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Abstract 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. Go to: At a glance Coronaviruses are positive strand RNA viruses that cause disease in humans, and domestic and companion animals. They are most notorious for causing the Severe Acute Respiratory Syndrome in 2002–2003. All coronaviruses follow the same basic strategy of replication. Coronaviruses all encode 15–16 replicase related proteins, 4–5 structural proteins and 1–8 group-specific or accessory proteins. Many of the replicase proteins are assembled into replication machinery in double membrane vesicles (DMVs) and on a reticular network of membranes that are derived from the endoplasmic reticulum. Coronaviruses are readily transmitted across species. This phenomenon was illustrated when the SARS-coronavirus crossed species from bats to intermediate hosts such as palm civets and then to humans. It also explains the large number of species, including humans, infected with viruses closely related to bovine coronavirus. In many coronavirus infections, disease severity increases during virus clearance, suggesting that the host immune response is both protective and pathogenic. Further, inhibition of specific aspects of the immune response results in less severe disease and less tissue destruction, without diminishing the kinetics of virus clearance. Like all successful viruses, coronaviruses have evolved both passive and active mechanisms to evade the interferon response. Replication in DMVs may contribute to passive evasion of the innate immune response by making double stranded RNA inaccessible to cellular sensors. Go to: Replication strategy and viral proteins Coronaviruses, a genus within the Coronaviridae family (order Nidovirales- see Box 1), are pleomorphic, enveloped viruses. The viral membrane contains the transmembrane (M) glycoprotein, spike (S) glycoprotein, and envelope (E) protein and surrounds a disordered or flexible, likely helical nucleocapsid 2, 3. The viral membrane is unusually thick, most likely because the C terminal region of the M protein forms an extra internal layer, as revealed b cryo-electron tomography 2. Coronaviruses are divided into three groups, subdivided into subgroups (Table 1), based initially on serologic, and more recently, on genetic analyses. With the identification of more distantly related viruses, the taxonomy of these viruses is likely to undergo further changes. Coronaviruses contain a single stranded, 5′-capped, positive strand RNA molecule ranging from 26–32 kb that contains at least 6 open reading frames (ORFs). The first ORF (ORF1a/b) comprises approximately 2/3 of the genome and encodes replicase proteins (Figure 1A). Translation begins in ORF1a and continues in ORF1b after a -1 frameshift signal. The large ORF1a and ORF1ab polypeptides, commonly referred to as pp1a and pp1ab, are processed primarily by the virally encoded chymotrypsin-like protease 3CLpro (also called Mpro or main protease) with additional cleavage performed by one or two viral papain-like proteases (PLP), depending upon species of coronavirus 4. The majority of the remaining 1/3 of the genome encodes four structural proteins: S, E, M and nucleocapsid (N) proteins. A subset of group 2 coronaviruses encode an additional hemagglutinin-esterase (HE) protein (Figure 1A, B). The HE protein, which may be involved in virus entry or egress, is not required for replication but appears to be important for infection of the natural host 5. Box 1The Nidoviruses Order: Nidovirales Family: Coronaviridae Genus: Coronavirus Torovirus\Bafinivirus Family: Roniviridae Genus: Okavirus Family: Arteriviridae Genus: Arterivirus Figure 1 Structure of coronavirus genome and virion Table 1 Representative coronavirus species and their receptors. Receptors for several coronaviruses have been identified (Table 1). The prototypic coronavirus, mouse hepatitis virus (MHV) uses CEACAM1a, a member of the murine carcinoembryonic antigen family, to enter cells. Deletion of this protein makes mice resistant to infection 6. Several group 1 coronaviruses use aminopeptidase N, consistent with their respiratory and enteric tract tropisms (reviewed in 7). SARS-CoV, a group 2 coronavirus, enters host cells through an interaction of the S protein with human angiotensin converting enzyme 2 (hACE2) 8. Strikingly, HCoV-NL63, which causes relatively mild disease, also uses hACE2, although it binds to a different part of the protein than SARS-CoV 9, 10. ACE2 is postulated to have a protective role in the inflamed lung and SARS-CoV S protein binding to ACE2 is believed to contribute to disease severity 11, 12. However, since