiological agent of the Severe Acute Respiratory Syndrome (SARS-CoV) 1. Previously these viruses were known to be important agents of respiratory and enteric infections of domestic and companion animals and to cause approximately 15% of all cases of the common cold. This Review focuses on recent advances in our understanding of the mechanisms of coronavirus replication, interactions with the host immune response and disease pathogenesis and highlights the recent identification of numerous novel coronaviruses and the propensity of this virus family to cross species barriers.

Infection of humans with the severe acute respiratory syndrome coronavirus (SARS-CoV) results in substantial morbidity and mortality, with death resulting primarily from respiratory failure. While the lungs are the major site of infection, the brain is also infected in some patients. Brain infection may result in long-term neurological sequelae, but little is known about the pathogenesis of SARS-CoV in this organ. We previously showed that the brain was a major target organ for infection in mice that are transgenic for the SARS-CoV receptor (human angiotensin-converting enzyme 2). Herein, we use these mice to show that virus enters the brain primarily via the olfactory bulb, and infection results in rapid, transneuronal spread to connected areas of the brain. This extensive neuronal infection is the main cause of death because intracranial inoculation with low doses of virus results in a uniformly lethal disease even though little infection is detected in the lungs. Death of the animal likely results from dysfunction and/or death of infected neurons, especially those located in cardiorespiratory centers in the medulla. Remarkably, the virus induces minimal cellular infiltration in the brain. Our results show that neurons are a highly susceptible target for SARS-CoV and that only the absence of the host cell receptor prevents severe murine brain disease.

Severe acute respiratory syndrome (SARS) was first identified in Guangdong Province in China (28). Over the ensuing 9 months, more than 8,000 cases were identified throughout the world, with a ϳ10% case fatality rate. A novel coronavirus, SARS coronavirus (SARS-CoV), was identified as the causative agent (6,17,29,32). Initial investigations indicated that the virus spread to humans from infected exotic animals such as Himalayan palm civets (Paguma larvata) and Chinese ferret badgers (Melogale moschata) (12); more recent work has suggested that the natural reservoirs for the virus are wild bat populations in China (19,24). Although SARS has not recurred in human populations to a significant extent since 2003, the potential severity of such a recurrence has spurred interest in developing an animal model for the human disease.SARS-CoV infects and replicates in mice, ferrets, hamsters, and several species of nonhuman primates (cynomolgus and rhesus macaques, African green monkeys, and common marmosets) (reviewed in reference 37). However, none of these animals develop a clinical disease that is reproducible and equivalent in severity to that observed in SARS patients. A mouse model would be useful for answering many questions about SARS pathogenesis and for testing vaccine efficacy, in part because reagents for the study of the immune response are widely available. However, other than aged or immunocompromised (STAT1 Ϫ/Ϫ ) mice (37), these animals do not develop significant clinical disease, and lethality has not been demonstrated in any murine model of SARS. With the goal of developing a more robust murine model, we generated transgenic (Tg) mice in which expression of hACE2 (human angiotensin-converting enzyme 2, the primary host cell receptor for SARS-CoV [23]) was targeted to epithelial cells. While human ACE2 and murine ACE2 (mACE2) molecules are very homologous, mACE2 does not support SARS-CoV binding as efficiently as hACE2 (22). Here we show that the transgenic expression of hACE2 in epithelia converts a mild SARS-CoV infection into a rapidly fatal disease. MATERIALS AND METHODSMice. All animal studies were approved by the University of Iowa and the Veterans Administration Institutional Animal Care and Use committees. Mice transgenic for expression of hACE2 (K18-hACE2 mice) were generated as follows (see Fig. 1A). The hACE2 coding sequence was PCR amplified from IMAGE consortium clone ID 5243048 (ATCC, Manassas, VA) and cloned into the pCR2.1-TOPO vector (Invitrogen, Carlsbad, CA). The lacZ coding sequence in the previously described pK18mTElacZ-K18i6x7pA construct (16) (a kind gift from Jim Hu, Hospital for Sick Children, Toronto, Canada) was then replaced by the hACE2 coding sequence to create pK18-hACE2. 5Ј of the hACE2 coding sequence, this plasmid contains 2.5 kb of upstream genomic sequence, the promoter, and the first intron (with a mutation in the 3Ј splice acceptor site to reduce exon skipping) of the human cytokeratin 18 (K18) gene as well as a translational enhancer sequence from alfalfa mosaic vi...

Studies of patients with severe acute respiratory syndrome (SARS) demonstrate that the respiratory tract is a major site of SARS-coronavirus (CoV) infection and disease morbidity. We studied host-pathogen interactions using native lung tissue and a model of well-differentiated cultures of primary human airway epithelia. Angiotensin converting enzyme 2 (ACE2), the receptor for both the SARS-CoV and the related human respiratory coronavirus NL63, was expressed in human airway epithelia as well as lung parenchyma. As assessed by immunofluorescence staining and membrane biotinylation, ACE2 protein was more abundantly expressed on the apical than the basolateral surface of polarized airway epithelia. Interestingly, ACE2 expression positively correlated with the differentiation state of epithelia. Undifferentiated cells expressing little ACE2 were poorly infected with SARS-CoV, while well-differentiated cells expressing more ACE2 were readily infected. Expression of ACE2 in poorly differentiated epithelia facilitated SARS spike (S) proteinpseudotyped virus entry. Consistent with the expression pattern of ACE2, the entry of SARS-CoV or a lentivirus pseudotyped with SARS-CoV S protein in differentiated epithelia was more efficient when applied to the apical surface. Furthermore, SARS-CoV replicated in polarized epithelia and preferentially exited via the apical surface. The results indicate that infection of human airway epithelia by SARS coronavirus correlates with the state of cell differentiation and ACE2 expression and localization. These findings have implications for understanding disease pathogenesis associated with SARS-CoV and NL63 infections.

Highlights d Longitudinal analysis of multifaceted immune memory following mild COVID-19 d Antibodies capable of neutralizing virus persist for at least 3 months in most subjects d Virus-specific memory B and T cells display hallmarks of anti-viral immunity d MBCs increase in number and express antibodies capable of neutralizing SARS-CoV-2