key: cord-339386-sxyeuiw1 authors: McIntosh, Kenneth; Perlman, Stanley title: 157 Coronaviruses, Including Severe Acute Respiratory Syndrome (SARS) and Middle East Respiratory Syndrome (MERS) date: 2015-12-31 journal: Mandell, Douglas, and Bennett's Principles and Practice of Infectious Diseases DOI: 10.1016/b978-1-4557-4801-3.00157-0 sha: doc_id: 339386 cord_uid: sxyeuiw1 nan The family Coronaviridae, within the order Nidovirales, contains two subfamilies, the Coronavirinae and the Torovirinae. Coronaviruses (CoVs) are a large group of viruses infecting mammals and birds and producing a wide variety of diseases. They have been divided into four genera, two of which contain viruses infecting humans (see later). All human coronaviruses (HCoVs) are primarily respiratory pathogens. During the winter of 2002 to 2003, an alarming new disease appeared, severe acute respiratory syndrome (SARS), which was quickly attributed to a new CoV, the SARS-CoV. The outbreak originated in southern People's Republic of China, with evidence that the virus was first derived from bats and was transmitted to man through an intermediate host, probably the palm civet (Paguma larvata) or raccoon dog (Nyctereutes procyonoides). [1] [2] [3] The SARS epidemic was controlled through a massive effort at case identification and containment, and the last known case occurred in mid-2004. In retrospect, the emergence of SARS is consistent with what is known about CoVs as a group: They are important pathogens in animals causing a wide variety of diseases through a wide variety of pathogenic mechanisms, and they have been noted to mutate frequently and infect new species. 4, 5 More recently, a related but different CoV producing severe respiratory disease has emerged, the Middle East respiratory syndrome coronavirus (MERS-CoV). MERS-CoV was grown in June 2012, from a sputum sample obtained from a man in Saudi Arabia who died of overwhelming pneumonia. The virus was quickly identified as a new CoV most closely related to several bat CoVs. 6 This report was followed by a number of other reports identifying a total of 537 infected individuals, all of whom had acute respiratory symptoms, severe in most, and fatal in 145 (as of May 11, 2014) . 7 Human-to-human transmission has been documented, especially in hospital settings, 7a but appears to be inefficient. In 1965, Tyrrell and Bynoe 8 cultured a virus obtained from the respiratory tract of a boy with a common cold by passage in human embryonic tracheal organ cultures. The media from these cultures consistently produced colds in volunteers. The agent was ether sensitive but not related to any known human virus. Subsequently, electron microscopy of fluids from infected organ cultures revealed particles that resembled infectious bronchitis virus of chickens. 9 At about the same time, Hamre and Procknow 10 recovered a cytopathic agent in tissue culture from medical students with colds. The prototype virus was named 229E and was found on electron microscopy to have a similar or identical morphology ( Fig. 157-1) . 9 Using techniques similar to those used by Tyrrell and Bynoe, McIntosh and colleagues 11 reported the recovery of several infectious bronchitis-like agents from the human respiratory tract, the prototype of which was named OC43 (OC for organ culture). At much the same time, mouse hepatitis virus and transmissible gastroenteritis virus of swine were shown to have the same morphology on electron microscopy. 12, 13 Shortly thereafter, the name coronavirus (the prefix corona denoting the crownlike appearance of the surface projections) was chosen to signify this new genus. 14 The number of animal CoVs quickly grew, including viruses causing diseases in rats, mice, chickens, turkeys, various other bird species, cattle, several wild ruminants, beluga whales, dogs, cats, rabbits, and pigs, with manifestations in the respiratory and gastrointestinal tracts, central nervous system (CNS), liver, reproductive tract, and others. Through sequencing and antigenicity studies, the animal and human CoVs (HCoVs) initially were divided into three groups: group 1, which • CoVs are members of the Nidovirales order, single-stranded, positive-sense RNA viruses with a large genome. They mutate and also recombine frequently. • Laboratory diagnosis is best accomplished by finding viral RNA through polymerase chain reaction. • There are no accepted effective antiviral drugs for CoVs. • Prevention is through epidemiologic methods. The SARS epidemic was halted through careful case finding, quarantine, and use of barrier precautions. 15 A CoV was independently and almost simultaneously isolated from SARS patients by several laboratories and found by sequencing to be only distantly related to previously characterized CoVs. [16] [17] [18] [19] [20] The SARS outbreak stimulated a rapid and intense public health response coordinated by the World Health Organization, and by July 2003, transmission had ceased throughout the world. Despite this effort, however, 8096 probable cases had occurred in 29 countries, with 774 deaths. 15 With the identification of the SARS-CoV, the HCoV field became much more active. Sensitive molecular methods were developed to detect RNA from viruses identical or closely related to HCoV-229E and HCoV-OC43 in the respiratory tract, and two new species were discovered: NL63, an alphacoronavirus, and HKU1, a betacoronavirus. [21] [22] [23] HCoV-NL63 was found independently by three groups, two in the Netherlands and, somewhat later, the third in New Haven, FIGURE 157-2 Phylogenetic relationships among members of the subfamily Coronavirinae. A rooted neighbor-joining tree was generated from amino-acid sequence alignments of Coronaviridae-wide conserved domains in replicase polyprotein 1 (ADRP, nsp3; Mpro, nsp5; RdRP, nsp12; Hel, nsp13; ExoN, nsp14; NendoU, nsp15; O-MT, nsp16) for 21 coronaviruses, each a representative of a currently recognized coronavirus species. Five of the six known human coronaviruses (HCoV-229E, HCoV-NL63, HCoV-HKU1, SARS-CoV, and MERS-CoV) are indicated. HCoV-OC43 is closely related to bovine coronavirus, which is shown in the figure. Equine torovirus Berne served as the outgroup. Virus names are given with strain specifications; species and genus names are in italics as per convention. The tree shows the four main monophyletic clusters, corresponding to genera Alpha-, Beta-, Gamma To be established numerous reports of CoV-like particles (CoVLPs) found by electron microscopy in human fecal matter, but these particles have been difficult to characterize further. More recent efforts to detect CoV RNA in feces using polymerase chain reaction (PCR) and primers for respiratory HCoVs have had limited success and have failed to associate CoVs with gastrointestinal disease. 