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PubMed1997

Interference of coronavirus infection by expression of immunoglobulin G (IgG) or IgA virus-neutralizing antibodies.

Immunoglobulin gene fragments encoding the variable modules of the heavy and light chains of a transmissible gastroenteritis coronavirus (TGEV)-neutralizing monoclonal antibody (MAb) have been cloned and sequenced. The selected MAb recognizes a highly conserved viral epitope and does not lead to the selection of neutralization escape mutants. The sequences of MAb 6A.C3 kappa and gamma 1 modules were identified as subgroup V and subgroup IIIC, respectively. The chimeric immunoglobulin genes encoding the variable modules from the murine MAb and constant modules of human gamma 1 and kappa chains were constructed by reverse transcriptase PCR. Chimeric immunoglobulins were stably or transiently expressed in murine myelomas or COS cells, respectively. The secreted recombinant antibodies had radioimmunoassay titers (i.e., the highest dilution giving a threefold increase over the background) higher than 10(3) and reduced the infectious virus more than 10(4)-fold. Recombinant dimeric immunoglobulin A (IgA) showed a 50-fold enhanced neutralization of TGEV relative to a recombinant monomeric IgG1 which contained the identical antigen binding site. Stably transformed epithelial cell lines which expressed either recombinant IgG or IgA TGEV-neutralizing antibodies reduced virus production by > 10(5)-fold after infection with homologous virus, although a residual level of virus production (< 10(2) PFU/ml) remained in less than 0.1% of the cells. This low-level persistent infection was shown not to be due to the selection of neutralization escape mutants. The implications of these findings for somatic gene therapy with recombinant antibodies are discussed.

Animals↗

PubMed1997

Coronavirus genomic and subgenomic minus-strand RNAs copartition in membrane-protected replication complexes.

The majority of porcine transmissible gastroenteritis coronavirus plus-strand RNAs (genome and subgenomic mRNAs), at the time of peak RNA synthesis (5 h postinfection), were not found in membrane-protected complexes in lysates of cells prepared by Dounce homogenization but were found to be susceptible to micrococcal nuclease (85%) or to sediment to a pellet in a cesium chloride gradient (61%). They therefore are probably free molecules in solution or components of easily dissociable complexes. By contrast, the majority of minus-strand RNAs (genome length and subgenomic mRNA length) were found to be resistant to micrococcal nuclease (69%) or to remain suspended in association with membrane-protected complexes following isopycnic sedimentation in a cesium chloride gradient (85%). Furthermore, 35% of the suspended minus strands were in a dense complex (1.20 to 1.24 g/ml) that contained an RNA plus-to-minus-strand molar ratio of approximately 8:1 and viral structural proteins S, M, and N, and 65% were in a light complex (1.15 to 1.17 g/ml) that contained nearly equimolar amounts of plus- and minus-strand RNAs and only trace amounts of proteins M and N. In no instance during fractionation were genome-length minus strands found segregated from sub-genome-length minus strands. These results indicate that all minus-strand species are components of similarly structured membrane-associated replication complexes and support the concept that all are active in the synthesis of plus-strand RNAs.

Animals↗

PubMed1997

In vivo study of interferon-alpha-secreting cells in pig foetal lymphohaematopoietic organs following in utero TGEV coronavirus injection.

