Performance of an ultra-fast deep-learning accelerated MRI screening protocol for prostate cancer compared to a standard multiparametric protocol.

OBJECTIVES: To establish and evaluate an ultra-fast MRI screening protocol for prostate cancer (PCa) in comparison to the standard multiparametric (mp) protocol, reducing scan time and maintaining adequate diagnostic performance. MATERIALS AND METHODS: This prospective single-center study included consecutive biopsy-naïve patients with suspected PCa between December 2022 and March 2023. A PI-RADSv2.1 conform mpMRI protocol was acquired in a 3 T scanner (scan time: 25 min 45 sec). In addition, two deep-learning (DL) accelerated sequences (T2- and diffusion-weighted) were acquired, serving as a screening protocol (scan time: 3 min 28 sec). Two readers evaluated image quality and the probability of PCa regarding PI-RADSv2.1 scores in two sessions. The diagnostic performance of the screening protocol with mpMRI serving as the reference standard was derived. Inter- and intra-reader agreements were evaluated using weighted kappa statistics. RESULTS: We included 77 patients with 97 lesions (mean age: 66 years; SD: 7.7). Diagnostic performance of the screening protocol was excellent with a sensitivity and specificity of 100%/100% and 89%/98% (cut-off ≥ PI-RADS 4) for reader 1 (R1) and reader 2 (R2), respectively. Mean image quality was 3.96 (R1) and 4.35 (R2) for the standard protocol vs. 4.74 and 4.57 for the screening protocol (p < 0.05). Inter-reader agreement was moderate (κ: 0.55) for the screening protocol and substantial (κ: 0.61) for the multiparametric protocol. CONCLUSION: The ultra-fast screening protocol showed similar diagnostic performance and better imaging quality compared to the mpMRI in under 15% of scan time, improving efficacy and enabling the implementation of screening protocols in clinical routine. CLINICAL RELEVANCE STATEMENT: The ultra-fast protocol enables examinations without contrast administration, drastically reducing scan time to 3.5 min with similar diagnostic performance and better imaging quality. This facilitates patient-friendly, efficient examinations and addresses the conflict of increasing demand for examinations at currently exhausted capacities. KEY POINTS: Time-consuming MRI protocols are in conflict with an expected increase in examinations required for prostate cancer screening. An ultra-fast MRI protocol shows similar performance and better image quality compared to the standard protocol. Deep-learning acceleration facilitates efficient and patient-friendly examinations, thus improving prostate cancer screening capacity.

Improved diffusion-weighted imaging of the prostate: Comparison of readout-segmented and zoomed single-shot imaging.

OBJECTIVES: Diffusion weighted imaging (DWI) is the most important sequence for detection and grading prostate cancer (PCa), but it is considerably prone to artifacts. New approaches like zoomed single-shot imaging (z-EPI) with advanced image processing or multi-shot readout segmentation (rs-EPI) try to improve DWI quality. This study evaluates objective and subjective image quality (IQ) of rs-EPI and z-EPI with and without advanced processing. MATERIALS AND METHODS: Fifty-six consecutive patients (67 ± 8 years; median PSA 8.3 ng/ml) with mp-MRI performed at 3 Tesla between February and October 2019 and subsequently verified PCa by targeted plus systematic MRI/US-fusion biopsy were included in this retrospective single center cohort study. Rs-EPI and z-EPI were prospectively acquired in every patient. Signal intensities (SI) of PCa and benign tissue in ADC, b1000, and calculated high b-value images were analyzed. Endpoints were signal-to-noise ratio (SNR), contrast-to-noise ratio (CNR), PCa contrast intensity (CI), and subjective IQ on a 5-point scale evaluated by three blinded readers. Wilcoxon signed rank test, Friedman test and Cohen's kappa coefficient was calculated. RESULTS: SNR, CNR, and PCa CI of z-EPI with and without advanced processing was superior to rs-EPI (p < 0.01), whereas no significant differences were observed between z-EPI with and without advanced processing. Subjective IQ was significantly higher for z-EPI with advanced processing compared rs-EPI for ADC, b1000, and calculated high b-values (p < 0.01). Compared to z-EPI without advanced processing, z-EPI with advanced processing was superior for ADC and calculated high b-values (p < 0.01), but no significant differences were shown for b1000 images. CONCLUSIONS: Z-EPI with and without advanced processing was superior to rs-EPI regarding objective imaging parameters and z-EPI with advanced processing was superior to rs-EPI regarding subjective imaging parameters for the detection of PCa.

