The epidemic of severe atypical pneumonia which was observed in the Chinese province of Guangdong and reported internationally on February 11, 2003 (WHO, WER 11/2003), was initially suspected to be linked to a newly emerging influenza virus: on February 19, 2003, researchers isolated an avian influenza A (H5N1) virus from a child in Hong Kong. This virus was similar to the influenza virus originating from birds that caused an outbreak in humans in Hong Kong in 1997, and new outbreaks of similar strains were expected. However, bird 'flu', possibly of poultry origin, was soon ruled out as the cause of the newly-termed Severe Acute Respiratory Syndrome, or SARS.

Investigations then focused on members of the Paramyxoviridae family, after paramyxovirus-like particles were found by electron microscopy of respiratory samples from patients in Hong Kong and Frankfurt am Main. Further investigations showed that human metapneumovirus (hMPV; van den Hoogen) was present in a substantial number of, but not in all, SARS patients reported at the time.

At about the same time, China reported the detection, by electron microscopy, of Chlamydia-like organisms in patients who had died from atypical pneumonia during the Guangdong outbreak. Again, this finding could not be confirmed by other laboratories in SARS patients from outside China.

On March 17, 2003, the WHO called upon eleven laboratories in nine countries to join a network for multicenter research into the etiology of SARS and to simultaneously develop a diagnostic test (http://www.who.int/csr/sars/project/en/). The member institutions communicated through regular telephone conferences (initially held on a daily basis) and via a secure website and exchanged data, samples and reagents to facilitate and speed up research into the etiology of SARS (World Health Organization Multicentre Collaborative Network for Severe Acute Respiratory Syndrome (SARS) Diagnosis).

The etiologic agent of SARS was identified in late March 2003, when laboratories in Hong Kong, the United States, and Germany found evidence of a novel coronavirus in patients with SARS. This evidence included isolation on cell culture, demonstration by electron microscopy, demonstration of specific genomic sequences by polymerase chain reaction (PCR) and by microarray technology, as well as indirect immunofluorescent antibody tests (Peiris, Drosten, Ksiazek ).

Three weeks later, on April 16, 2003, following a meeting of the collaborating laboratories in Geneva, the WHO announced that this new coronavirus, never before seen in humans or animals, was the cause of SARS (Kuiken. This announcement came after research done by the then 13 participating laboratories from ten countries had demonstrated that the novel coronavirus met all four of Koch’s postulates necessary to prove the causation of disease:

1. The pathogen must be found in all cases of the disease;
2. It must be isolated from the host and grown in pure culture;
3. It must reproduce the original disease when introduced into a susceptible host;
4. It must be found in the experimental host so infected.

Proof of the last two requirements was provided after inoculation of cynomolgus macaques (Macaca fascicularis) with Vero-cell cultured virus that had previously been isolated from a SARS case. The infection caused interstitial pneumonia resembling SARS, and the virus was isolated from the nose and throat of the monkeys, as shown by polymerase chain reaction with reverse transcription (RT-PCR) and by virus isolation. The isolated virus was identical to that inoculated (Fouchier). A detailed account of the history of discovery of this novel agent can be found in Drosten 2003b.

The coronaviruses (order Nidovirales, family Coronaviridae, genus Coronavirus) are members of a family of large, enveloped, positive-sense single-stranded RNA viruses that replicate in the cytoplasm of animal host cells (Siddell).

The genomes of coronaviruses range in length from 27 to 32 kb, the largest of any of the RNA viruses. The virions measure between about 100 and 140 nanometers in diameter. Most but not all viral particles show the characteristic appearance of surface projections, giving rise to the virus' name (corona, Latin = crown). These spikes extend a further 20 nanometers from the surface.

The Coronaviridae family has been divided up into three groups, originally on the basis of serological cross-reactivity, but more recently on the basis of genomic sequence homology (see online database ICTVdB). Groups 1 (canine, feline infectious peritonitis, porcine transmissible gastroenteritis and porcine respiratory viruses, human coronavirus 229E) and 2 (bovine, murine hepatitis, rat sialodacryoadenitis viruses, human coronavirus OC43) contain mammalian viruses, while group 3 contains only avian viruses (avian infectious bronchitis, turkey coronavirus).

In animals, coronaviruses can lead to highly virulent respiratory, enteric, and neurological diseases, as well as hepatitis, causing epizootics of respiratory diseases and/or gastroenteritis with short incubation periods (2-7 days), such as those found in SARS (Holmes). Coronaviruses are generally highly species-specific. In immunocompetent hosts, infection elicits neutralizing antibodies and cell-mediated immune responses that kill infected cells.