28, 29 Toroviruses were, like CoVs, first described in animals. They were first detected in the feces of cattle (Breda virus) and horses (Berne virus). 30, 31 Shortly thereafter, Beards and colleagues 32 examined human fecal material and reported finding particles with a similar appearance that aggregated in the presence of antiserum to the bovine and equine viruses. The human toroviruses, like the bovine toroviruses, do not grow in tissue culture, and thus almost all existing information about them is based on electron micrographic data. Unlike animal toroviruses, 33,34 PCR-amplified torovirus RNA sequences have not been found in human stool samples. A report of genome sequences amplified from particles purified from human stool 35 was subsequently retracted and considered to reflect laboratory contamination from porcine strains. 36 At this time, there are no reports definitively showing the existence of human toroviruses. The CoV nucleic acid is RNA, approximately 30 kilobases in length, of positive sense, single stranded, polyadenylated, and infectious. The RNA, the largest known viral RNA ( Fig. 157-3) , codes for (in order from the 5′ end) a large polyprotein that is cleaved by virus-encoded Connecticut. 24 In all three cases, positive samples were from infants and children with respiratory disease. Notably, HCoV-NL63 and HCoV-229E were estimated to originate from a common precursor and diverge approximately 1000 years ago. 25 HCoV-HKU1 was found in Hong Kong in an adult with respiratory disease. These two new HCoV strains subsequently have been found worldwide and appear to have pathogenicity similar to that of HCoV-229E and HCoV-OC43, with the possible exception that NL63 is more frequently found in children with croup. 26, 27 The MERS-CoV was found when a man was admitted in June 2012 to a hospital in Jeddah, Saudi Arabia with overwhelming acute pneumonia with renal failure, and a sample of sputum grew a cytopathic virus that, on sequencing, proved to be a CoV, classified as a Betacoronavirus, and most closely related to two bat CoVs, HKU-4 and HKU-5. 6 Over the next 23 months 536 additional cases (145 fatal) were found, all but a few of them sporadic or hospital-based and in individuals living or traveling in the Middle East. In the remainder of this chapter, the group of respiratory HCoVs first discovered in the 1960s and containing HCoVs 229E, OC43, NL63, and HKU-1 are referred to as community-acquired HCoVs to distinguish them from the SARS-CoV and the MERS-CoV. In view of the prominence of CoVs in animal enteric diseases, there have been extensive efforts to identify enteric HCoVs. There are FIGURE 157-3 Genome organization of representative human coronaviruses. All coronavirus genomes have the same basic structure and mechanism of replication. 4 The 5′ end of each genome encodes a leader sequence, which is attached to each virus-specific messenger RNA transcript by a novel mechanism of discontinuous replication. The first two thirds of each genome encode replicase-associated genes. Gene 1 is translated as two large polyproteins, with the first expressed from ORF1a and the second from ORF1a/b following a −1 frameshift event. These polyproteins are then cleaved into individual proteins by two virus-encoded proteases. The major structural genes, the hemagglutinin-esterase (HE), surface (S), envelope (E), transmembrane (M), and nucleocapsid (N) proteins, are indicated in green. The nonreplicase, accessory genes located at the 3′ end of the genome are indicated with open boxes. The functions of these proteins are largely not known, and there is no sequence homology between accessory proteins of different coronaviruses. Some of these proteins are virion associated, but none are required for virus replication. The open reading frames (ORFs) encoding these proteins are numbered in order of appearance from the 5′ end of the genome, with the exception that ns12.9 of HCoV-OC43. I is an internal protein expressed from an alternative reading frame located within the N gene. It is equivalent to SARS-CoV-specific protein 9b and the MERS-CoV-specific protein 8b. (Figure prepared by 4 (DPP-4), which, like ACE2 and APN, is an ectopeptidase that is abundantly expressed in the respiratory tract. 46 All the community-acquired respiratory HCoVs grow only with difficulty in tissue culture. Despite this, both 229E and NL63 were discovered because they produced a detectable cytopathic effect, the first in human embryonic kidney 10 and the second in LLC-MK2 cells. 21 Both the SARS-CoV and the MERS-CoV were initially isolated and grew readily in Vero cells. 6, 17 HCoVs OC43 and HKU-1 have been grown in tissue culture after laboratory adaptation or in primary human airway epithelial cells. [47] [48] [49] Detection of all these viruses in clinical specimens is most conveniently and sensitively achieved using PCR. Likewise, the enteric CoVs have been difficult to cultivate in vitro. All but a few strains have been detected only by electron microscopy of human fecal material. [50] [51] [52] 53, 54 Some strains have been characterized by immune electron microscopy and found to be related to HCoV-OC43. 55 Two strains obtained from an outbreak of necrotizing enterocolitis in Texas and passaged in intestinal organ cultures were reported to contain four or five proteins with apparent molecular weights similar to those of other CoVs but not related antigenically to known human strains or mouse hepatitis virus A59. 56 The evidence favors the view that these isolates, as well as particles antigenically related to HCoV-OC43, are members of the family Coronaviridae, although their association with human disease is not yet proven. Surveys of children with diarrhea using PCR imply that diarrhea associated with the four well-described HCoV strains is unusual. 28, 29 EPIDEMIOLOGY Evidence of community-acquired respiratory CoV infections has been found wherever in the world it has been sought. In temperate climates, respiratory CoV infections occur more often in the winter and spring than in the summer and fall. The contribution of CoV infections to the total number of upper respiratory illnesses may be as high as 35% during times of peak viral activity. Overall, the proportion of adult colds produced by CoVs may be reasonably estimated at 15%. Early studies of HCoV-OC43 and 229E in the United States demonstrated periodicity, with large epidemics occurring at 2-to 3-year intervals. 57 Strain HCoV-229E tended to be epidemic throughout the United States, whereas strain HCoV-OC43 appeared in localized outbreaks. Similar studies of NL63 and HKU1 have not been done, but it seems from the available data that they also vary widely in incidence from year to year and place to place. Reinfection is common and may be due to the rapid diminution of antibody levels after infection. 