SARS coronavirus

I. Organism Information

A. Taxonomy Information
  1. Species:
    1. SARS coronavirus (NCBI_Taxonomy):
      1. Ontology: UMLS:A7875071, SNOMED:A787507
      2. GenBank Taxonomy No.: 227859
      3. Description: Synonyms: Severe acute respiratory sundrome coronavirus, Human coronavirus (strain SARS) (NCBI_Taxonomy) Severe acute respiratory syndrome (SARS) first emerged in China's Guangdong Province in November 2002. During the following 3 months, it spread rapidly across the world, infecting individuals in several countries and thus resulting in the first human pandemic of the 21st century (Gu et al., 2007). Within months, the disease had spread globally, affecting over 8000 patients in 29 countries with 774 fatalities. Over half of these infections can be directly traced to one index patient who arrived in Hong Kong on Feb 21, 2003, after acquiring the disease in Guangdong (Poon et al., 2004). Additional sporadic cases occurred in the period between the winter of 2003 and early spring of 2004 (Gu et al., 2007). A novel coronavirus was identified as the etiological agent of SARS. This virus (SARS-CoV) belongs to a family of large, positive, single-stranded RNA viruses. Nevertheless, genomic characterization showed that the SARS-CoV is only moderately related to other known coronaviruses (Gu et al., 2007). The most probable explanation was that it was an animal virus that had recently acquired the ability for human-human transmission (Poon et al., 2004).
      4. Variant(s):
        • SARS coronavirus Tor2 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 227984
          • Parent: SARS coronavirus
          • Description: The National Microbiology Laboratory in Canada obtained the Tor2 isolate from a patient in Toronto and succeeded in growing a coronavirus-like agent in African green monkey kidney (Vero E6) cells. This coronavirus was named publicly by the World Health Organization and member laboratories as the "SARS virus" after tests of causation according to Koch's postulates, including monkey inoculation (Marra et al., 2003). Pandemic strain (Poon et al., 2004). Source: Patient; Location and date of sampling: Toronto, March 2003; Proposed group affiliation: Epidemic (Kan et al., 2005).
        • SARS coronavirus Urbani (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 228330
          • Parent: SARS coronavirus
          • Description: SARS-CoV (Urbani strain) (NCBI_Taxonomy). Source: Patient; Location and date of sampling: Vietnam, March 2003; Proposed group affiliation: Epidemic (Kan et al., 2005). Because of the death of Dr. Carlo Urbani during the investigation, we propose that our first isolate be named the Urbani strain of SARS-associated coronavirus (Ksiazek et al., 2003).
        • SARS coronavirus BJ202 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 321149
          • Parent: SARS coronavirus
          • Description: In this work, severe acute respiratory syndrome associated coronavirus (SARS-CoV) genome BJ202 (AY864806) was completely sequenced. The genome was directly accessed from the stool sample of a patient in Beijing (Shang et al., 2006).
        • SARS Coronavirus CDC#200301157 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 292360
          • Parent: SARS coronavirus
          • Description: Isolation source: sputum of SARS patient clinical specimen #809940 ; Country: USA; Note: passaged two times on GMP grade Vero cells batch FA139414, passage 141 (NCBI_CoreNucleotide).
        • SARS coronavirus CUHK-AG01 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 239241
          • Parent: SARS coronavirus
          • Description: To identify the SARS-coronavirus sequence characteristic of the Amoy Gardens outbreak, we obtained clinical samples from patient 1 and from two other residents infected with SARS (patients 2 and 3) randomly selected from the unit where patient 1 had stayed or from the adjacent unit (Chim et al., 2003). Serum from patient 1 (isolate CUHK-AG01) (Chim et al., 2003).
        • SARS coronavirus CUHK-AG03 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 239242
          • Parent: SARS coronavirus
          • Description: To identify the SARS-coronavirus sequence characteristic of the Amoy Gardens outbreak, we obtained clinical samples from patient 1 and from two other residents infected with SARS (patients 2 and 3) randomly selected from the unit where patient 1 had stayed or from the adjacent unit (Chim et al., 2003). Nasopharyngeal aspirate from patient 2 (CUHK-AG02) (Chim et al., 2003).
        • SARS coronavirus CUHK-AG03 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 239243
          • Parent: SARS coronavirus
          • Description: To identify the SARS-coronavirus sequence characteristic of the Amoy Gardens outbreak, we obtained clinical samples from patient 1 and from two other residents infected with SARS (patients 2 and 3) randomly selected from the unit where patient 1 had stayed or from the adjacent unit (Chim et al., 2003). Stools from patient 3 (CUHK-AG03) (Chim et al., 2003).
        • SARS coronavirus CUHK-L2 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 260550
          • Parent: SARS coronavirus
          • Description: We recently confirmed a case of SARS that presented in Hong Kong before the report of the case cluster at the Metropole Hotel (Chim et al., 2003). This patient, designated A, presented to a hospital in Hong Kong on February 17, 2003, with a 2-day history of fever, chills, rigors, dry cough, and intense malaise (Chim et al., 2003). The CUHK-L2 sequence represents the third SARS-CoV genotype directly traceable to southern China early in the course of the SARS epidemic, with a transitory sequence that bridges the two major genotypes reported earlier (Chim et al., 2003).
        • SARS coronavirus Frankfurt 1 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 229992
          • Parent: SARS coronavirus
          • Description: Frankfurt 1, obtained from a 32-year-old male physician who was admitted with typical symptoms of SARS to the isolation ward of the Frankfurt University hospital on 15 March 2003 (Thiel et al., 2003).
        • SARS coronavirus GZ50 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 231522
          • Parent: SARS coronavirus
          • Description: Source: Patient; Location and date of sampling: Guangzhou, March 2003; Proposed group affiliation: Epidemic (Kan et al., 2005). SARS-CoV strain GZ50 was isolated from the nasopharyngeal wash fluid of a female patient who suffered from SARS in Guangzhou, late February 2003 (Qu et al., 2005).
        • SARS coronavirus Hong Kong/03/2003 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 227998
          • Parent: SARS coronavirus
          • Description: Two virus isolates, identified as a coronavirus, were isolated from two patients. One was from an open lung biopsy sample from a male Hong Kong Chinese resident aged 53 years and the other from a nasopharyngeal aspirate of a woman aged 42 years with good previous health (Peiris et al., 2003).
        • SARS coronavirus HSR 1 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 235088
          • Parent: SARS coronavirus
          • Description: We have recently obtained a SARS-CoV isolate from a frozen sputum sample collected from an Italian patient affected by a respiratory disease of unknown cause; onset of illness began during the patient's travel to Vietnam in March 2003. The viral strain has been designated as SARS-CoV HSR1 (Vicenzi et al., 2004).
        • SARS coronavirus Sin0409 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 266147
          • Parent: SARS coronavirus
          • Description: Sequences related to Singapore September 2003 case (Vega et al., 2004). Singapore encountered an unusual incident where a stable lab SARS-CoV isolate commonly used for in vitro experimentation accidentally infected a laboratory worker. We sequenced both the originating laboratory isolate (SIN_WNV) and the viral sample directly from the patient's sputum (SIN0409) (Vega et al., 2004).
        • SARS coronavirus Sin_WNV (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 266148
          • Parent: SARS coronavirus
          • Description: Sequences related to Singapore September 2003 case (Vega et al., 2004). Singapore encountered an unusual incident where a stable lab SARS-CoV isolate commonly used for in vitro experimentation accidentally infected a laboratory worker. We sequenced both the originating laboratory isolate (SIN_WNV) and the viral sample directly from the patient's sputum (SIN0409) (Vega et al., 2004).
        • SARS coronavirus Sino1-11 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 255730
          • Parent: SARS coronavirus
          • Description: SARS viral strains Sino1, Sino2, Sino3 and Sino6 were isolated from pharyngeal swabs of clinically confirmed SARS patients at Peking Union Hospital (Zhang et al., 2005).
        • SARS coronavirus Sino3-11 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 255729
          • Parent: SARS coronavirus
          • Description: SARS viral strains Sino1, Sino2, Sino3 and Sino6 were isolated from pharyngeal swabs of clinically confirmed SARS patients at Peking Union Hospital (Zhang et al., 2005).
        • SARS coronavirus SoD (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 255194
          • Parent: SARS coronavirus
          • Description: Complete nucleotide cDNA sequence (29715 nucleotides) of SARS-associated coronavirus (strain SoD) isolated for the first time in the territory of the Russian Federation was determined (Onishchenko et al., 2004).
        • SARS coronavirus WHU (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 247149
          • Parent: SARS coronavirus
          • Description: SARS-CoV isolation was undertaken from a blood speciment of a fatal SARS case in Hubei province beloning to the original case cluster from Beijing, China as described by Ksiazek et al (Yan et al., 2004). The virus isolate was named after WHU strain (Yan et al., 2004).
        • SARS coronavirus ZJ01 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 230471
          • Parent: SARS coronavirus
          • Description: A SARS-associated coronavirus isolate named ZJ01 was obtained from throat swab samples taken from a patient in Hangzhou, Zhejing province (Li et al., 2003).
        • SARS coronavirus ZJ0301 (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 344702
          • Parent: SARS coronavirus
          • Description: Isolation source: cultured virus in Vero cell inoculated with throat swab from the first patient with severe acute respiratory syndrome (SARS) in Zhejiang; Country: China: Hangzhou. Collection date: April 21, 2003 (NCBI_CoreNucleotide).
        • SARS coronavirus ZS-A (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 249082
          • Parent: SARS coronavirus
          • Description: Source: Patient; Location and date of sampling: Zhongshan, early phase of epidemic, January 2004; Proposed group affiliation: High pathogenicity (Kan et al., 2005).
        • SARS coronavirus ZS-B (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 249081
          • Parent: SARS coronavirus
          • Description: Source: Patient; Location and date of sampling: Zhongshan, early phase of epidemic, January 2004; Proposed group affiliation: High pathogenicity (Kan et al., 2005).
        • SARS coronavirus ZS-C (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 249088
          • Parent: SARS coronavirus
          • Description: Source: Patient; Location and date of sampling: Zhongshan, early phase of epidemic, January 2004; Proposed group affiliation: High pathogenicity (Kan et al., 2005).
        • SARS coronavirus TJF (NCBI_Taxonomy):
          • GenBank Taxonomy No.: 284672
          • Parent: SARS coronavirus
          • Description: Of 93 blood specimens and 15 fecal swabs on which we performed RT-PCR, 1 or the same 2 pigs tested positive. We subsequently obtained 2 viral isolates from its blood and fecal samples, designated TJB and TJF, respectively (Chen et al., 2005).
B. Lifecycle Information :
  1. Virion :
    1. Size: Coronaviruses are enveloped viruses with round and sometimes pleiomorphic virions of approximately 80 to 120 nm in diameter (Lai et al., 2003).
    2. Shape: Spherical capsid (Stadler et al., 2003).
    3. Picture(s):
      1. Schematic drawing of SARS coronavirus (WHO):


    4. Description: Like other coronaviruses, the SARS CoV is an enveloped, positive-stranded RNA virus that contains a large genome, comprised of 29,740 bases that constitute 14 open reading frames (Yeung et al., 2006). The genome RNA is complexed with the basic nucleocapsid (N) protein to form a helical capsid found within the viral membrane. The membranes of all coronaviruses contain at least three viral proteins. These are spike (S), the type I glycoprotein that forms the peplomers on the virion surface, giving the virus its corona- or crown-like morphology in the electron microscope; the membrane (M) protein, a protein that spans the membrane three times and has a short N-terminal ectodomain and a cytoplasmic tail; and small membrane protein (E), a highly hydrophobic protein (Lai et al., 2003).
  2. The arrows in this scanning electron micrograph point to the exportation of virus particles from the cell surface pseudopodia. (CDC_PHIL):