Several coronaviruses can cause fatal systemic diseases in animals, including feline infectious peritonitis virus (FIPV), hemagglutinating encephalomyelitis virus (HEV) of swine, and some strains of avian infectious bronchitis virus (IBV) and mouse hepatitis virus (MHV). These coronaviruses can replicate in liver, lung, kidney, gut, spleen, brain, spinal cord, retina, and other tissues (Holmes). Coronaviruses cause economically important diseases in domestic animals.

Human coronaviruses (HCoVs) were previously only associated with mild diseases. They are found in both group 1 (HCoV-229E) and group 2 (HCoV-OC43) and are a major cause of normally mild respiratory illnesses (Makela). They can occasionally cause serious infections of the lower respiratory tract in children and adults and necrotizing enterocolitis in newborns (McIntosh, El-Sahly, Folz, Sizun). The known human coronaviruses are able to survive on environmental surfaces for up to 3 hours (Sizun). Coronaviruses may be transmitted from person-to-person by droplets, hand contamination, fomites, and small particle aerosols (Ijaz).

SARS-related CoV seems to be the first coronavirus that regularly causes severe disease in humans.

In April 2003, a Canadian group of researchers from the Michael Smith Genome Sciences Centre in Vancouver, British Columbia, and the National Microbiology Laboratory in Winnipeg, Manitoba, were the first to complete the genome sequencing of the new coronavirus (Marra), followed two days later by colleagues from the CDC (Rota).

The genome sequence data of SARS Co-V reveal that the novel agent does not belong to any of the known groups of coronaviruses, including two human coronaviruses, HCoV-OC43 and HCoV-229E (Drosten, Peiris, Marra, Rota), to which it is only moderately related. The SARS-CoV genome appears to be equidistant from those of all known coronaviruses. Its closest relatives are the murine, bovine, porcine, and human coronaviruses in group 2 and avian coronavirus IBV in group 1. For links to the most recent sequence data and publications, see the NCBI web page http://www.ncbi.nlm.nih.gov/genomes/SARS/S ARS.html.

It has been proposed that the new virus defines a fourth lineage of coronavirus (Group 4, Marra). The sequence analysis of SARS-CoV seems to be consistent with the hypothesis that it is an animal virus for which the normal host is still unknown and that has recently either developed the ability to productively infect humans or has been able to cross the species barrier (Ludwig). The genome shows that SARS-CoV is neither a mutant of a known coronavirus, nor a recombinant between known coronaviruses.

As the virus passes through human beings, SARS-CoV is apparently maintaining its consensus genotype and seems thus well-adapted to the human host (Ruan). However, genetic analysis is able to distinguish between different strains of SARS-CoV, which is of great value for epidemiological studies and may also have clinical implications (Tsui).

Negative-stain transmission electron microscopy of patient samples and of cell culture supernatants reveals pleomorphic, enveloped coronavirus-like particles with diameters of between 60 and 130 nm. (Ksiazek, Peiris).

Examination of infected cells by thin-section electron microscopy shows coronavirus-like particles within cytoplasmic membrane-bound vacuoles and the cisternae of the rough endoplasmic reticulum. Extracellular particles accumulate in large clusters, and are frequently seen lining the surface of the plasma membrane (MMWR 2003; 52: 241-248).

The SARS-CoV genome contains five major open reading frames (ORFs) that encode the replicase polyprotein; the spike (S), envelope (E), and membrane (M) glycoproteins; and the nucleocapsid protein (N).

The main function of the S protein is to bind to species-specific host cell receptors and to trigger a fusion event between the viral envelope and a cellular membrane. Much of the species specificity of the initial infection depends upon specific receptor interactions. In addition, the spike protein has been shown to be a virulence factor in many different coronaviruses. Finally, the S protein is the principal viral antigen that elicits neutralizing antibody on behalf of the host.

The M protein is the major component of the virion envelope. It is the major determinant of virion morphogenesis, selecting S protein for incorporation into virions during viral assembly. There is evidence that suggests that the M protein also selects the genome for incorporation into the virion.

One remarkable feature about coronavirus RNA synthesis is the very high rate of RNA-RNA recombination.

SARS Co-V has been detected in multiple specimens including extracts of lung and kidney tissue by virus isolation or PCR; bronchoalveolar lavage specimens by virus isolation, electron microscopy and PCR; and sputum or upper respiratory tract swab, aspirate, or wash specimens by PCR (Ksiazek, Drosten).