58 Infection occurs at all ages but is most common in children. The ratio of symptomatic to total infections varies between 50% and 90%, depending on the age of the population studied, the method of virus detection, and the definition of "infection. " 59, 60 Among adult volunteers 72% of those infected with HCoV-229E developed colds. 61 The first report of a new CoV causing severe pneumonia appeared in ProMed Mail on September 20, 2012. A man from Jeddah, Saudi Arabia had developed pneumonia in June and died of respiratory and renal failure, and a virus was grown from a sputum sample that was subsequently sequenced and found to be a betacoronavirus thought at the time to be most closely related to bat CoVs HKU4 and HKU5. 6 Between then and May 2014, a total of 537 cases occurred, all infected by this virus, now termed the Middle East respiratory syndrome coronavirus (MERS-CoV). One hundred forty-five of the cases were fatal. 7 More than 400 of these have been acquired and diagnosed in the Kingdom of Saudi Arabia, with most of the remainder in the United Arab Emirates, Qatar, Jordan, and Kuwait. Cases originating in the Arabian peninsula have also occurred in travelers to Egypt, Tunisia, Germany, Italy, Great Britain, Malaysia, the Philippines, and the United States, with a few secondary cases sometimes occurring in those locations through close family or hospital spread. In the United States,these include two unrelated MERS cases, and a third case related to one of these. 61a While infections with severe respiratory involvement have proteases to form several nonstructural proteins, including an RNAdependent RNA polymerase, methyltransferases, and a helicase, followed by either four or five structural proteins intermingled with a variable number of nonstructural and minor structural proteins. 37 The first of the major structural proteins is a surface hemagglutinin-esterase protein, present on HCoVs OC43 and HKU1 and some animal betacoronaviruses, that may play some role in the attachment or release of the particle, or both, at the cell surface. This gene contains sequences similar to the hemagglutinin of influenza C virus, likely evidence of an interfamily recombinational event that occurred many years ago. The next gene encodes the surface glycoprotein that forms the petal-shaped surface projections and is responsible for attachment and the stimulation of neutralizing antibody. This is followed by a small envelope protein, a membrane glycoprotein, and a nucleocapsid protein that is complexed with the RNA. There are several other open reading frames whose coding functions are not clear. The strategy of replication of CoVs is similar to that of other nidoviruses, in that all messenger RNAs form a nested set with common polyadenylated 3′ ends, with only the unique portion of the 5′ end being translated. 4 As in other RNA viruses, mutations are common in nature, although the mutation rate is much lower, approximately 2 × 10 −6 per site per replication cycle. 38 Unlike other RNA viruses, CoVs encode a 3′-5′ exonuclease that has proofreading activities, playing a critical role in maintaining replication fidelity in cell cultures and in animals. 39 CoVs are also capable of genetic recombination if two viruses infect the same cell at the same time. All CoVs develop exclusively in the cytoplasm of infected cells (Fig. 157-4) . They bud into cytoplasmic vesicles from membranes of the pre-Golgi endoplasmic reticulum. These virus-filled vesicles are then extruded by the exocytic secretory pathway. 40 The resultant virus particles have a diameter of 70 to 80 nm on thin-section electron microscopy and 60 to 220 nm on negative staining. They are pleomorphic, with widely spaced, petal-shaped projections 20 nm long (see Fig. 157-1) . The cellular receptor for 229E and most other alphacoronaviruses is aminopeptidase N (APN). 41 Interestingly, NL63, the other known human alphacoronavirus, uses as its cellular receptor angiotensinconverting enzyme II (ACE2), 42 the same receptor as is used by the SARS-CoV. 43 Mouse hepatitis virus, a betacoronavirus related to strain OC43, uses as its receptor a member of the carcinoembryonic antigen family. 44 HCoV-OC43 and BCoV, which is closely related to HCoV-OC43, bind to 9-O-acetylated neuraminic acid as part of the entry process. 45 The host cell receptor for MERS-CoV is dipeptidyl peptidase as 50%. There was no mortality in children or young adults younger than the age of 24 years. In response to the global spread and associated severe disease, the World Health Organization coordinated a rapid and effective control program that included isolation of cases, careful attention to contact, droplet and airborne infection control procedures, quarantine of exposed persons in some settings, and efforts to control spread between countries through travel advisories and travel alerts. Presumably as a result of these efforts, global transmission ceased by July 2003. A few subsequent cases of SARS were detected, but all were either a result of laboratory spread or individual cases related to presumed contact with civet cats or other intermediate hosts. The last known case occurred in mid-2004. Spread of SARS to humans is thought to have occurred primarily through droplet or contact transmission, with a possible role for fomites. In most instances, an individual case transmitted to very few others, although several well-documented instances of small-particle airborne transmission occurred, resulting in super-spreading events. 67 Spread in hospital settings appeared to be surprisingly efficient, but it could be effectively suppressed with the enforcement of droplet and contact precautions. 68 Containment measures were efficacious, in part, because patients were most contagious only after lower respiratory disease developed. 69 The chain of spread was finally broken in the People's Republic of China, the last country to experience endemic spread, in June 2003. It now seems almost certain that the human epidemic began with the spread of a closely related bat virus first to palm civets or other animals sold in live wild game markets and then to humans in Guangdong Province in the People's Republic of China, and that the virus adapted itself through mutation and possibly recombination, until it transmitted readily among humans. [70] [71] [72] The virus that spread worldwide came largely from a single infected individual who traveled from occurred at all ages, the elderly and those with underlying conditions (diabetes, renal disease, immunosuppression) have been most often severely or fatally affected. The WHO and the CDC have published a case definition, as well as surveillance instructions to aid in epidemiologic control of the MERS-CoV. 62, 63 The putative bat origin of this virus was strengthened by the finding that the virus grew readily in primary bat tissue culture. 