    Description: 24 hours after Vero E6 culture cells were infected with the SARS-CoV (coronavirus) it was evident that there was a prolific increase in the number of cell surface pseudopodia from which virus particle were being exported in great numbers (CDC_PHIL).
C. Genome Summary:
  1. Genome of SARS coronavirus
    1. Description: Based on extensively scientific cooperation and almost two-year's studies, remarkable achievements have been made in the understanding of the phylogenetic property and the genome organization of SARS-CoV, as well as the detailed characters of the major proteins involved in SARS-CoV life cycle (Chen et al., 2006). Since the initial full genomic sequencing of the SARS-CoV strain Tor2, which was achieved by Marra et al. in 2003, the complete or partial genome of SARS-CoV has been sequenced at several sites around the world. As of October 7, 2005, the NCBI GenBank database harbored 126 complete genome sequences for SARS-CoV, including those of both human and animal origin (Kim et al., 2006). The genome of SARS-CoV is a positive sense single-stranded ribonucleic acid (RNA) consisting of 29,751 bases (Satija et al., 2007). Generally speaking, the genome of coronavirus is the largest (about 30 kb in length) found in any RNA viruses, which encodes 23 putative proteins, including 4 major structure proteins: nucleocapsid (N), spike (S), membrane (M) and envelope (E) proteins. The N, S and M mature proteins contribute to generating the host immune response (Shih et al., 2005). The genomic organization of SARS-CoV is similar to that of a typical coronavirus, containing the characteristic gene order (5)-replicase, spike, envelope, membrane, and nucleocapsid-3)) and short untranslated regions at both termini (Shih et al., 2005). Analogous to other coronaviruses, the first 2/3 of the SARS-CoV genome encodes the viral replicase genes (ORFs 1a and 1b), which translates into two large polyproteins, pp1a (486 kDa) and pp1ab (790 kDa) (Tan et al., 2005). Proteolytic processings of these polyproteins are mediated by viral cysteine proteinases and produces a minimum of 13 non-structural proteins (also called nsp's), some of which are responsible for replicating the viral genome and/or generating a nested set of subgenomic mRNAs to express all the ORFs downstream of ORF 1b (Tan et al., 2005). The remaining 12 ORFs encode the 4 structural proteins: spike, membrane, nucleocapsid and enveloped; and eight accessory proteins (Satija et al., 2007).
    2. SARS coronavirus Tor2 (Marra et al., 2003):
      1. GenBank Accession Number: Refseq: NC_004718
      2. Size: 29,751 nt (NCBI_Genome)
      3. Gene Count: 13 (NCBI_Genome)
      4. Description: At the 5' end of the genome, we detected a putative 5' leader sequence with similarity to the conserved coronavirus core leader sequence, 5'-CUAAAC-3' (Marra et al., 2003). The 3' untranslated region (3'UTR) sequence contains a 32-base pair region corresponding to the conserved s2m motif (Marra et al., 2003). Recognizable ORFs include the replicase 1a and 1b translation products, the S glycoprotein, the E protein, the M protein, and the N protein. We have, in addition, conducted a preliminary analysis of the nine novel ORFs in an attempt to ascribe to them a possible functional role (Marra et al., 2003). The replicase 1a ORF (base pairs 265 to 13,398) and replicase 1b ORF (base pairs 13,398 to 21,485) occupy 21.2 kb of the SARS virus genome. Conserved in both length and amino acid sequence to other coronavirus replicase proteins, the genes encode a number of proteins that are produced by proteolytic cleavage of a large polyprotein (Marra et al., 2003). The Spike (S) glycoprotein (base pairs 21,492 to 25,259) encodes a surface projection glycoprotein precursor predicted to be 1255 amino acids in length (Marra et al., 2003). ORF 3 (base pairs 25,268 to 26,092) encodes a predicted protein of 274 amino acids that lacks significant BLAST, FASTA, or PFAM similarities to any known protein (Marra et al., 2003). ORF 4 (base pairs 25,689 to 26,153) encodes a predicted protein of 154 amino acids. This ORF overlaps entirely with ORF 3 and the E protein (Marra et al., 2003). The gene encoding the small envelope (E) protein (base pairs 26,117 to 26,347) yields a predicted protein of 76 amino acids protein (Marra et al., 2003). The gene encoding the membrane (M) glycoprotein (base pairs 26,398 to 27,063) yields a predicted protein of 221 amino acids (Marra et al., 2003). ORF 7 (base pairs 27,074 to 27,265) encodes a predicted protein of 63 amino acids. BLAST and FASTA searches yield no significant matches indicative of function (Marra et al., 2003). Similarly, ORF 8 (base pairs 27,273 to 27,641), encoding a predicted protein of 122 amino acids, has no significant BLAST or FASTA matches to known proteins (Marra et al., 2003). ORF 9 (base pairs 27,638 to 27,772) encodes a predicted protein of 44 amino acids (Marra et al., 2003). Similarly, ORF 10 (base pairs 27,779 to 27,898), encoding a predicted protein of 39 amino acids, exhibits no significant matches in BLAST and FASTA searches but is predicted to encode a transmembrane helix by TMpred, with the N terminus located within the viral particle (Marra et al., 2003). ORF 11 (base pairs 27,864 to 28,118), encoding a predicted protein of 84 amino acids, exhibits only very short (9 or 10 residues) matches to a region of the human coronavirus S glycoprotein precursor (Marra et al., 2003). The gene encoding the nucleocapsid protein (base pairs 28,120 to 29,388) yields a predicted protein of 422 amino acids (Marra et al., 2003). ORF 13 (base pairs 28,130 to 28,426) encodes a predicted protein of 98 amino acids. BLAST analysis fails to identify similar sequences, and no transmembrane helices are predicted. ORF 14 (base pairs 28,583 to 28,795) encodes a predicted protein of 70 amino acids (Marra et al., 2003).

II. Epidemiology Information

SARS has been described as a Chinese plague because it emerged from the colourful markets of wild animals and the exotic kitchens of Guangdong, southern China in mid-November 2002 (Tai, 2002). The first train of transmission of SARS occurred in Fosham City, Guangdong Province, China. During the period from November 16, 2002, until February 9, 2003, there were 305 cases reported in Guangdong Province. SARS was spread to Hong Kong on February 22, 2003, by a patient from Guangdong Province who, before his hospitalization, stayed in the Metropole Hotel in Hong Kong for 1 d. Ten secondary cases occurred in hotel guests, and these infected persons led directly to tertiary cases in two Hong Kong hospitals and outbreaks in Singapore, Toronto, and Hanoi (Cherry et al., 2004). SARS spread rapidly around the world during March 2003 by infected persons who traveled by airplane (Cherry et al., 2004). During the 2003 global epidemic, 8098 cases of probable SARS with 774 (9.6%) deaths were reported in 29 countries. The countries with the greatest number of reported cases included mainland China (N = 2674), Hong Kong Special Administrative Region (N = 977), Taiwan (N = 218), Singapore (N = 161), and Canada (N = 151) (Parashar et al., 2004). Worldwide, about 21% (1706/8096) of SARS victims were healthcare workers (HCWs). The percentage of HCWs was highest in Vietnam (57%), Canada (43%) and Singapore (41%), followed by Hong Kong (22%), Taiwan (20%) and mainland China (19%) (Tai, 2002).

A. Outbreak Locations:
  1. MAINLAND CHINA: Cumulative number of cases with onset of illness from 1 November 2002 to 31 July 2003: 5327 (WHO). On February 11, the Chinese Ministry of Health notified WHO that 305 cases of acute respiratory syndrome of unknown etiology had occurred in six municipalities in Guangdong province in southern China during November 16, 2002--February 9, 2003 (CDC, 2003). SARS remained isolated in China from November 2002 until 21 February 2003, when a physician with SARS traveled from Guangdong province to a hotel in Hong Kong, infecting 10 other guests. The movements of these 11 individuals resulted in the spread of SARS worldwide and sparked all of the major epicenters outside of China (Christian et al., 2004). Beijing experienced the largest outbreak of SARS, with >2,500 cases reported between March and June 2003. Several instances of superspreading were recognized during the Beijing epidemic, including two associated with imported cases, from Guangdong and Hong Kong, that each proved critical to the rapid increase in cases (Shen et al., 2004). After the World Health Organization (WHO) declared the end of the SARS epidemic, 4 new cases of SARS were reported from December 16, 2003, to January 1, 2004, in Guangzhou in Guangdong Province. These cases were not linked to any laboratory accidents (Wang et al., 2005).
  2. HONG KONG: Cumulative number of cases with onset of illness from 1 November 2002 to 31 July 2003: 1755 (WHO). During late February, an outbreak of a similar respiratory illness was reported in Hong Kong among workers at another hospital; this cluster was linked to a patient who had traveled previously to southern China (CDC, 2003). In the 2 week period from 11 March to 25 March 2003 a total of 156 patients were admitted to the Prince of Wales Hospital with SARS, of whom 138 were identified as either secondary or tertiary cases stemming from our index patient (Sung et al., 2004). Of particular interest was the point source outbreak, which involved the Amoy Gardens housing complex, Kowloon Bay, Hong Kong. The primary case in this outbreak was a 33 year old man who lived in Shenzhen. He had chronic renal disease and he frequently visited his brother in Amoy Gardens when he made visits to the Prince of Wales Hospital, where his renal disease was being treated. On March 14 and 19, 2003, he visited his brother who lived in a flat in Block E of the housing complex, which had 15 high-rise units (blocks). The patient had diarrhea at the time of his visits and used the toilet in his brother's unit. During the following month, 321 SARS cases occurred in Amoy Gardens, with 41% occurring in Block E residents (Cherry et al., 2004).
  3. TAIWAN: Cumulative number of cases with onset of illness from 1 November 2002 to 31 July 2003: 346 (WHO). In Taiwan, the outbreak had two phases. The first phase consisted of sporadic SARS cases in travelers without nosocomial transmission. In the second phase, transmission at one municipal hospital ignited a number of subsequent nosocomial outbreaks when SARS patients were transferred to other facilities (McDonald et al., 2004). The first case of SARS in Taiwan occurred in a businessman working in Guangdong Province, who developed a febrile illness on February 25, 2003, four days after returning to Taiwan and was admitted on March 8, 2003 (Hsueh et al., 2005). In mid-April in Taipei City, a large nosocomial outbreak of SARS occurred in a regional hospital, leading to subsequent countrywide spreads of SARS (Hsueh et al., 2005). An infected hospital laundry attendant continued working despite worsening symptoms of diarrhea and pneumonia. Between the onset of his illness and eventual recognition of SARS, exposures to the worker and to the hospital led to at least 137 probable cases, including 45 in healthcare workers (McDonald et al., 2004). The epidemic began to subside on May 20, 2003 and no new cases were found after June 15. On July 5, 2003, the WHO removed Taiwan from the list of areas with recent local transmission of SARS (Hsueh et al., 2005).
  4. CANADA: Cumulative number of cases with onset of illness from 1 November 2002 to 31 July 2003: 251 (WHO). The first phase resulted from a case of unrecognized SARS in an infected contact of a recent traveler to Hong Kong (McDonald et al., 2004). The index case of SARS in Canada was an elderly woman who returned to Toronto from Hong Kong on February 23, 2003. She had stayed at a hotel in Kowloon, which was later identified as being the focus on a cluster of SARS cases (Booth et al., 2005). The community hospital that cared for the son of the index patient was the site of Toronto's largest nosocomial outbreak of SARS (Booth et al., 2005). In total, 128 cases of SARS resulted from transmission of the virus within this hospital (42% HCWs, 28% patients or visitors, and 30% household contacts). During this time, two patients, not recognized as having have SARS, were transferred to ICUs at other institutions because the hospital experiencing the outbreak was unable to cope with the influx of activity and loss of staff. These transfers led to further nosocomial outbreaks in the Toronto critical care community. One of these patients was transferred to the ICU of a second community hospital (Booth et al., 2005). In addition to the patient's wife, 14 further cases of SARS resulted from transmission within this ICU (10 hospital staff and four patients (Booth et al., 2005). The second patient transferred from the original hospital was admitted to the ICU of a tertiary-care university hospital (Booth et al., 2005). The ICU was closed and 69 HCWs were quarantined. Seven of these individuals developed SARS, none of whom became critically ill (Booth et al., 2005). Toronto's third nosocomial outbreak in the critical care setting occurred in a university medical/surgical ICU during the intubation of a patient with SARS (Booth et al., 2005). After enhanced infection control precautions and public health measures were implemented in March 2003, the Canadian outbreak began to subside in April (Wong et al., 2005). On May 14, the World Health Organization (WHO) took Toronto off the list as a SARS-affected area in the absence of newly reported cases for at least 2 incubation periods after the last SARS case-patient was isolated. In accordance with public health principles, the enhanced measures were selectively relaxed in low-risk settings in Toronto area hospitals in early May 2003, although full precautions were still recommended for patients with febrile respiratory illnesses. In the third week of May, a cluster of febrile respiratory illness at a Toronto area rehabilitation hospital was reported to the health department. Traceback of these SARS cases identified the index patient as a postoperative patient who was transferred from hospital X to the rehabilitation hospital. This link uncovered clusters of unrecognized SARS infections on a surgical ward and a psychiatry ward at hospital X. Investigation determined that the ventilation system did not contribute to the spread of SARS at that hospital. On May 23, 2003, hospital X was closed to nonobstetric admissions other than newly identified SARS cases, and SARS precautions were reintroduced (Wong et al., 2005).
  5. SINGAPORE: Cumulative number of cases with onset of illness from 1 November 2002 to 31 July 2003: 238 (WHO). The index case of SARS in Singapore occured in a previously healthy 23 year old woman of Chinese ethnicity who had stayed on the 9th floor of a hotel during a vacation to Hong Kong, February 20-25, 2003 (Hsu et al., 2003). The index patient was admitted to Tan Tock Seng Hospital (TTSH) on 1 March 2003 for atypical pneumonia following a trip to Hong Kong on 25 February 2003 (Leong et al., 2006). The disease was subsequently transmitted to other patients, healthcare workers and visiting friends and relatives, and was initially limited within the hospital. As the epidemic progressed, the disease eventually reached the community (Leong et al., 2006). The first major extension of this illness outside the health-care setting was from a recent probable SARS patient to two taxi drivers and the patient's coworkers in a wholesale market (CDC, 2003). Eventually, the SARS outbreak in Singapore was declared over on 31 May 2003 (Leong et al., 2006).
  6. VIETNAM: Cumulative number of cases with onset of illness from 1 November 2002 to 31 July 2003: 63 (WHO). On February 26 [2003], a man aged 47 years who had traveled in mainland China and Hong Kong became ill with a respiratory illness and was hospitalized shortly after arriving in Hanoi, Vietnam. Health-care providers at the hospital in Hanoi subsequently developed a similar illness (CDC, 2003). Transmission of SARS-CoV among staff, visitors, and patients of Hospital A, and their close contacts outside the hospital, ultimately resulted in 62 cases of SARS in Northern Vietnam (Reynolds et al., 2006).
  7. UNITED STATES: Cumulative number of cases with onset of illness from 1 November 2002 to 31 July 2003: 27 (WHO). Travel to an affected area was the most commonly reported epidemiologic link (83% of cases). Mainland China was the most frequent destination (39% of travelers), followed by Hong Kong (38%), and Toronto (18%); 22% of case-patients traveled to more than one affected area (Schrag et al., 2004). One case of possible household transmission was reported, and no laboratory-confirmed infections occurred among healthcare workers (Schrag et al., 2004). A total of 156 reported U.S. SARS cases from the 2003 epidemic remain under investigation, with 137 (88%) cases classified according to previous surveillance criteria as suspect SARS and 19 (12%) classified as probable SARS. Because convalescent serum specimens have not been obtained from the 19 probable and 137 suspect cases that remain under investigation, whether these persons had SARS-CoV disease is unknown (CDC, 2003).
B. Transmission Information:
  1. Ontology: UMLS:C1444005 From: Human To: Human
    Mechanism: The main way that SARS seems to spread is by close person-to-person contact. The virus that causes SARS is thought to be transmitted most readily by respiratory droplets (droplet spread) produced when an infected person coughs or sneezes. Droplet spread can happen when droplets from the cough or sneeze of an infected person are propelled a short distance (generally up to 3 feet) through the air and deposited on the mucous membranes of the mouth, nose, or eyes of persons who are nearby. The virus also can spread when a person touches a surface or object contaminated with infectious droplets and then touches his or her mouth, nose, or eye(s). In addition, it is possible that the SARS virus might spread more broadly through the air (airborne spread) or by other ways that are not now known (CDC). Blood and fecal-oral transmission has been suggested to be the route of transmission for one index case (Weiss et al., 2005).