infection with HCoV-NL63 produces mild disease, binding to ACE2 by itself can not be sufficient for this process. The N protein is important for encapsidation of viral RNA, and acts as an IFN antagonist (see below). Additionally, it causes upregulation of FGL-2, a prothrombinase that contributes to fatal hepatic disease in mice infected with MHV-3 13 and modifies TGF-β (transforming growth factor-β) signaling in SARS-CoV-infected cells 14. The E proteins are small integral membrane proteins with roles in virus morphogenesis, assembly and budding. In the absence of E, virus release is inhibited completely (in the case of TGEV) or partially (in the case of SARS-CoV, MHV) 15–17. The E protein also possesses ion channel activity, which is required for optimal virus replication 18, 19. Interspersed between and within these structural genes are one to eight genes encoding accessory proteins, depending on the virus strain. These show no sequence similarity with other viral or cellular proteins and are not required for virus replication in cultured cells 20–22, although their conservation within virus species isolated at different times and locales (e.g., for SARS-CoV, 23), suggests an important role for these proteins in replication in the natural host. Several accessory proteins were shown to be virion-associated 24–27 although whether these proteins are truly structural is controversial 28. The non-replicase proteins are expressed from a set of “nested” subgenomic mRNAs that have common 3′ ends and a common leader encoded at the 5′ end of genomic RNA). Proteins are produced generally only from the first ORF of each subgenomic (sg) mRNA. Sg RNAs are produced during minus strand RNA synthesis, with transcription termination (and subsequent acquisition of a leader RNA) occurring at transcription regulatory sequences (TRS), located between ORFs. These minus strand sg RNAs serve as the template for the production of sg mRNAs (Figure 2). The latter process is very efficient, resulting in a high ratio of sg mRNA to minus strand sg RNA 29. Figure 2 Following entry into the cell and uncoating, the positive-sense RNA genome is translated to generate replicase proteins from ORF1a/b. These proteins use the genome as a template to generate full-length negative-sense RNAs, which subsequently serve as ... Go to: Coronavirus replication and double membrane vesicles (DMVs) One consequence of the SARS epidemic was increased efforts to understand coronavirus replication and thereby identify additional possible targets for anti-viral therapy. ORF1 of coronaviruses encodes 15 or 16 proteins involved in viral replication (Figure 1A); the range occurs because group 3 coronaviruses do not express nsp1. The structure of many of these proteins has been solved by x-ray crystallography or nuclear magnetic resonance, facilitating structure-function studies 30–40. Functions were predicted 41 and later confirmed for many of these proteins (Box 2), including the proteases nsp3 (PL1pro and PL2pro (papain-like proteases)) and nsp5 (3CLpro or Mpro), the RNA-dependent RNA polymerase nsp12 and the helicase nsp13. A second RNA polymerase, nsp8, may function as a primase 42. The nsp3 protease has additional roles in assembly of virus replication structures (see below) and possesses poly(ADP-ribose) binding capabilities, and deubiquitinating activity in its protease domain, although the role of the latter in virus replication is not yet known 43. Box 2Coronavirus non-structural proteins and their functions Protein	Functions Nsp1	Host mRNA degradation Translation inhibition Cell cycle arrest Inhibition of IFN signaling Nsp2	Unknown Nsp3	Papain-like proteases (PL1pro, PL2pro) (Polyprotein processing) Poly(ADP-ribose) binding DMV formation? IFN antagonist Nucleic acid binding Deubiquitinating activity Nsp4	DMV formation? Nsp5	Main protease (Mpro, 3CLpro) (Polyprotein processing) Nsp6	DMV formation? Nsp7	ssRNA binding Nsp8	Primase Nsp9	Part of replicase complex Nsp10	Part of replicase complex Nsp11	Unknown Nsp12	RNA-dependent RNA polymerase (RdRp) Nsp13	Helicase Nucleoside triphosphatase activity RNA 5′-triphosphatase activity Nsp14	3′-to-5′ exoribonuclease (ExoN) RNA cap formation (guanine-N7)-methyltransferase Nsp15	Endonuclease (NendoU) Nsp16	RNA cap formation (2′O-methytransferase) Nsps 7, 8, 9 and 10 are postulated to have a role in subgenomic and genomic RNA replication and all four proteins are essential for viral replication 44. Nsps 7 and 8 form a hexadecameric structure, with RNA binding activity 31, while the structure of nsp9 also suggests that it binds RNA 45. Mutations in nsp10 inhibit minus strand RNA synthesis, but this effect