64 Nevertheless, and while bats sampled in the Middle East, Africa, and Europe were found to carry viruses closely related to the MERS-CoV, 65 epidemiologic studies suggested there was likely to be at least one intermediate host. Serologic and virologic studies indicated that camels in the Middle East and Africa were frequently infected by viruses very similar to some of those found in human MERS cases. 65a Acquisition of MERS from camels appears likely, although the proportion of camel-acquired cases (versus those acquired from person-to-person contact or through another animal intermediate) is not clear. Case clusters indicate that person-to-person hospital spread is more common than spread within families, and that casual-contact spread is unusual. 7a The SARS epidemic began in Guangdong Province in the People's Republic of China in mid-November 2002. 66 It came to worldwide attention in March 2003 when cases of severe, acute pneumonia were reported to the World Health Organization from Hong Kong, Hanoi, and Singapore. Disease spread in hospitals to health care workers, visitors, and patients, among family members, and, on occasion, in hotels, apartment complexes, markets, and airplanes. Worldwide spread was rapid but focal ( Fig. 157-5) . The largest numbers of cases were reported from the People's Republic of China, Hong Kong, Taiwan, Singapore, and Toronto, Canada. The overall case-fatality rates in these locations ranged from 7% to 17%, but persons with underlying medical conditions and those older than 65 years of age had mortality rates as high involvement in other organ systems, including diarrhea, leukopenia, thrombocytopenia, and, most notably, pan-lymphopenia. 83 Virus has been detected in respiratory secretions, blood, stool, and urine specimens and tissue from the lung and kidney. On the basis of PCR testing, virus titer is highest during the second week of illness 84 and can often be detected into the third week of illness and sometimes for as long as several months. 18, 85 Pulmonary symptoms may worsen late in the course of the illness, with the development of adult respiratory distress syndrome. 84 There may also be late evidence of liver and kidney involvement. The pulmonary pathology of infection by the SARS-CoV has been described extensively, 17,86,87 but little has been published about the pathology in other organ systems. [87] [88] [89] The extrapulmonary pathologic changes found most consistently at autopsy are extensive necrosis of the white pulp of the spleen and a generalized small vessel arteritis. 87, 88 In the lung, there is hyaline membrane formation, interstitial infiltration with lymphocytes and mononuclear cells, and desquamation of pneumocytes in the alveolar spaces. Giant cells are a constant finding and usually have macrophage markers. In bronchoalveolar lavage, biopsy, and autopsy specimens viral particles have been noted in type I and II pneumocytes. 90 At this point, little is known about the pathogenesis of MERS-CoV because the infection was only recently described and no pathological specimens are yet available. It is anticipated that the pathologic changes in the lungs of patients with severe disease will be similar to those observed in patients with SARS or other patients with acute respiratory distress syndrome (ARDS). Almost all the antigenically distinct respiratory CoV strains that were isolated in the 1960s have been administered to volunteers, and all these produce illness with similar characteristics. 8,91 A summary of these characteristics is given in Table 157 -1, in which a comparison is made with colds produced by rhinoviruses in similarly inoculated volunteers. The incubation period of CoV colds was longer and their duration somewhat shorter, but the symptoms were similar. Asymptomatic infection was sometimes seen and, indeed, has been a feature Guangdong Province to Hong Kong and infected a large number of individuals before himself succumbing to the disease. In contrast, the virus that was epidemic in the People's Republic of China was more variable. Although an etiologic role is not proven, enteric CoVs (or CoVLPs) have been most frequently associated with gastrointestinal disease in neonates and infants younger than 12 months. Particles have been found in the stools of adults with the acquired immunodeficiency syndrome. 73, 74 Asymptomatic shedding is common, particularly in tropical climates 75 and in populations living in poor hygienic conditions. 76 The viruses can be detected for prolonged periods 51,53,77 and without any apparent seasonal pattern. 78 Community-acquired respiratory CoVs (HCOV-229E, OC43, NL63, HKU-7) generally replicate in ciliated and nonciliated (HCoV-229E) epithelial cells of the nasopharynx, 79 probably producing both direct cell degeneration 80 and an outpouring of chemokines and interleukins, with a resultant common-cold symptom complex similar to that produced by rhinovirus infection. 81 The incubation period is, on average, 2 days, and the peak of respiratory symptoms, as well as viral shedding, is reached at approximately 3 or 4 days after inoculation. 61 The pattern of virus replication of CoVs must be at least in part determined by virus-receptor interaction. The two best-defined receptors for the respiratory CoVs are aminopeptidase N for strain HCoV-229E and angiotensin-converting enzyme II for NL63. The pathogenicity of SARS is more complex and involves systemic spread. The route of infection of the SARS-CoV is probably through the respiratory tract. After an incubation period that is usually 4 to 7 days, but can be as long as 10 to 14 days, the disease begins, starting usually with fever and other systemic (influenza-like) symptoms, with cough and dyspnea developing a few days to a week later. 82 Although the lung is the focus of the disease process, there are often signs of levels decreased, suggesting that disease was partly immune mediated. 84 Clinical improvement was associated with the onset of a virusspecific antibody response. 119 Pediatric disease was, interestingly, significantly less severe than adult disease, although the features were similar. 121 Disease during pregnancy was severe, with high mortality in both the mother and fetus. 122 Congenital transmission was not described. The nature of the illness associated with enteric CoV infection is much less clear. One study found a significant association of gastroenteritis in infants 2 to 12 months of age with the presence of CoVLPs in the stool. 55 Another study, confined to infants in a neonatal intensive care unit, found highly significant associations between the presence of CoVLPs in the stool and the presence of water-loss stools, bloody stools, abdominal distention, and bilious gastric aspirates. 