Highlights d A SARS-CoV-2 infectious cDNA clone and reporter viruses are generated d SARS-CoV-2 and SARS-CoV neutralization assays show limited cross neutralization d SARS-CoV-2 shows a gradient infectivity from the proximal to distal respiratory tract d Ciliated airway cells and AT-2 cells are primary targets for SARS-CoV-2 infection

The emergence of Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) and Middle East Respiratory Syndrome (MERS)-CoV underscores the threat of cross-species transmission events leading to outbreaks in humans. In this study, we examine the disease potential for SARS-like CoVs currently circulating in Chinese horseshoe bat populations. Utilizing the SARS-CoV infectious clone, we generated and characterized a chimeric virus expressing the spike of bat coronavirus SHC014 in a mouse adapted SARS-CoV backbone. The results indicate that group 2b viruses encoding the SHC014 spike in a wild type backbone can efficiently utilize multiple ACE2 receptor orthologs, replicate efficiently in primary human airway cells, and achieve in vitro titers equivalent to epidemic strains of SARS-CoV. Additionally, in vivo experiments demonstrate replication of the chimeric virus in mouse lung with notable pathogenesis. Evaluation of available SARS-based immune-therapeutic and prophylactic modalities revealed poor efficacy; both monoclonal antibody and vaccine approaches failed to neutralize and protect from CoVs utilizing the novel spike protein. Importantly, based on these findings, we synthetically rederived an infectious full length SHC014 recombinant virus and demonstrate robust viral replication both in vitro and in vivo. Together, the work highlights a continued risk of SARS-CoV reemergence from viruses currently circulating in bat populations.

The host innate immune response is an important deterrent of severe viral infection in humans and animals. Nuclear import factors function as key gatekeepers that regulate the transport of innate immune regulatory cargo to the nucleus of cells to activate the antiviral response. Using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model, we demonstrate that SARS-COV ORF6 protein is localized to the endoplasmic reticulum (ER)/Golgi membrane in infected cells, where it binds to and disrupts nuclear import complex formation by tethering karyopherin alpha 2 and karyopherin beta 1 to the membrane. Retention of import factors at the ER/Golgi membrane leads to a loss of STAT1 transport into the nucleus in response to interferon signaling, thus blocking the expression of STAT1-activated genes that establish an antiviral state. We mapped the region of ORF6, which binds karyopherin alpha 2, to the C terminus of ORF6 and show that mutations in the C terminus no longer bind karyopherin alpha 2 or block the nuclear import of STAT1. We also show that N-terminal deletions of karyopherin alpha 2 that no longer bind to karyopherin beta 1 still retain ORF6 binding activity but no longer block STAT1 nuclear import. Recombinant SARS-CoV lacking ORF6 did not tether karyopherin alpha 2 to the ER/Golgi membrane and allowed the import of the STAT1 complex into the nucleus. We discuss the likely implications of these data on SARS-CoV replication and pathogenesis.

Coronaviruses are prone to emergence into new host species most recently evidenced by SARS-CoV-2, the causative agent of the COVID-19 pandemic 1 . Small animal models that recapitulate SARS-CoV-2 disease are desperately needed to rapidly evaluate medical countermeasures (MCMs) 2 , 3 . SARS-CoV-2 cannot infect wildtype laboratory mice due to inefficient interactions between the viral spike (S) protein and the murine ortholog of the human receptor, ACE2 4 . We used reverse genetics 5 to remodel the interaction between S and mACE2 resulting in a recombinant virus (SARS-CoV-2 MA) that could utilize mACE2 for entry. SARS-CoV-2 MA replicated in both the upper and lower airways of both young adult and aged BALB/c mice. Importantly, disease was more severe in aged mice, and showed more clinically relevant phenotypes than those seen in HFH4-hACE2 transgenic mice. We then demonstrated the utility of this model through vaccine challenge studies in immune competent mice with native expression of mACE2. Lastly, we show that clinical candidate interferon (IFN) lambda-1a can potently inhibit SARS-CoV-2 replication in primary human airway epithelial cells in vitro , and both prophylactic and therapeutic administration diminished replication in mice. Our mouse-adapted SARS-CoV-2 model demonstrates age-related disease pathogenesis and supports the clinical use of pegylated IFN lambda-1a treatment in human COVID-19 infections 6 .