may be indirect since subsequent studies showed that nsp10 is required for proper function of the main viral protease (Mpro) 46. Nsp14, a bifunctional protein, is a 3′ to 5′ exonuclease (ExoN) with a role in maintaining fidelity of RNA transcription 47 and a (guanine-N7)-methyl transferase (N7-MTase), involved in RNA cap formation 48. Coronaviruses also encode a novel uridylate-specific endoribonuclease (NendoU) (nsp15) that distinguishes nidoviruses in general from other RNA viruses and is critical for virus replication 49. NendoU cleavage results in 2′–3′ cyclic phosphate ends, but its function in the virus life cycle remains unknown. Nsp16 is an S-adenosyl-L-methionine-dependent RNA (nucleoside-2′O)-methyl transferase (2′O-MTase) and like nsp14, is involved in cap formation 50. Nsp15 has been postulated to function with nsp14 and nsp16 in RNA processing or cap production, but this remains to be proven. RNA replication is believed to occur on double membrane vesicles (DMVs) 51 (Figure 4). Newly synthesized genomic RNA is then incorporated into virions on membranes located between the endoplasmic reticulum and Golgi apparatus (endoplasmic reticulum-Golgi intermediate compartment (ERGIC)) (reviewed in 52). Initial studies suggested that these DMVs assembled using components of the autophagy pathway 53, but other studies showed replication proceeded normally and DMVs were produced in macrophages lacking ATG5, a key component of autophagosomes 54. Thus, whether autophagy is involved at all or whether its involvement is cell-specific remains uncertain. In addition, the Unfolded Protein Response (UPR) is induced during coronavirus infections and may contribute to DMV formation 55. Figure 4 Coronavirus-induced membrane alterations as platforms for viral replication Recent results show that DMVs are likely to originate from the endoplasmic reticulum. Using electron tomography of cryofixed SARS-CoV-infected Vero E6 cells and three-dimensional reconstruction imaging, Knoops et al. showed that DMVs were not isolated vesicles but rather were part of a reticulovesicular network of modified ER membranes 56. At later times after infections, these networks appeared to merge into large single membrane vesicles. Proteins involved in virus replication (nsp3, nsp5 and nsp8-see box 2) were located mainly outside of DMVs, in adjacent reticular structures. dsRNA, representing either replicative intermediates or “dead-end” double stranded RNA was detected primarily within DMVs and surprisingly, no obvious connections between the interior of these vesicles and the cytosol were detected 56. Thus, it remains unknown how newly synthesized RNA might be transported to sites of virus assembly, assuming that RNA transcription occurs within DMVs. Formation of DMVs requires membrane curvature and this may be initiated by insertion of specific viral proteins into membranes. Based on studies of equine arteritis virus 57, a non-coronavirus member of the nidovirus order (Box 1), nsp3 and nsp4 are likely to be sufficient for DMV formation. Mutations in nsp4 result in aberrant formation of DMVs, further supporting a role for these proteins in establishing sites of virus replication 58. Nsp 6, like nsp3 and nsp4, also contains multiple transmembrane regions and may be involved in membrane modification 59–61. Of note, nsp3 and nsp6 encode an odd number of hydrophobic domains, but both the N and C termini of these proteins are in the cytoplasm, suggesting that one hydrophobic region does not span the membrane 60; whether this region contributes to membrane curvature or has another function will require further investigation. Go to: Coronavirus-mediated human and animal diseases Prior to the SARS epidemic in 2002–2003, two human coronaviruses, HCoV-OC43 and HCoV-229E, were recognized as important causes of upper respiratory tract infections and were occasionally associated with more severe pulmonary disease in the elderly, newborn and immunocompromised 62. SARS-CoV, unlike HCoV-OC43 and HCoV-229E, causes a severe respiratory disease, with a nearly 10% mortality observed in 2002–2003 1. Notable features of the disease were an apparent worsening of symptoms as the virus was cleared (suggesting an immunopathological basis of disease), a propensity to cause more severe disease, with 50% mortality in the elderly, and a lack of contagion until lower respiratory tract symptoms were apparent. This latter feature made control of the epidemic by quarantine feasible, since it simplified identification of infected patients. Unlike HCoV-OC43 and HCoV-229E, SARS-CoV also caused systemic disease, with evidence of infection of the gastrointestinal