High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum (Drosten). SARS-associated coronavirus RNA was detected in nasopharyngeal aspirates by RT-PCR in 32% at initial presentation (mean 3.2 days after onset of illness) and in 68% at day 14 (Peiris 2003b). In stool samples, viral RNA was detected in 97% of patients two weeks after the onset of illness. 42% of urine samples were positive for viral RNA (Peiris 2003b).

Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase, suggesting that the virus may be shed in feces for prolonged periods of time (Drosten).

Work is on-going to evaluate the stability of SARS-CoV and its resistance against various environmental factors and disinfectants.

Preliminary results, obtained by members of the WHO multicenter collaborative network on SARS diagnosis (see: http://www.who.int/csr/sars/sur vival_2003_05_04/en/index.html), show that the virus is stable in feces and urine at room temperature for at least 1-2 days. The stability seems to be higher in stools from patients with diarrhea (the pH of which is higher than that of normal stool).

In supernatants of infected cell cultures, there is only a minimal reduction in the concentration of the virus after 21 days at 4°C and -80°C. After 48 hours at room temperature, the concentration of the virus is reduced by one log only, indicating that the virus is more stable than the other known human coronaviruses under these conditions. However, heating to 56°C inactivates SARS-CoV relatively quickly. Furthermore, the agent loses its infectivity after exposure to different commonly-used disinfectants and fixatives.

Research teams in Hong Kong and Shenzhen detected several coronaviruses that were closely related genetically to the SARS coronavirus in animals taken from a southern Chinese market that was selling wild animals for human consumption. They found the virus in masked palm civets (Paguma larvata) as well as some other species. All six of the civets included in the study were found to harbor SARS coronavirus, which was isolated in cell culture or detected by a PCR molecular technique. Serum from these animals also inhibited the growth of SARS coronavirus isolated from humans. Vice versa, human serum from SARS patients inhibited the growth of SARS isolates from these animals. Sequencing of viruses isolated from these animals demonstrated that, with the exception of a small additional sequence, the viruses are identical to the human SARS virus (Cyranoski; Enserink 2003b).

The study provides a first indication that the SARS virus exists outside a human host. However, at present, no evidence exists to suggest that these wild animal species play a significant role in the epidemiology of SARS outbreaks. The civets sold on Chinese markets are born in the wild and then captured and raised on farms. They could therefore have acquired the virus from a wild animal or from other animals or even humans during captivity. More research is needed before any firm conclusions can be reached (WHO Update 64, 23 May).

Efforts are underway at various institutions to assess potential anti-SARS-CoV agents in vitro. According to the data available so far, Ribavirin, a "broad spectrum" agent, which is active against various RNA viruses (Tam) which has been used extensively in SARS patients (Koren), seems to lack in vitro efficacy. Convalescent plasma and normal human immunoglobulin, not containing specific anti-SARS-CoV antibodies, have also been used in SARS patients (Wong). In addition, interferons may be promising candidate drugs (Cinatl 2003b). In the light of the widespread utilization of traditional Chinese medicine in SARS patients in the Far East it is interesting that glycyrrhizin, a compound found in liquorice roots, was recently reported to have a good in vitro activity against SARS-CoV (Cinatl).

Further research includes detailed physico-chemical analysis of SARS-CoV proteins to allow the development of novel compounds based on targeted drug design (Anand; Thiel).

There are currently no commercial veterinary vaccines to prevent respiratory coronavirus infections, except for infectious bronchitis virus infections in chickens. Although an effective vaccine cannot be expected to be available soon, the relative ease with which SARS-CoV can be propagated in vitro and the availability of vaccines against animal coronaviruses, such as avian infectious bronchitis virus, transmissible gastroenteritis coronavirus of pigs, and feline infectious peritonitis virus, are encouraging. The S protein is generally thought to be a good target for vaccines because it will elicit neutralizing antibody.

The apparent genetic stability of SARS-CoV is certainly encouraging with regard to the development of a vaccine (Brown). It should be noted, however, that in experimental infections with human coronavirus 229E, infection did not provide long-lasting immunity. Likewise, several animal coronaviruses can cause re-infections, so lasting immunity may be difficult to achieve. However, re-infections seem to be generally mild or sub-clinical. Before immunization strategies are devised, the immune pathogenesis of feline infectious peritonitis warrants careful investigation into whether immune enhancement also plays a role in SARS.