53 A further study of symptomatic infants shedding CoVLPs pointed to possible differences between CoVLP-associated diarrhea and rotavirus diarrhea: Although fever and vomiting were of similar incidence, stools were more often occult blood positive (18% in CoVLP-associated vs. 0% in rotavirus-associated disease), less often watery (66% vs. 92%), and more often mucoid (32% vs. 8%). 77 Finally, CoVs have been associated with at least three outbreaks of necrotizing enterocolitis in newborns, 52, 53, 56 and the best characterized strains 56 were isolated in infants with this illness. Surveys seeking HCoV RNA by PCR using primers that would detect the known community-acquired respiratory HCoVs in stool have been quite disappointing. In one study of 878 fecal samples from children with gastrointestinal complaints tested over 2 years, all four HCoV species were found, but in all but 4 of 22 HCoV-positive cases, either rotavirus or norovirus was also present. 28 In addition, about half of the children in this survey had respiratory and gastrointestinal symptoms. In the same study, 112 asymptomatic children were sampled and 2 were positive. Another study sampled 151 symptomatic children, and 2 were found to have HKU1 RNA. 29 Molecular studies using primers that would broadly detect new CoVs should be considered to resolve questions about the role of CoVs in human gastrointestinal disease. 123 Like many other viruses, CoVs have been sought as possible etiologic agents in multiple sclerosis. The search has been stimulated by the capacity of JHM, a well-studied strain of mouse hepatitis virus, to produce in mice and rats an immune-mediated chronic demyelinating encephalitis histologically similar to multiple sclerosis. 124 HCoV-OC43 125, 126 and HCoV-229E 127 have been detected in brain tissue from multiple sclerosis patients using virus isolation, 125 in situ hybridization, immunohistology, 126 and PCR. 127 Moreover, T-cell lines established from patients with multiple sclerosis by stimulation with myelin basic protein or HCoV-229E were found to be cross-reactive with the opposite antigen, suggesting that molecular mimicry might be a possible pathogenic mechanism for the disease association. 128 An adolescent boy with acute demyelinating encephalitis was found to have HCoV-OC43 RNA in both the respiratory tract and the cerebrospinal fluid. 129 Despite these intriguing reports, compelling evidence is lacking to establish an etiologic or pathogenetic association of CoVs with CNS disease in humans. Although some human respiratory CoVs grow in tissue culture directly from clinical samples and although antigen detection systems have been developed for both HCoV-OC43 and HCoV-229E, 130, 131 laboratory diagnosis of CoV respiratory infections is best accomplished by molecular methods. Reverse-transcriptase PCR (RT-PCR) systems have been developed using many different primers and detectors. From a clinical point of view, a single generic test for respiratory CoVs would be desirable, and such tests have been developed. However, when tested side by side with specific systems, the generic systems have a somewhat lower sensitivity. 95 Systems that combine primers and probes specific for several CoVs have also had considerable success. 132 of both serologic surveys and PCR-based studies of natural infection of infants, children, and adults. 92, 93 More serious respiratory tract illness is probably also caused by all four strains of community-acquired HCoV. The evidence for this is not conclusive, but it seems likely that all strains can produce pneumonia and bronchiolitis in infants, 24, 27, 94, 95 otitis and exacerbations of asthma in children and young adults, [96] [97] [98] pneumonia in healthy adults, 99 exacerbations of asthma and chronic bronchitis in adults, 100, 101 both serious bronchitis and pneumonia in the elderly, 102, 103 and pneumonia in the immunocompromised host. 104, 105 HCoVs are found in asymptomatic individuals of all ages, and, when accompanied by illness, are also sometimes accompanied by infections with other potential respiratory pathogens. These characteristics (infection without disease, coinfection during disease) are features of many respiratory pathogens, including particularly rhinoviruses, adenoviruses, human metapneumovirus, human bocavirus, and parainfluenza viruses, but also (although less frequently) respiratory syncytial virus and influenza virus. Because infections with respiratory HCoVs are so common, however, it is possible that they are responsible for a significant portion of these serious lower respiratory tract diseases, even though the basic pathogenicity of HCoVs (judging from volunteer studies) is similar to that of rhinoviruses, and clearly less than that of respiratory syncytial virus, influenza viruses, and certain adenovirus types. There is some evidence that HCoV-OC43 is more pathogenic in the elderly than HCoV-229E 106 and also some evidence that infection with NL63 in children is different from the other respiratory HCoVs in that several series have found an excess of children with croup. 26, 27 Information about the clinical presentation of patients infected with the MERS-CoV is limited. It is clear that there is a spectrum of illness with some infections consisting of mild upper respiratory symptoms only, and others characterized by cough and fever with progression to respiratory failure over about a week. 6, 7, 62, 107 Renal failure, as well as pericarditis and adult respiratory distress syndrome has been part of the reported clinical picture. The MERS-CoV host cell receptor, DPP-4, is expressed at high levels in the kidney, 108 raising the possibility that direct infection of this organ contributes to renal disease. A case definition that will lead to further epidemiologic studies has been published by the World Health Organization. 109 Severe Acute Respiratory Syndrome Coronavirus The first symptom in most cases of SARS was fever, usually accompanied by headache, malaise, or myalgia. This was followed, usually in a few days, but as long as a week later, by a nonproductive cough and, in more severe cases, dyspnea. Approximately 25% of patients had diarrhea. Interestingly, upper respiratory symptoms such as rhinorrhea and sore throat usually did not occur. 82, 84, [110] [111] [112] [113] The chest radiograph was frequently abnormal, showing scattered air-space opacification, usually in the periphery and lower zones of the lung. 114 Spiral computed tomography demonstrated both ground-glass opacification and consolidation, often in a subpleural distribution. [115] [116] [117] Lymphopenia was common, 84, 86, 110 with normal or somewhat depressed neutrophils. Paradoxically, neutrophilia was associated with poor outcomes. 