tract, liver, kidney and brain, among other tissues 63. While the virus spread primarily via respiratory droplets, infection of the gastrointestinal tract may have facilitated other routes of spread. The recognition that SARS was caused by a coronavirus intensified the search for other pathogenic coronaviruses associated with human disease, which led to the identification of HCoV-NL63 and HCoV-HKU1. These viruses were isolated from hospitalized patients, either young children with severe respiratory disease (HCoV-NL63) 64, 65 or elderly patients with underlying medical problems (HCoV-HKU1) 65, 66. HCoV-NL63 has infected human populations for centuries, since phylogenetic studies show that it diverged from HCoV-229E nearly 1000 years ago 67. HCoV-NL63 and HCoV-HKU1 have worldwide distributions and generally cause relatively mild upper respiratory tract diseases, with the exception that HCoV-NL63 is also an etiological agent of croup 68. HCoV-NL63 can be propagated in tissue culture cells and recently an infectious cDNA clone of this virus has been engineered, facilitating future studies 69. In contrast, HCoV-HKU1 cannot be grown in tissue culture cells, which makes it imperative that an infectious cDNA clone be developed for future studies. While it took the SARS epidemic to reveal the severe disease-forming capabilities of human coronaviruses, it was well known that animal coronaviruses could cause life-threatening disease. Transmissible gastroenteritis virus (TGEV), which causes diarrhea in piglets, infectious bronchitis virus (IBV), a cause of severe upper respiratory tract and kidney disease in chickens and bovine coronavirus (BCoV), which causes respiratory tract disease and diarrhea in cattle (“winter dysentery” and “shipping fever”), are all economically important pathogens. Feline infectious peritonitis virus (FIPV), a virulent feline coronavirus (FCoV), causes an invariably fatal systemic disease in domestic cats and other felines. Unlike most strains of FCoV, which are endemic causes of mild diarrhea, FIPV arises sporadically, most likely by mutation or deletion in felines persistently infected with enteric strains of FCoV 70 and is macrophage-tropic. Perhaps the most convincing explanation for FIPV-mediated disease was suggested by the observation that progressive waves of virus replication, lymphopenia and ineffectual T cell responses occurred in FIP 71. In conjunction with previous studies, these results raised the possibility that FIPV infection of macrophages and dendritic cells caused aberrant cytokine/chemokine expression and lymphocyte depletion, resulting in enhanced virus loads and consequently, a fatal outcome. However, while this explanation is appealing, additional work will be needed to prove its validity. Of note, anti-FIPV antibody-mediated enhancement has been implicated in pathogenesis but this has been shown only after immunization with S protein expressing vaccines 72; it has not been shown to play a role in a natural feline infection. Go to: Cross species transmission and role of bat coronaviruses A striking feature of the 2002–2003 SARS epidemic was the ability of the SARS-CoV to cross species from Himalayan palm civets (Pagkuma larvata), raccoon dogs (Nyctereutes procyonoides) and Chinese ferret badgers (Melogale moschata) to infect human populations 73 (Figure 3A). Transmission occurred in live animal retail (wet) markets, where animal handlers became infected. In retrospect, it appears that variants of SARS-CoV related to the epidemic strain infected human populations in the wet markets fairly frequently as shown by the high seropositivity rate detected in animal handlers even though they did not develop a SARS-like illness 73. The epidemic began when a physician caring for personnel in the wet markets became infected and was able to infect multiple contacts in “superspreading events” 74. Figure 3 Cross-species transmission of coronaviruses Genetic analyses of virus isolates from infected palm civets and humans during the epidemic showed that the virus underwent rapid adaptation in both hosts 75, 76, primarily in the receptor binding domain (RBD) of the S protein, to allow more efficient infection of human cells77; in particular mutations K479N and S487T in the RBD were key to adaptation to the human receptor (hACE2). These results were recently confirmed using cell lines expressing civet ACE2 or hACE2 78. The observation that SARS-CoV could not be detected in either farmed or wild palm civets 79, together with evidence for adaptive changes detected in virus isolated from infected animals, suggested that palm civets and other animals in wet markets were not the primary