Non-infectious UV-inactivated transmissible gastroenteritis virus (TGEV) was previously shown to induce interferon alpha (IFN alpha) secretion following in vitro incubation with blood mononuclear cells. In this study, pig foetuses at different stages of gestation were injected in utero with (a) partially UV-inactivated wild TGEV or (b) fully UV-inactivated wild or dm49-4 mutant TGEV coronavirus. Nucleated cells from foetal liver, bone marrow, spleen and blood were isolated 10 or 20 h after injection and assayed ex vivo for IFN alpha secretion by ELISPOT and ELISA techniques. The administration of TGEV induced IFN alpha-secreting cells in foetal lymphohaematopoietic organs at mid-gestation. In contrast, IFN alpha was not detected in control sham-operated foetuses. A specific point mutation in the amino acid sequence of the viral membrane glycoprotein M of TGEV mutant dm49-4 was associated with lower or absent IFN alpha in utero inducibility by mutant virus as compared with wild virus. Flow cytometry analysis did not show differences in leukocyte surface marker expression between control and TGEV- or between dm49-4 and wild virus-treated foetus cells, with the exception of a reduction in percentages of polymorphonuclear cells in TGEV-treated lymphohaematopoietic tissues, which is probably due to IFN alpha secretion. The present data provided in vivo evidence of IFN alpha secretion at the cell level in foetal lymphohaematopoietic organs. Such IFN alpha-secreting cells in lymphohaematopoietic tissues may be the source of IFN alpha detected during foetal infections.

Animals↗

PubMed1998

The viral spike protein is not involved in the polarized sorting of coronaviruses in epithelial cells.

Coronaviruses are assembled by budding into a pre-Golgi compartment from which they are transported along the secretory pathway to leave the cell. In cultured epithelial cells, they are released in a polarized fashion; depending on the virus and cell type, they are sorted preferentially either to the apical domain or to the basolateral plasma membrane domain. In this study, we investigated the role of the coronavirus spike protein, because of its prominent position in the virion the prime sorting candidate, in the directionality of virus release. Three independent approaches were taken. (i) The inhibition of N glycosylation by tunicamycin resulted in the synthesis of spikeless virions. The absence of spikes, however, did not influence the polarity in the release of virions. Thus, murine hepatitis virus strain A59 (MHV-A59) was still secreted from the basolateral membranes of mTAL and LMR cells and from the apical sides of MDCK(MHVR) cells, whereas transmissible gastroenteritis virus (TGEV) was still released from the apical surfaces of LMR cells. (ii) Spikeless virions were also studied by using the MHV-A59 temperature-sensitive mutant Albany 18. When these virions were produced in infected LMR and MDCK(MHVR) cells at the nonpermissive temperature, they were again preferentially released from basolateral and apical membranes, respectively. (iii) We recently demonstrated that coronavirus-like particles resembling normal virions were assembled and released when the envelope proteins M and E were coexpressed in cells (H. Vennema, G.-J. Godeke, J. W. A. Rossen, W. F. Voorhout, M. C. Horzinek, D.-J. E. Opstelten, and P. J. M. Rottier, EMBO J. 15:2020-2028, 1996). The spikeless particles produced in mTAL cells by using recombinant Semliki Forest viruses to express these two genes of MHV-A59 were specifically released from basolateral membranes, i.e., with the same polarity as that of wild-type MHV-A59. Our results thus consistently demonstrate that the spike protein is not involved in the directional sorting of coronaviruses in epithelial cells. In addition, our observations with tunicamycin show that contrary to the results with some secretory proteins, the N-linked oligosaccharides present on the viral M proteins of coronaviruses such as TGEV also play no role in viral sorting. The implications of these conclusions are discussed.

Animals↗

PubMed2000

Downstream ribosomal entry for translation of coronavirus TGEV gene 3b.

Gene 3b (ORF 3b) in porcine transmissible gastroenteritis coronavirus (TGEV) encodes a putative nonstructural polypeptide of 27.7 kDa with unknown function that during translation in vitro is capable of becoming a glycosylated integral membrane protein of 31 kDa. In the virulent Miller strain of TGEV, ORF 3b is 5'-terminal on mRNA 3-1 and is presumably translated following 5' cap-dependent ribosomal entry. For three other strains of TGEV, the virulent British FS772/70 and Taiwanese TFI and avirulent Purdue-116, mRNA species 3-1 is not made and ORF 3b is present as a non-overlapping second ORF on mRNA 3. ORF 3b begins at base 432 on mRNA 3 in Purdue strain. In vitro expression of ORF 3b from Purdue mRNA 3-like transcripts did not fully conform to a predicted leaky scanning pattern, suggesting ribosomes might also be entering internally. With mRNA 3-like transcripts modified to carry large ORFs upstream of ORF 3a, it was demonstrated that ribosomes can reach ORF 3b by entering at a distant downstream site in a manner resembling ribosomal shunting. Deletion analysis failed to identify a postulated internal ribosomal entry structure (IRES) within ORF 3a. The results indicate that an internal entry mechanism, possibly in conjunction with leaky scanning, is used for the expression of ORF 3b from TGEV mRNA 3. One possible consequence of this feature is that ORF 3b might also be expressed from mRNAs 1 and 2.