  2. Ontology: UMLS:C1444006, SNOMED:A6945720 From: Animal To: Human
    Mechanism: The virus - now named SARS-coronavirus (SARS-CoV) - probably circulated among wild mammals, which was subsequently transmitted to humans (Wong et al., 2005). Tracing the source of infection has been complicated, given the sporadic nature of index cases without a clear history of contact with animals (Wang et al., 2005). The virus probably originated from wild game food animals caged in the wet markets of southern China, of which the palm civet is the most likely amplification host and one of the likely candidates for the introductory host (Wong et al., 2005). Genome sequence analysis data strongly suggest that sporadic cases of SARS in Guangzhou in 2003-2004 were caused by SARS-CoV of animal origin (Wang et al., 2005). A restaurant serving palm civets positive for this virus was the source of infection for 2 of 4 confirmed SARS patients during the resurgence of SARS in the winter of 2003-2004 (Wang et al., 2005). Clearly, SARS cases contracted at the restaurant were the result of recent interspecies transfer from a putative palm civet virus reservoir, rather than the result of circulation of SARS-CoV in the human population (Wang et al., 2005).

C. Environmental Reservoir:
  1. Palm Civets (Paguma larvata) (Shi et al., 2007):
    1. Ontology: UMLS:C0325069, SNOMED:A5018342
    2. Description: Evidence so far indicated that the origin of the 2003 SARS epidemic and the 2004 mild SARS cases was from masked palm civets. It is still uncertain whether the masked palm civets were infected during trading in animal market or were naturally infected in the wild or under farmed conditions. The very high nt sequence identity among the masked palm civet SARS-CoV-like viruses (>99.6%) clearly suggested that the virus has not resided in the masked palm civet population for a very long time. Nevertheless, it is quite clear that masked palm civets are highly susceptible to the SARS-CoV where the virus can be amplified to very high titers (Shi et al., 2007).
    3. Survival Information: Our present study demonstrated equal susceptibility of farmed civets to infection by two different human SARS-CoV isolates under experimental conditions, and all animals displayed clinical signs of infection. Our results support the notion that civets may play an important role for transmission of SARS-CoV from animals to humans. However, further field study of wild civets is required to assess whether civets are a natural reservoir host of SARS-CoV (Wu et al., 2005).
  2. Horseshoe Bats (Rhinolopholus) (Shi et al., 2007):
    1. Ontology: UMLS:C1265469, SNOMED:A4936394
    2. Description: Horseshoe bats have been found to be a natural reservoir of the SARS-CoV-like virus (Shi et al., 2007). The genetic diversity of bat-derived sequences supports the notion that bats are a natural reservoir host of the SARS cluster of coronaviruses (Li et al., 2005). A plausible mechanism for emergence from a natural bat reservoir can be readily envisaged. Fruit bats including R. leschenaulti, and less frequently insectivorous bats, are found in markets in southern China. An infectious consignment of bats serendipitously juxtaposed with a susceptible amplifying species, such as P. larvata, at some point in the wildlife supply chain could result in spillover and establishment of a market cycle while susceptible animals are available to maintain infection (Li et al., 2003).
    3. Survival Information: Bats that harbor the viruses rarely display clinical signs or overt symptoms of pathology (Shi et al., 2007).
  3. Other potential reservoirs (Shi et al., 2007):
    1. Ontology: UMLS:C0003070
    2. Description: Apart from masked palm civets and bats, 29 other animal species from 13 families, 8 animal orders and 2 classes that have been tested for SARS-CoV. Among them, seven species were shown to harbor the virus under certain circumstances (Shi et al., 2007). Two independent surveys have shown that raccoon dogs from live animal markets harbored SARS-CoV at a very high prevalence (100%) (Shi et al., 2007). A survey conducted on 5 January 2004 in a Guangzhou market before the mass culling of masked palm civets demonstrated that red foxes, domestic cats, and Lesser rice field rats may also harbor the virus. Real-time RT-PCR revealed that 4 out of 20 cats, 3 out of 5 red foxes, and 1 out of 6 Lesser rice field rats were positive for SARS-CoV (Shi et al., 2007). After culling of animals and disinfection of the market place, follow up surveillance was conducted 2 weeks later and only one greylag goose out of 119 animals tested positive for SARS-CoV. At this time cats did not test positive for the virus. Also, the same team reported that in 2004, one wild boar out of 102 animals tested positive. Since the environment of the animal market was heavily contaminated with SARS-CoV-like viruses during that period, it remains unknown whether those animals that tested positive by PCR were truly susceptible hosts or simply mechanical carriers of SARS-CoV genetic materials. In this regard, it is worth to note that under experimental conditions, goose was not susceptible to SARS-CoV infection. In another study carried out in a village close to Beijing, 2 of 108 pigs sampled were positive for the SARS-CoV by antibody analysis and by RT-PCR during a survey which also included 60 cattle, 20 dogs, 11 cats, 11 chickens and 30 ducks. Based on the sequence and available epidemiological data, the authors suggested that the pigs were infected from feed materials contaminated by human SARS patients and were not a natural host of the SARS-CoV (Shi et al., 2007).
D. Intentional Releases:
  1. Intentional Release information :
    1. Description:
    2. Emergency contact: Report to state or local health department: All persons requiring hospitalization for radiographically confirmed pneumonia who report at least one of the three risk factors for exposure to SARS-CoV; Any cluster of unexplained pneumonia, especially among healthcare workers; Any positive SARS-CoV test result (CDC). Health departments should immediately report any SARS-CoV positive test result to CDC. Health departments should also inform CDC of other cases or clusters of pneumonia that are of particular concern by calling 770-488-7100 (CDC).
    3. Delivery mechanism: Unlike smallpox, access to SARS coronavirus is not heavily restricted; it is conceivable that the virus could be obtained from the wild. Many deaths from SARS during the 2002-03 epidemic were in parts of the world where security of the remains is not guaranteed. As a result, several nations have access to specimens from which large quantities of SARS coronavirus could be cultivated. Furthermore, the virus could be obtained from the animal species that seem to be its natural reservoirs (Weberet al., 2004).
    4. Containment: During the 2003 global SARS outbreak, patients in the United States were isolated until they were no longer infectious. This practice allowed patients to receive appropriate care, and it helped contain the spread of the illness. Seriously ill patients were cared for in hospitals. Persons with mild illness were cared for at home. Persons being cared for at home were asked to avoid contact with other people and to remain at home until 10 days after the resolution of fever, provided respiratory symptoms were absent or improving (CDC). In the United States, where there was limited transmission of SARS-CoV during the 2003 SARS outbreak, neither individual nor population-based quarantine of contacts was recommended. CDC advised persons who were exposed but not symptomatic to monitor themselves for symptoms and advised home isolation and medical evaluation if symptoms appeared. Individual quarantine was an integral part of the control measures used in countries more severely affected by the 2003 SARS outbreak. Quarantine of large groups was used only in selected settings where extensive transmission was occurring (CDC).

III. Infected Hosts

  1. Human:
    1. Taxonomy Information:
      1. Species:
        1. Human :
          • Ontology: UMLS:C0086418
          • GenBank Taxonomy No.: 9606
          • Scientific Name: Homo sapiens (NCBI_Taxonomy)
          • Description: Severe acute respiratory syndrome (SARS) emerged in late 2002 from Guangdong Province, China and spread worldwide to over 30 countries by midsummer 2003. Before the epidemic ended, the novel coronavirus causing SARS (SARS-CoV) infected over 8000 persons around the world, killed nearly 10% of those that contracted the disease and inflicted great socioeconomic strain (Cameron et al., 2007). The SARS coronavirus is believed to have jumped from an animal host to people in the animal markets in towns in the Guangdong province of southern China (Vijayanand et al., 2004). SARS-CoV infection results in severe and potentially fatal lung disease. Although the majority of patients recovered after 1-2 weeks of debilitating febrile illness, a substantial proportion (up to one-third) developed severe inflammation of the lung, requiring ventilator support and intensive care. Many patients in this group deteriorated into acute respiratory distress syndrome (ARDS). The mortality of this group of patients is high (Lo et al., 2006). The SARS outbreak was ultimately contained by a concerted effort that included patient isolation, intensive control of infection in hospitals, traditional quarantine measures, and the issuing of a travel advisory that was enforced by the World Health Organization (Yeung et al., 2006). Currently, there is no known SARS transmission anywhere in the world. The most recent human cases of SARS-CoV infection were reported in China in April 2004 in an outbreak resulting from laboratory-acquired infections. CDC and its partners, including the World Health Organization, continue to monitor the SARS situation globally (CDC).

    2. Infection Process:
      1. Infectious Dose: We have not seen published data on the infectious dose of the SARS virus, either by inhalation or by contact with mucous membranes and conjunctiva. It may be that the deposition of just one virus particle in the lungs can initiate infection, as appears to be the case for the smallpox virus (Nicas et al., 2004).
      2. Description: SARS-CoV produces an acute viral infection in humans with an incubation period ranging from 2-10 days (Chan et al., 2006). The first few cases of the 2003 pandemic and the 2004 Guangdong outbreak had a history of eating or occupational contacts with wild game animals (Wong et al., 2005). The main way that SARS seems to spread is by close person-to-person contact. The virus that causes SARS is thought to be transmitted most readily by respiratory droplets (droplet spread) produced when an infected person coughs or sneezes. Droplet spread can happen when droplets from the cough or sneeze of an infected person are propelled a short distance (generally up to 3 feet) through the air and deposited on the mucous membranes of the mouth, nose, or eyes of persons who are nearby. The virus also can spread when a person touches a surface or object contaminated with infectious droplets and then touches his or her mouth, nose, or eye(s). In addition, it is possible that the SARS virus might spread more broadly through the air (airborne spread) or by other ways that are not now known (CDC). Blood and fecal-oral transmission has been suggested to be the route of transmission for one index case (Weiss et al., 2005). In the Amoy Gardens outbreak, evidence suggests that virus-laden droplets traveled hundreds of feet through the air to cause human infection (McKinney et al., 2006). For the first time, we have demonstrated that SARS-CoV is detectable in sweat gland cells in the skin. This suggests another route of transmission for SARS-CoV, since this virus may be excreted in sweat and infect other people who are in direct contact with the patient's skin (Ding et al., 2004). SARS has been documented, under certain circumstances, to be highly communicable in hospital settings. Attack rates among workers with direct patient care roles have been observed as high as 10.0 and 11.8% in Canada and Hong Kong, respectively (Reynolds et al., 2006). Respiratory droplet transmission to health care workers might have been augmented by a nebulizer used by SARS patients. In addition, airborne transmission to health care workers was suspected during the manipulation of patients' airway by suction, intubation, bronchoscopy, or cardiopulmonary resuscitation (Wong et al., 2005). Fomites should be considered as a possible mode of transmission in laboratories (Lim et al., 2006).

    3. Disease Information:
      1. SARS (i.e., Severe Acute Respiratory Syndrome) :
        1. Pathogenesis Mechanism: SARS CoV takes hold in the airways and other organs by exploiting the renin-angiotension pathway via its main putative receptor, angiotensin-converting enzyme 2 (ACE2) expressed on many cell types including pneumocytes, enterocytes and endothelial cells (Cameron et al., 2007). Coronavirus, including SARS-CoV, associate with cellular receptors to mediate infection of their target cells via the surface spike protein (S protein (Lau et al., 2005). Apart from direct membrane fusion at the target cell surface, SARS-CoV might gain cell entry via pH-dependent endocytosis, which is also mediated by the S protein (Cameron et al., 2007). The mechanism of injury caused by SARS-CoV infection remains unknown. A SARS disease model was proposed, consisting of three phases: viral replication, immune hyperactivity, and pulmonary destruction. SARS pathology of the lung has been associated with diffuse alveolar damage, epithelial cell proliferation, and an increase of macrophages. Multinucleate giant-cell infiltrates of macrophage or epithelial origin have been associated with putative syncytium-like formation that is characteristic of many coronavirus infections. The lymphopenia, hemophagocytosis in the lung, and white-pulp atrophy of the spleen observed in SARS patients are reminiscent of those reported for fatal influenza virus subtype H5N1 disease in 1997. Strikingly, the presence of hemophagocytosis supports a cytokine deregulation (Weiss et al., 2005). Proinflammatory cytokines released by stimulated macrophages in the alveoli may have a role in the pathogenesis of SARS (Weiss et al., 2005). Lymphopenia and increasing viral load in the first 10 days of SARS suggest immune evasion by SARS-CoV (Weiss et al., 2005).


        2. Incubation Period: The incubation period for SARS is likely to be varied, with the frequency distribution being nonnormal (Meltzer et al., 2004). The incubation period of SARS CoV infection ranges from 2 to 10 days but may last as long as 16 days (Cameron et al., 2007). The median incubation period was six days (Lee et al., 2003).


        3. Prognosis: Between November 2002 and July 2003, the coronavirus responsible for severe acute respiratory syndrome (SARS-CoV) infected >8500 people and cause >800 deaths in 32 countries (Denison et al., 2004). About one-third of patients have prompt resolution of fever and pneumonitis with treatment and even without specific treatment in a minority. However, the remaining run a much more stormy course - 19 to 34% of SARS patients required admission to ICU, 13 to 26% required assisted ventilation, 20 to 22.6% developed ALI or ARDS and 3.6 to 10.1% died at day 21 to day 28 (Chan et al., 2003). Based on the data received by the WHO, the case fatality rate for SARS was <1% for patients aged 24 years or younger, 6% for 25-44 years, 15% for 45-64 years, and >50% for patients aged 65 years or older (Chan et al., 2003). Most deaths were attributed to complications related to sepsis, ARDS and multiorgan failure, which occurred commonly in the elderly with comorbidities (Chan et al., 2003). There is no evidence to suggest that a difference in mortality rate exists between different ethnic groups. A number of clinical/laboratory parameters have been shown to have a prognostic value. Poor prognostic factors include advanced age, chronic hepatitis B treated with lamivudine, severe hepatitis, high initial LDH, high peak LDH, high neutrophil count on presentation, diabetes mellitus or other co-morbid conditions, low CD4 and CD8 lymphocyte counts at presentation and a high initial SARS-CoV viral load (Chan et al., 2006).


