Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) is a serious emerging human infectious disease. In this report, we immunized ferrets (Mustela putorius furo) with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV spike (S) protein. Immunized ferrets developed a more rapid and vigorous neutralizing antibody response than control animals after challenge with SARS-CoV; however, they also exhibited strong inflammatory responses in liver tissue. Inflammation in control animals exposed to SARS-CoV was relatively mild. Thus, our data suggest that vaccination with rMVA expressing SARS-CoV S protein is associated with enhanced hepatitis.

Severe acute respiratory syndrome (SARS) caused by a newly identified coronavirus (SARS-CoV) remains a threat to cause epidemics as evidenced by recent sporadic cases in China. In this communication, we evaluated the efficacy and safety of two SARS vaccine candidates based on the recombinant modified vaccinia Ankara (MVA) expressing SARS-CoV spike or nucleocapsid proteins in ferrets. No clinical signs were observed in all the ferrets challenged with SARS-CoV. On the other hand, vaccination did not prevent SARS-CoV infection in ferrets. In contrast, immunized ferrets (particularly those immunized with rMVA-spike) exhibited significantly stronger inflammatory responses and focal necrosis in liver tissue after SARS-CoV challenge than control animals. Thus, our data suggest that enhanced hepatitis is linked to vaccination with rMVA expressing SARS-CoV antigens.

Severe Acute Respiratory Syndrome (SARS), an emerging disease characterized by atypical pneumonia, has recently been attributed to a novel coronavirus. The genome of SARS Coronavirus (SARS-CoV) has recently been sequenced, and a number of genes identified, including that of the nucleocapsid protein (N). It is noted, however, that the N protein of SARS-CoV (SARS-CoV N) shares little homology with nucleocapsid proteins of other members of the coronavirus family [Science 300 (2003) 1399; Science 300 (2003) 1394]. N proteins of other coronavirus have been reported to be involved in forming the viral core and also in the packaging and transcription of the viral RNA. As data generated from some viral systems other than coronaviruses suggested that viral N-N self-interactions may be necessary for subsequent formation of the nucleocapsid and assembly of the viral particles, we decided to investigate SARS-CoV N-N interaction. By using mammalian two-hybrid system and sucrose gradient fractionations, a homotypic interaction of N, but not M, was detected by the two-hybrid analysis. The mammalian two-hybrid assay revealed an approximately 50-fold increase in SEAP activity (measurement of protein-protein interaction) in N-N interaction compared to that observed in either M-M or mock transfection. Furthermore, mutational analyses characterized that a serine/arginine-rich motif (SSRSSSRSRGNSR) between amino acids 184 and 196 is crucial for N protein oligomerization, since deletion of this region completely abolished the N protein self-multimerization. Finally, the full-length nucleocapsid protein expressed and purified from baculovirus system was found to form different levels of higher order structures as detected by Western blot analysis of the fractionated proteins. Collectively, these results may aid us in elucidating the mechanism pertaining to formation of viral nucleocapsid core, and designing molecular approaches to intervene SARS-CoV replication.

The human coronavirus, associated with severe acute respiratory syndrome (SARS-CoV), was identified and molecularly characterized in 2003. Sequence analysis of the virus indicates that there is only 20% amino acid (aa) identity with known coronaviruses. Previous studies indicate that protein-protein interactions amongst various coronavirus proteins are critical for viral assembly. Yet, little sequence homology between the newly identified SARS-CoV and those previously studied coronaviruses suggests that determination of protein-protein interaction and identification of amino acid sequences, responsible for such interaction in SARS-CoV, are necessary for the elucidation of the molecular mechanism of SARS-CoV replication and rationalization of anti-SARS therapeutic intervention. In this study, we employed mammalian two-hybrid system to investigate possible interactions between SARS-CoV nucleocapsid (N) and the membrane (M) proteins. We found that interaction of the N and M proteins takes place in vivo and identified that a stretch of amino acids (168-208) in the N protein may be critical for such protein-protein interactions. Importantly, the same region has been found to be required for multimerization of the N protein (He et al., 2004) suggesting this region may be crucial in maintaining correct conformation of the N protein for self-interaction and interaction with the M protein.

In March 2003, a novel coronavirus was isolated from patients exhibiting atypical pneumonia and subsequently proven to be the causative agent of the disease now referred to as severe acute respiratory syndrome (SARS). The complete genome of the SARS coronavirus (SARS-CoV) has since been sequenced. The SARS-CoV nucleocapsid (SARS-CoV N) shares little homology with other members of the coronavirus family. To determine if the N protein is involved in the regulation of cellular signal transduction, an ELISA-based assay on transcription factors was used. We found that the amount of transcription factors binding to promoter sequences of c-Fos, ATF2, CREB-1, and FosB was increased by the expression of SARS-CoV N. Since these factors are related to AP-1 signal transduction pathway, we investigated whether the AP-1 pathway was activated by SARS-CoV N protein using the PathDetect system. The results demonstrated that the expression of N protein, not the membrane protein (M), activated AP-1 pathway. We also found that SARS-CoV N protein does not activate NF-kappaB pathway, demonstrating that activation of important cellular pathways by SAS-CoV N protein is selective. Thus our data for the first time indicate that SARS-CoV has encoded a strategy to regulate cellular signaling process.

The mechanisms underlying severe thrombocytopenia in dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS) are not completely understood. We present here the first evidence that dengue type 2 virus binds to human platelets only in the presence of virus-specific antibody, supporting a role for immune-mediated clearance of platelets in the pathogenesis of thrombocytopenia in DHF/DSS. Antibody-enhanced binding of virus of platelets was also demonstrated with a panel of eight murine monoclonal antibodies specific for the dengue E protein. The degree of binding was dependent on the antibody used but not on the antibody IgG subclass, indicating that factors other than the platelet Fc receptor are involved in binding of virus-antibody complexes to the platelet surface. Confirmation that antibody-dependent virus binding to platelets is not primarily mediated by the platelet Fc receptor was obtained by demonstrating good binding even when platelets were pretreated with the Fc gamma RII-specific antibody IV.3.

The severe acute respiratory syndrome virus (SARS) is a coronavirus that instigated regional epidemics in Canada and several Asian countries in 2003. The newly identified SARS coronavirus (SARS-CoV) can be transmitted among humans and cause severe or even fatal illnesses. As preventive vaccine development takes years to complete and adverse reactions have been reported to some veterinary coronaviral vaccines, anti-viral compounds must be relentlessly pursued. In this study, we analyzed the effect of aurintricarboxylic acid (ATA) on SARS-CoV replication in cell culture, and found that ATA could drastically inhibit SARS-CoV replication, with viral production being 1000-fold less than that in the untreated control. Importantly, when compared with IFNs alpha and beta, viral production was inhibited by more than 1000-fold as compared with the untreated control. In addition, when compared with IFNs alpha and beta, ATA was approximately 10 times more potent than IFN alpha and 100 times more than interferon beta at their highest concentrations reported in the literature previously. Our data indicated that ATA should be considered as a candidate anti-SARS compound for future clinical evaluation.

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