Highlights d Remdesivir binding of active site of polymerase is conserved across all human CoVs d Remdesivir inhibits SARS-CoV-2 in primary and continuous human lung cell cultures d Remdesivir potency depends on cell-type-specific metabolism to its active form d Therapeutic remdesivir reduces viral loads and improves outcomes in mice

No single animal model for severe acute respiratory syndrome (SARS) reproduces all aspects of the human disease. Young inbred mice support SARS-coronavirus (SARS-CoV) replication in the respiratory tract and are available in sufficient numbers for statistical evaluation. They are relatively inexpensive and easily accessible, but their use in SARS research is limited because they do not develop illness following infection. Older (12- to 14-mo-old) BALB/c mice develop clinical illness and pneumonitis, but they can be hard to procure, and immune senescence complicates pathogenesis studies. We adapted the SARS-CoV (Urbani strain) by serial passage in the respiratory tract of young BALB/c mice. Fifteen passages resulted in a virus (MA15) that is lethal for mice following intranasal inoculation. Lethality is preceded by rapid and high titer viral replication in lungs, viremia, and dissemination of virus to extrapulmonary sites accompanied by lymphopenia, neutrophilia, and pathological changes in the lungs. Abundant viral antigen is extensively distributed in bronchial epithelial cells and alveolar pneumocytes, and necrotic cellular debris is present in airways and alveoli, with only mild and focal pneumonitis. These observations suggest that mice infected with MA15 die from an overwhelming viral infection with extensive, virally mediated destruction of pneumocytes and ciliated epithelial cells. The MA15 virus has six coding mutations associated with adaptation and increased virulence; when introduced into a recombinant SARS-CoV, these mutations result in a highly virulent and lethal virus (rMA15), duplicating the phenotype of the biologically derived MA15 virus. Intranasal inoculation with MA15 reproduces many aspects of disease seen in severe human cases of SARS. The availability of the MA15 virus will enhance the use of the mouse model for SARS because infection with MA15 causes morbidity, mortality, and pulmonary pathology. This virus will be of value as a stringent challenge in evaluation of the efficacy of vaccines and antivirals.

Outbreaks from zoonotic sources represent a threat to both human disease as well as the global economy. Despite a wealth of metagenomics studies, methods to leverage these datasets to identify future threats are underdeveloped. In this study, we describe an approach that combines existing metagenomics data with reverse genetics to engineer reagents to evaluate emergence and pathogenic potential of circulating zoonotic viruses. Focusing on the severe acute respiratory syndrome (SARS)-like viruses, the results indicate that the WIV1-coronavirus (CoV) cluster has the ability to directly infect and may undergo limited transmission in human populations. However, in vivo attenuation suggests additional adaptation is required for epidemic disease. Importantly, available SARS monoclonal antibodies offered success in limiting viral infection absent from available vaccine approaches. Together, the data highlight the utility of a platform to identify and prioritize prepandemic strains harbored in animal reservoirs and document the threat posed by WIV1-CoV for emergence in human populations.A lthough previously associated with upper respiratory infections, the emergence of severe acute respiratory coronavirus (SARS-CoV) in [2002][2003], and more recently, Middle East respiratory syndrome (MERS)-CoV underscores the threat of cross-species transmission leading to virulent pandemic viral infections (1, 2). Whereas prevailing research suggests that SARSCoV emerged from viruses in the Chinese horseshoe bat, identifying a progenitor strain that used human angiotensin converting enzyme 2 (ACE2) had proven elusive (3, 4). However, recent metagenomics studies isolated several SARS-like virus sequences that share ≥90% genome-wide homology and represented the closest sequences to the epidemic strains (5, 6). Importantly, researchers also isolated replication competent virus; WIV1-CoV, part of the Rs3306 cluster, could use ACE2 orthologs and mediated low-level replication in human cells (5). Overall, the evidence indicates that SARS-CoV likely emerged from Chinese horseshoe bats and that similar viruses are still harbored in these populations.The identification of WIV1-CoV and its capacity to use ACE2 orthologs offers a warning for possible reemergence and provides an opportunity to prepare for a future CoV outbreak. To achieve this goal, a new platform is required to translate metagenomics findings; the approach must generate critical diagnostic reagents, define emergence potential of novel strains, and determine efficacy of current therapeutics. Building on this premise, we developed a framework to examine circulating CoVs using reverse genetic systems to construct full-length and chimeric viruses. The results indicate that viruses using WIV1-CoV spike are poised to emerge in human populations due to efficient replication in primary human airway epithelial cell cultures. However, additional adaptation, potentially independent of the spike protein receptor-binding domain, is required for pathogenesis and epidemic disease. Importantly,...

