Detection of SARS Coronavirus in Patients with Suspected SARS

Although the 2003 severe acute respiratory syndrome (SARS) outbreak was controlled, repeated transmission of SARS coronavirus (CoV) over several years makes the development of a SARS vaccine desirable. We performed a comparative evaluation of two SARS vaccines for their ability to protect against live SARS-CoV intranasal challenge in ferrets. Both the whole killed SARS-CoV vaccine (with and without alum) and adenovirus-based vectors encoding the nucleocapsid (N) and spike (S) protein induced neutralizing antibody responses and reduced viral replication and shedding in the upper respiratory tract and progression of virus to the lower respiratory tract. The vaccines also diminished haemorrhage in the thymus and reduced the severity and extent of pneumonia and damage to lung epithelium. However, despite high neutralizing antibody titres, protection was incomplete for all vaccine preparations and administration routes. Our data suggest that a combination of vaccine strategies may be required for effective protection from this pathogen. The ferret may be a good model for SARS-CoV infection because it is the only model that replicates the fever seen in human patients, as well as replicating other SARS disease features including infection by the respiratory route, clinical signs, viral replication in upper and lower respiratory tract and lung damage. INTRODUCTIONSevere acute respiratory syndrome (SARS) caused 8098 reported cases and 774 deaths in 26 countries (WHO, 2004a) in a single autumn-to-spring period from 2002 to 2003, and had significant effects on the global economy. Serological evidence suggests zoonotic transmission of SARS coronavirus (CoV) into the human population for several years before this outbreak (Zheng et al., 2004); transmission to humans has continued, resulting in at least four independent non-laboratory associated cases in (Che et al., 2006Fleck, 2004; Guan et al., 2005; WHO, 2004b). The aetiological agent of SARS has been identified as a novel human CoV by sequencing of its genome (Marra et al., 2003;Rota et al., 2003) and by experimental infection of macaques to fulfil Koch's postulates (Fouchier et al., 2003). It is of particular concern as a zoonosis because it can replicate in a large number of animals including cats, pigs, ferrets, foxes, monkeys and rats (Chen et al., 2005; et al., 1996;Olsen, 1993). Although antibodies to CoV N proteins have no virus-neutralizing activity, there is evidence that the protein may provide protection in vivo by induction of cell-mediated immunity, although it has also been suggested to induce eosinophilic infiltrates resulting in immunopathology (Deming et al., 2006;Enjuanes et al., 1995; Stohlman et al., 1995;Wesseling et al., 1993). N protein has been shown to generate CoV-specific CD8 + T cells (Boots et al., 1991;Seo et al., 1997; Stohlman et al., 1993 Stohlman et al., , 1995; it also provides protection in animals in response to infection by animal CoV (Collisson et al., 2000;Seo et al., 1997). In addition, vaccination with SARS N protein decre...

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“…Ferrets shed virus from the upper respiratory tract in a similar way to human SARS patients (Chan et al, 2004). As shown in Fig.…”

Section: Nasal Virus Sheddingmentioning

confidence: 72%

Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines

See

Petric

Lawrence

et al. 2008

Journal of General Virology

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“…Northern blotting showed that ACE2 mRNA is expressed abundantly in endothelial cells of the heart, kidneys, and testis (37). In addition, RT-PCR and immunocytochemistry showed that ACE2 mRNA and protein are expressed on oral, nasal, and intestinal mucosa, alveolar epithelial cells in lung, stomach, skin, lymph nodes, thymus, bone marrow, spleen liver, kidney, and brain, and arterial and venous endothelial cells and arterial smooth muscle cells in many organs (38)(39)(40)(41). These sites for ACE2 expression include most of the sites for SARS-CoV replication in autopsy specimens (26).…”

