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Identifying inhibitors of the SARS coronavirus proteinase.

The Severe Acute Respiratory Syndrome (SARS) is a serious respiratory illness that has recently been reported in parts of Asia and Canada. In this study, we use molecular dynamics (MD) simulations and docking techniques to screen 29 approved and experimental drugs against the theoretical model of the SARS CoV proteinase as well as the experimental structure of the transmissible gastroenteritis virus (TGEV) proteinase. Our predictions indicate that existing HIV-1 protease inhibitors, L-700,417 for instance, have high binding affinities and may provide good starting points for designing SARS CoV proteinase inhibitors.

Antiviral Agents↗

General characteristics and viral susceptibility of a newborn pig kidney (NPK) continuous culture.

We have developed a fibroblastic-like continuous culture of newborn pig kidney (NPK). The current cell line was serially passaged 160 times and appeared to be well suited for production and assay of a number of viruses affecting pigs, such as pig parvovirus, pseudorabies and transmissible gastroenteritis. The cell line appeared aneuploid, with a modal chromosome number of 36 and induced tumors, classified as fibrosarcoma, in athymic mice.

Animals↗

Antiviral effect of trimeric 2',5'-oligoadenylic acid and some of its analogues.

The antiviral effect of 2',5'-trioligoadenylate (2',5'-A3) and some of its analogues was studied using several model cell culture systems and viruses: mice L929 fibroblast cells inoculated with vaccine virus, testicular piglet cells inoculated with Aueszki disease virus (strain BUK-628), and the same culture inoculated with a reference strain of transmissible gastroenteritis virus, strain Purdue-115. Our results suggest that both 2',5'-trioligoadenylate and its analogues are promising antiviral substances against DNA- and RNA-containing viruses.

Adenine Nucleotides↗

Neutralization of enteric coronaviruses with Escherichia coli cells expressing single-chain Fv-autotransporter fusions.

We report here that fusions of single-chain antibodies (scFvs) to the autotransporter beta domain of the IgA protease of Neisseria gonorrhoeae are instrumental in locating virus-neutralizing activity on the cell surface of Escherichia coli. E. coli cells displaying scFvs against the transmissible gastroenteritis coronavirus on their surface blocked in vivo the access of the infectious agent to cultured epithelial cells. This result raises prospects for antiviral strategies aimed at hindering the entry into target cells by bacteria that naturally colonize the same intestinal niches.

Animals↗

High interferon titer in newborn pig intestine during experimentally induced viral enteritis.

We looked for the presence of interferon in the digestive tract of newborn piglets infected with a virulent strain of transmissible gastroenteritis virus (Coronaviridae). High levels of type 1 interferon activity were found early in the disease in jejunal and ileal parts of the intestine as well as in the serum. Enterocytes appeared to be involved in the interferon synthesis. These findings raise the question of the role of interferon in the pathogenesis of viral enteritis.

Animals↗

Rapid diagnosis of porcine epidemic diarrhea virus infection by polymerase chain reaction.

The diagnosis of porcine epidemic diarrhea virus (PEDV) infection in the laboratory is rather fastidious because of difficulties in virus propagation. The feasibility of virus propagation in vivo is also limited by the handling of a number of samples at the same time. In this study, the detection of PEDV by reverse transcription polymerase chain reaction (RT-PCR) is described. The RT-PCR could detect up to 10(4) TCID50/ml of PEDV and did not show any cross reaction with transmissible gastroenteritis virus or porcine rotavirus. Using this method, the detection of PEDV in experimentally inoculated piglets was possible as early as one day after inoculation. These results suggest that the RT-PCR could be applicable for a rapid diagnosis of PEDV infection.

Animals↗

Plant-based vaccines: unique advantages.

Numerous studies have shown that viral epitopes and subunits of bacterial toxins can be expressed and correctly processed in transgenic plants. The recombinant proteins induce immune responses and have several benefits over current vaccine technologies, including increased safety, economy, stability, versatility and efficacy. Antigens expressed in corn are particularly advantageous since the seed can be produced in vast quantities and shipped over long distances at ambient temperature, potentially allowing global vaccination. We have expressed the B-subunit of Escherichia coli heat-labile enterotoxin and the spike protein of swine transmissible gastroenteritis virus at high levels in corn, and demonstrate that these antigens delivered in the seed elicit protective immune responses.

Animals↗

[Concentration of viruses and virus-specific antigens by ultrafiltration with membranes developed in the GDR].

