A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening.
(33/203)
AIM: To constructed a three-dimensional (3D) model for the 3C like (3CL) proteinase of SARS coronavirus (SARS-CoV), and to design inhibitors of the 3CL proteinase based on the 3D model. METHODS: Bioinformatics analyses were performed to search the homologous proteins of the SARS-CoV 3CL proteinase from the GenBank and PDB database. A 3D model of the proteinase was constructed by using homology modeling technique. Targeting to the 3D model and its X-ray crystal structure of the main proteinase (Mpro) of transmissible gastroenteritis virus (TGEV), virtual screening was performed employing molecular docking method to identify possible 3CL proteinase inhibitors from small molecular databases. RESULTS: Sequence alignment indicated that the SARS-CoV 3CL proteinase was extremely homologous to TGEV Mpro, especially the substrate-binding pocket (active site). Accordingly, a 3D model for the SARS-CoV 3CL proteinase was constructed based on the crystal structure of TGEV Mpro. The 3D model adopts a similar fold of the TGEV Mpro, its structure and binding pocket feature are almost as same as that of TGEV Mpro. The tested virtual screening indicated that 73 available proteinase inhibitors in the MDDR database might dock into both the binding pockets of the TGEV Mpro and the SARS-CoV 3CL proteinase. CONCLUSIONS: Either the 3D model of the SARS-CoV 3CL proteinase or the X-ray crystal structure of the TGEV Mpro may be used as a starting point for design anti-SARS drugs. Screening the known proteinase inhibitors may be an appreciated shortcut to discover anti-SARS drugs. (+info)
Transmissible gastroenteritis coronavirus packaging signal is located at the 5' end of the virus genome.
(34/203)
To locate the transmissible gastroenteritis coronavirus (TGEV) packaging signal, the incorporation of TGEV subgenomic mRNAs (sgmRNAs) into virions was first addressed. TGEV virions were purified by three different techniques, including an immunopurification using an M protein-specific monoclonal antibody. Detection of sgmRNAs in virions by specific reverse transcription-PCRs (RT-PCRs) was related to the purity of virus preparations. Interestingly, virus mRNAs were detected in partially purified virus but not in virus immunopurified using stringent conditions. Analyses by quantitative RT-PCR confirmed that virus mRNAs were not present in highly purified preparations. Lack of sgmRNA encapsidation was probably due to the absence of a packaging signal (Psi) within these mRNAs. This information plus that from the encapsidation of a collection of TGEV-derived minigenomes suggested that Psi is located at the 5' end of the genome. To confirm that this was the case, a set of minigenomes was expressed that included an expression cassette for an mRNA including the beta-glucuronidase gene (GUS) plus variable sequence fragments from the 5' end of the virus genome potentially including Psi. Insertion of the first 649 nucleotides (nt) of the TGEV genome led to the specific encapsidation of the mRNA, indicating that a Psi was located within this region which was absent from all of the other virus mRNAs. The presence of this packaging signal was further confirmed by showing the expression and rescue of the mRNA including the first 649 nt of the TGEV genome under control of the cytomegalovirus promoter in TGEV-infected cells. This mRNA was successfully amplified and encapsidated, indicating that the first 649 nt of TGEV genome also contained the 5' cis-acting replication signals. The encapsidation efficiency of this mRNA was about 30-fold higher than the genome encapsidation efficiency, as estimated by quantitative RT-PCR. In contrast, viral mRNAs presented significantly lower encapsidation efficiencies (about 100-fold) than those of the virus genome, strongly suggesting that TGEV mRNAs in fact lacked an alternative TGEV Psi. (+info)
Construction, characterization, and immunogenicity of an attenuated Salmonella enterica serovar typhimurium pgtE vaccine expressing fimbriae with integrated viral epitopes from the spiC promoter.
(35/203)
Transmissible gastroenteritis virus (TGEV) is a porcine coronavirus that causes diarrhea, leading to near 100% mortality in neonatal piglets with corresponding devastating economic consequences. For the protection of neonatal and older animals, oral live vaccines present the attractive property of inducing desired mucosal immune responses, including colostral antibodies in sows--an effective means to passively protect suckling piglets. Newly attenuated Salmonella vaccine constructs expressing TGEV S protein epitopes were studied and evaluated for improved humoral immune response to TGEV. The macrophage-inducible Salmonella ssaH and spiC/ssaB promoters were compared for their ability to express the TGEV C and A epitopes in the context of the heterologous 987P fimbriae on Salmonella vaccines. Compared to the ssaH promoter, the Salmonella cya crp vector elicited significantly higher levels of mucosal and systemic antibodies in orally immunized mice when the chimeric fimbriae were expressed from the spiC promoter. The Salmonella spiC promoter construct induced the highest level of chimeric fimbriae after being taken up by the J774A.1 macrophagelike cells. The Salmonella cya crp vaccine vector was shown to incorporate into 987P partially degraded chimeric subunits lacking the TGEV epitopes. In contrast, its isogenic pgtE mutant produced fimbriae consisting exclusively of intact chimeric subunits. Mice immunized orally with the Salmonella pgtE vaccine expressing chimeric fimbriae from the spiC promoter elicited significantly higher systemic and mucosal antibody titers against the TGEV epitopes compared to the parental vaccine. This study indicates that the Salmonella cya crp pgtE vector and the spiC promoter can be used successfully to improve immune responses toward heterologous antigens. (+info)
Bovine viral diarrhea virus isolated from fetal calf serum enhances pathogenicity of attenuated transmissible gastroenteritis virus in neonatal pigs.