        4. Diagnosis Overview: The presenting features in adults are pronounced. These include persistent high fever, chills and rigor, malaise, myalgia, headache and dry cough. Most patients had some degree of dyspnoea at presentation, which increased towards the end of the first week of illness. Sputum production, sore throat and rhinorrhoea were less commonly reported symptoms. The reported proportion of patients with gastrointestinal symptoms varies among different clusters (Chan et al., 2006). In general, the radiographical features of SARS are similar to those found in community-acquired pneumonia caused by other organisms [30]. Nevertheless, several characteristic features are frequently observed in SARS, including the predominant involvement of the lung periphery and the lower zones, whereas cavitation, hilar lymphadenopathy and pleural effusion are rarely found [3,30]. Radiographical changes progress from a unilateral focal air-space opacity, to multifocal or bilateral involvement during the later phase of disease (Chan et al., 2006). The evolution of diagnostics for SARS has been different from that of most viruses. The need for a BSL-3 facility for virus isolation and difficulty in culturing the virus from infected individuals late in the outbreak (possibly as a result of genetic drift of the virus) precluded the use of culture for diagnosis in most outbreak settings. Furthermore, the late seroconversion, 2 to 4 weeks after infection for most patients, all but eliminates a serological diagnosis for detecting recent infections. The first serological tests used SARS-CoV-infected cells on microscope slides and immunofluorescent staining or enzyme-linked immunosorbent assay tests of crude cell lysates containing SARS-CoV antigens to detect serum antibodies. When used together with multiple specimens, virus isolation and serology proved useful for identifying patients at the beginning of the outbreak; however, they proved less satisfactory for diagnosing new cases in the first few days after onset of symptoms. Initial culturing of a CoV from patients in Hong Kong enabled the early identification of SARS-CoV by electron microscopy and led to the development of the first nucleic acid amplification tests (NAATs) for detecting SARS-CoV RNA. Identification of a CoV by electron microscopy led to the search for conserved regions of the CoV genome and the development of RT-PCR assays for SARS-CoV RNA. Molecular diagnostic testing using NAAT quickly became the mainstay of SARS diagnosis (Mahoney et al., 2005).


        5. Symptom Information :
          • Syndrome -- Severe Acute Respiratory Syndrome (SARS):
            • Description: The disease manifests primarily as an acute community- or hospital-acquired pneumonia that does not respond to antimicrobial coverage for typical and atypical pathogens (Wong et al., 2005). Interestingly, although the syndrome was named for these severe respiratory manifestations that lead to impressive overall mortality, the summary of cases suggests that SARS may be a systemic infection with severe respiratory disease as the major manifestation (Denison et al., 2004). No individual symptom or cluster of symptoms has proven specific (Vijayanand et al., 2004). The clinical course of SARS can be divided into two phases: Phase I refers to active viral replication where patients experience high fever, myalgia and other systemic symptoms that generally improve after a few days; and Phase II refers to the stage of immunopathological injury where patients experience a recurrence of fever, increasing hypoxaemia and radiological progression of pneumonia with falls in viral load (Chan et al., 2006).
            • Observed: A number of studies have been conducted to search for asymptomatic cases of SARS-CoV infection. It is now quite certain that asymptomatic infection is very rare in adults; however, data on children and elderly are less comprehensive (Chan et al., 2006). It affects all age groups with a slight bias for females (Wong et al., 2005).


              • Symptoms Shown in the Syndrome:

            • Abdominal pain (Chan et al., 2006):
              • Ontology: UMLS:C0000737
            • Acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (Chan et al., 2006):
              • Ontology: UMLS:C0242488, C0035222
              • Description: The overriding clinical feature of SARS is the rapidity with which many patients develop symptoms of acute respiratory distress syndrome (ARDS) (Stockman et al., 2006) Acute respiratory distress syndrome and acute lung injury were first described in 1967, and are characterised by the abrupt onset of clinically significant hypoxaemia with presence of diffuse pulmonary infiltrates (Wheeler et al., 2007). When the hypoxaemia in acute lung injury is severe (partial arterial pressure of oxygen [PaO2]/fractional concentration of oxygen in inspired air [FIO2] < 200), the disorder is termed the acute respiratory distress syndrome (Wheeler et al., 2007).
              • Observed: 20% of patients developed evidence of ARDS (acute respiratory distress syndrome) over a period of 3 weeks (Chan et al., 2006).
            • Anorexia (Peiris et al., 2003):
              • Ontology: UMLS:C0003123
            • Arthralgia (Booth et al., 2003):
              • Ontology: UMLS:C0003862
            • Chest pain (Booth et al., 2003):
              • Ontology: UMLS:C0008031
            • Chest rales (Peiris et al., 2003):
              • Ontology: UMLS:C0034642
            • Chills/rigor (Chan et al., 2006):
              • Ontology: UMLS:C0085593, C0424790
            • Cough - nonproductive (Chan et al., 2006):
              • Ontology: UMLS:C0850149
            • Cough - sputum production (Chan et al., 2006):
              • Ontology: UMLS:C0239134
              • Description: The prodromal symptoms that usually persisted for 3-5 d are cough and sputum production, occurring at the 2nd and 3rd d, and disappeared later at day 13 and 7 respectively (Lu et al., 2005).
              • Observed: 4.9% (Booth et al., 2003)
            • Diarrhoea (Chan et al., 2006):
              • Ontology: UMLS:C0011991
              • Description: The diarrhoea was mainly watery without blood or mucus, lasting for 3.7 +/- 2.7 days (Chan et al., 2006).
              • Observed: The reported proportion of patients with gastrointestinal symptoms varies among different clusters. For the first hospital outbreak in Hong Kong, 20% of patient had diarrhoea at presentation, and another 18% developed diarrhoea during the course of illness (Chan et al., 2006). In the Toronto outbreak, 24% of the 144 patients had diarrhoea at presentation. A remarkably high proportion (70%) of patients in the rapidly evolving community outbreak, which occurred at the Amoy Gardens Residential Estate in Hong Kong, had diarrhoea at presentation (Chan et al., 2006). 130/647 (20.1%) (Peiris et al., 2003).
            • Dizziness (Booth et al., 2003):
              • Ontology: UMLS:C0012833
            • Dyspnoea (Chan et al., 2006):
              • Ontology: UMLS:C0013404
            • Elevated CK (creatinine kinase) (Chan et al., 2006):
              • Ontology: UMLS:C1657311
            • Elevated LDH (lactate dehydrogenase) (Chan et al., 2006):
              • Ontology: UMLS:C0151754
              • Description: LDH is a non-specific enzyme found ubiquitously in cells. The high level demonstrated probably reflects the degree of tissue necrosis and hence severity of the pneumonia (Leong et al., 2006).
              • Observed: 42% (Tsui et al., 2003).
            • Fever (Chan et al., 2006):
              • Ontology: UMLS:C0015967
            • Ground glass opacification on radiograph (Rainer et al., 2007):
              • Description: During the initial phases of infection the virus causes pauci-inflammatory alveolar and interstitial edema that result in imaging abnormalities dominated by ground glass opacities (Ketai et al., 2006).
              • Observed: 98% (Rainer et al., 2007).
            • Headache (Chan et al., 2006):
              • Ontology: UMLS:C0018681
            • Leucopenia/leucocytosis (Wong et al., 2003):
              • Ontology: UMLS:C0023530, C0023518
              • Description: Transient leucopenia (leucocyte count <4x10(9)/l) was found in 100 (64%) of patients during their first week of illness. Ninety six patients (61%) developed leucocytosis (leucocyte count <11x10(9)/l), mostly during the second and third week of illness (Wong et al., 2003).
              • Observed: Transient leucopenia: 100 (64%) (Wong et al., 2003). Leucocytosis: 96 (61%) (Wong et al., 2003).
            • Lymphopenia (Wong et al., 2003):
              • Ontology: UMLS:C0024312
              • Description: Lymphopenia (absolute lymphocyte count < 1000/mm3) (Wong et al., 2003). Most patients had a normal lymphocyte count at the onset of disease. Progressive lymphopenia occurred early in the course of illness and reached its lowest point in the second week in most cases (Wong et al., 2003).
              • Observed: 153 (98% of patients) (Wong et al., 2003). 221 (68%) (Tsui et al., 2003).
            • Malaise (Chan et al., 2006):
              • Ontology: UMLS:C0231218
            • Mylagia (Chan et al., 2006):
              • Ontology: UMLS:C0026893
            • Nausea or vomiting (Booth et al., 2003):
              • Ontology: UMLS:C0027498
            • Neutropenia/Neutrophilia (Wong et al., 2003):
              • Ontology: UMLS:C0027947, C0151683
              • Description: Biochemical studies demonstrated elevated transaminase and neutropenia/lymphopenia associated with illness vefore onset of severe respiratory signs and symptoms (Denison et al., 2004).
              • Observed: Four patients (2.5%) developed transient neutropenia (absolute neutrophil count < 0.5x109/l) lasting for one to two days. We noted neutrophilia (absolute neutrophil count < 7.5x109/l) in 129 (82%) of patients (Denison et al., 2004).
            • Pneumomediastinum (Chan et al., 2006):
              • Ontology: UMLS:C0025062
              • Description: In patients in whom the initial consolidation is abutting on to the mediastinum, adhesions and cyst formation might occur at the interface between the mediastinal pleura and the pulmonary pleura. Any rupture of these cysts will result in spontaneous pneumomediastinum (Peiris et al., 2003).
              • Observed: In a case series, 12% of patients developed spontaneous pneumomediastinum (Chan et al., 2006).
            • Reactive hepatitis (Chan et al., 2006):
              • Ontology: UMLS:C1399450
              • Description: Those with severe hepatitis had worse clinical outcome (Chan et al., 2006).
              • Observed: Reactive hepatitis is a common complication of SARS-CoV infection with 24% of patients having elevated ALT (alanine aminotransferase) levels on admission, and 69% had raised ALT levels during the subsequent course of their illness (Chan et al., 2006).
            • Rhinorrhoea (Chan et al., 2006):
              • Ontology: UMLS:C1260880
            • Sore throat (Booth et al., 2003):
              • Ontology: UMLS:C0242429
              • Description: Neither the pharyngeal wall nor the tonsillar area demonstrated hyperemia or inflammatory reaction. Instead, the pharyngeal wall revealed "drying" features, without moisture on the mucous membrane (Chen et al., 2003).
              • Observed: 91/552 (16.5%) (Peiris et al., 2003). 12.5% (Booth et al., 2003).
            • Tachycardia (Peiris et al., 2003):
              • Ontology: UMLS:C0039231
            • Tachypnea (Peiris et al., 2003):
              • Ontology: UMLS:C0231835
            • Thrombocytopenia (Wong et al., 2003):
              • Ontology: UMLS:C0040034
              • Description: Most patients' platelet count was normal at the onset of illness. Progressive thrombocytopenia occurred and reached a low point at the end of the first week. Thrombocytopenia was self limiting and resolved by the fourth week of illness (Wong et al., 2003).
              • Observed: Eighty seven patients (55%) developed thrombocytopenia (platelet count < 140 000/mm3) during the course of their illness. Most of them had mild thrombocytopenia, and only three (2%) patients had a platelet count below 50 000/mm3 (Wong et al., 2003).

