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Application of Reverse Genetics to Studies of SCoV

Mark R. Denison, M.D

Because of the potential for re-emergence of SARS, it is important to move forward with research in diagnostics, vaccines, and therapeutics for SCoV. Experience with the development and use of reverse genetics to study other coronaviruses resulted in establishment of reverse genetics for SCoV within months of the onset of the worldwide epidemic (Yount et al., 2003). How should the understanding of other coronaviruses, the rapid advances in research with SCoV, and the development of reverse genetics for SCoV be harnesssed to achieve these goals and attack these critical questions in SCoV replication, pathogenesis, and disease? Certainly, the use of SCoV reverse genetics, along with robust tissue culture systems and emerging animal models, creates the potential to rapidly answer questions concerning: (1) determinants of virus growth in culture; (2) potential mechanisms of transpecies adaptation; (3) sensitivity to and escape from biochemical and immune interference with replication; (4) determinants of virulence and pathogenesis; (5) mechanisms of genome recombination and mutation; (6) functions of and requirements for replicase, structural, and accessory proteins; and (7) development of stably attenuated viruses for use as seed stocks for inactivated vaccine or testing as live-attenuated vaccines.

How then should these critical issues be investigated while recognizing the potential of SCoV to cause severe disease, as well as the potential for rapid spread? First, there is significant experience with other coronaviruses in attenuation of virus replication and pathogenesis, both using virus passage and by direct engineering of changes. Although coronavirus genome organization, proteins, and replication appear more tolerant of changes then previously thought, all changes of gene order, gene deletion, insertion, or mutagenesis so far reported have led to viruses impaired in replication, pathogenesis, or both. Many of the attenuating changes in MHV and other coronaviruses are conserved in SCoV and thus could be tested for likely attenuation in SCoV culture and animal models. Second, where there is clear conservation of sequences, motifs, proteins, or putative functions between SCoV and model viruses such as MHV, new or untested changes might be most rapidly analyzed under BSL2 conditions in those model viruses, and then directly applied to SARS once their phenotypes are determined. Third, all work with SCoV will be performed only under BSL3 conditions. This would also apply to chimeric viruses, whether engineered by introduction into the SCoV background, or by introducing SCoV proteins or sequences with known or predicted pathogenic consequences into other coronavirus backgrounds. Finally, it is important to develop strains of SCoV that are attenuated and stabilized against reversion and recombination, to be used as the basis for studies of other replication and pathogenesis determinants and construction of virus chimeras. Such attenuated variants would provide additional safeguards while allowing application of powerful genetic tools to the study of SCoV emergence, biology, disease, treatment, and prevention. Overall, newly invigorated programs in other human and animal coronaviruses, combined with the new research in SCoV, will shed important new light on this important virus family and perhaps lead to better understanding of the potential for resurgence of SCoV or the emergence of other coronaviruses into human populations.

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