Section: Discussionmentioning

confidence: 99%

CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus

Jeffers

Tusell

Gillim-Ross

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

580

570

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Severe acute respiratory syndrome (SARS) is caused by a novel coronavirus called SARS-CoV (1-3). The virus causes atypical pneumonia with diffuse alveolar damage with an overall mortality of Ϸ10% that ranges from 0% in children and 50% in persons over 65 (2). Coronaviruses bind to their glycoprotein receptors by the Ϸ200-kDa spike glycoprotein, S, on the viral envelope. Identification of virus receptors can provide insight into mechanisms of virus entry, tissue tropism, pathogenesis, and host range.Several types of receptors were previously identified for coronavirus S glycoproteins. The receptors for the murine coronavirus mouse hepatitis virus are murine carcinoembryonic antigen cell adhesion molecule 1 (CEACAM1) and related murine glycoproteins in the carcinoembryonic antigen family in the Ig superfamily (4). The receptors for human coronavirus 229E (HCoV-229E), transmissible gastroenteritis virus of swine, and feline coronavirus in genetic group 1 are aminopeptidase N (APN) glycoproteins (5-8).Angiotensin-converting enzyme 2 (ACE2) was found to be an efficient receptor for the S glycoprotein of SARS-CoV (9, 10). Because the Vero line of rhesus monkey kidney cells is highly susceptible to infection with SARS-CoV, Li et al. (9) used a codon-optimized soluble SARS-CoV spike glycoprotein (amino acids 12-672) fused to the Fc domain of human IgG1 to immunoprecipitate a putative receptor glycoprotein from Vero cell membranes and identified the simian ACE2 protein by mass spectrometry. They showed that expression of recombinant human ACE2 greatly enhanced the susceptibility of human 293T cells to infection by SARS-CoV. Wang et al. (10) independently demonstrated that human ACE2 is a receptor for SARS-CoV by transducing HeLa cells with a retrovirus library of cDNAs from Vero E6 cells and by using flow cytometry to select transduced cells that bound to purified soluble SARS-CoV S glycoprotein (amino acids 14-502) with a 6-histidine tag. They found that the simian cDNA in these cells, which encoded triosephosphate isomerase, enhanced expression of human ACE2 by inserting into the HeLa cell genome immediately upstream of the ACE2 ORF. Murine NIH 3T3 cells expressing recombinant human ACE2, but not those expressing recombinant triosephosphate isomerase, were susceptible to infection by HIV pseudovirus expressing SARS-CoV S protein.In this report, we describe the discovery of an additional receptor for SARS-CoV, CD209L (also called L-SIGN, DC-SIGNR, and DC-SIGN2) (11). Our strategy for identifying a SARS-CoV receptor was to transduce a human lung cDNA library carried by a retroviral vector into Chinese hamster ovary (CHO) cells and use flow cytometry to select transduced CHO cells that bound soluble, codon-optimized, c-myc-tagged SARSCoV S 590 glycoprotein (amino acids 1-590) expressed in 293T cells. The SARS-CoV S 590 -binding cells were then challenged with infectious SARS-CoV, and infection was demonstrated by detection of subgenomic viral RNA synthesis and immunofluorescence with antiviral antibody. The finding that ...

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“…At this juncture, the newer generation of real-time PCR performed on NPAs is expected to have a sensitivity of y80% [41,42]. Later in the disease, viral RNA could also be detected in other specimens, such as stool and urine [36]. Following optimisation, the detection limit of real-time PCR is around 10 RNA copies per reaction.…”

Section: Virological Diagnosis Of Severe Acute Respiratory Syndromementioning

confidence: 99%

“…In equivocal cases, such as the recent laboratory-acquired situations, they could be valuable for establishing the diagnosis. Viral culture is still insensitive [36] and, again, suffers from the drawback of taking too long for bedside decisions. Hence, much effort has been put into the development and evaluation of rapid tests that yield a result within hours.…”

Section: Virological Diagnosis Of Severe Acute Respiratory Syndromementioning

confidence: 99%

“…The evaluated specimen types include nasopharyngeal aspirate (NPA) or swab, throat swab, faeces and urine [36][37][38][39][40][41][42][43]. In the upper respiratory tract, the viral load is low in the 7 days and peaks at around day 10 of illness [22,36,40]. Thus, diagnostic tests for SARS should be compared with reference to the timing of collection.…”

Section: Virological Diagnosis Of Severe Acute Respiratory Syndromementioning

confidence: 99%

See 1 more Smart Citation

Diagnosis and pharmacotherapy of severe acute respiratory syndrome: what have we learnt?

Tsang¹

2004

European Respiratory Journal

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Detection of SARS Coronavirus in Patients with Suspected SARS

Cited by 294 publications

References 14 publications

Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines

Severe acute respiratory syndrome vaccine efficacy in ferrets: whole killed virus and adenovirus-vectored vaccines

CD209L (L-SIGN) is a receptor for severe acute respiratory syndrome coronavirus

Diagnosis and pharmacotherapy of severe acute respiratory syndrome: what have we learnt?

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