SUMMARY Coronaviruses are a family of enveloped, single-stranded, positive-strand RNA viruses classified within the Nidovirales order. This coronavirus family consists of pathogens of many animal species and of humans, including the recently isolated severe acute respiratory syndrome coronavirus (SARS-CoV). This review is divided into two main parts; the first concerns the animal coronaviruses and their pathogenesis, with an emphasis on the functions of individual viral genes, and the second discusses the newly described human emerging pathogen, SARS-CoV. The coronavirus part covers (i) a description of a group of coronaviruses and the diseases they cause, including the prototype coronavirus, murine hepatitis virus, which is one of the recognized animal models for multiple sclerosis, as well as viruses of veterinary importance that infect the pig, chicken, and cat and a summary of the human viruses; (ii) a short summary of the replication cycle of coronaviruses in cell culture; (iii) the development and application of reverse genetics systems; and (iv) the roles of individual coronavirus proteins in replication and pathogenesis. The SARS-CoV part covers the pathogenesis of SARS, the developing animal models for infection, and the progress in vaccine development and antiviral therapies. The data gathered on the animal coronaviruses continue to be helpful in understanding SARS-CoV.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) that first emerged in late 2019 is responsible for a pandemic of severe respiratory illness. People infected with this highly contagious virus can present with clinically inapparent, mild, or severe disease. Currently, the virus infection in individuals and at the population level is being monitored by PCR testing of symptomatic patients for the presence of viral RNA. There is an urgent need for SARS-CoV-2 serologic tests to identify all infected individuals, irrespective of clinical symptoms, to conduct surveillance and implement strategies to contain spread. As the receptor binding domain (RBD) of the spike protein is poorly conserved between SARS-CoVs and other pathogenic human coronaviruses, the RBD represents a promising antigen for detecting CoV-specific antibodies in people. Here we use a large panel of human sera (63 SARS-CoV-2 patients and 71 control subjects) and hyperimmune sera from animals exposed to zoonotic CoVs to evaluate RBD's performance as an antigen for reliable detection of SARS-CoV-2-specific antibodies. By day 9 after the onset of symptoms, the recombinant SARS-CoV-2 RBD antigen was highly sensitive (98%) and specific (100%) for antibodies induced by SARS-CoVs. We observed a strong correlation between levels of RBD binding antibodies and SARS-CoV-2 neutralizing antibodies in patients. Our results, which reveal the early kinetics of SARS-CoV-2 antibody responses, support using the RBD antigen in serological diagnostic assays and RBD-specific antibody levels as a correlate of SARS-CoV-2 neutralizing antibodies in people.

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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...

Summary Many viruses induce hepatitis in humans, highlighting the need to understand the underlying mechanisms of virus-induced liver pathology. The murine coronavirus, mouse hepatitis virus (MHV), causes acute hepatitis in its natural host and provides a useful model for understanding virus interaction with liver cells. The MHV accessory protein, ns2, antagonizes the type I interferon response and promotes hepatitis. We show that ns2 has 2′,5′-phosphodiesterase activity, which blocks the interferon inducible 2′,5′-oligoadenylate synthetase (OAS)-RNase L pathway to facilitate hepatitis development. Ns2 cleaves 2′,5′-oligoadenylate, the product of OAS, to prevent activation of the cellular endoribonuclease RNase L and consequently block viral RNA degradation. An ns2 mutant virus was unable to replicate in the liver or induce hepatitis in wild-type mice, but was highly pathogenic in RNase L deficient mice. Thus, RNase L is a critical cellular factor for protection against viral infection of the liver and the resulting hepatitis.