reservoir for the virus. Since SARS-like CoV were isolated from Chinese horseshoe bats 23, 80, which were also present in the live animal markets, the virus may have recently spread from bats to other mammals such as palm civets and then to humans (Figure 3A). Consistent with recent spread, antibodies to SARS-CoV were detected at extremely low levels (0.008%) in population studies in Hong Kong 81. Bat SARS-like CoV is unable to replicate in cells expressing bat ACE2, although productive infection of cells expressing hACE2 occurs if the RBD of the bat S protein is replaced with that of a human isolate 82, 83. Collectively, these observations suggest that virus spread from bats to other species, but host cell entry does not occur via ACE2 in bats as it does in palm civets and humans. Besides SARS-CoV, there are other examples of coronavirus cross-species transmission. BCoV and HCoV-OC43 are very similar and one estimate is that the virus crossed species approximately 100 years ago 84. BCoV has continued to cross species since a related virus (99.5% similarity) has also been isolated from an alpaca with enteritis and from captive wild ruminants 85, 86 (Figure 3B). Canine (CCoV), feline, and porcine viruses also show evidence of recombination. Recombination events between early CCoV and FCoV strains (CCoV-1 and FCoV-1) and an unknown coronavirus resulted in two sets of novel viruses (CCoV-II) and FCoV-II). Sequence data suggest that TGEV resulted from a cross-species transmission of CCoV-II from an infected canine 87 (Figure 3C). Molecular surveillance studies have identified at least 60 novel bat coronaviruses in China 88, North America 50, Europe 89, 90 and Africa 91. These bat CoVs may have originated from a common source but subsequently diverged as they adapted to growth in different species of bat. They are now only distantly related to other coronaviruses. These studies also identified several novel avian group 3 coronaviruses 92 that were related to a novel coronavirus isolated from Asian leopard cats and Chinese ferret badgers sold in illegal wild animal markets in China 93, suggesting that this virus, like SARS-CoV, was able to cross species. Another novel group 3 coronavirus, isolated from a deceased beluga whale, was shown to be only distantly related to IBV-like and novel avian coronaviruses, suggesting that it comprised a third subgroup 94. Thus group 3 coronaviruses, which formerly included only avian viruses, now consist of at least 3 subgroups and include viruses that infect mammalian hosts. Go to: Immunopathology in coronavirus infections It is generally accepted that the host response is responsible for many of the disease manifestations in infections caused by coronaviruses 95, 96. This was shown initially in mice infected with the neurotropic JHM and A59 strains of mouse hepatitis virus (JHMV and MHV-A59). Many attenuated strains of JHMV cause a subacute or persistent infection in the central nervous system (CNS), with persistence in glia, especially oligodendrocytes. A consequence of host efforts to clear the virus is myelin destruction (demyelination). However, JHMV infection of mice lacking T or B cells (sublethally irradiated mice or mice with severe combined immunodeficiency or genetically deficient in recombination activating gene 1 (RAG1−/−)) results, eventually, in death in all mice but without demyelination. Adoptive transfer of CD4 or CD8 T splenocytes 7 or 30 days after immunization with JHMV to infected RAG1−/− or SCID mice results in virus clearance and demyelination 95–97. Myelin destruction is also observed if anti-JHMV antibody is transferred to infected RAG1−/− mice in the absence of T cells 98 or if mice are infected with virus expressing the macrophage chemoattractant, CCL2, in the absence of other interventions 99. In all cases, infiltrating macrophages appear to be critical for virus clearance and subsequent demyelination; these results suggest that the process of macrophage infiltration can be initiated by T cells, anti-JHMV antibody or overexpression of a single macrophage chemoattractant. These results have been extended to mice with encephalitis caused by virulent strains of JHMV. While CD4 and CD8 T cells are both required for virus clearance 100, partial abrogation of the CD4 T cell response (by mutating the immunodominant CD4 T cell epitope, rJ.MY135Q)) results in disease amelioration, and virulence is regained if another CD4 T cell epitope is reintroduced in the rJ.MY135Q genome 101. Thus acute encephalitis, like chronic demyelination, is at least partially immune-mediated. Similar processes may occur in SARS-CoV-infected humans, since pulmonary disease often