Animals↗

PubMed2005

A point mutation within the replicase gene differentially affects coronavirus genome versus minigenome replication.

During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.

Animals↗

PubMed1994

Coronavirus M proteins accumulate in the Golgi complex beyond the site of virion budding.

The prevailing hypothesis is that the intracellular site of budding of coronaviruses is determined by the localization of its membrane protein M (previously called E1). We tested this by analyzing the site of budding of four different coronaviruses in relation to the intracellular localization of their M proteins. Mouse hepatitis virus (MHV) and infectious bronchitis virus (IBV) grown in Sac(-) cells, and feline infectious peritonitis virus (FIPV) and transmissible gastroenteritis virus (TGEV) grown in CrFK cells, all budded exclusively into smooth-walled, tubulovesicular membranes located intermediately between the rough endoplasmic reticulum and Golgi complex, identical to the so-called budding compartment previously identified for MHV. Indirect immunofluorescence staining of the infected cells showed that all four M proteins accumulated in a perinuclear region. Immunogold microscopy localized MHV M and IBV M in the budding compartment; in addition, a dense labeling in the Golgi complex occurred, MHV M predominantly in trans-Golgi cisternae and trans-Golgi reticulum and IBV M mainly in the cis and medial Golgi cisternae. The corresponding M proteins of the four viruses, when independently expressed in a recombinant vaccinia virus system, also accumulated in the perinuclear area. Quantitative pulse-chase analysis of metabolically labeled cells showed that in each case the majority of the M glycoproteins carried oligosaccharide side chains with Golgi-specific modifications within 4 h after synthesis. Immunoelectron microscopy localized recombinant MHV M and IBV M to the same membranes as the respective proteins in coronavirus-infected cells, with the same cis-trans distribution over the Golgi complex. Our results demonstrate that some of the M proteins of the four viruses are transported beyond the budding compartment and are differentially retained by intrinsic retention signals; in addition to M, other viral and/or cellular factors are probably required to determine the site of budding.

Animals↗

PubMed2003

A 3D model of SARS_CoV 3CL proteinase and its inhibitors design by virtual screening.

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.

Computational Biology↗

PubMed1996

A murine and a porcine coronavirus are released from opposite surfaces of the same epithelial cells.

Epithelial cells are important target cells for coronavirus infection. Earlier we have shown that transmissible gastroenteritis coronavirus (TGEV) and mouse hepatitis coronavirus (MHV) are released from different sides of porcine and murine epithelial cells, respectively. To study the release of these viruses from the same cells, we constructed a porcine LLC-PK1 cell line stably expressing the recombinant MHV receptor cDNA (LMR cells). The MHV and TGEV receptor glycoproteins were shown by immunofluorescence to appear at the surface of the cells and to be functional so that the cells were susceptible to both MHV and TGEV infection. Both coronaviruses entered polarized LMR cells only through the apical surface. Remarkably, while the cells remained susceptible to TGEV for long periods, infectability by MHV decreased with time after plating of the cells onto filters. This was not due to a lack of expression of the MHV receptor, since this glycoprotein was still abundant on the apical surface of these cells. TGEV and MHV appeared to exit LMR cells from opposite sides. Whereas TGEV was released preferentially at the apical membrane, MHV was released preferentially at the basolateral surface. These results show that vesicles containing the two coronaviruses are targeted differently in LMR cells. We propose that the viruses are sorted at the Golgi complex into different transport vesicles that carry information directing them to one of the two surface domains. The apical release of TGEV and the basolateral release of MHV might be factors contributing to the difference in virus spread found between TGEV and MHV in their respective natural hosts, the former causing mainly a localized enteric infection, the latter spreading through the body to other organs.