        6. Treatment Information:
          • Assisted Ventilation/Oxygen Delivery (Wong et al., 2005): Oxygen delivery by low-flow nasal cannula rather than the high-flow face mask should be used to reduce the risk of cross infection by aerosolization of infectious respiratory secretion. Other modes of non-invasive ventilation such as continuous positive airway pressure (CPAP) and bilevel positive airway pressure (BIPAP) should only be performed in negative pressure isolation room with adequate personal protective equipment for the health care workers. A low-tidal-volume strategy for lung protection is used for those who required mechanical ventilation (Wong et al., 2005).
            • Applicable: Acute respiratory failure (ARF) is common in SARS cases, with high rates of intensive care (20% - 38%), assisted ventilation (59% - 100%), and mortality among invasively ventilated patients (34% - 53% at 28 days). Non-invasive ventilation (NIV) is successful in treated ARF due to various causes, and is associated with reduced rates of intubation and nosocomial infection (Yam et al., 2005).
            • Contraindicator:
            • Complication:
            • Success Rate: In conclusion, analysis of patients from the Hong Kong Hospital Authority SARS Database revealed that, compared to invasive mechanical ventilation, early application of non-invasive ventilation as initial support for SARS-related acute respiratory failure appeared to be associated with significantly reduced need for intubation and mortality (Yam et al., 2005).
            • Drug Resistance:
          • Corticosteroids (Stockman et al., 2006): Corticosteroids were commonly prescribed to SARS patients with worsening pulmonary disease or progressing abnormalities on chest X-rays (Stockman et al., 2006). Steroids were also often used in SARS treatment to try to reduce the inflammation of the lungs (Stockman et al., 2006). The theoretical rational is the similarity of the radiological and histological features between bronchiolitis obliterans organising pneumonia (BOOP) and SARS pneumonia. Since corticosteroid has been shown to be effective against BOOP, it was postulated that it could be useful in reducing the complication of adult respiratory distress syndrome (ARDS) in SARS (Auyeung et al., 2005).
            • Applicable: Treatment regimens varied widely but can be classified into two groups, early treatment and rescue treatment given at a later stage of illness (Stockman et al., 2006) During the Phase II of clinical course when patients progressed to develop pneumonia and hypoxaemia, intravenous administration of rescue-pulsed methylprednisolone has been shown to suppress cytokine-induced lung injury (Chan et al., 2006).
            • Contraindicator: Corticosteroids are contraindicated in patients with known hypersensitivity to any of the other corticosteroids. Specific corticosteroid preparations are contraindicated in patients with known hypersensitivity to any ingredient in the respective formulation. Fludrocortisone is contraindicated in all conditions except those which require a high degree of mineralocorticoid activity (AHFS Drug Information 2006).
            • Complication: It is difficult to make a clear recommendation about whether corticosteroids should be used to treat SARS-associated lung injury in any stage of illness, particularly as the drug is immunosuppressive and may delay viral clearance if given before viral replication is controlled. Of added concern are infectious complications, avascular necrosis, and steroid induced psychosis - recognized adverse effects of corticosteroid use. Fungal superinfection and aspergillosis have been noted in case reports and autopsy findings of SARS patients given corticosteroids at high doses or for prolonged periods (Stockman et al., 2006).
            • Success Rate: In an uncontrolled and nonrandomized study, 95/107 (89%) of patients treated with high-dose methylprednisolone (0.5-1mg/kg prednisolone on day 3 of illness, followed by hydrocortisolone 100 mg every 8 h, and pulse-doses of methylprednisolone 0.5 g IV for 3 d) after the first week of illness recovered from progressive lung disease (Stockman et al., 2006).
            • Drug Resistance:
          • Intravenous immunoglobulin (IVIG) or convalescent plasma (Stockman et al., 2006): IVIg has immunomodulatory properties and may down-regulate cytokine expression, and was used quite extensively in Singapore during the SARS outbreak in 2003 (Chan et al., 2006). Convalescent plasma, donated by patients who had recovered from SARS, contains neutralizing antibodies and may be useful clinically for treating other SARS patients (Chan et al., 2006).
            • Applicable:
            • Contraindicator:
            • Complication: Commercial immune globulin intravenous human products have been reported to be associated with renal dysfunction, acute renal failure, osmotic nephrosis and death (Stockman et al., 2006).
            • Success Rate: Five studies of either IVIG or convalescent plasma treatment given in addition to steroids and ribavirin were reported for treatment of SARS. These studies were inconclusive, because the effect of convalescent plasma or IVIG could not be discerned from effects of patient comorbidities, stage of illness, or effect of other treatments (Stockman et al., 2006).
            • Drug Resistance:
          • Lopinavir and ritonavir (LPV/r) (Stockman et al., 2006): Lopinavir and ritonavir is a fixed combination of 2 human immunodeficiency virus (HIV) protease inhibitors (PIs) (AHFS Drug Information 2006).
            • Applicable: Genomic analysis of the SARS-CoV has revealed several enzymatic targets including protease (Chan et al., 2006). The combination of the protease inhibitors lopinavir and ritonavir was used less frequently during the SARS outbreak compared with ribavirin (Groneberg et al., 2005).
            • Contraindicator: Known hypersensitivity to lopinavir, ritonavir, or any ingredient in the formulation. Concomitant use with drugs that are highly dependent on the cytochrome P-450 (CYP) 3A or isoenzyme for metabolism and for which elevated plasma concentrations are associated with serious and/or life-threatening events (e.g., astemizole, terfenadine [antihistamines no longer commercially available in the US], cisapride, ergot derivatives [e.g., dihydroergotamine, ergonovine, ergotamine, methylergonovine], midazolam, pimozide, triazolam) (AHFS Drug Information 2006).
            • Complication: Side effects of combined treatment include diarrhea of mild to moderate severity. In HIV patients, the treatment has been associated with new-onset diabetes and hyperglycaemia (Stockman et al., 2006).
            • Success Rate: The addition of LPV/r (400 mg of lopinavir/100 mg of ritonavir) as initial therapy was associated with significant reduction in overall death rate (2.3% compared with 15.6%) and intubation rate (0% compared with 11%) when compared with a matched historical cohort that received ribavirin alone as the initial antiviral therapy. Other reported beneficial effects include a reduction in corticosteroid use, fewer nosocomial infections, a decreasing viral load and rising peripheral lymphocyte count. In contrast, the subgroup that had received LPV/r as rescue therapy after receiving pulsed methylprednisolone treatment for worsening respiratory symptoms was not better than the matched cohort. The improved clinical outcome in patients that received LPV/r as part of the initial therapy may be due to the fact that both peak (9.6 mg/ml) and trough (5.5 mg/ml) serum concentrations of lopinavir could inhibit the virus (Chan et al., 2006).
            • Drug Resistance:
          • NO (nitric oxide) (Chan et al., 2006): Inhaled NO has been reported to have beneficial effects in SARS (Chan et al., 2006).
            • Applicable:
            • Contraindicator:
            • Complication:
            • Success Rate: Keyaerts and colleagues also report their findings on the use of inhaled NO gas to treat a number of people with SARS. Their results suggest an associated immediate improvement in oxygenation and a lasting effect after termination of the inhalation of NO, which is known to be a potent mediator of airway inflammation (Groneberg et al., 2005).
            • Drug Resistance:
          • Ribavirin (Stockman et al., 2006): Ribavirin, a nucleoside analogue that has activity against a number of viruses in vitro, was widely used for treating SARS patients after observing the lack of clinical response to broad-spectrum antibiotics and oseltamivir (Chan et al., 2006). Because the urgency of the international outbreak did not allow time for efficacy studies, physicians in Canada and Hong Kong treated the earliest patients with intravenous ribavirin, based on its broad-spectrum antiviral activity (Stockman et al., 2006).
            • Applicable: IV and/or oral ribavirin has been used empirically in some adults and a limited number of children with severe acute respiratory syndrome (SARS), alone or in conjunction with corticosteroids (AHFS Drug Information 2006).
            • Contraindicator: Oral ribavirin is contraindicated in patients with known hypersensitivity to ribavirin or any component of the formulations (AHFS Drug Information 2006). Oral ribavirin is contraindicated in women who are or may become pregnant and also is contraindicated in male partners of such women. Oral ribavirin is contraindicated in patients with hemoglobinopathies (e.g., thalassemia, sickle-cell anemia). Oral ribavirin is contraindicated in patients with creatinine clearances less than 50mL/minute. Concomitant use of oral ribavirin and peginterferon alfa or interferon alfa is contraindicated in patients with autoimmune hepatitis. Concomitant use of ribavirin tablets and peginterferon alfa-2a is contraindicated in cirrhotic patients with chronic hepatitis C viris (HCV) monoinfection (without coexisting HIV infection) who have hepatic decompensation (Child-Pugh score greater than 6; class B and C) prior to or during treatment. Concomitant use of ribavirin tablets and peginterferon alfa-2a is contraindicated in cirrhotic patients with chronic HIV infection who are coinfected with HIV and have hepatic decompensation (Child-Pugh score 6 or greater) prior to or during treatment (AHFS Drug Information 2006).
            • Complication: If ribavirin is used in the empiric treatment of SARS pending more definitive treatment recommendations, the risks associated with the drug (e.g., severe hemolytic anemia, teratogenic potential) and the lack of definite evidence of therapeutic benefit should be considered. In a recent retrospective analysis of SARS patients in Toronto, ribavirin was used for empiric therapy in 88% of patients but was temporally associated with clinically important toxicity and was discontinued in 18% (AHFS Drug Information 2006). Knowles and co-workers reported common adverse events in 110 people with suspected or probable SARS who were treated with ribavirin. 61% of these people had evidence of haemolytic anaemia; hypocalcaemia and hypomagnesaemia were reported in 58% and 46% of the people, respectively (Groneberg et al., 2005).
            • Success Rate: Despite thirty reports of SARS-infected patients treated with ribavirin, there is no convincing evidence that it led to recovery (Stockman et al., 2006). It is now known that ribavirin has no significant in vitro activity against SARS-CoV (Chan et al., 2006).
            • Drug Resistance: Development of in vitro or in vivo resistance to the antiviral activity of ribavirin has not been fully evaluated. Unlike some other currently available antiviral agents (e.g., acyclovir, amantadine), resistance to ribavirin does not appear to develop during repeated exposure of most susceptible viruses to the the drug. The lack of development of resistance in susceptible viruses may result from ribavirin's multiple mechanisms of antiviral action (AHFS Drug Information 2006).
          • Type I interferon (IFN) (Stockman et al., 2006): Type I IFNs such as IFN-a are produced early as part of the innate immune response to virus infections (Chan et al., 2006).
            • Applicable: Type 1 interferons have been shown to inhibit SARS-coronavirus replication in in-vitro studies. Because of initial reports describing these in-vitro results, interferons were used clinically during the latter part of the outbreak (Groneberg et al., 2005).
            • Contraindicator:
            • Complication: Flu-like symptoms are the most commonly reported side effect after long term treatment with interferon, however hypertension, myocardial infarctions and depression have been reported (Stockman et al., 2006).
            • Success Rate: Use of IFN-a 1 plus corticosteroids was associated with improved oxygen saturation, more rapid resolution of radiographical lung opacities and lower levels of CPK (creatine kinase) in SARS patients (Chan et al., 2006). Two studies of IFN-alpha given with steroids and/or ribavirin were reported. No significant difference was seen in outcome between IFN-alpha treatment group and those treated with other regimens. Results of both studies were inconclusive due to a lack of a consistent treatment regimen or suitable control group (Stockman et al., 2006).
            • Drug Resistance:

        7. Other Information:
          • Elderly: Older subjects with SARS often show atypical presenting features such as decrease in general well-being, poor feeding and delirium. The fact that older SARS patients might be afebrile further hinders early recognition (Chan et al., 2006).
          • Children and Adolescents: Young children (< 12 years of age) often run a more benign clinical course mimicking other viral upper respiratory tract infections, whereas teenagers tend to have a clinical course similar to that of adult SARS patients. SARS in children 1-12 years of age was more coldlike, with rhinorrhea being a fairly consistent finding, along with headache, chills, myalgia and rigors (Denison et al., 2004). In children older than 12 years old, SARS more closely approximated adult disease, with more common myalgia, dyspnea and clinical pneumonia with hypoxemia in addition to the findings above (Denison et al., 2004) No fatality has been reported in young children and teenage patients (Chan et al., 2006). Fever was present in all cases reported, cough was prominent and significant lymphopenia was present in a majority of cases (Denison et al., 2004).
          • Pregnant Women: Finally 2 reports of SARS in pregnant women and children did not find evidence of SARS clinical disease, characteristic laboratory changes or positive diagnostic tests. However, there were spontaneous abortions during the first trimester in 4 of 7 women with SARS in 1 study and in 5 women with SARS in the second or third trimester. Of these 5 women, 3 required emergency cesarean sections and the remaining 2 who carried to term developed oligohydramnios with intrauterine growth retardation of the infant. Because of the severity of the disease in the women and because they were on both intravenous ribavirin and steroids, and in the absence of virologic evidence, the findings may be related either to hypoxemia and placental insufficiency or to direct toxic effects of the therapeutic interventions (Denison et al., 2004).

    4. Prevention:
      1. Current prevention strategies (Peiris et al., 2003):
        • Ontology: UMLS:C0205394
        • Description: In the absence of a vaccine, preventing the transmission of SARS involves triage, early case detection and isolation of patients to prevent transmission within hospitals, public education, contact tracing and quarantine of contacts to prevent community transmission, and surveillance at border crossings through health-declaration forms and the monitoring of persons for fever. Nosocomial spread has been one of the major epidemiologic features of SARS outbreaks, and the elimination of hospital transmission through enhanced infection-control practices is an important control measure (Peiris et al., 2003). Precautions against transmission through respiratory droplets and contact precautions are critical in preventing the bulk of hospital transmission. In addition, precautions against airborne transmission have been recommended and are clearly needed when aerosol-generating procedures are being undertaken (Peiris et al., 2003).
      2. Potential therapeutic vaccines and neutralizing antibodies (Taylor et al., 2006):
        • Ontology: UMLS:C0301521, C0475463, SNOMED:A3076630, A3162828
        • Description: Spike-specific monoclonal and polyclonal antibodies that neutralize the virus have been developed and passive transfer of immune serum into naive mice protected them from infection with SARS-CoV. This suggests that neutralizing antibody alone can prevent viral infection. Neutralization may not require host recognition of the Fc region of the antibody, but the need to develop humanized forms of these types of antibodies may be critical if they are to be considered for use as a treatment. A human monoclonal antibody, derived from a phage display library, was administered to ferrets and protected the ferrets from lung disease and the shedding of virus in pharyngeal secretions. Neutralizing antibodies from convalescent patients have been identified and characterized. Usually neutralizing epitopes are located in the spike protein of the virus. Recent evidence has determined that virus neutralization is sensitive to deglycosylation of the spike protein, suggesting that conformational epitopes are important in antibody recognition (Taylor et al., 2006).
      3. Potential inactivated SARS-CoV vaccine (Taylor et al., 2006):
        • Ontology: UMLS:C0042212, C0042212
        • Description: Several reports have showed that SARS-CoV inactivated with formaldehyde, UV light, and Beta-propiolactone can induce virus-neutralizing antibodies in immunized animals, and the first inactivated SARS-CoV vaccine is being tested in the clinical trials in China (Jiang et al., 2005).
      4. Potential recombinant virus and virus-like particle vaccine (Taylor et al., 2006):
        • Ontology: UMLS:C0281606
        • Description: Recombinant viruses may be used to elicit responses to introduced SARS-CoV genes. The first type of recombinant virus is a defective or non-pathogenic vector that expresses SARS-CoV proteins. The second type is one that is stimulated to assemble virus-like particles (VLP) in vitro. VLPs containing the structural envelope proteins including spike (S), envelope (E) and membrane protein (M) have been assembled by coinfecting insect cells with three baculoviruses expressing one of the three structural proteins. Structural proteins expressed by the live attenuated bovine parainfluenza virus type 3 (BHPIV3) were evaluated for efficacy in hamsters and African green monkeys. High titer neutralizing antibodies were obtained after only one intranasal immunization with this vector. Single immunization with BHPIV3 expressing S alone provided complete protection upon challenge with SARS-CoV. Recombinant live attenuated modified vaccinia virus Ankara (MVA) was used to deliver the SARS spike protein (rMVA-S) into Balb/c mice. Neutralizing antibodies were obtained and a reduction in the viral titer was observed after challenge with live SARS-CoV. Only ferrets that were challenged with SARS-CoV after vaccination with rMVA-S showed enhanced liver disease as demonstrated by increases in ALT values and the presence of mononuclear hepatitis upon histological examination. These data suggest enhanced disease due to vaccination with a SARS protein. Adenoviruses expressing the S, M or N proteins were used in combination to vaccinate rhesus macaques. The immunized animals all had antibody responses to the S protein and T-cell responses to the N protein. Highly infectious HIV particles expressing the S protein have been made, primarily to study the host-cell distribution of the putative SARS-CoV receptor [35]. Additionally, investigating the requirements for viral receptor binding and entry will also enhance our understanding of the requirements for viral control. Recombinant HIV particles that express the SARS spike protein may provide insight into cell tropism and receptor expression profiles [35]. Another retrovirus, murine leukemia virus, was used to generate infectious particles containing most of the S protein. Convalescent serum was able to neutralize infection of the recombinant virus in Vero cells (Taylor et al., 2006).
      5. Potential DNA vaccine (Taylor et al., 2006):
        • Ontology: UMLS:C0376613
        • Description: High cytotoxic T-lymphocyte (CTL) and antibody responses were observed after mice were injected three times with a recombinant plasmid vector expressing the N protein. Mice immunized with a plasmid containing the S protein produced anti-SARS-CoV IvG and developed neutralizing antibodies and a T-cell mediated response resulting in a six-fold reduction in viral titer in the lungs. Plasmids encoding either the S1 or S2 regions of the spike protein elicited antibody production in mice. Neither the S1 or S2 antibodies alone were capable of neutralizing the virus; however, cooperatively they enabled neutralization of the virus, suggesting that both regions of the spike protein are important for host-cell viral entry. The nucleocapsid protein may also stimulate an effective immune response. DNA vaccination with calreticulin fused to the N protein generated SARS-specific humoral and cellular immunity in C57BL/6 mice. Calreticulin was used because it was found to enhance major histocompatibility complex class I presentation of fusion proteins to CD8 (+) T cells. Recombinant viruses may be generated from the full-length infectious cDNA clone of SARS-CoV. This clone may provide a source for genetic manipulation of the genome. Once the viral virulence factors are understood, attenuated strains may be obtained by engineered mutation of the virus. Vaccine development may proceed through the undertaking of a systematic approach to understanding the correlates of immunity raised by SARS-CoV. Much of the focus has centered towards the humoral response and neutralizing epitopes, but cell-mediated immunity may also be important. CTL epitopes within SARS-CoV that may be presented by 99% of the human leukocyte antigen supertypes were identified by advanced bioinformatics. Further characterization of these epitopes, including their recognition by convalescent serum, should advance the understanding of important immunological features in the control of SARS-CoV (Taylor et al., 2006).

