Complementar y research action to suport SARS-related diagnostic tests, therapeutic interventions and vaccine development
Severe Acute Respiratory Syndrome (SARS) is a life-threatening form of pneumonia caused by an emerging Coronavirus (SARS-CoV) that likely circulates in bats. The prevention and/ or containment of future outbreaks of this virus, or any of its relatives, will depend on our understanding of their biology, pathogenesis and evolution. To aid in designing an overall strategy, the SARS-DTV project focused its attention on the development of reliable diagnostic tools, specific antiviral compounds, and a SARS-CoV vaccine.
Due to their high mutation frequency, RNA viruses are the most common source of unanticipated virus outbreaks, which are caused either by novel variants of known viruses of by the introduction into the human population of previously unknown viruses, often transmitted from an animal source.[+] Read More
Plaque assay to determine the infectivity titer of SARSCoV on Vero-E6 [monkey kidney] cells. A dilution of a SARS-CoV stock was used to infect a cell monolayer, which was subsequently covered with a solid medium to prevent spread of newly produced virus throughout the cell layer. The local spread of the virus gives rise to clusters of dead cells [plaques], which were visualized after 2 days [using crystal violet staining of living cells ] and counted to determine the virus titer.
The 2003 SARS outbreak, caused by a Coronavirus, was a clear example of the latter situation. Still, less virulent Coronaviruses (causing common colds) were already known to circulate among humans and several others were detected in the post-SARS era, together with many novel Coronaviruses circulating in bats and a variety of other animal species. This underlines the widespread nature and zoonotic potential of the Coronavirus group. The SARSDTV project aimed to dissect the biology of SARS-CoV and increase our general knowledge of Coronaviruses, in order to be well prepared when the SARS virus (or any of its relatives) would (re-)emerge.
The SARS-DTV consortium consists of a balanced mixture of coronavirologists and specialists in other fields from Europe and Asia. The overall objective of the SARS-DTV network was to contribute to the development of candidate anti-SARS-CoV drugs, vaccines and diagnostic assays, by collaborative and complementary research in five Work Packages (WPs). In the 'Molecular Targets' WP, SARS-CoV proteins - in particular the Spike protein and the replicative enzymes that are primary targets for antiviral drug development - were studied both in vitro and in vivo. A combination of structural biology, biochemistry and molecular virology was used.
In the WP 'Correlates of Humoral and Cellular Protection', vaccine development and SARS immunopathology were addressed. The WP 'Model Systems' developed a reverse genetics system for SARS-CoV in order to establish biosafe cell culture-based model systems to study virus replication and its inhibition by specific compounds. The 'Diagnostics and Standardization' WP aimed to develop rapid and reliable diagnostic methods and to promote the exchange of SARS-CoV-specific data and reagents between partners. Finally, the 'Management' WP took care of internal management issues, as well as contacts with the EC and the general public.
With other Coronaviruses, SARS-CoV uses replicative machinery that is unique among RNA viruses because of the large number of enzymatic subunits and the use of several enzymes that are rare or lacking in other virus groups. Both individually expressed subunits and the integral enzyme complex in the living infected cell were studied. This resulted, for example, in two novel crystal structures of important SARS-CoV enzymes (NendoU and ADRP), in the discovery of a unique secondary RNA-dependent RNA polymerase activity (the nsp8 'primase'), in the more detailed description of several other viral enzymes, and in the ultrastructural characterisation of an elaborate network of modified membranes with which viral RNA synthesis is associated in the infected cell.
Furthermore, an initial screening for antiviral lead compounds targeting SARS-CoV enzymes was performed. In addition, a 30 000-compound library was purchased for (ongoing and future) screening campaigns using biochemical assays developed during the characterisation of various enzymes. In a similar screening approach, but now targeting the viral life cycle as a whole, over 2 000 potential antiviral compounds were screened and several were found to show antiviral activity. Derivatives of selected anti-Coronavirus compounds were synthesised to assess their antiviral activity in more detail. Furthermore, an animal model was used to study the in vivo activity of chloroquine against Coronaviruses.
In the consortium's analysis of SARS-CoV Spike protein functions, the interactions with the ACE-2 receptor protein and the fusion process of viral envelope and cellular membrane were investigated. Efforts were made to obtain small molecules (e.g. peptides) that might be used to inhibit receptor-binding and entry of the virus.
In the immunological WP, antigenic sites on SARSCoV proteins were characterised and the role of the humoral and cellular immune response in SARS infection was studied. Using an antibodyphage library approach, genes-encoding neutralising human monoclonal antibodies were cloned and expressed as IgG molecules. Virus neutralisation epitopes and escape were studied using these reagents and transient expression systems required to rapidly produce anti-SARSCoV antibodies were developed. In terms of cellular immunology, SARS-CoV T cell epitopes were defined and fine mapping was performed (55 new CD4 and CD8 epitopes). The magnitude of the responses to the novel epitopes in patient samples was determined.
Partners in the SARS-DTV 'Model Systems' WP successfully developed a vaccinia virus-based reverse genetic systems for SARS-CoV and other Coronaviruses. This important new research tool will enable the development (in progress) of biosafe, cell culture-based systems that can be used for antiviral testing. Furthermore, these systems allow the site-directed mutagenesis of Coronavirus RNA and protein sequences, a technique commonly essential for probing the importance of these elements and functions in the viral life cycle and in virus-host interactions. Several Coronavirus replicase subunits and also the nucleocapsid protein are currently targeted using this approach.
SARS-DTV partners developed prototype immunochromatography assays for the detection of SARS-CoV antigens in patient specimens, which could be developed into assays for rapid bedside testing. Also, rapid tests to detect SARS-CoV-specific nucleic acid sequences were developed, including a novel isothermal amplification method. Finally, the SARS-DTV partners have created a Biobase and Database for central storage and exchange of reagents and information, respectively.
Clearly, because a second episode of SARS has not occurred (yet), the partners' newly acquired knowledge and SARS-DTV prototypic assays and reagents have not been put to the test, in practice. In the meantime, also supported by SARS-DTV reagents and technical advancements, basic research of Coronaviruses continues with a clear purpose and enhanced possibilities. Researchers' knowledge of Coronaviruses was already expanded enormously over the past five years, largely as spin-off of the SARS outbreak. As during the 2003 SARS outbreak, previous studies on Coronaviruses will be the basis for a swift and adequate response in the case of any new outbreaks. In that case, assays to detect and identify the virus are in place, several targets for antiviral drug development have been clearly defined, and candidate vaccines will be available.