Animals↗

PubMed1998

Lactogenic immunity in transgenic mice producing recombinant antibodies neutralizing coronavirus.

Protection against coronavirus infections can be provided by the oral administration of virus neutralizing antibodies. To provide lactogenic immunity, eighteen lines of transgenic mice secreting a recombinant IgG1 monoclonal antibody (rIgG1) and ten lines of transgenic mice secreting recombinant IgA monoclonal antibodies (rIgA) neutralizing transmissible gastroenteritis coronavirus (TGEV) into the milk were generated. Genes encoding the light and heavy chains of monoclonal antibody (MAb) 6A.C3 were expressed under the control of regulatory sequences derived from the mouse genomic DNA encoding the whey acidic protein (WAP) and beta-lactoglobulin (BLG), which are highly abundant milk proteins. The MAb 6A.C3 binds to a highly conserved epitope present in coronaviruses of several species. This MAb does not allow the selection of neutralization escaping virus mutants. The antibody was expressed in the milk of transgenic mice with titers of one million as determined by RIA, and neutralized TGEV infectivity by one million fold corresponding to immunoglobulin concentrations of 5 to 6 mg per ml. Matrix attachment regions (MAR) sequences were not essential for rIgG1 transgene expression, but co-microinjection of MAR and antibody genes led to a twenty to ten thousand-fold increase in the antibody titer in 50% of the rIgG1 transgenic animals generated. Co-microinjection of the genomic BLG gene with rIgA light and heavy chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and BLG genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of BLG co-integration. Antibody expression levels were transgene copy number independent and integration site dependent. The generation of transgenic animals producing virus neutralizing antibodies in the milk could be a general approach to provide protection against neonatal infections of the enteric tract.

Animals↗

PubMed2006

Mutational analysis of aminopeptidase N, a receptor for several group 1 coronaviruses, identifies key determinants of viral host range.

Feline coronavirus (FCoV), porcine transmissible gastroenteritis coronavirus (TGEV), canine coronavirus (CCoV), and human coronavirus HCoV-229E, which belong to the group 1 coronavirus, use aminopeptidase N (APN) of their natural host and feline APN (fAPN) as receptors. Using mouse-feline APN chimeras, we identified three small, discontinuous regions, amino acids (aa) 288 to 290, aa 732 to 746 (called R1), and aa 764 to 788 (called R2) in fAPN that determined the host ranges of these coronaviruses. Blockade of infection with anti-fAPN monoclonal antibody RG4 suggested that these three regions lie close together on the fAPN surface. Different residues in fAPN were required for infection with each coronavirus. HCoV-229E infection was blocked by an N-glycosylation sequon present between aa 288 to 290 in murine APN. TGEV required R1 of fAPN, while FCoV and CCoV required both R1 and R2 for entry. N740 and T742 in fAPN and the homologous R741 in human APN (hAPN) were key determinants of host range for FCoV, TGEV, and CCoV. Residue N740 in fAPN was essential only for CCoV receptor activity. A conservative T742V substitution or a T742R substitution in fAPN destroyed receptor activity for the pig, dog, and cat coronaviruses, while a T742S substitution retained these receptor activities. Thus, the hydroxyl on T742 is required for the coronavirus receptor activity of fAPN. In hAPN an R741T substitution caused a gain of receptor activity for TGEV but not for FCoV or CCoV. Therefore, entry and host range of these group 1 coronaviruses depend on the ability of the viral spike glycoproteins to recognize small, species-specific amino acid differences in the APN proteins of different species.

Amino Acid Substitution↗

PubMed1998

Coronavirus pseudoparticles formed with recombinant M and E proteins induce alpha interferon synthesis by leukocytes.