The coronavirus mouse hepatitis virus (MHV) induces a minimal type I interferon (IFN) response in several cell types in vitro despite the fact that the type I IFN response is important in protecting the mouse from infection in vivo. When infected with MHV, mice deficient in IFN-associated receptor expression (IFNAR ؊/؊ ) became moribund by 48 h postinfection. MHV also replicated to higher titers and exhibited a more broad tissue tropism in these mice, which lack a type I IFN response. Interestingly, MHV induced IFN-␤ in the brains and livers, two main targets of MHV replication, of infected wild-type mice. MHV infection of primary cell cultures indicates that hepatocytes are not responsible for the IFN-␤ production in the liver during MHV infection. Furthermore, macrophages and microglia, but not neurons or astrocytes, are responsible for IFN-␤ production in the brain. To determine the pathway by which MHV is recognized in macrophages, IFN-␤ mRNA expression was quantified following MHV infection of a panel of primary bone marrow-derived macrophages generated from mice lacking different pattern recognition receptors (PRRs). Interestingly, MDA5, a PRR thought to recognize primarily picornaviruses, was required for recognition of MHV. Thus, MHV induces type I IFN in macrophages and microglia in the brains of infected animals and is recognized by an MDA5-dependent pathway in macrophages. These findings suggest that secretion of IFN-␤ by macrophages and microglia plays a role in protecting the host from MHV infection of the central nervous system.The type I interferon (IFN) response, consisting of IFN-␣/␤, represents one of the first lines of defense against viral infection. During viral infection of a cell, pathogen-associated molecular patterns, such as double-stranded RNA (dsRNA), are exposed and recognized by pattern recognition receptors (PRRs). These PRRs include Toll-like receptors (TLRs), such as TLR3, that reside on the cell membrane or in endosomal compartments and the cytoplasmic receptors RIG-I (retinoic acid-inducible gene I) and MDA5 (melanoma differentiationassociated protein 5) (8). Recognition by PRRs leads to the activation of IRF-3 (IFN regulatory factor 3), which dimerizes and translocates to the nucleus. In the nucleus it associates with the promoter region of the IFN-␤ gene along with NF-B, AP-1, and CBP/p300, driving IFN-␤ transcription. Once IFN-␤ is produced, it is secreted from infected cells and is able to bind to the IFN-associated receptor (IFNAR) on neighboring cells, amplifying the production of IFN-␤ and inducing the expression of IFN-␣ and many IFN-stimulated genes (37, 41). This series of events generates an antiviral milieu that helps to limit viral spread. Therefore, it is not surprising that many viruses have developed mechanisms to subvert or alter the type I IFN response, making it more difficult for the host to combat viral infection (8,20).Mouse hepatitis virus (MHV), a group 2 coronavirus (CoV), is a positive-strand RNA virus. Depending on the strain, MHV can infect both the ...

In addition to a characteristic set of essential genes coronaviruses contain several so-called group-specific genes. These genes differ distinctly among the three coronavirus groups and are specific for each group. While the essential genes encode replication and structural functions, hardly anything is known about the products and functions of the group-specific genes. As a first step to elucidate their significance, we deleted the group-specific genes from the group 2 mouse hepatitis virus (MHV) genome via a novel targeted recombination system based on host switching (L. Kuo, G. J.Godeke, M. J. Raamsman, P. S. Masters, and P. J. M. Rottier, 2000, J. Virol. 74, 1393-1406). Thus, we obtained recombinant viruses from which the two clusters of group-specific genes were deleted either separately or in combination in a controlled genetic background. As all recombinant deletion mutant viruses appeared to be viable, we conclude that the MHV group-specific genes are nonessential, accessory genes. Importantly, all deletion mutant viruses were attenuated when inoculated into their natural host, the mouse. Therefore, deletion of the coronavirus group-specific genes seems to provide an attractive approach to generate attenuated live coronavirus vaccines.

Since the first reports of a novel SARS-like coronavirus in December 2019 in Wuhan, China, there has been intense interest in understanding how SARS-CoV-2 emerged in the human population. Recent debate has coalesced around two competing ideas: a “laboratory escape” scenario and zoonotic emergence. Here, we critically review the current scientific evidence that may help clarify the origin of SARS-CoV-2.