worsens at 1–2 weeks after onset of respiratory symptoms concomitant with the onset of virus clearance 1. While worsening clinical disease occurring as a consequence of virus clearance has not been duplicated in any animal model of SARS, the severe disease observed in older patients can be mimicked in SARS-CoV-infected aged mice 102–104. This has been attributed, in part, to a suboptimal T cell response resulting in delayed kinetics of virus clearance. A suboptimal T cell response, occurring as a consequence of infection of macrophages or dendritic cells, may also be critical for the immunopathological lethal disease observed in FIPV-infected felines 71. Thus in many instances, host efforts to clear a coronavirus infection result in some level of tissue destruction. Go to: Evasion of the innate immune response While anti-viral T cells and antibodies are critical for virus clearance and the prevention of recrudescence (reviewed in 96), the efficacy of the innate immune response determines the extent of initial virus replication and thus the load that the host must overcome to clear the infection. Coronaviruses, like all other successful viruses, have developed strategies to counter the innate immune response. Interferon (IFN) expression is a critical component of this initial response, and coronaviruses have developed “passive” and “active” tools to prevent IFN induction and signaling. Interferon is not induced in fibroblasts infected with either SARS-CoV or MHV 105–107. However, in both instances, treatment of cells with poly I:C or other interferon-inducing agents, results in IRF-3 (Interferon Regulating Factor) activation and IFN induction 105, 106. Thus, in these cells, viruses appear to be invisible to intracellular viral sensors (RIG-I, MDA-5 and TLR3), perhaps because double stranded RNA, a potent stimulator of the innate immune system, is buried in DMV (Figure 4). Additionally, viral proteins, in particular nsp1, nsp3, N protein and the SARS-CoV accessory proteins ORF6 and ORF3b, also prevent IFN induction 108–110, 111–113. The N protein of MHV inhibits AP-1 signaling and PKR function, while the N protein of SARS-CoV also inhibits NF-κB activation 108–110, when expressed in transfection assays. Whether these inhibitory functions of the N protein are coronavirus or cell-type specific and whether they occur in infected cells remains to be determined. The ORF6 protein (p6) inhibits IFN signaling by binding to karyopherin α2, thereby tethering karyopherin β to cytoplasmic membranes 114. This, in turn, prevents nuclear translocation of proteins containing classical nuclear import signals 115, including STAT1, a critical component of both IFNα/β and IFNγ signaling pathways. Of note, deletion of p6 does not increase the IFN sensitivity of SARS-CoV 116, probably because mechanisms of IFN antagonism are redundant. SARS-CoV and MHV nsp1 function, at least in part, by degrading host cell mRNA and inhibiting translation 111–113, 117. Nsp1 also inhibits IFN signaling in both SARS-CoV and MHV-infected cells, in part by inhibiting STAT1 phosphorylation 112, 113. Mutation of nsp1 attenuates SARS-CoV and MHV growth in mice and tissue culture cells in the presence of an intact IFN system but not when IFN function is deficient 111–113. Nsp3, is also an IFN antagonist, inhibiting phosphorylation and nuclear importation of IRF-3 118. Although both MHV and SARS-CoV inhibit IFN-α/β induction and signaling, IFN-α/β is detected in infected mice and humans 119, 120 and mice deficient in IFN-α/βR receptor expression are exquisitely sensitive to MHV infection 113, 121, showing that IFN-α/β has a major role in the anti-virus immune response. Reconciling these disparate results, recent studies showed that IFN-α is produced in large amounts in SARS-CoV and MHV-infected plasmacytoid DCs, via a TLR7-dependent mechanism 122; further, IFN-β is expressed by macrophages and microglia, but not dendritic cells after MHV infection 123. Macrophages and to a lesser extent, DCs are the major targets for IFN-α/β in MHV-infected mice 124. In addition to IFN, multiple chemokines and cytokines are also induced as part of the host response to coronaviruses including MHV, SARS-CoV and FIPV. Cytokines such as IL-1, IL-6 and IL-12 and chemokines such as IL-8, CCL2 and CXCL10 were elevated in SARS patients. Using genomics and proteomics, Cameron et al found that IFN-α/β and IFN-γ, as well as chemokines such as CXCL10 and CCL2, were elevated at early times post infection in all patients and diminished in those who recovered, accompanied by a robust anti-virus antibody response 119. However, levels of CXCL10, CCL2 and other proinflammatory mediators