Influence of brain tumors on the MR spectra of healthy brain tissue.

The neurochemical environment of nontumorous white matter tissue was investigated in 135 single voxel spectra of "healthy" white matter regions of 43 tumor patients and 129 spectra of 52 healthy subjects. Spectra were acquired with short TE and TR values. With the data of tumor patients, it was examined whether differences were caused by the tumor itself or aggressive tumor therapies as confounding factors. Comparing the spectra of both classes, an excellent differentiation was possible based on the metabolite peak of N-acetylaspartate (P ≈ 0) and myoinositol (P < 0.03). The area under curve of the receiver operating characteristic was calculated as 0.86 and 0.62, respectively. With linear discriminant analysis using combinations of integrals, a prediction was possible, whether a spectrum belonged to the patient or the healthy subject class with an overall accuracy above 80%. The confounding factors could be ruled out as source of the differences. The results show strong evidence for an influence of malignant growth on the biochemical environment of nontumorous white matter tissue. Because of the T(1) weighting, the measured differences between both classes were most likely concentration changes interfered by T(1) effects. The underlying processes will be subject of future studies.

Characterization of epitopes for neutralizing monoclonal antibodies to classical swine fever virus E2 and Erns using phage-displayed random peptide library.

Infection of cells with classical swine fever virus (CSFV) is mediated by the interaction of envelope glycoproteins E2 and Erns with receptor molecules on the cell surface. These proteins are also the major antigens for eliciting neutralizing antibodies and conferring protective immunity. Here we report the identification of multiple neutralizing epitopes on these proteins by screening a phage-displayed random peptide library with CSFV-specific neutralizing monoclonal antibodies. Two different E2-specific neutralizing mAbs (a18 and 24/10) were found to bind to a common motif SPTxL, which is similar to the sequence SPTTL of the E2 protein (aa 289-293), indicating that this is likely to be an immunodominant epitope. Similarly, an immunodominant epitope corresponding to the sequence DKN of Erns (aa 117-119) was identified for two independent Erns-specific neutralizing antibodies, b4-22 and 24/16, respectively. Another binding motif, CxNNxTC, was identified for mAb 24/16, but not for b4-22. Sequencing analysis of the genes coding for the light chain of these mAbs was conducted to ensure that all mAbs were derived from different hybridomas, rather than from different subclones of a common parent line. Inhibition studies using immunofluorescent antibody assay and virus neutralization test demonstrated that the mimotope peptides truly mimicked the antibody binding determinants on the viral proteins. The detailed mapping data for these neutralizing epitopes will be useful for development of improved diagnostic tests and perhaps a peptide-based vaccine for this important swine disease.

The major envelope protein, GP5, of a European porcine reproductive and respiratory syndrome virus contains a neutralization epitope in its N-terminal ectodomain.