    5. Model System:
      1. African green monkey (Chlorocebus aethiops) (Weiss et al., 2005):
        1. Ontology: UMLS:C0026438, SNOMED:A4711197
        2. Model Host: African green monkey
        3. Model Pathogens:
        4. Description: SARS coronavirus (SARS-CoV) administered intranasally and intratracheally to rhesus, cynomolgus and African Green monkeys (AGM) replicated in the respiratory tract but did not induce illness. The titer of serum neutralizing antibodies correlated with the level of virus replication in the respiratory tract (AGM>cynomolgus>rhesus). Moderate to high titers of SARS-CoV with associated interstitial pneumonitis were detected in the lungs of AGMs on day 2 and were resolving by day 4 post-infection. Following challenge of AGMs 2 months later, virus replication was highly restricted and there was no evidence of enhanced disease. These species will be useful for the evaluation of the immunogenicity of candidate vaccines, but the lack of apparent clinical illness in all three species, variability from animal to animal in level of viral replication, and rapid clearance of virus and pneumonitis in AGMs must be taken into account by investigators considering the use of these species in efficacy and challenge studies (McAuliffe et al., 2004).
      2. Cynomolgus macaque (Macaca fascicularis) (Weiss et al., 2005):
        1. Ontology: UMLS:C0024399, SNOMED: A2883669
        2. Model Host: Cynomolgus macaque
        3. Model Pathogens:
        4. Description: The cynomolgus macaque (Macaca fascicularis) was the first animal subjected to experimental infection with SARS-CoV (Weiss et al., 2005). Initial studies of macaque models were promising. The histology of infected lung tissue is similar to that in humans. Both acute and organized stages of diffuse alveolar damage were seen when the macaques were sacrificed on the sixth day after a heavy dose of the virus. SARS-CoV was detected in the alveolar epithelial cells and in the intra-alveolar syncytial cells. However, detailed morphological studies and viral distribution in other organs in these animal studies are lacking. In studies involving longer observation times, the disease in macaque models appears self-limiting and different from the genuine human disease (Lo et al., 2006). Rowe et al. have demonstrated that cynomolgus as well as rhesus macaques develop a mild self-limited respiratory infection very different from that observed in humans/ (Weiss et al., 2005) Rhesus and cynomolgus macaques were challenged with 107 PFU of a clinical isolate of the severe acute respiratory syndrome (SARS) coronavirus. Some of the animals developed a mild self-limited respiratory infection very different from that observed in humans with SARS (Rowe et al., 2004). The usefulness of the macaque as a model of the disease remains to be established (Lo et al., 2006).
      3. Domestic cat (Felis domesticus) (Martina et al., 2003):
        1. Ontology: UMLS:C0007450, SNOMED:A4709169
        2. Model Host: Domestic cate
        3. Model Pathogens:
        4. Description: Domestic cats (Felis domesticus) support SARS-CoV replication. Natural asymptomatic infection in cats was first documented during the community outbreak at Emory Gardens, Hong Kong. Martina et al. studied the susceptibility of cats to experimental SARS-CoV infection. Cats inoculated intratracheally with 106 TCID50 do not develop clinical signs, although cats shed virus from the pharynx starting at 2 days p.i. and continuing until day 10. The virus was isolated from nasal swabs on days 4 and 6 p.i., whereas rectal swabs were negative. SARS-CoV was isolated from the trachea and lungs, although titers were low (1 X 103 TCID50/ml), peaking at days 6 to 8 p.i. Some infected cats developed mild pulmonary lesions. Cats seroconvert by day 28 p.i. (Weiss et al., 2005).
      4. Ferret (Mustela putorius furo) (Martina et al., 2003):
        1. Ontology: UMLS:C0015859, SNOMED:A2872878
        2. Model Host: Ferret
        3. Model Pathogens:
        4. Description: To date, there is evidence that ferrets support SARS-CoV replication and that lungs of infected ferrets show some pulmonary lesions milder than those observed in cynomolgus macaques (although no pathological analysis was reported in this study). However, ferrets did not develop fever or respiratory signs, but they were lethargic (Weiss et al., 2005). Photomicrographs of the evolution of histopathological findings following primary infection with SARS-CoV in ferrets have not been published so it is not yet possible to compare the findings in ferrets with those in other animal models. Therefore, based on the evaluation of published reports, it would be premature to conclude that ferrets are a superior model for SARS (Subbarao et al., 2006).
      5. Golden Syrian hamster (Mesocricetus auratus) (Roberts et al., 2006):
        1. Ontology: UMLS:C0018561, SNOMED:A3321185
        2. Model Host: Golden Syrian hamster
        3. Model Pathogens:
        4. Description: Golden Syrian hamsters are excellent models for SARS-CoV infection because the virus replicates to high titer in the respiratory tract [108 50% tissue culture infectious doses (TCID50) per gram of lung tissue following intranasal administration of 103 TCID50 of virus] with associated interstitial pneumonitis, pulmonary consolidation and diffuse alveolar damage. Infection is not accompanied by overt clinical illness and the virus is cleared seven to ten days pi. As in mice, SARS-CoV infection of golden Syrian hamsters elicits a robust neutralizing antibody response and previously infected hamsters are protected from subsequent infection. A correlation between the level of virus recovered from the lungs and the extent of pneumonitis has been demonstrated in SARS-CoV infected hamsters. The fact that SARS-CoV reproducibly replicates to extremely high titer in the respiratory tract of hamsters with associated pneumonitis makes this an excellent model for the evaluation of vaccines, immunoprophylaxis and immunotherapy for SARS (Subbarao et al., 2006). Hamsters are suitable for vaccine efficacy, immunoprophylaxis and treatment studies (Roberts et al., 2006).
      6. Marmoset (Callithrix jacchus) (Weiss et al., 2005):
        1. Ontology: UMLS:C0006765, SNOMED:A3880780
        2. Model Host: Marmoset
        3. Model Pathogens:
        4. Description: We inoculated common marmosets (Callithrix jacchus) with the objective of developing a small nonhuman primate model of SARS. Two groups of C. jacchus were inoculated intratracheally with cell culture supernatant containing SARS-CoV. In a time course pathogenesis study, animals were evaluated at 2, 4, and 7 days after infection for morphological changes and evidence of viral replication. All animals developed a multifocal mononuclear cell interstitial pneumonitis, accompanied by multinucleated syncytial cells, edema, and bronchiolitis in most animals. Viral antigen localized primarily to infected alveolar macrophages and type-1 pneumocytes by immunohistochemistry. Viral RNA was detected in all animals from pulmonary tissue extracts obtained at necropsy. Viral RNA was also detected in tracheobronchial lymph node and myocardium, together with inflammatory changes, in some animals. Hepatic inflammation was observed in most animals, predominantly as a multifocal lymphocytic hepatitis accompanied by necrosis of individual hepatocytes. These findings identify the common marmoset as a promising nonhuman primate to study SARS-CoV pathogenesis (Greenough et al., 2005).
      7. Mouse (Mus musculus) - BALB/C strain (Subbarao et al., 2006):
        1. Ontology: UMLS:C0025929
        2. Model Host: Mouse
        3. Model Pathogens:
        4. Description: When SARS-CoV is administered intranasally to lightly anesthetized 4-8-week-old BALB/c or B6 mice, the virus replicates to high titer in the upper and lower respiratory tract (nasal turbinates and lung tissues, respectively) without associated signs of morbidity or mortality. Virus replication in the respiratory tract peaks at day two or three post-infection (pi) but it is not accompanied by substantial pulmonary inflammation or pneumonitis. The virus is cleared from the lungs by day five to seven pi. Transient weight loss and pneumonitis are seen in strain 129S mice following SARS-CoV infection. Old (12-14-month-old) BALB/c mice develop signs of clinical illness (weight loss, ruffled fur and mild dehydration) and histopathological evidence of disease (bronchiolitis, patchy interstitial pneumonitis and diffuse alveolar damage) following SARS-CoV infection; this age-related increase in morbidity in BALB/c mice is reminiscent of observations in humans in the 2003 SARS outbreak in which age was a significant risk factor for severe disease and poor outcome. Because SARS-CoV replicates to high titer in the respiratory tract of mice and findings in mice are highly reproducible, the mouse model can be used for evaluation of vaccines, immunoprophylaxis and antiviral drugs. Additionally, the available range of immunologic reagents and knockout mice make it possible to carry out studies of pathogenesis in mice that develop pneumonitis in association with viral replication (Subbarao et al., 2006).
      8. Mouse (Mus musculus) - C57B26 (B6) strain (Glass et al., 2004):
        1. Ontology: UMLS:C0025929
        2. Model Host: Mouse
        3. Model Pathogens:
        4. Description: This study demonstrates that B6 mice can be productively infected by SARS-CoV in the bronchial and bronchiolar epithelium of the respiratory tract, and that virus is rapidly cleared through a mechanism independent of NK cells, NK-T cells, and T and B lymphocytes. Virus is able to spread to the brain at late time points when it has already been cleared by the lung, and may spread to multiple other organs. SARS-CoV induces dramatic up-regulation of a subset of inflammatory chemokines and the chemokine receptor CXCR3, but interestingly this occurs without detectable expression of classic proinflammatory and immunoregulatory cytokines and without evoking marked leukocyte infiltration of the lung. Overall, infected B6 mice do not develop overt disease, but their weight gain is slowed relative to mock-infected controls (Glass et al., 2004). With regard to the first main goal of the study, it is clear that the wild-type B6 mouse does not provide a robust model of lethal pulmonary infection with SARS-CoV. However, with regard to the second main goal, our work has succeeded in validating an acute viral infection model for SARS-CoV in B6 mice that could be relevant to subclinical human infection, and in delimiting the range of immunologic control mechanisms. Moreover, these results suggest that it may eventually be possible to develop a pulmonary disease model for SARS in the mouse by experimentally inactivating innate antiviral control systems (Glass et al., 2004).
      9. Mouse (Mus musculus) - 1259 Sv/Ev and STAT1-/- strains (Hogan et al., 2004):
        1. Ontology: UMLS:C0025929
        2. Model Host: Mouse
        3. Model Pathogens:
        4. Description: Intranasal inhalation of the severe acute respiratory syndrome coronavirus (SARS CoV) in the immunocompetent mouse strain 129SvEv resulted in infection of conducting airway epithelial cells followed by rapid clearance of virus from the lungs and the development of self-limited bronchiolitis. Animals resistant to the effects of interferons by virtue of a deficiency in Stat1 demonstrated a markedly different course following intranasal inhalation of SARS CoV, one characterized by replication of virus in lungs and progressively worsening pulmonary disease with inflammation of small airways and alveoli and systemic spread of the virus to livers and spleens (Hogan et al., 2004). Prolonged viral replication, dissemination of virus to liver and spleen and accompanying pathology are seen in STAT1-/- mice; these mice, therefore, also are suitable for studies of pathogenesis and evaluation of antiviral drugs (Roberts et al., 2006). The results for the STAT1-/- animals highlight the importance of innate immunity in controlling SARS CoV infection and suggest potential therapeutic strategies that augment the innate immune response in the context of interferon action (Hogan et al., 2004).
      10. Rhesus monkey (Macaca mulatta) (Qin et al., 2005):
        1. Ontology: UMLS:C0024400, SNOMED:A2883670
        2. Model Host: Rhesus monkey
        3. Model Pathogens:
        4. Description: A new SARS animal model was established by inoculating SARS coronavirus (SARS-CoV) into rhesus macaques (Macaca mulatta) through the nasal cavity. Pathological pulmonary changes were successively detected on days 5-60 after virus inoculation. All eight animals showed a transient fever 2-3 days after inoculation. Immunological, molecular biological, and pathological studies support the establishment of this SARS animal model. Firstly, SARS-CoV-specific IgGs were detected in the sera of macaques from 11 to 60 days after inoculation. Secondly, SARS-CoV RNA could be detected in pharyngeal swab samples using nested RT-PCR in all infected animals from 5 days after virus inoculation. Finally, histopathological changes of interstitial pneumonia were found in the lungs during the 60 days after viral inoculation: these changes were less marked at later time points, indicating that an active healing process together with resolution of an acute inflammatory response was taking place in these animals. This animal model should provide insight into the mechanisms of SARS-CoV-related pulmonary disease and greatly facilitate the development of vaccines and therapeutics against SARS (Qin et al., 2005).
  2. Other Mammals:
    1. Taxonomy Information:
      1. Species:
        1. Chinese Ferret-Badger :
          • Ontology: UMLS:C1222895
          • GenBank Taxonomy No.: 204267
          • Scientific Name: Melogale moschata (NCBI_Taxonomy)
          • Description: Sera from five animals had neutralizing antibody to the animal coronavirus; these were from three palm civets, a raccoon dog, and a Chinese ferret badger, respectively (Guan et al., 2003).
        2. Horseshoe bat :
          • Ontology: UMLS:C1265469
          • GenBank Taxonomy No.: 49422
          • Scientific Name: Rhinolophus (NCBI_Taxonomy)
          • Description: Here we report that species of bats are a natural host of coronaviruses closely related to those responsible for the SARS outbreak. These viruses, termed SARS-like coronaviruses (SL-CoVs), display greater genetic variation than SARS-CoV isolated from humans or from civets. The human and civet isolates of SARS-CoV nestle phylogenetically within the spectrum of SL-CoVs, indicating that the virus responsible for the SARS outbreak was a member of this coronavirus group (Li et al., 2005). It also appears that SARS-like CoVs from bats are restricted to different species of Rhinolophus (Tang et al., 2006). We found, in a study of horseshoe bat species in different regions of mainland People's Republic of China in 2004, that each of the 4 species surveyed had evidence of infection by a SARS-like-CoV: 2 species (R. pearsoni and R. macrotis) had positive results by both serologic and PCR tests, and 2 (R. pussilus and R. ferrumequinum) had positive results by either serologic or PCR tests, respectively (Wang et al., 2006). A group in Hong Kong found that, when analyzed by PCR, 23 (39%) of 59 anal swabs of wild Chinese horseshoe bats (R. sinicus) contained genetic materal closely related to SARS-CoV. They also found that as many as 84% of the horseshoe bats examined contained antibodies to a recombinant N protein of SARS-CoV (Wang et al., 2006).
        3. Masked palm civet :
          • Ontology: UMLS:C0325072
          • GenBank Taxonomy No.: 9675
          • Scientific Name: Paguma larvata (NCBI_Taxonomy)
          • Description: Massive numbers of palm civets were culled to remove sources for the reemergence of severe acute respiratory syndrome (SARS) in Guangdong Province, China, in January 2004, following SARS coronavirus detection in market animals. The virus was identified in all 91 palm civets and 15 raccoon dogs of animal market origin sampled prior to culling, but not in 1,107 palm civets later sampled at 25 farms, spread over 12 provinces, which were claimed to be the source of traded animals (Kan et al., 2005). Epidemiologic investigations showed that 2 of 4 patients with severe acute respiratory syndrome (SARS) identified in the winter of 2003-2004 were a waitress at a restaurant in Guangzhou, China, that served palm civets as food and a customer who ate in the restaurant a short distance from animal cages (Wang et al., 2005). SARS cases at the restaurant were the result of recent interspecies transfer from the putative palm civet reservoir, and not the result of continued circulation of SARS-CoV in the human population (Wang et al., 2005). Civets' high susceptibility to SARS-CoV infection and wide presence in markets and restaurants strongly indicates an important role for civets in the 2002-2003 and 2003-2004 SARS outbreaks (Wang et al., 2005). However, subsequent studies suggested that the civet may have served only as an amplification host for SARS-CoV and provided the environment for major genetic variations permitting efficient animal-to-human and human-to-human transmissions (Lau et al., 2005). Experimental infection of civets with two different human isolates of SARS-CoV resulted in overt clinical symptoms, rendering them unlikely to be the natural reservoir hosts. These data suggest that although P. larvata may have been the source of the human infection that precipitated the SARS outbreak, infection in this and other common species in animal markets was more likely a reflection of an "artificial" market cycle in naive species than an indication of the natural reservoir of the virus (Li et al., 2005).
        4. Raccoon dog :
          • Ontology: UMLS:C0034499
          • GenBank Taxonomy No.: 34880
          • Scientific Name: Nyctereutes procyonoides (NCBI_Taxonomy)
          • Description: A virus was also detected by virus isolation and direct RT-PCR from the fecal swab of a raccoon dog (Nyctereutes procyonoides) (Guan et al., 2003). Of the 15 raccoon dogs, 12 tested positive with both throat and rectal swabs, while 3 tested positive with throat swabs only (Kan et al., 2005).