A cellulose acetate membrane, jointly developed by the Institute of Dairy Research in Oranienburg and the Institute of Polymer Chemistry in Teltow, was successfully used to concentrate by factors between ten and 40 virus suspensions of transmissible gastroenteritis and foot and mouth disease with a protein level of roughly 0.1 per cent in a 400-ml cell with magnetic agitation. The procedure took about two hours, with working pressures being between 0.2 MPa and 0.4 MPa. Virus yield in the residue was 100 per cent. Good results were obtained also from virus-specific antigens and calibration proteins with molecular weights of somewhat in excess of 50,000 Dalton.

Antigens, Viral↗

Sequence analysis of the porcine epidemic diarrhea virus genome between the nucleocapsid and spike protein genes reveals a polymorphic ORF.

In order to investigate the genome organization of the porcine epidemic diarrhea virus (PEDV) further, cDNA clones covering the region between the nucleocapsid and the spike (S) protein genes were independently constructed and sequenced for the two virulent isolates Br1/87 and CV777. Of the three major ORFs identified, two were found to encode the major and minor coronavirus membrane proteins M and sM. A potentially single ORF, designated ORF3 according to the pattern of the viral subgenomic mRNAs, could be identified between the S and sM genes. A striking variability, essentially generated by short deletions clustered in a few loci, was observed in the ORF3 of both isolates. The largest predicted polypeptide of 223 amino acids showed homology with polypeptides potentially encoded by other members of the same genetic subset, including two shorter polypeptides of human respiratory virus HCV 229E and one of transmissible gastroenteritis virus TGEV. This homology suggests that the two HCV ORFs may have originated from a single precursor. The function of these polypeptides is not known, but the predicted products of the PEDV ORF3 and related ORFs share features suggestive of a membrane-associated protein.

Amino Acid Sequence↗

Diarrhea: the nemesis of the artificially reared, early weaned piglet and a strategy for defense.

Rearing early weaned piglets artificially for the purpose of increasing the efficiency of the sow is an attractive management concept. However, high death losses resulting from diarrhea in artificially reared piglets have dampered enthusiasm for early weaning. Enterotoxigenic Escherichia coli, transmissible gastroenteritis virus and rotavirus are the three main enteropathogens responsible for causing the diarrhea. The enteropathogens infect the small intestine, which produces a secretory or malabsorptive diarrhea. In nature, the nursing piglet is protected from the enteropathogens by antibody bathing his gut. The source of the antibody is the dam's colostrum and milk. It should be possible to protect artificially reared, early weaned piglets from enteropathogens by feeding them diets that contain antibodies to putative enteropathogens.

Animal Husbandry↗

Molecular characterization of a novel coronavirus associated with epizootic catarrhal enteritis (ECE) in ferrets.

A novel coronavirus, designated as ferret enteric coronavirus (FECV), was identified in feces of domestic ferrets clinically diagnosed with epizootic catarrhal enteritis (ECE). Initially, partial sequences of the polymerase, spike, membrane protein, and nucleocapsid genes were generated using coronavirus consensus PCR assays. Subsequently, the complete sequences of the nucleocapsid gene and the last two open reading frames at the 3' terminus of the FECV genome were obtained. Phylogenetic analyses based on predicted partial amino acid sequences of the polymerase, spike, and membrane proteins, and full sequence of the nucleocapsid protein showed that FECV is genetically most closely related to group 1 coronaviruses. FECV is more similar to feline coronavirus, porcine transmissible gastroenteritis virus, and canine coronavirus than to porcine epidemic diarrhea virus and human coronavirus 229E. Molecular data presented in this study provide the first genetic evidence for a new coronavirus associated with clinical cases of ECE.

Amino Acid Sequence↗

Exceptional flexibility in the sequence requirements for coronavirus small envelope protein function.

The small envelope protein (E) plays a role of central importance in the assembly of coronaviruses. This was initially established by studies demonstrating that cellular expression of only E protein and the membrane protein (M) was necessary and sufficient for the generation and release of virus-like particles. To investigate the role of E protein in the whole virus, we previously generated E gene mutants of mouse hepatitis virus (MHV) that were defective in viral growth and produced aberrantly assembled virions. Surprisingly, however, we were also able to isolate a viable MHV mutant (DeltaE) in which the entire E gene, as well as the nonessential upstream genes 4 and 5a, were deleted. We have now constructed an E knockout mutant that confirms that the highly defective phenotype of the DeltaE mutant is due to loss of the E gene. Additionally, we have created substitution mutants in which the MHV E gene was replaced by heterologous E genes from viruses spanning all three groups of the coronavirus family. Group 2 and 3 E proteins were readily exchangeable for that of MHV. However, the E protein of a group 1 coronavirus, transmissible gastroenteritis virus, became functional in MHV only after acquisition of particular mutations. Our results show that proteins encompassing a remarkably diverse range of primary amino acid sequences can provide E protein function in MHV. These findings suggest that E protein facilitates viral assembly in a manner that does not require E protein to make sequence-specific contacts with M protein.