A set of neutralizing monoclonal antibodies (mAbs) directed against the GP(5) protein of European type porcine reproductive and respiratory syndrome virus (PRRSV) has been produced previously (Weiland et al., 1999). This set reacted with a plaque-purified virus (PPV) subpopulation of Dutch isolate Intervet-10 (I-10), but not with the European prototype PRRSV LV. In order to map the neutralization epitope in the GP(5) protein of the PPV strain, the ORF5 nucleotide sequence of PPV was determined. When the amino acid sequence derived from this nucleotide sequence was compared with that of PRRSV LV, four amino acid differences were found. Using site-directed mutagenesis, we showed that a proline residue at position 24 of the GP(5) sequence of the PPV strain enabled recognition by the neutralizing mAbs. Pepscan analysis demonstrated that the epitope recognized by the neutralizing mAbs stretched from residues 29 to 35. Surprisingly, the reactivity of the mAbs in the Pepscan system was independent of the presence of a proline in position 24. Moreover, residue 24 is located within the predicted signal peptide, implying that either the signal peptide is not cleaved or is cleaved due to the presence of Pro(24) such that the epitope remains intact. Our results demonstrate the presence of a neutralization epitope in the N-terminal ectodomain of the GP(5) protein of PRRSV and imply a role for the ectodomain of GP(5) in the infection of PRRSV.

Detection of European porcine reproductive and respiratory syndrome virus in porcine alveolar macrophages by two-colour immunofluorescence and in-situ hybridization-immunohistochemistry double labelling.

Two groups of five pigs aged 6 weeks were each infected oronasally with one of two different European isolates of porcine reproductive and respiratory syndrome virus (PRRSV). The animals were killed sequentially at 4, 7, 14 or 21 days post-inoculation for examination. The methods used consisted of histopathology, and mono- and double-labelling techniques based on in-situ hybridization, immunofluorescence and immunohistochemistry. Porcine alveolar macrophages (PAMs) contained large amounts of PRRSV antigen and PRRSV RNA, as shown by double labelling with (1) either PRRSV immunofluorescence or PRRSV-specific in-situ hybridization with digoxigenin-labelled riboprobes, and (2) immunolabelling with Mac 387 antibody for calprotectin. Expression of PRRSV-RNA was not detectable in cytokeratin-positive hypertrophic and proliferating pneumocytes or in cells of alveolar ducts or bronchiolar epithelium. The use of two-colour immunofluorescence with confocal laser scanning microscopy and double labelling with in-situ hybridization-immunohistochemistry showed that PAMs were the only pulmonary target cells. This contradicts earlier reports that epithelial pulmonary cells may also be infected by PRRSV.

Monoclonal antibodies directed against conserved epitopes on the nucleocapsid protein and the major envelope glycoprotein of equine arteritis virus.

We recently developed a highly effective immunization procedure for the generation of monoclonal antibodies (MAbs) directed against the porcine reproductive and respiratory syndrome virus (E. Weiland, M. Wieczorek-Krohmer, D. Kohl, K. K. Conzelmann, and F. Weiland, Vet. Microbiol. 66:171-186, 1999). The same method was used to produce a panel of 16 MAbs specific for the equine arteritis virus (EAV). Ten MAbs were directed against the EAV nucleocapsid (N) protein, and five MAbs recognized the major viral envelope glycoprotein (G(L)). Two of the EAV G(L)-specific MAbs and one antibody of unknown specificity neutralized virus infectivity. A comparison of the reactivities of the MAbs with 1 U.S. and 22 newly obtained European field isolates of EAV demonstrated that all N-specific MAbs, the three nonneutralizing anti-G(L) MAbs, and the weakest neutralizing MAb (MAb E7/d15-c9) recognized conserved epitopes. In contrast, the two MAbs with the highest neutralization titers bound to 17 of 23 (MAb E6/A3) and 10 of 23 (MAb E7/d15-c1) of the field isolates. Ten of the virus isolates reacted with only one of these two MAbs, indicating that they recognized different epitopes. The G(L)-specific MAbs and the strongly neutralizing MAb of unknown specificity (MAb E6/A3) were used for the selection of neutralization-resistant (NR) virus variants. The observation that the E6/A3-specific NR virus variants were neutralized by MAb E7/d15-c1 and that MAb E6/A3 blocked the infectivity of the E7/d15-c1-specific NR escape mutant confirmed that these antibodies reacted with distinct antigenic sites. Immunoelectron microscopy revealed for the first time that the antigenic determinants recognized by the anti-G(L) MAbs were localized on the virion surface. Surprisingly, although the immunofluorescence signal obtained with the neutralizing antibodies was relatively weak, they mediated binding of about three times as much gold granules to the viral envelope than the nonneutralizing anti-G(L) MAbs.