remained elevated and anti-SARS-CoV antibody titers were low in those patients who developed severe disease. SARS-CoV-infected pulmonary epithelial cells were the source for at least some of the cytokines/chemokines such as CCL2, IL-6, IL-1β and TNF detected in infected patients 125. Others have suggested that a strong Th2 (IL4, IL-5 and IL-10) response correlated with a poor outcome 126. It has been postulated that an over exuberant cytokine response contributed to a poor outcome in patients with SARS in 2002–3 (reviewed in 95, 127, 128). Collectively, these results do not strongly prove or disprove a role for an exuberant cytokine/chemokine response in severe SARS, in part because virus titers could not be determined concomitantly, and serum but not pulmonary cytokine or chemokine levels were measured. Go to: Animal models for SARS Since human SARS has disappeared, the role of an exuberant (but perhaps appropriate for level of virus) immune response will need to be addressed using animal models of SARS. Mice, cats, ferrets, macaques and civet cats are all susceptible to SARS-CoV but none, with the exception of aged mice, develop severe disease (reviewed in 129). In efforts to develop models that more closely mimic human disease, mice transgenic for the expression of hACE2 were developed and infected with SARS-CoV 130, 131. Although these mice develop modestly more severe pulmonary disease than non transgenic mice, they also develop an overwhelming neuronal infection, accompanied by high cytokine/chemokine expression but minimal cellular infiltration in the brain 132. Although the severity of the brain infection observed in hACE2 transgenic mice is greater than that seen in patients, infection of this organ has been detected in some studies and patients who survived SARS had a greater incidence of neurological and psychiatric sequelae than anticipated 63, 133, 134. The high susceptibility of these mice to infection with SARS-CoV makes them useful for vaccine and therapeutic trials. Another approach to developing an animal model for SARS was to adapt the virus by passage 10–15 times through the lungs of BALB/c mice or rats 103, 135, 136. Three to six mutations were detected in the adapted viruses, with changes most commonly observed in the S protein and nsp5 (3CLpro). The adapted virus caused extensive pulmonary infection with disease being most severe in aged animals. These viruses will be useful for studies of pathogenesis and for vaccine and therapeutic trials. Some models have been tested on the genomic and proteomic level. Studies of SARS-CoV infected macaques showed that several chemokines/cytokines such as IL6, IL-8, CXCL10 and CCL2, as well as IFN-α/β/γ were upregulated 137. These animals recovered, showing that the same inflammatory mediators associated with severe human disease are also produced as part of the inflammatory response in animals that mount an appropriate response. Genomics studies of mice infected with the Urbani strain of SARS-CoV showed continued expression of inflammatory mediators, such as IL-6, TNF, CXCL10 and CCL2, accompanied by slower kinetics of virus clearance and worse outcomes in aged compared to young animals 138, paralleling disease patterns in patients with SARS 119. These two studies also showed changes in expression of proteins involved in cell growth, cycling, development and death, and cell-to-cell signaling. It will be important to determine whether these changes are useful as a “fingerprint” for SARS or whether they represent generalized responses to pulmonary stress. Go to: Conclusions and Future Directions: What is next in understanding coronavirus biology and pathogenesis? Perhaps the most important insight made over the past several years is that coronaviruses have and will likely continue to cross species and cause disease in unrelated hosts. This disease may be mild, as seemed to occur when most SARS-like CoVs were transmitted to animal handlers in wet markets in China, but may be severe, as illustrated by the transmission that triggered the SARS epidemic. Further, SARS-CoV appeared to use an entirely new receptor when it crossed species from bats to palm civets and humans. As part of this transmission to a new species, the virus also needed to evolve strategies to evade the innate immune response of the new hosts. One future goal will be to further delineate how the virus evades the immune response and better understand its interaction with the T and B cell responses, both in the original host (bats), in which disease appears to be mild, and in humans and experimentally infected animals. While coronaviruses use host proteins as part of their