Amino Acid Sequence↗

CS31A capsule-like antigen as an exposure vector for heterologous antigenic determinants.

CS31A is a multimeric surface protein surrounding certain enterotoxigenic and septicemic bovine Escherichia coli strains. The possibility of using CS31A as a carrier of foreign antigenic determinants was investigated. Introduction of heterologous viral epitopes into the CS31A major subunit, ClpG, was successfully performed in the V3 region of the molecule which encodes for an immunodominant linear epitope. E. coli K-12 strains producing the hybrid CS31A molecules or the purified chimeric proteins were used for mice immunization. By using the C epitope derived from the S protein of the porcine transmissible gastroenteritis virus, significant antiviral antibody titers were elicited and seroneutralization of the virus was demonstrated, confirming that the molecular environment in V3 is favorable for antigen presentation. These results indicate that synthesis of CS31A hybrid proteins by the wild-type strain 31A could become a powerful tool for the development of oral vaccines directed against mucosal pathogens.

Amino Acid Sequence↗

[Test of an attenuated viral strain of TGE as a vaccine].

A live vaccine was produced using a local strain P of the virus of the transmissive gastroenteritis, arrenuated in cell cultures. The vaccinated pregnant sows had high-titer serum and colostrum virus-neutralizing antibodies. In the first days following farrowing there were in the colostrum ummunoglobulins of the Igg class that prevailed, however, IgA and IgM proved more effective in the virus-neutralizing test. Newborn pigs acquired passive immunity at about the 24th hour after the intake of colostrum. They withstood a challenge with a virulent virus.

Animals↗

Antiviral and antigenic properties of recombinant porcine interferon gamma.

Recombinant porcine interferon gamma (rPoIFN gamma) induced a dose-dependent inhibition of the cytopathic effect produced by vesicular stomatitis virus (VSV) challenge of both homologous and heterologous (bovine) cell lines. In addition, an antiviral effect of rPoIFN gamma was demonstrable against the coronavirus transmissible gastroenteritis virus (TGEV) infection of porcine epithelial cells and of pulmonary macrophages. A rabbit anti-PoIFN gamma antiserum was prepared and shown to specifically neutralize the antiviral effects of natural and recombinant porcine IFN gamma preparations. This antiserum could also neutralize recombinant bovine IFN gamma but not recombinant human IFN gamma. These results suggest antigenic homology of porcine and bovine IFN gamma but antigenic differences between these molecules and human IFN gamma.

Animals↗

Viral susceptibility of a cell line derived from the pig oviduct.

Seventeen of 24 RNA viruses and eight of nine DNA viruses replicated in a cell line derived from a pig fallopian tube. The following RNA viruses grew poorly in it: the virus of transmissible gastroenteritis of pig and the swine-influenza, Sendai and bovine para-influenza type 3 viruses. Among other RNA viruses an untyped swine para-myxovirus and some picornaviruses, rhabdoviruses and togaviruses attained high titers and produced an extensive cytopathic effect. Among the DNA viruses a porcine adeno, equine rhinopneumonitis, infectious bovine rhinotraceheitis, pseudorabies and porcine cytomegalo viruses replicated in pig fallopian tube cells as well as in other cells generally used to grow them.

Adenoviridae↗

The nucleoprotein is required for efficient coronavirus genome replication.

The construction of a set of transmissible gastroenteritis coronavirus (TGEV)-derived replicons as bacterial artificial chromosomes is reported. These replicons were generated by sequential deletion of nonessential genes for virus replication, using a modified TGEV full-length cDNA clone containing unique restriction sites between each pair of consecutive genes. Efficient activity of TGEV replicons was associated with the presence of the nucleoprotein provided either in cis or in trans. TGEV replicons were functional in several cell lines, including the human cell line 293T, in which no or very low cytopathic effect was observed, and expressed high amounts of heterologous protein.

Base Sequence↗

Isolation of porcine respiratory coronavirus from pigs affected with porcine reproductive and respiratory syndrome.

Four cytopathogenic viruses were isolated in CPK cells derived from porcine kidneys from tonsils and lungs of 3 of 15 pigs affected with porcine reproductive and respiratory syndrome virus. Physicochemically and morphologically, the isolates were similar to a coronavirus. The isolates were not distinguished from transmissible gastroenteritis virus (TGEV) by a neutralization test using polyclonal antibodies, but differentiated from TGEV by monoclonal antibodies capable of discriminating between TGEV and porcine respiratory coronavirus (PRCV), indicating that the isolates were PRCV. In a serological survey of 30 serum samples each collected from about 50 days old pigs in the 2 affected farms, 29 (97%) and 15 (50%) sera were positive for neutralizing antibody against the isolate with the titers ranging from 2 to 64, respectively.

Animals↗