Transmissible gastroenteritis virus (TGEV), an enteric coronavirus of swine, is a potent inducer of alpha interferon (IFN-alpha) both in vivo and in vitro. Incubation of peripheral blood mononuclear cells with noninfectious viral material such as inactivated virions or fixed, infected cells leads to early and strong IFN-alpha synthesis. Previous studies have shown that antibodies against the virus membrane glycoprotein M blocked the IFN induction and that two viruses with a mutated protein exhibited a decreased interferogenic activity, thus arguing for a direct involvement of M protein in this phenomenon. In this study, the IFN-alpha-inducing activity of recombinant M protein expressed in the absence or presence of other TGEV structural proteins was examined. Fixed cells coexpressing M together with at least the minor structural protein E were found to induce IFN-alpha almost as efficiently as TGEV-infected cells. Pseudoparticles resembling authentic virions were released in the culture medium of cells coexpressing M and E proteins. The interferogenic activity of purified pseudoparticles was shown to be comparable to that of TGEV virions, thus establishing that neither ribonucleoprotein nor spikes are required for IFN induction. The replacement of the externally exposed, N-terminal domain of M with that of bovine coronavirus (BCV) led to the production of chimeric particles with no major change in interferogenicity, although the structures of the TGEV and BCV ectodomains markedly differ. Moreover, BCV pseudoparticles also exhibited interferogenic activity. Together these observations suggest that the ability of coronavirus particles to induce IFN-alpha is more likely to involve a specific, multimeric structure than a definite sequence motif.

Amino Acid Sequence↗

PubMed1996

The major subunit ClpG of Escherichia coli CS31A fibrillae as an expression vector for different combinations of two TGEV coronavirus epitopes.

Previously, two B-cell epitopes from the entero-pathogenic transmissible gastroenteritis virus (TGEV), namely the C epitope (TGEV-C) amino acids (aa) 363-371 and the A epitope (TGEV-A) aa 522-531 of the spike S protein (TGEV-S), have been separately expressed on the CS31A fibrillae at the surface of Escherichia coli following insertion into a same region of ClpG. However, the resulting chimeras induced a marginal TGEV-neutralizing antibody (Ab) response in mice. Here, with the view to improving this response, we introduced TGEV-C alone or in different tandem association with TGEV-A (A::C or C::A) in twelve putatively exposed regions of ClpG. Among the 28 resulting engineered proteins only 15, carrying up to 51 extra aa, had not essentially disturbed the correct CS31A fibrillae formation process. Six partially permissive sites accepting only TGEV-C and three highly permissive sites tolerating A::C or C::A tandem peptide, were identified throughout ClpG. Intact bacteria or extracted CS31A hybrid fibrillae expressing TGEV epitopes at any of the permissive sites, were recognized by Ab directed against the foreign parent protein, providing a direct argument for exposure of the corresponding CIpG region at the cell surface and for antigenicity of the epitopes in the polymeric CS31A fibrillae context. The potential of CS31A fibrillae as carriers of the TGEV peptides indicates that there may be three positions (N terminus, aa 202-204 and 202-218) in ClpG which may turn out to be important fusion sites and therefore be relevant for the eventual design of TGEV vaccines. Unexpectedly, TGEV-A, whatever its position in ClpG, mediated the partial proteolytic degradation of the hybrid proteins, suggesting that it functions as a substrate for a cellular protease, and thereby that its suitability as a vaccine antigen candidate is doubtful.

Amino Acid Sequence↗

PubMed1995

Sequence comparison of porcine respiratory coronavirus isolates reveals heterogeneity in the S, 3, and 3-1 genes.