(36/203)
A bovine viral diarrhea virus (BVDV-C) was isolated from swine tissue culture cells used to attenuate the transmissible gastroenteritis virus (TGEV) after 68 passes. Piglets given a pure culture of BVDV-C developed clinical signs similar to those of a mild TGEV infection and recovered by 10 days postexposure. Villous blunting and fusion was observed in the small intestine, and a lymphocyte depletion was observed in Peyer's patches in the ileum. Piglets given a combination of BVDV-C and attenuated TGEV developed clinical signs similar to those of a virulent TGEV infection and were euthanized. The combined infection induced a generalized lymphocyte depletion throughout the lymphatic system and villous atrophy in the intestinal tract. Piglets exposed to a another type I strain of BVDV (NY-1) either alone or in combination with the attenuated TGEV had mild clinical signs similar to those of a TGEV infection. Moderate villous atrophy in the ileum and a lymphocyte depletion in the mesenteric lymph node were observed in these piglets postmortem. The data indicate a potential problem for diagnostic laboratories in relation to a diagnosis of virulent TGEV infections and in the field for young piglets exposed to a BVDV-contaminated TGEV vaccine. (+info)
Single amino acid changes in the viral glycoprotein M affect induction of alpha interferon by the coronavirus transmissible gastroenteritis virus.
(37/203)
Transmissible gastroenteritis virus, an enteropathogenic coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vitro and in vivo. Previous studies have shown that virus-infected fixed cells or viral suspensions were able to induce an early and strong IFN-alpha synthesis by naive lymphocytes. Two monoclonal antibodies directed against the viral membrane glycoprotein M (29,000; formerly E1) were found to markedly inhibit virus-induced IFN production, thus assigning to M protein a potential effector role in this phenomenon (B. Charley and H. Laude, J. Virol. 62:8-11, 1988). The present report describes the selection and characterization of a collection of 125 mutant viruses which escaped complement-mediated neutralization by two IFN induction-blocking anti-M protein monoclonal antibodies. Two of these mutants, designated H92 and dm49-4, were found to exhibit a markedly reduced interferogenic activity. IFN synthesis by lymphocytes incubated with purified suspensions of these mutants was 30- to 300-fold lower than that of the parental virus. The transcription of IFN-alpha genes following induction by each mutant was decreased proportionally, as evidenced by Northern (RNA) blot analysis. The sequence of the M gene of 20 complement-mediated neutralization-resistant mutants, including the 2 defective mutants, was determined by direct sequencing of genome RNA. Thirteen distinct amino acid changes were predicted, all located at positions 6 to 22 from the N terminus of the mature M protein and within the putative ectodomain of the molecule. Two substitutions, Thr-17 to Ile and Ser-19 to Pro, were assumed to generate the defective phenotypes of mutants dm49-4 and H92, respectively. The alteration of an Asn-Ser-Thr sequence in dm49-4 virus led to the synthesis of an M protein devoid of a glycan side chain, which suggests a possible involvement of this structure in IFN induction. Overall, these data supported the view that an interferogenic determinant resides in the N-terminal, exposed part of the molecule and provided further evidence for the direct role of M protein in the induction of IFN-alpha by transmissible gastroenteritis virus. The acronym VIP (viral interferogenic protein) is proposed as a designation for this particular class of proteins. (+info)
Efficacy of antiserum produced in goats and pigs to passively protect piglets against virulent transmissible gastroenteritis virus.
(38/203)
The protective effect of sera produced in swine and goats exposed to virulent transmissible gastroenteritis virus (TGEV) or modified-live TGEV was tested in hysterectomy-derived, colostrum-deprived three-day-old pigs. Pigs were given serum with their daily ration of milk, and their immunity to virulent TGEV was determined. The pigs were observed for ten days for clinical signs of TGEV infection. One of nine pigs receiving goat serum was protected whereas all three pigs receiving three doses of swine serum per day were protected. Because virus was not isolated from the goats after oral/intranasal vaccination, it is suggested the virus did not replicate in either the respiratory or digestive tract of the goat. (+info)
Transmissible gastroenteritis virus antibody production in vitro by porcine peripheral blood leukocytes.
(39/203)
The purpose of this study was to demonstrate the in vitro production of transmissible gastroenteritis virus (TGEV)-specific antibodies by peripheral blood leukocytes (PBL) harvested from piglets infected with TGEV. Piglets were infected with the virulent Purdue strain of TGEV and at intervals postinfection their PBL were cultivated in the presence of TGEV antigen, control antigen or pokeweed mitogen (PWM). The culture supernatants were tested for TGEV antibodies by a fixed cell enzyme immunoassay. Antibodies were never found in the supernatants of unprimed PBL cultures from control piglets, nor in cultures stimulated with control antigen, and antibodies were produced more frequently in response to stimulation of primed PBL with viral antigen than with PWM. In PBL cultures stimulated with viral antigen, TGEV antibodies of the IgG class were produced more frequently than IgA class antibodies. Optimal antibody responses were produced by PBL harvested two weeks after infection and cultivated at a concentration of 10(7) cells/mL for five days. (+info)
Binding of transmissible gastroenteritis coronavirus to brush border membrane sialoglycoproteins.
(40/203)
Transmissible gastroenteritis coronavirus (TGEV) is a porcine pathogen causing enteric infections that are lethal for suckling piglets. The enterotropism of TGEV is connected with the sialic acid binding activity of the viral surface protein S. Here we show that, among porcine intestinal brush border membrane proteins, TGEV recognizes a mucin-type glycoprotein designated MGP in a sialic acid-dependent fashion. Virus binding assays with cryosections of the small intestine from a suckling piglet revealed the binding of TGEV to mucin-producing goblet cells. A nonenteropathogenic mutant virus that lacked a sialic acid binding activity was unable to bind to MGP and to attach to goblet cells. Our results suggest a role of MGP in the enteropathogenicity of TGEV. (+info)