The discovery of the SARS-associated coronavirus was the result of an unprecedented global collaborative exercise coordinated by the WHO (World Health Organization Multicentre Collaborative Network for Severe Acute Respiratory Syndrome (SARS) Diagnosis). The rapid success of this approach results from a collaborative effort - rather than a competitive approach - by high-level laboratory investigators making use of all available techniques, from cell culture through electron microscopy (Hazelton and Gelderblom) to molecular techniques, in order to identify a novel agent. It demonstrates how an extraordinarily well orchestrated effort may be able to address the threat of emerging infectious diseases in the 21st century. (Hawkey). The SARS experience also sadly underlines that non-collaborative approaches may seriously impede scientific progress and sometimes have grave consequences (Enserink 2003b). It may be surprising that despite the remarkable world-wide coopera-tive research efforts that allowed such significant progress in such a short time, the apparent success in ending the SARS outbreak (no new cases have been notified since 15 June 2003, suggesting that SARS-CoV no longer circulates within the human population) is undoubtedly due to "old-fashioned" infection control measures. It is completely unclear at present (early September 2003) whether SARS will reappear. Clinically "silent" infections and long-term car-riage can not be ruled out completely and may result in further out-breaks, perhaps in a season-dependent manner. Interestingly, the an-nual peak incidence of influenza virus infections is from March to July in southern China (Huang), which is similar to the epidemic curve of the 2003 SARS outbreak. It is also likely that SARS-CoV or a closely related coronavirus persist in an unidentified animal reser-voir from where it may again spill over into the human population. Therefore, it is vital that vigilance for new SARS cases be maintained (see "Alert, verification and public health management of SARS in the post-outbreak period", http://www.who.int/csr/sars/postoutbreak/en/ ).

Sustained control of SARS will require the development of reliable diagnostic tests to diagnose patients in the early stages of illness and to monitor its spread, as well as of vaccines and antiviral compounds to prevent or treat the disease (Breiman). Vaccines are successful in preventing coronavirus infections in animals, and the development of an effective vaccine against this new coronavirus is a realistic possibility. As is the case for the development of any vaccine, time is needed. Suitable animal models must demonstrate efficacy, and time is necessary in order to be able to demonstrate the safety of the new vaccine in humans. While involvement by commercial enterprises is clearly wanted and necessary, it is to be hoped that patent issues will not stand in the way of scientific progress (Gold).

With the availability of different and improved laboratory methods, a number of important questions regarding the natural history of the SARS-associated coronavirus are now being addressed:

• What is the origin of SARS-CoV? What is the animal reservoir, if any? If SARS-CoV was present in an unknown animal species, did it jump to humans because of a unique combination of random mutations? Or can SARS-CoV now infect both its original host and humans?
• Which factors determine the period of time between infection and the onset of infectiousness?
• When, during the course of infection, is virus shedding highest? What is the concentration of the virus in various body compartments? In what way does the "viral load" relate to the severity of the illness or the likelihood of transmission?
• Do healthy virus carriers exist? If so, do they excrete the virus in amounts and concentrations sufficient to cause infection?
• Does virus shedding occur following clinical recovery? If so, for how long? Is this epidemiologically relevant?
• Why are children less likely to develop SARS ? Do they have a lower clinical manifestation index, or do they possess a (relative) (cross-?) immunity against SARS-CoV?
• What is the role of potential co-factors such as Chlamydia spp. and hMPV? Are they related to a clinically more severe illness or to a higher degree of infectiousness ("super-spreaders")?
• Are there environmental sources of SARS-CoV infection, such as foodstuff, water, sewage?
• How stable is SARS-CoV under different conditions? How can efficient disinfection be achieved? How long can the virus "survive" in the environment on both dry surfaces and in suspension, including in fecal matter?
• How important is genetic diversity among SARS-CoV strains?

Medicine; Director: H. Schmitz; full-size picture: http://SARSReference.com/archive/coronavirus_em.jpg)

 
Figure 2. Cytopathic effect in Vero cell culture caused by SARS-associated coronavirus 24 hours post inoculation; for comparison: uninfected cell culture. ( Source: Institute for Medical Virology, Director: H. W. Doerr; full-size picture: http://SARSReference.com/archive/cytopathiceffect.jpg,
http://SARSReference.com/archive/uninfectedcells.jpg )

Figure 3. Phylogenetic tree of the SARS-associated coronavirus (Source: S. Günther, Department of Virology, Bernhard Nocht Institute for Tropical

Medicine; Director: H. Schmitz; full-size picture:

http://SARSReference.com/archive/phylogenetictree.jpg)

References