Four new porcine respiratory coronavirus (PRCV) isolates were genetically characterized. Subgenomic mRNA patterns and the nucleotide sequences of the 5' ends of the S genes, the open reading frame (ORF) 3/3a genes, and the ORF 3-1/3b genes of these PRCV isolates were determined and compared with those of other PRCV and transmissible gastroenteritis virus (TGEV) isolates. The S, ORF 3/3a, and ORF 3-1/3b genes are under intense study because of their possible roles in determining tissue tropism and virulence. Northern (RNA) blot analysis of subgenomic mRNAs revealed that mRNA 2, which encodes for the S gene, of the PRCV isolates migrated faster than the mRNA 2 of TGEV. The PRCV isolates AR310 and LEPP produced eight subgenomic mRNA species, the same number as produced by the virulent Miller strain of TGEV. However, the PRCV isolates IA1894 and ISU-1 produced only seven subgenomic mRNA species. All four of the PRCV isolates were found to have a large in-frame deletion in the 5' end of the S gene; however, the size and location of the deletion varied. Analysis of the ORF 3/3a gene nucleotide sequences from the four PRCV isolates also showed a high degree of variability in this area. The ORF 3 gene of the PRCV isolates AR310 and LEPP was preceded by a CTAAAC leader RNA-binding site, and the ORF 3 gene was predicted to yield a protein of 72 amino acids, the same size as that of the virulent Miller strain of TGEV. The PRCV isolates AR310 and LEPP are the first PRCV isolates found to have an intact ORF 3 gene. The ORF 3a gene of the PRCV isolate IA1894 was preceded by a CTAAAC leader RNA-binding site and was predicted to yield a truncated protein of 54 amino acids due to a 23-nucleotide deletion. The CTAAAC leader RNA-binding site and ATG start codon of ORF 3 gene of the PRCV isolate ISU-1 were removed because of a 168-nucleotide deletion. Analysis of the ORF 3-1/3b gene nucleotide sequences from the four PRCV nucleotides isolates also showed variability.

Amino Acid Sequence↗

PubMed1998

Transgenic mice secreting coronavirus neutralizing antibodies into the milk.

Ten lines of transgenic mice secreting transmissible gastroenteritis coronavirus (TGEV) neutralizing recombinant monoclonal antibodies (rMAbs) into the milk were generated. The rMAb light- and heavy-chain genes were assembled by fusing the genes encoding the variable modules of the murine MAb 6A.C3, which binds an interspecies conserved coronavirus epitope essential for virus infectivity, and a constant module from a porcine myeloma with the immunoglobulin A (IgA) isotype. The chimeric antibody led to dimer formation in the presence of J chain. The neutralization specific activity of the recombinant antibody produced in transiently or stably transformed cells was 50-fold higher than that of a monomeric rMAb with the IgG1 isotype and an identical binding site. This rMAb had titers of up to 10(4) by radioimmunoassay (RIA) and neutralized virus infectivity up to 10(4)-fold. Of 23 transgenic mice, 17 integrated both light and heavy chains, and at least 10 of them transmitted both genes to the progeny, leading to 100% of animals secreting functional TGEV neutralizing antibody during lactation. Selected mice produced milk with TGEV-specific antibody titers higher than 10(6) as determined by RIA, neutralized virus infectivity by 10(6)-fold, and produced up to 6 mg of antibody per ml. Antibody expression levels were transgene copy number independent and integration site dependent. Comicroinjection of the genomic beta-lactoglobulin gene with rMAb light- and heavy-chain genes led to the generation of transgenic mice carrying the three transgenes. The highest antibody titers were produced by transgenic mice that had integrated the antibody and beta-lactoglobulin genes, although the number of transgenic animals generated does not allow a definitive conclusion on the enhancing effect of beta-lactoglobulin cointegration. This approach may lead to the generation of transgenic animals providing lactogenic immunity to their progeny against enteric pathogens.

Amino Acid Sequence↗

PubMed1995

Investigation of the control of coronavirus subgenomic mRNA transcription by using T7-generated negative-sense RNA transcripts.

