Investigation Of Animal Reservoirs Of SARS CoV And Immune Characteristics Of The Structural Proteins

Severe acute respiratory syndrome (SARS), firstly emerged in China in November2002, then spread widely and affected persons in all age groups. The causative agenthas subsequently been identified as a novel coronavirus, SARS-associatedcoronavirus, SARS CoV. In despite that much effort has been done about this kind ofvirus, we still not very clear about its nature reservoir and the cross-host transmissionmechanisms. And very little was known about the interaction between SARS CoVand host cells. Effective vaccines were very important for prevention of there-emergence of SARS CoV. In this paper, we have mainly investigated the animalreservoirs of SARS CoV and study the immune characterization of its structuralproteins. Our study may help us to elucidate the relationship between SARS virusfound in human and that in animals, and to explain the interspecies transmissionmechanism. They could also provide important information for understanding thepathogenesis of SARS-like CoV and developing effective cross-protective vaccines.In chapter 1, we gave an overview to outline the character of SARS CoV, major onthe derivation and evolution of this virus as well as the function of structural proteins.We also introduced that baculovirus can transduce into some mammalian cells andexpress exogenous genes under mammalian-cell activate promoter elements.The suspected zoonotic origin of SARS CoV illustrates the need for additionalanimal studies on the mechanisms of its interspecies transmission and adaptation tonew hosts. Therefore, we sampled 110 farmed civets at different area from 2003 to2005. PCR and ELISA detection showed that much more SARS like CoV positive civets in Wufeng and Yichang than those in other farms. In addition, to search for thewildlife reservoir of SARS CoV, we used an ELISA to screen individuals from twoorders of wild and domestic mammals in Hubei Province, China, where bats haverecently been found infected with bat SARS-like CoV. We found serological evidenceof exposure to SARS-like CoVs in 4/24 (16.67%) of Chinese ferret badgers (Melogalemoschata); 1/7(14.29%) of hog badgers(Actonyx collaris); 6/18 (33.33%) ofEdward's rat (Leopoldamys edwardsi); and 2/37(5.41%) of palm civets (Pagumalarvata). This is the first demonstration of exposure to SARS-like CoVs in wildmammals other than bats outside wildlife markets in China, and it suggests that alarge diversity of SARS-Iike CoVs exists in wild mammals in China (Chapter 2).The pathogenesis of SARS CoV remains poorly understood. In chapter 3, tworecombinant baculovirus were generated to express the spike (S) protein of SL-CoVisolated from bats (vAcRpS) and the envelope (E) and membrane (M) proteins ofSARS CoV (vAcME), respectively. Co-infection of insect cells with these tworecombinant baculoviruses led to self-assembly of virus-like particles as demonstratedby electron microscopy. Incorporation of RpS into VLPs was confirmed by westernblot and immunogold labeling. Such VLPs up-regulated the level of CD40, CD80,CD86, CD83, and enhanced the secretion of IL-6, IL-10 and TNF-αin immaturedendritic cells(DCs). Immune responses were compared in immature DCs inoculatedwith VLPs or with VLPs formed by S, E and M proteins of human SARS CoV. Theformer showed a stronger ability to stimulate DCs in terms of inducing cytokinesecretion, as evidenced by 2 to 6 fold higher production of IL-6 and TNF-α. Our datahave demonstrated for the first time that SL-CoV VLPs formed by membrane proteinsof different origins, one from SL-CoV isolated from bats (RpS) and the other twofrom human SARS CoV (E and M), activated immature DCs and enhanced theexpression of co-stimulatory molecules and the secretion of cytokines. Finding in thisstudy may provide important information for understanding the pathogenesis ofSARS-like CoV.Continuous efforts have been made to developing a prophylactic vaccine againstSARS CoV. In chapter 4, two modified recombinant baculovirus were constructed, containing mammalian-cell activate promoter elements, human elongation factor1α-subunit (EF-1α) and human cytomegalovirus (CMV) immediate-early promoters.The gene of N or S protein of SL-CoV was cloned under the control of CMVpromoter. Mice were subcutaneously and intraperitonealy injected with these tworecombinant baculoviruses. Our results showed that both humoral and cellularimmune responses were induced in vaccinated groups comparing with control groups.The secretion level of IFN-γwas much higher than that of IL-4 in both recombinantbaculovirus-immunized groups, suggesting a strong Th1 bias of cellular immuneresponses. Additionally, marked increase of CD4 T cell immune responses and highlevel of anti-SARS CoV specific IgG were also detected in recombinantbaculovirus-immunized groups. Our data demonstrate that recombinant baculoviruscan be served as an effective vaccine strategy. In addition, because effective SARSvaccines should consider not only preventing the reemergence of SARS CoV but alsoproviding cross-protection against SL-CoV, findings in this study may provide criticalinformation for developing such cross-protective vaccines.