    2. Infection Process:

      No infection process information is currently available here.

    3. Disease Information:

      No disease information is currently available here.

    4. Prevention:

      No prevention information is currently available here.

    5. Model System:

      No model system information is currently available here.

IV. Labwork Information

A. Biosafety Information:
  1. Biosafety information for : SARS coronavirus (CDC):
    • Biosafety Level: Universal Precautions (CDC)
    • Applicable: (blood, serum and plasma) and urine specimens (CDC).
    • Precautions:
      • Handle these specimens using Universal Precautions, which includes use of gloves, gown, mask, and eye protection (CDC). Any procedure with the potential to generate fine-particulate aerosols (e.g., vortexing or sonication of specimens in an open tube) should be performed in a biological safety cabinet (BSC). Use sealed centrifuge rotors or sample cups, if available, for centrifugation. Ideally, rotors and cups should be loaded and unloaded in a BSC. Perform any procedures outside a BSC in a manner that minimizes the risk of exposure to an inadvertent sample release (CDC).
    • Disposal:
      • After specimens are processed, decontaminate work surfaces and equipment. Use any EPA-registered hospital disinfectant (CDC).
  2. Biosafety information for : SARS coronavirus (CDC):
    • Biosafety Level: Biosafety Level 2 (CDC)
    • Applicable: Other specimens (e.g., respiratory secretions, stool, or tissue for procedures performed in microbiology or pathology laboratories). The following activities may be performed in BSL-2 facilities with standard BSL-2 work practices: Pathologic examination and processing of formalin-fixed or otherwise inactivated tissues; Molecular analysis of extracted nucleic acid preparations; Electron microscopic studies with glutaraldehyde-fixed grids; Routine examination of bacterial and mycotic cultures; Routine staining and microscopic analysis of fixed smears; Final packaging of specimens for transport to diagnostic laboratories for additional testing. Specimens should already be in a sealed, decontaminated primary container.The following activities involving manipulation of untreated specimens should be performed in BSL-2 facilities and in a Class II BSC: Aliquoting and/or diluting specimens; Inoculating bacterial or mycological culture media; Performing diagnostic tests that do not involve propagation of viral agents in vitro or in vivo; Nucleic acid extraction procedures involving untreated specimens; Preparation and chemical- or heat-fixing of smears for microscopic analysis (CDC).
    • Precautions:
      • Laboratory workers should wear personal protective equipment (PPE), including disposable gloves and laboratory coats. Any procedure or process that cannot be conducted in a BSC should be performed while wearing gloves, gown, eye protection, and respiratory protection. Acceptable methods of respiratory protection include: a properly fit-tested, NIOSH-approved filter respirator (N-95 or higher level) or a powered air-purifying respirator (PAPR) equipped with high-efficiency particulate air (HEPA) filters. Accurate fit-testing is a key component of effective respirator use. Personnel who cannot wear fitted respirators because of facial hair or other fit limitations should wear loose-fitting hooded or helmeted PAPRs. Appropriate physical containment devices (e.g., centrifuge safety cups; sealed rotors) should also be used. Ideally, rotors and cups should be loaded and unloaded in a BSC (CDC).
    • Disposal:
      • All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are placed in a durable, leakproof container and closed for transport from the laboratory. Materials to be decontaminated off-site from the facility are packaged in accordance with applicable local, state, and federal regulations, before removal from the facility (CDC).
  3. Biosafety information for : SARS coronavirus (CDC):
    • Biosafety Level: Biosafety Level 3 (CDC)
    • Applicable: SARS-CoV propagation in cell culture. Initial characterization of viral agents recovered in cultures of SARS specimens (CDC).
    • Precautions:
      • Any procedure or process that cannot be conducted in a BSC should be performed while wearing gloves, gown, eye protection, and respiratory protection. Acceptable methods of respiratory protection include: a properly fit-tested, NIOSH-approved filter respirator (N-95 or higher level) or PAPR equipped with HEPA filters. Accurate fit-testing is a key component of effective respirator use. Personnel who cannot wear fitted respirators because of facial hair or other fit limitations should wear loose-fitting hooded or helmeted PAPRs. Centrifugation should be carried out using sealed centrifuge cups or rotors that are unloaded in a BSC (CDC).
    • Disposal:
      • All cultures, stocks, and other regulated wastes are decontaminated before disposal by an approved decontamination method, such as autoclaving. Materials to be decontaminated outside of the immediate laboratory are placed in a durable, leakproof container and closed for transport from the laboratory. Infectious waste from BSL-3 laboratories should be decontaminated before removal for off-site disposal (CDC).
  4. Biosafety information for : SARS coronavirus (CDC):
    • Biosafety Level: Animal Biosafety Level 3 (CDC)
    • Applicable: Inoculation of animals for potential recovery of SARS-CoV from SARS samples. Protocols involving animal inoculation for characterization of putative SARS (CDC).
    • Precautions:
      • Uniforms or scrub suits are worn by personnel entering the animal room. Wrap-around or solid-front gowns should be worn over this clothing. Front-button laboratory coats are unsuitable. The gown must be removed and left in the animal room. Before leaving the animal facility, scrub suits and uniforms are removed and appropriately contained and decontaminated prior to laundering or disposal. Personal protective equipment used is based on risk assessment determinations (CDC). Biological safety cabinets and other physical containment devices are used whenever conducting procedures with a potential for creating aerosols (CDC).
    • Disposal:
      • All wastes from the animal room (including animal tissues, carcasses, contaminated bedding, unused feed, sharps, and other refuse animal tissues) are transported from the animal room in leak-proof, covered containers for appropriate disposal in compliance with applicable institutional or local requirements. Incineration is recommended (CDC). All wastes from the animal room must be autoclaved prior to incineration or other appropriate terminal treatment (CDC).
B. Culturing Information:
  1. Culture in Vero E6 (green monkey kidney) cells (Ng et al., 2003):
    1. Description: In this study, SARS CoV was found to replicate extremely well in Vero E6 cells reaching high titres. Despite the fact that it was a human host isolate, it grew well in the monkey kidney cells in vitro (Ng et al., 2003). SARS-CoV produces cytopathic effects (CPE) in Vero E6 cells, providing a simple model for in vitro antiviral evaluation (Keyaerts et al., 2005).

    2. Medium:
      1. Vero E6 cells were propagated at 37 C in 5% CO2 in minimal essential medium (MEM) supplemented with 10% fetal calf serum, 1% l-glutamine, and 1.4% sodium bicarbonate. Virus-infected cells were maintained at 37 C in 5% CO2 in MEM supplemented with 2% FCS (Keyaerts et al., 2005).
    3. Optimal Temperature: 37 C (Keyaerts et al., 2005).
    4. doubling-time: O.ur observations suggest that one replication cycle of the SARS-CoV takes 7 h to complete and that onset of intracellular RNA replication is at 6 h post-infection (Keyaerts et al., 2005) From 1 to 5 h post-infection, the intracellular viral load remained nearly constant. A statistical significant (P <0.05) increase in the number of intracellular RNA copies was first observed at 6 h post-infection. After this initial amplification, the titer of the intracellular viral RNA increased exponentially, reaching a titer of 9.6 x 10(6) RNA copies per 10(6) cells at 12 h post-infection (Keyaerts et al., 2005).
  2. Culture in FRhK (fetal rhesus kidney) cells (Peiris et al., 2003):
    1. Description: Viruses were isolated on fetal rhesus kidney cells from the lung biopsy and nasopharyngeal aspirate, respectively, of these two patients. The initial cytopathic effect noted was the appearance of rounded refractile cells appearing 2-4 days after inoculation. The cytopathic effect did not progress in the initial culture tubes but on subsequent passage, and appeared in 24 h (Peiris et al., 2003).

    2. Medium:
      1. Confluent cells were maintained at 34 C in 25-mL flasks containing 10 mL appropriate maintenance medium supplemented with fetal bovine serum (FBS), 100 U/mL penicillin, and 100 ug/mL streptomycin (Kaye et al., 2006). Maintained in modified Eagle medium (MEM) supplemented with 10% FBS (Kaye et al., 2006). An isolate of SARS-CoV, strain HKU 39849, was passaged on 2 occasions in Vero E6 cells to establish a high-titer stock that was used in all infectivity experiments (Kaye et al., 2006). Confluent cells were infected with SARS-CoV, which resulted in a multiplicity of infection of 1.7, or were mock-infected with medium only (Kaye et al., 2006).
    3. Optimal Temperature: 34 C (Kaye et al., 2006).
  3. Culture in LoVo human colonic cells (Chan et al., 2004):
    1. Description: LoVo cells were found to be highly permissive for productive infection with a high viral titre (>3x10(7) viral copies/ml) produced in culture supernatant following a few days of incubation. SARS-CoV established a stable persistent chronic infection that could be maintained after multiple passages. Being a cell line of human origin, LoVo cells could be a useful in vitro model for studying the biology and persistent infection of SARS-CoV (Chan et al., 2004).