83 The decrease in lymphocytes in the blood was most marked for CD4 cells but was seen in all T-cell phenotypes, including CD3 and CD8, as well as natural killer cells. Creatine kinase was often abnormal, as were lactic dehydrogenase and aspartate aminotransferase. Levels of proinflammatory cytokines were elevated at early times during infection in patients with severe clinical disease 118 and decreased in those patients who resolved the infection. 119 Approximately 25% of patients developed severe pulmonary disease that progressed to adult respiratory distress syndrome. Adult respiratory distress syndrome with SARS-CoV infection was most likely to develop in patients older than 50 years or with underlying disease such as diabetes, cardiac disease, and chronic hepatitis. 84, 110, 111, 120 The overall mortality rate was between 9% and 12%, with the highest rates in the elderly and adults with underlying liver disease. In some patients, clinical deterioration occurred during the second week of illness, as virus ribavirin. 134 It is now known that ribavirin has little activity against SARS-CoV in vitro, and there is no evidence that either intervention improved outcomes. 135, 136 Lopinavir/ritonavir and intravenous immune globulin were also used in some patients, again without conclusive evidence that they were helpful or harmful. There is anecdotal and at least partially controlled evidence of the benefit of either interferon-α or interferon-β treatment. Further, treatment of SARS-CoV-infected cynomolgus monkeys with pegylated interferon-α resulted in improved outcomes, 137 lending credence to the use of this therapy if SARS recurred. Treatment of MERS-CoV infection depends entirely on supportive measures. No antiviral drugs are recommended, although several studies have indicated that MERS-CoV is more sensitive to interferonα or interferon-β than SARS-CoV. 138, 139 Standard droplet precautions should be used, with aerosol precautions during certain high-risk procedures. 109 Rigorous application of hospital infection control procedures, particularly those directed at contact and droplet spread, was shown to have a major beneficial effect on the spread of the SARS-CoV. 68 The containment of the global SARS outbreak is a testament to the power of the cooperation and collaboration engendered by the World Health Organization to address a major public health threat. Similar precautions are recommended for patients with suspected or confirmed MERS-CoV infections. 109 Vaccines for animal CoVs have been developed and widely used with variable efficacy. In one instance, a vaccine for feline infectious peritonitis appeared to lead to enhanced disease with subsequent natural infection. If SARS does return or MERS reaches epidemic proportions, an effective vaccine would be extremely helpful in control efforts, and a variety of vaccination strategies, including inactivated, subunit, and live-attenuated vaccines, are being pursued. 135, 140 In addition, hospitals have been advised on improvement of infection control procedures so that in future epidemics of respiratory viruses, they will not be a major source of spread of infection, as occurred in the 2002 epidemic of SARS. The MERS-CoV was originally isolated in Vero and LLC-MK2 cells, and there are several published methodologies for detection and identification by PCR. 133 Current recommendations from WHO are that definition of a possible case should be immediately reported to national authorities, and clinical, epidemiologic, and microbiologic investigations should be carried out. 63 Although SARS-CoV was grown from respiratory tract specimens in Vero E6 and fetal rhesus monkey kidney cells, the more sensitive and rapid RT-PCR assays were most widely used to detect infection. Virus was detected by RT-PCR in upper and lower respiratory tract, blood, stool, and urine specimens. Early in the illness, specimens were found positive only in approximately one third of patients. 84 Use of samples from multiple sources increased the yield. Virus was detected most frequently during the second week of illness. 18, 84, 85 Antibody tests have been developed using tissue culture-grown virus and indirect immunofluorescence or enzyme-linked immunosorbent assay. Immunoglobulin M antibody can be detected in most patients for a limited period of time, and immunoglobulin G antibody appears first approximately 10 days after onset of fever in patients with good outcomes and becomes essentially universal after 4 weeks. 84 Laboratory diagnosis of the gastrointestinal CoVs depends now entirely on electron microscopy of stool specimens and detection of characteristic particles in negatively stained specimens. Such testing is best performed in laboratories with extensive previous experience. Given the severity of SARS, clinicians throughout the world empirically treated most patients with corticosteroids and intravenous or oral The complete reference list is available online at Expert Consult. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China Coronaviruses post-SARS: update on replication and pathogenesis Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia Hospital outbreak of Middle East respiratory syndrome coronavirus Cultivation of a novel type of common-cold virus in organ cultures The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture A new virus isolated from the human respiratory tract Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease Severe acute respiratory syndrome Coronavirus as a possible cause of severe acute respiratory syndrome A novel coronavirus associated with severe acute respiratory syndrome Identification of a novel coronavirus in patients with severe acute respiratory syndrome Characterization of a novel coronavirus associated with severe acute respiratory syndrome Identification of a new human coronavirus A previously undescribed coronavirus associated with respiratory disease in humans Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia Mosaic structure of human coronavirus NL63, one thousand years of evolution Croup is associated with the novel coronavirus NL63 Detection of human coronaviruses in children with acute gastroenteritis Unique and conserved features of genome and proteome of SARScoronavirus, an early split-off from the coronavirus group 2 lineage Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing Morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229E) Human aminopeptidase N is a receptor for human coronavirus 229E Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC Association of coronavirus infection with neonatal necrotizing enterocolitis Medical reviews: coronaviruses A prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection Effects of a "new" human respiratory virus in volunteers Available at www.cdc .gov.coronavirus/MERS/INDEX Middle East