The subgenomic mRNAs of the coronavirus transmissible gastroenteritis virus (TGEV) are not produced in equimolar amounts. We have developed a reporter gene system to investigate the control of this differential subgenomic mRNA synthesis. Transcription of mRNAs by the TGEV polymerase was obtained from negative-sense RNA templates generated in situ from DNA containing a T7 promoter. A series of gene cassettes was produced; these cassettes comprised the reporter chloramphenicol acetyltransferase (CAT) gene downstream of transcription-associated sequences (TASs) (also referred to as intergenic sequences and promoters) believed to be involved in the synthesis of TGEV subgenomic mRNAs 6 and 7. The gene cassettes were designed so that negative-sense RNA copies of the CAT gene with sequences complementary to the TGEV TASs, or modified versions, at the 3' end would be synthesized in situ by T7 RNA polymerase. Using this system, we have demonstrated that CAT was expressed from mRNAs derived from the T7-generated negative-sense RNA transcripts only in TGEV-infected cells and only from transcripts possessing a TGEV negative-sense TAS. Analysis of the CAT mRNAs showed the presence of the TGEV leader RNA sequence at the 5' end, in keeping with observations that all coronavirus mRNAs have a 5' leader sequence corresponding to the 5' end of the genomic RNA. Our results indicated that the CAT mRNAs were transcribed from the in situ-synthesized negative-sense RNA templates without the requirement of TGEV genomic 5' or 3' sequences on the T7-generated negative-sense transcripts (3'-TAS-CAT-5'). Modification of the TGEV TASs indicated (i) that the degree of potential base pairing between the 3' end of the leader RNA and the TGEV negative-sense TAS was not the sole determinant of the amount of subgenomic mRNA transcribed and (ii) that other factors, including nucleotides flanking the TAS, are involved in the regulation of transcription of TGEV subgenomic mRNAs.

Animals↗

PubMed2004

Verification of sensitivity and specificity of group a rotavirus detection in piglets faeces with monoclonal blocking ELISA methods.

Monoclonal antibodies to group A rotavirus Vp6 protein were prepared and used for verification of three blocking enzyme-linked immunosorbent assay (ELISA) modifications to detect rotavirus A. Selected competitive blocking ELISA (CB-ELISA) and electron microscopy (EM) were used for examination of 194 field faecal samples of piglets affected with diarrhoea. Rotavirus was detected in 43 samples (22.2%) by CB-ELISA method, whereas in 26 (13.4%) samples by EM examination. However, of 26 samples positive by EM, rotavirus A was detected by CB-ELISA in 19 (73.1%) samples; indicating the share of group A rotavirus in all cases of gastroenteritis caused by rotavirus. The sensitivity and specificity of the CB-ELISA was verified both by inclusion of control samples containing transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhoea virus (PEDV) in each analysis and by comparative examination of samples with the commercial ELISA kit. The CB-ELISA sensitivity was positively affected by examination of samples in the presence of chelating agent.

Animals↗

PubMed2006

[Cryptosporidium hominis diarrhea outbreak and transmission linked to diaper infant use].

BACKGROUND AND OBJECTIVE: On the basis of several cases of cryptosporidiosis detected in a child day-care center, we stablished the extent of the outbreak and investigated causes of parasite transmission. PATIENTS AND METHOD: A retrospective cohort study was designed on all children attending day-care center and care givers to determine their infection status and identify risk factors associated to the outbreak. RESULTS: 24 cases of cryptosporidiosis were detected, with an attack rate of 0.46 (24/52); 12 of them were parasitologycally confirmed. All care givers were negative for Cryptosporidium and none of them reported symptoms of acute gastroenteritis. Transmission pattern was compatible with person to person modes. Among the factors investigated, two were associated with the risk of disease: diaper wear (relative risk = 2.06; p = 0.059); and diarrhea in relatives (relative risk = 2.05; p = 0.01). In all confirmed cases, Cryptosporidium hominis (previously known as C. parvum, genotype 1), was identified. CONCLUSIONS: Cryptosporidiosis should be considered as a possible cause of outbreaks of gastroenteritis at day-care centers. Increasing care on diaper changing practices, specially over children with diarrhea, may be the key factor to prevent transmission of Cryptosporidium.

Child Day Care Centers↗