    2. Medium:
      1. Cell Line: LoVo; ATCC number: CCL-229; Growth Medium: Ham's F12K medium, 10% foetal bovine serum (Chan et al., 2004). The CUHK-W1 strain of SARS-CoV was grown in Vero cells and the third passage at a concentration of 5 x 10(6) 50% tissue culture infective dose (TCID50)/ml was kept at -70 C for experiments. Cell lines at 60-70% confluence in 25-square centimeter flasks were inoculated with 300 ul of virus suspension to provide a multiplicity of infection (MOI) of 10. Inoculated cell cultures were incubated at 37 C (Chan et al., 2004).
    3. Optimal Temperature: 37 C (Chan et al., 2004).
    4. Note: SARS-CoV did not produce observable cytopathic effects on LoVo cells at the microscopic level. This is in stark contrast to Vero cells, were SARS-CoV produced a lytic infection with characteristic refractile rounding cytopathic effects. The fact that SARS-CoV did not produce lytic infection in this intestinal cell line is reminscence of what we have observed from endoscopic intestinal biopsies obtained from a SARS patient, and in fatal case of SARS (Chan et al., 2004).
  4. Culture in Calu-3 human lung epithelial cells (Tseng et al., 2005):
    1. Description: Calu-3 cells were originally isolated from a human pulmonary adenocarcinoma and are characterized as nonciliated human lung/bronchial epithelial cells (Sims et al., 2007). Here, we report that SARS-CoV can productively infect human bronchial epithelial Calu-3 cells, causing cytopathic effects, a process reflective of its natural course of infection in the lungs (Tseng et al., 2005).

    2. Medium:
      1. Calu-3 cells were grown in DMEM medium (Dulbecco's modified Eagle's minimal essential medium) supplemented with 20% fetal calf serum (FCS) (D-20) medium (Tseng et al., 2005). Confluent Calu-3 cell cultures grown in flasks, chamber slides, or 24-well plates were infected with SARS-CoV at indicated MOIs by using standard protocols (Tseng et al., 2005).
    3. Optimal Temperature: 37 C (Tseng et al., 2005).
C. Diagnostic Tests :
  1. Organism Detection Tests:
    1. Electron microscopy of tissue-culture samples (Ksiazek et al., 2003):
      1. Ontology: UMLS:C0026019, SNOMED:A4740732
      2. Time to Perform: unknown
      3. Description: Negative-stain electron-microscopical specimens were prepared by drying culture supernatant, mixed 1:1 with 2.5 percent paraformaldehyde onto Formvarcarbon-coated grids and staining with 2 percent methylamine tungstate. Thin-section electron-microscopical specimens were prepared by fixing a washed cell pellet with 2.5 percent glutaraldehyde and embedding it in epoxy resin (Ksiazek et al., 2003). Electron-microscopical examination of bronchoalveolar-lavage fluid from one patient revealed many coronavirus-infected cells (Ksiazek et al., 2003).
      4. False Positive:
      5. False Negative:
    2. Spike protein-based immunofluorescence assay (IFA) (Manopo et al., 2005):
      1. Ontology: UMLS:C0079604
      2. Time to Perform: unknown
      3. Description: IFA is considered as the "gold standard" for the detection of SARS-CoV infection. The technique is simple and inexpensive to perform with acceptable sensitivity and specificity in the detection of SARS-CoV infection. IFA is also known for its ability to detect antibody responses, as early as 8 days. Therefore, we have developed an IFA technique using recombinant baculovirus expressing the C domain of Spike protein in Sf-9 cells. Our Spike protein-based IFA using the antigenic protein C had a sensitivity and specificity of 100%, which was comparable to the conventional and commercial IFAs (Manopo et al., 2005).
      4. False Positive: There was no false positive result from the 42 serum samples collected from non-SARS patients sera or from the 100 serum samples collected from normal donors, indicating 100% specificity (Manopo et al., 2005).
      5. False Negative: Our IFA could identify all of the 21 positive sera, giving a sensitivity of 100% (Manopo et al., 2005).

  2. Immunoassay Tests:
    1. ELISA for nucleocapsid protein, nucleocapsid protein fragments, and intraviral domain of the membrane protein (Carattoli et al., 2005):
      1. Ontology: UMLS:C0014441, SNOMED:A7873393
      2. Time to Perform: unknown
      3. Description: . Because of the critical role played by serological assays for SARS diagnosis, an in-house ELISA based on SARS-CoV recombinant antigens was developed (Carattoli et al., 2005) Recombinant proteins were purified and used as coating antigens in ELISA set up to analyze the 6 SARS-positive human sera (Carattoli et al., 2005).
      4. False Positive: The specificity of the assay appears to be high as no positive reaction was detected in the sera of 20 healthy subjects and 73 patients with non-SARS, low-tract respiratory infections (Carattoli et al., 2005).
      5. False Negative: High-titre positive reactions were detected in all SARS positive sera (Carattoli et al., 2005).
    2. Double-antigen sandwich ELISA for recombinant N protein (Chen et al., 2005):
      1. Ontology: UMLS:C0014441, SNOMED:A7873393
      2. Time to Perform: unknown
      3. Description: This double-antigen sandwich ELISA, which employs recombinant N protein as the serodiagnostic antigen and uses enzyme-conjugated antigen instead of enzyme-conjugated secondary antibody, provides a safe, specific, and sensitive means of detecting or confirming SARS infection (Chen et al., 2005). Compared with real-time RT-PCR, the sandwich ELISA was less effective for detecting SARS-CoV during the early stage of illness, but it was effective for detecting antibodies in patients who had been ill for more than 10 days (Chen et al., 2005).
      4. False Positive: Patient type: Non-SARS with respiratory illness; No. of samples tested: 22; No. (%) positive: 0. Patient type: Non-SARS with fever; No. of samples tested: 84; No. (%) positive: 0 (Chen et al., 2005).
      5. False Negative: Patient type: Confirmed SARS; No. of samples tested: 407; No. (%) positive: 286 (70.2) (Chen et al., 2005).
    3. Western Blot assay (He et al., 2004):
      1. Ontology: UMLS:C0005863
      2. Time to Perform: unknown
      3. Description: The N195 protein was used to develop a Western blot assay to detect antibodies against SARS coronavirus in 274 clinically blinded samples (He et al., 2004). The N195 protein was identified as a suitable protein to be used as an antigen in Western blot and other possible assays for the detection of SARS coronavirus infection (He et al., 2004).
      4. False Positive: Specificity: 98.3% (He et al., 2004).
      5. False Negative: Sensitivity: 90.9% (He et al., 2004)

  3. Nucleic Acid Detection Tests: :
    1. Two-step real-time RT-PCR assay targeting 3'-NCR (non-coding region) (Houng et al., 2004):
      1. Ontology: UMLS:C0599161
      2. Time to Perform: unknown
      3. Description: In this study, we report the development of a real-time RT-PCR assay based on the highly conserved 3'-NCR of the genome as a quantitative SARS diagnostic system. It was retrospectively demonstrated that the assay could be used as an efficient diagnostic to identify SARS-CoV positive samples derived from SARS probable, or suspected patients during Taiwan's 2003 SARS outbreak (Houng et al., 2004).
      4. Primers:
      5. False Positive: The 3'-NCR based SARS-CoV assay demonstrated 100% diagnostic specificity testing samples of patients with acute respiratory disease from a non-SARS epidemic region (Houng et al., 2004).
      6. False Negative: The assay's detection sensitivity could reach 0.005 pfu or 6-8 GE (genomic equivalences) per assay. It was preliminarily demonstrated that the assay could efficiently detect SARS-CoV from clinical specimens of SARS probable and suspected patients identified in Taiwan (Houng et al., 2004).
    2. One step quantitative RT-PCR assay targeting ORF1b (Poon et al., 2004):
      1. Ontology: UMLS:C0599161
      2. Time to Perform: 1-hour-to-1-day
      3. Description: Here, we report a one step quantitative RT-PCR assay for SARS diagnosis (Poon et al., 2004). RNA was reverse transcribed and amplified by a TaqMan EZ RT-PCR Core Reagents kit in a 7000 Sequence Detection System (Applied Biosystems). The ORF1b region of SARS-CoV was the target for SARS detection (Poon et al., 2004).
      4. Primers:
      5. False Positive: Specificity: 100% (Poon et al., 2004).
      6. False Negative: Sensitivity: 86.2% (Poon et al., 2004).

    3. Reverse transcriptase (RT) loop-mediated isothermal amplification (LAMP) assay targeting Rep gene (Hong et al., 2004):
      1. Time to Perform: 1-hour-to-1-day
      2. Description: The LAMP is a novel approach for nucleic acid amplification that amplifies DNA with high specificity, selectivity, and rapidity under isothermal conditions, thereby obviating the need for a thermal cycler. The amplification efficiency of the LAMP method is extremely high because there is no time loss for thermal change to its isothermal reaction (Hong et al., 2004). Therefore, the LAMP assay has emerged as a powerful tool to facilitate point-of-care genetic testing at the bedside (Hong et al., 2004). A set of six primers comprising two outer, two inner, and two loop primers that recognize eight distinct regions on the target sequence were designed by using the LAMP primer designing support softward program (NET Laboratory, Kanagawa, Japan) (Hong et al., 2004)
      3. False Positive: The sensitivity and specificity of the RT-LAMP assay with regard to RT-PCR were 100 and 87%, respectively (Hong et al., 2004).
      4. False Negative: The sensitivity and specificity of the RT-LAMP assay with regard to RT-PCR were 100 and 87%, respectively (Hong et al., 2004).

  4. Other Types of Diagnostic Tests:
    1. SARS Coronavirus POL assay from EraGen Biosciences (Madison, WI) (Mahoney et al., 2005):
      1. Time to Perform: unknown
      2. Description:
      3. False Positive: Specificity: 86.8% (Mahoney et al., 2005)
      4. False Negative: Sensitivity: 81.8% (Mahoney et al., 2005)
    2. RealART HPA Coronavirus kit from Artus (Hamburg, Germany) (Mahoney et al., 2005):
      1. Time to Perform: unknown
      2. Description:
      3. False Positive: Specificity: 86.8% (Mahoney et al., 2005)
      4. False Negative: Sensitivity: 81.8% (Mahoney et al., 2005)
    3. SARS Coronavirus LightCycler kit from Roche (Branchburg, NJ) (Mahoney et al., 2005):
      1. Time to Perform: unknown
      2. Description:
      3. False Positive: Specificity: 100% (Mahoney et al., 2005)
      4. False Negative: Sensitivity: 36.4% (Mahoney et al., 2005)
    4. Euroimmun SARS-CoV IgG Indirect Immunoflourescence test (IFT) (Euroimmun, Lubeck, Germany) (Mahoney et al., 2005):
      1. Time to Perform: unknown
      2. Description:

V. References

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NCBI_Taxonomy: SARS coronavirus PC4-115 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=296837 ].
NCBI_CoreNucleotide: SARS coronavirus HC/SZ/61/03, complete genome [ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=42556132 ].
NCBI_Taxonomy: SARS coronavirus HC/SZ/61/03 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=260069 ].
NCBI_CoreNucleotide: SARS coronavirus ES260 spike glycoprotein gene, complete cds [ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=99963902 ].
NCBI_Taxonomy: SARS coronavirus ES260 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=385686 ].
NCBI_CoreNucleotide: SARS coronavirus ES191 spike glycoprotein gene, complete cds [ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=99963900 ].
NCBI_Taxonomy: SARS coronavirus ES191 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=385685 ].
NCBI_CoreNucleotide: SARS coronavirus CS24 spike glycoprotein gene, complete cds [ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=99963906 ].
NCBI_Taxonomy: SARS coronavirus CS24 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=385684 ].
NCBI_CoreNucleotide: SARS coronavirus CS21 spike glycoprotein gene, complete cds [ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=99963904 ].
NCBI_Taxonomy: SARS coronavirus CS21 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=385683 ].
NCBI_Taxonomy: SARS coronavirus civet020 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=285949 ].
NCBI_Taxonomy: SARS coronavirus civet019 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=285948 ].
NCBI_Taxonomy: SARS coronavirus civet014 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=285947 ].
NCBI_Taxonomy: SARS coronavirus civet010 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=285946 ].
NCBI_Taxonomy: SARS coronavirus C029 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305418 ].
NCBI_Taxonomy: SARS coronavirus C028 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305417 ].
NCBI_Taxonomy: SARS coronavirus C025 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305416 ].
NCBI_Taxonomy: SARS coronavirus C019 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305415 ].
NCBI_CoreNucleotide: SARS coronavirus C018 spike glycoprotein gene, complete cds [ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=56554890 ].
NCBI_Taxonomy: SARS coronavirus C018 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305414 ].
NCBI_Taxonomy: SARS coronavirus C017 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305413 ].
NCBI_Taxonomy: SARS coronavirus C014 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305412 ].
NCBI_Taxonomy: SARS coronavirus C013 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305411 ].
NCBI_Taxonomy: SARS coronavirus B040 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305410 ].
NCBI_Taxonomy: SARS coronavirus B039 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=299335 ].
NCBI_Taxonomy: SARS coronavirus B033 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305409 ].
NCBI_Taxonomy: SARS coronavirus B029 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305408 ].
NCBI_CoreNucleotide: SARS coronavirus B024 spike glycoprotein gene, complete cds [ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=56554874 ].
NCBI_Taxonomy: SARS coronavirus B024 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305407 ].
NCBI_Taxonomy: SARS coronavirus B012 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305406 ].
NCBI_Taxonomy: SARS coronavirus A022 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=304858 ].
NCBI_CoreNucleotide: SARS coronavirus A021 spike glycoprotein gene, complete cds [ http://www.ncbi.nlm.nih.gov/entrez/viewer.fcgi?db=nuccore&id=56554866 ].
NCBI_Taxonomy: SARS coronavirus A021 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305403 ].
NCBI_Taxonomy: SARS coronavirus A013 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305402 ].
NCBI_Taxonomy: SARS coronavirus A001 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=305401 ].
NCBI_Taxonomy: Bat SARS coronavirus Rp2 [ http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?lvl=0&id=349343 ].
NCBI_Taxonomy: Bat SARS coronavirus Rp1 [