Respiratory Syndrome (MERS): Case Definitions Interim Surveillance Recommendations for Human Infection with Middle East Respiratory Syndrome Coronavirus Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe Middle East respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in Saudi Arabia Effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (SARS) A review of studies on animal reservoirs of the SARS coronavirus Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China Clinical progression and viral load in a community outbreak of coronavirusassociated SARS pneumonia: a prospective study Multiple organ infection and the pathogenesis of SARS Time course and cellular localization of SARS-CoV nucleoprotein and RNA in lungs from fatal cases of SARS Role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction Respiratory viruses and exacerbations of asthma in adults A prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection Clinical impact of human coronaviruses 229E and OC43 infection in diverse adult populations Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome Clinical disease in children associated with newly described coronavirus subtypes SARS: systematic review of treatment effects The spike protein of SARS-CoVa target for vaccine and therapeutic development Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats Evolutionary insights into the ecology of coronaviruses Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China Coronaviruses post-SARS: update on replication and pathogenesis Isolation of a novel coronavirus from a man with pneumonia in Saudi Arabia Middle East Respiratory Syndrome Coronavirus (MERS-CoV) Summary and Literature Update-as of 9 Hospital outbreak of Middle East respiratory syndrome coronavirus Cultivation of a novel type of common-cold virus in organ cultures The morphology of three previously uncharacterized human respiratory viruses that grow in organ culture A new virus isolated from the human respiratory tract Recovery in tracheal organ cultures of novel viruses from patients with respiratory disease Growth in suckling-mouse brain of "IBV-like" viruses from patients with upper respiratory tract disease Morphologic characteristics and nucleic acid type of transmissible gastroenteritis virus of pigs Severe acute respiratory syndrome Coronavirus as a possible cause of severe acute respiratory syndrome A novel coronavirus associated with severe acute respiratory syndrome Identification of a novel coronavirus in patients with severe acute respiratory syndrome The genome sequence of the SARS-associated coronavirus Characterization of a novel coronavirus associated with severe acute respiratory syndrome Identification of a new human coronavirus A previously undescribed coronavirus associated with respiratory disease in humans Characterization and complete genome sequence of a novel coronavirus, coronavirus HKU1, from patients with pneumonia Evidence of a novel human coronavirus that is associated with respiratory tract disease in infants and young children Mosaic structure of human coronavirus NL63, one thousand years of evolution Croup is associated with the novel coronavirus NL63 The association of newly identified respiratory viruses with lower respiratory tract infections in Korean children Detection of human coronaviruses in children with acute gastroenteritis Human coronaviruses are uncommon in patients with gastrointestinal illness Studies with an unclassified virus isolated from diarrheic calves Purification and partial characterization of a new enveloped RNA virus (Berne virus) An enveloped virus in stools of children and adults with gastroenteritis that resembles the Breda virus of calves Identification and characterization of a porcine torovirus Phylogenetic and evolutionary relationships among torovirus field variants: evidence for multiple intertypic recombination events Characterization of torovirus from human fecal specimens Notice of retraction to "The novel hemagglutinin-esterase genes of human torovirus and Breda virus Unique and conserved features of genome and proteome of SARScoronavirus, an early split-off from the coronavirus group 2 lineage Infidelity of SARS-CoV Nsp14-exonuclease mutant virus replication is revealed by complete genome sequencing A live, impaired-fidelity coronavirus vaccine protects in an aged, immunocompromised mouse model of lethal disease Morphogenesis of avian infectious bronchitis virus and a related human virus (strain 229E) Human aminopeptidase N is a receptor for human coronavirus 229E Human coronavirus NL63 employs the severe acute respiratory syndrome coronavirus receptor for cellular entry Angiotensin-converting enzyme 2 is a functional receptor for the SARS coronavirus Receptor for mouse hepatitis virus is a member of the carcinoembryonic antigen family of glycoproteins Crystal structure of mouse coronavirus receptor-binding domain complexed with its murine receptor Dipeptidyl peptidase 4 is a functional receptor for the emerging human coronavirus-EMC The adaptation of two human coronavirus strains (OC38 and OC43) to growth in cell monolayers Detection of the new human coronavirus HKU1: a report of 6 cases Culturing the unculturable: human coronavirus HKU1 infects, replicates, and produces progeny virions in human ciliated airway epithelial cell cultures Pleomorphic virus-like particles in human faeces Chronic enterocyte infection with coronavirus: one possible cause of the syndrome of tropical sprue? Association of coronavirus infection with neonatal necrotizing enterocolitis Pleomorphic, enveloped, virus-like particles associated with gastrointestinal illness in neonates Human enteric coronaviruses: antigenic relatedness to human coronavirus OC43 and possible etiologic role in viral gastroenteritis Isolation and propagation of a human enteric coronavirus Medical reviews: coronaviruses The time course of the immune response to experimental coronavirus infection of man A prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection Human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls Effects of a "new" human respiratory virus in volunteers Available at www.cdc.gov. coronavirus/MERS/INDEX Middle East Respiratory Syndrome (MERS): Case Definitions Interim Surveillance Recommendations for Human Infection with Middle East Respiratory Syndrome Coronavirus Human coronavirus EMC does not require the SARS-coronavirus receptor and maintains broad replicative capability in mammalian cell lines Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe Middle East respiratory syndrome coronavirus quasispecies that include homologues of human isolates revealed through whole-genome analysis and virus cultured from dromedary camels in Saudi Arabia Description and clinical treatment of an early outbreak of severe acute respiratory