replication strategies, it has also become clear that multiple immune, metabolic, stress, cell cycling and other pathways are activated by infection. Assessing the biological function of these pathways in virus replication and in disease outcome will be critical. Determining the extent to which virus-host interactions are coronavirus-specific and organ-specific will be possible, using genomics and proteomics, as well as new reagents such as Collaborative Cross mice. The Collaborative Cross, a panel of approximately 1000 recombinant inbred mouse strains derived from eight founder strains, will be very useful for analyses of complex genetic traits 139. Using sophisticated microscopy and biochemical approaches, details of coronavirus replication within infected cells have been revealed to a greater extent than ever before. However, these new results have led to a new set of questions about the relationship between sites of viral RNA replication and virus assembly. Further, while putative functions have been assigned to many of the proteins encoded by the large ORF1 replicase gene, the precise roles of these proteins in virus replication still require additional investigation. Progress in these fields will take advantage of new methodologies that allow detailed observations of both fixed and living cells at high resolution. Finally, no effective treatments exist for any coronavirus infections including SARS 140 and vaccines, even for animal coronaviruses, are not very effective; further, live attenuated vaccines are prone to recombination with circulating coronaviruses. One future goal will be to translate new information about the structure and function of coronavirus proteins into specific anti-virus therapies. Also, development of live, attenuated, safe vaccines that do not recombine in the wild is another goal, made more feasible as more is learned about basic coronavirus biology. Over the past few years, the development of new technologies has simplified the identification of novel coronaviruses; the next major goals will be to understand viral pathogenesis and to design effective coronavirus vaccines and therapies. ​ Figure 5 Inefficient activation of the Type 1 IFN response, and immunopathological disease, in coronavirus infections Go to: Acknowledgments Supported in part by research (PO1 AI060699 and RO1 NS36592) and training (T32 AI007533) grants from the National Institutes of Health (USA). Go to: Glossary Collaborative Cross Mice A panel of 1000 recombinant inbred mouse strains derived from 8 genetically diverse founder strains. The crosses were designed for complex trait analysis and will be useful for identifying and establishing the role of host genes in SARS pathogenesis Granuloma A collection of modified macrophages that resemble epithelial cells and is usually surrounded by a layer of lymphocytes that may include multinucleated giant cells. Observed in many chronic inflammatory conditions Prothrombinase Molecule that cleaves thrombin, thereby initiating coagulation cascade Primase (nsp 8) RNA-dependent RNA polymerase that produces RNA primers required for initiation of RNA synthesis by main viral RNA polymerase (nsp12) Double membrane vesicle (DMV) Structure observed in electron micrographs of infected cells that is believed to be site of virus replication Go to: Biographies • Stanley Perlman: Stanley Perlman is a professor of microbiology and of pediatrics at the University of Iowa, USA. He received a Ph.D. in biophysics from the Massachusetts Institute of Technology and an M.D. from the University of Miami. After completing clinical training in pediatrics and infectious diseases, he began studies of the pathogenesis of neurotropic strains of murine coronaviruses. His current work continues these studies and also uses molecular virology and mouse models to probe the host response to the SARS-coronavirus. • Jason Netland: Jason Netland received his Ph.D. in Immunology from the University of Iowa and is now pursuing postdoctoral work at the University of Washington, USA. His Ph.D thesis entailed studies of SARS-coronavirus replication in mice. His interests are in the immune response to viral pathogens. Go to: References 1. Peiris JS, Guan Y, Yuen KY. Severe acute respiratory syndrome. Nat Med. 2004;10:S88–97. [PubMed] 2. Barcena M, et al. Cryo-electron tomography of mouse hepatitis virus: Insights into the structure of the coronavirion. Proc Natl Acad Sci U S A. 2009;106:582–7. This paper and ref 3 use novel methodologies to obtain detailed models of the structure of the intact coronavirion. [PMC free article] [PubMed] 3. Neuman BW, et al. 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