A novel method was developed to assemble a full-length infectious cDNA of the group II coronavirus mouse hepatitis virus strain A59 (MHV-A59). Seven contiguous cDNA clones that spanned the 31.5-kb MHV genome were isolated. The ends of the cDNAs were engineered with unique junctions and assembled with only the adjacent cDNA subclones, resulting in an intact MHV-A59 cDNA construct of ϳ31.5 kb in length. The interconnecting restriction site junctions that are located at the ends of each cDNA are systematically removed during the assembly of the complete full-length cDNA product, allowing reassembly without the introduction of nucleotide changes. RNA transcripts derived from the full-length MHV-A59 construct were infectious, although transfection frequencies were enhanced 10-to 15-fold in the presence of transcripts encoding the nucleocapsid protein N. Plaque-purified virus derived from the infectious construct replicated efficiently and displayed similar growth kinetics, plaque morphology, and cytopathology in murine cells as did wild-type MHV-A59. Molecularly cloned viruses recognized the MHV receptor (MHVR) for docking and entry, and pretreatment of cells with monoclonal antibodies against MHVR blocked virus entry and replication. Cells infected with molecularly cloned MHV-A59 virus expressed replicase (gene 1) proteins identical to those of laboratory MHV-A59. Importantly, the molecularly cloned viruses contained three marker mutations that had been derived from the engineered component clones. Full-length infectious constructs of MHV-A59 will permit genetic modifications of the entire coronavirus genome, particularly in the replicase gene. The method has the potential to be used to construct viral, microbial, or eukaryotic genomes approaching several million base pairs in length and used to insert restriction sites at any given nucleotide in a microbial genome.The Nidovirales order includes mammalian and avian positive-polarity, single-stranded RNA viruses in the arterivirus and coronavirus families (43). The Coronaviridae family is further subdivided into the Coronavirus and Torovirus genera (14,45). Despite remarkable size differences in the genomic RNA (13 to 32 kb), the polycistronic genome organization and regulation of gene expression from a nested set of subgenomic mRNAs are similar for all members of the order (29, 45). Coronaviruses contain the largest single-stranded, plus-polarity RNA genome in nature, ranging in size from about 27 to 31 kb in length, and are divided into three subgroups based upon antigenic and sequence comparisons (29,43). The group I coronaviruses include human coronavirus strain 229E (HCV 229E) and transmissible gastroenteritis virus (TGEV). The group II coronaviruses contain mouse hepatitis virus (MHV), bovine coronavirus, and HCV OC43. Bovine coronavirus is an important pathogen of cattle while HCV infection is associated with a significant percentage of common colds in winter. The group III coronaviruses contain infectious bronchitis virus (IBV).The MHV-A59 and MHV-JHM strains...