syndrome (SARS) in Guangzhou, PR China Severe acute respiratory syndrome-Singapore Effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (SARS) Viral loads in clinical specimens and SARS manifestations A review of studies on animal reservoirs of the SARS coronavirus Cross-host evolution of severe acute respiratory syndrome coronavirus in palm civet and human Molecular evolution of the SARS coronavirus during the course of the SARS epidemic in China Detection of coronavirus-like particles in homosexual men with acquired immunodeficiency and related lymphadenopathy syndrome Stool viruses, coinfections, and diarrhea in HIV-infected patients. Berlin Diarrhea/Wasting Syndrome Study Group Coronaviruslike particles and other agents in the faeces of children in Efate, Vanuatu Coronavirus-like particles in adults in Melbourne, Australia Coronaviruslike particles in human gastrointestinal disease: epidemiologic, clinical, and laboratory observations An eight-year study of the viral agents of acute gastroenteritis in humans: ultrastructural observations and seasonal distribution with a major emphasis on coronavirus-like particles Isolation and characterization of current human coronavirus strains in primary human epithelia cultures reveals differences in target cell tropism Ultrastructure of human nasal epithelium during an episode of coronavirus infection Signs and symptoms in common colds Epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in Hong Kong Haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis Clinical progression and viral load in a community outbreak of coronavirusassociated SARS pneumonia: a prospective study Detection of SARS-CoV RNA in stool samples of SARS patients by nest RT-PCR and its clinical value A major outbreak of severe acute respiratory syndrome in Hong Kong Lung pathology of fatal severe acute respiratory syndrome The clinical pathology of severe acute respiratory syndrome (SARS): a report from China Multiple organ infection and the pathogenesis of SARS Time course and cellular localization of SARS-CoV nucleoprotein and RNA in lungs from fatal cases of SARS Antigenic relationships amongst coronaviruses A case-control study of acute respiratory tract infection in general practice patients in The Netherlands Role of respiratory viruses in acute upper and lower respiratory tract illness in the first year of life: a birth cohort study Coronavirus infection in acute lower respiratory tract disease of infants Genetic variability of human coronavirus OC43-, 229E-, and NL63-like strains and their association with lower respiratory tract infections of hospitalized infants and immunocompromised patients The association of viral and bacterial respiratory infections with exacerbations of wheezing in young asthmatic children Recurrent wheezy bronchitis and viral respiratory infections Detection of rhinovirus, respiratory syncytial virus, and coronavirus infections in acute otitis media by reverse transcriptase polymerase chain reaction Coronavirus infections in military recruits: three-year study with coronavirus strains OC43 and 229E Respiratory viruses and exacerbations of asthma in adults Pan-viral screening of respiratory tract infections in adults with and without asthma reveals unexpected human coronavirus and human rhinovirus diversity Rhinovirus and coronavirus infection-associated hospitalizations among older adults A prospective, community-based study on virologic assessment among elderly people with and without symptoms of acute respiratory infection Coronavirus 229E-related pneumonia in immunocompromised patients Clinical impact of community-acquired respiratory viruses on bronchiolitis obliterans after lung transplant Clinical impact of human coronaviruses 229E and OC43 infection in diverse adult populations The United Kingdom public health response to an Dipeptidyl-peptidase IV from bench to bedside: an update on structural properties, functions, and clinical aspects of the enzyme DPP IV World Health Organization. Global Alert and Response (GAR): Infection Prevention and Control Clinical features and short-term outcomes of 144 patients with SARS in the greater Toronto area Acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome Severe acute respiratory syndrome in children: Experience in a regional hospital in Hong Kong Severe acute respiratory syndrome among children Severe acute respiratory syndrome: radiographic appearances and pattern of progression in 138 patients Imaging of severe acute respiratory syndrome in Hong Kong Severe acute respiratory syndrome: radiographic and CT findings Thin-section CT of severe acute respiratory syndrome: evaluation of 73 patients exposed to or with the disease Early enhanced expression of interferon-inducible protein-10 (CXCL-10) and other chemokines predicts adverse outcome in severe acute respiratory syndrome Interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome Critically ill patients with severe acute respiratory syndrome Clinical presentations and outcome of severe acute respiratory syndrome in children Severe acute respiratory syndrome and pregnancy Viral discovery and sequence recovery using DNA microarrays Corona virus induced subacute demyelinating encephalomyelitis in rats: a morphological analysis Two coronaviruses isolated from central nervous system tissue of two multiple sclerosis patients Detection of coronavirus RNA and antigen in multiple sclerosis brain Human coronavirus gene expression in the brains of multiple sclerosis patients Long-term human coronavirus-myelin cross-reactive T-cell clones derived from multiple sclerosis patients Detection of coronavirus in the central nervous system of a child with acute disseminated encephalomyelitis Diagnosis of human coronavirus infection by immunofluorescence: method and application to respiratory disease in hospitalized children Surveillance of community-acquired viral infections due to respiratory viruses in Rhone-Alpes (France) during winter 1994 to 1995 Clinical disease in children associated with newly described coronavirus subtypes Detection of a novel human coronavirus by real-time reversetranscription polymerase chain reaction Development of a standard treatment protocol for severe acute respiratory syndrome Treatment and vaccines for severe acute respiratory syndrome SARS: systematic review of treatment effects Pegylated interferon-alpha protects type 1 pneumocytes against SARS coronavirus infection in macaques Efficient replication of the novel human betacoronavirus EMC on primary human epithelium highlights its zoonotic potential Human cell tropism and innate immune system interactions of human respiratory coronavirus EMC compared to those of severe acute respiratory syndrome coronavirus The spike protein of SARS-CoVa target for vaccine and therapeutic development