To the serum dilutions, virus was added; 25 l virus stock (107 PFU ml?1) was added to 15 ml of medium and from that a 1/10 dilution was made and 100 l/well of the last dilution was added to the serum dilutions. group\specific proteins is termed 3a and the 3a protein has also been shown to be expressed in SARS\CoV infected cells [4, 5] and could be detected in tissues obtained from SARS patients [5, 6, 7]. Antibodies against 3a have also been detected in different cohorts of SARS patients [8, 9, 10, 11]. The 3a protein consists of 274 amino acids (aa) and contains three putative transmembrane domains and it is expressed on the cell surface [4, 12]. The topology of 3a on the cell surface was determined experimentally: its first 34 aa, i.e. before the first transmembrane domain, is facing the extracellular matrix and its C\terminal after the third transmembrane domain (i.e. aa 134C274) is facing the cytoplasm [4]. As 3a is a novel coronavirus structural protein [12, 13], its N\terminal ectodomain would be expected to protrude out of the virion. Interestingly, in two separate cohorts of SARS patients, one from Taiwan [14] and one from Hong Kong [15], B cells recognizing the N\terminal region of 3a were isolated from patients. In addition, it was recently reported that the N terminal of 3a elicits strong and potentially protective humoral responses in infected patients [11]. In this study, rabbit polyclonal antibodies targeted against the N\terminal ectodomain and the C\terminal cytoplasmic domain of the 3a protein were tested for their abilities to inhibit SARS\CoV propagation in Vero E6 culture. 2.?Materials and methods 2.1. Cell\line and virus Rac-1 The Vero E6 cells and SARS\CoV isolate used in this study have been previously described [16]. Culturing of 293T cells have been previously described [4]. 2.2. Synthesis of peptide and production of rabbit polyclonal antibodies A peptide ((C)AQPVKIDNASPAST), which corresponds to amino acids 15C28 of SARS\CoV 3a protein, was synthesized by BioGenes GmbH (Berlin, Germany). The peptide was conjugated to a carrier, Limulus Polyphemus Hemocyanine (LPH) from horseshoe crab, and used to immunize two rabbits using standard protocols. All procedures were performed by BioGenes GmbH. The immunization schedule is showed in Table 1 . All the TC-S 7010 (Aurora A Inhibitor I) sera were tested by Western blot analysis. Table Table 1 Schedule for the immunization of rabbits (#2 and #3) with a synthetic peptide corresponding to 15–28 amino acids of SARS\CoV 3a protein thead th valign=”top” rowspan=”1″ colspan=”1″ Day /th th valign=”top” rowspan=”1″ colspan=”1″ Immunization no. /th th valign=”top” rowspan=”1″ colspan=”1″ Bleed no. /th /thead 01Pre\immune72C143C284C35C1495C63C2776C91C31207C127C41418C148C5 Open in a separate window A rabbit polyclonal antibody raised against bacterially\expressed GST\3a (134\274aa) has been previously described [4]. This antibody targets the C\terminal cytoplasmic domain of 3a and the 6th bleed was used in this study. A neutralizing antibody (rabbit anti\S10) that targeted the SARS\CoV spike (S) protein was also used in the neutralizing assays [17]. The last bleed obtained after 16 immunizations was used. 2.3. Western blot analysis and immunofluorescence experiments In order to express recombinant 3a protein in mammalian cells, Vero E6 cells were transfected with a cDNA construct (pXJ\3a) for expressing TC-S 7010 (Aurora A Inhibitor I) full\length 3a protein, as previously described [4]. Transfected cells were then subjected to Western blot analysis and immunofluorescence experiments as previously described [4]. Briefly, TC-S 7010 (Aurora A Inhibitor I) cells were grown to 80% confluence in a 6 cm dish and transfected with 1 g of the plasmid. The cells were harvested after 16 h and washed with PBS and then lysed in 1 ml of lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 0.5% NP40, 0.5% deoxycholic acid, 0.005% SDS, 1 mM PMSF). After 6 rounds of alternate freezing and thawing, the cells suspension was centrifuged at 13 000 rpm for 20 min at 4 C. The lysates were used for Western blot analysis. Western blot analysis was also performed on Vero E6 cells infected with SARS\CoV at a multiplicity of infection (MOI) of 1 1. At 24 h post\infection, the cells were washed with PBS and lysed in 350 l of lysis buffer and the lysate was subjected to Western blot analysis in a similar manner. For immunofluorescence experiments, the cells were grown on coverslips and transfected as described above. About 16 h later, the cells were fixed with 4% paraformaldehyde and/or.