Pseudorabies virus glycoprotein K requires the UL20 gene product for processing.

Glycoprotein K (gK) of pseudorabies virus (PrV) has recently been identified as a virion component which is dispensable for viral entry but required for direct cell-to-cell spread. Electron microscopic data suggested a possible function of gK in virus egress by preventing immediate fusion of released virus particles with the plasma membrane (B. G. Klupp, J. Baumeister, P. Dietz, H. Granzow, and T. C. Mettenleiter, J. Virol. 72:1949-1958, 1998). For more detailed analysis, a PrV mutant with a deletion of the UL53 (gK) open reading frame (ORF) from codons 48 to 275 was constructed, and the protein was analyzed with two monoclonal antibodies directed against PrV gK. The salient findings of this report are as follows. (i) From the PrV UL53 ORF, a functional gK is translated only from the first in-frame methionine. From the second in-frame methionine, a nonfunctional product is expressed which is not incorporated into virions. (ii) When constitutively expressed in a stable cell line without other viral proteins, gK is only incompletely processed. After superinfection with gK-deletion mutants, proper processing is restored and mature gK is incorporated into virions. (iii) The UL20 gene product is specifically required for processing of gK. gK is not correctly processed in a UL20 deletion mutant of PrV, and superinfection of gK-expressing cells with PrV-UL20(-) does not restore processing. However, all other known structural viral glycoproteins appear to be processed normally in PrV-UL20(-)-infected cells. (iv) Coexpression of gK and UL20 restored gK processing at least partially. Thus, our data show that the UL20 gene product is required for proper processing of PrV gK.

Porcine reproductive and respiratory syndrome virus (PRRSV): kinetics of infection in lymphatic organs and lung.

Pigs were infected by the oronasal route with European isolates of the porcine reproductive and respiratory syndrome virus (PRRSV; I10 and Cobbelsdorf). The kinetics of infection in lymphatic organs and the lung were analysed by immunofluorescence detection of virus antigen, re-isolation of the virus and reverse transcription--polymerase chain reaction (RT-PCR) for PRRSV-specific RNA. The kinetics of PRRSV infection proceeded in three phases, irrespective of the varying infestation of lymphatic organs within the first days post-infection (p.i.). First, an early acute infection of lymphatic organs developed within the first week and was characterized by a high number of antigen-positive macrophages. Second, a delayed acute infection of the lung was observed, which was most pronounced during the second and third week p.i. when a high number of infected alveolar macrophages was observed. The acute infection of lymphatic organs had resolved at this time. Infected cells in the lung were predominantly located in pneumonic lesions. Third, a persistent infection was demonstrated by RT-PCR and immunohistology when the experiments were terminated at day 49 p.i. The virus persisted in lymphatic organs, especially in the tonsils, and in the lung. At this stage, indications for a re-occurrence of acute infection were observed in restricted areas of the lung.

Localization of pestiviral envelope proteins E(rns) and E2 at the cell surface and on isolated particles.

The glycoproteins E(rns) of classical swine fever virus (CSFV) and E(rns) and E2 of bovine viral diarrhoea virus (BVDV) are shown to be located at the surface of infected cells by the use of indirect immunofluorescence and by cytofluorometric analysis. The positive immunostaining of the cell surface was further analysed by immunogold electron microscopy and it could be shown that only extracellular virions were labelled. Gold granules were not seen at the cellular plasma membrane. In contrast to BVDV E2, the CSFV E2 of virions sticking to the plasma membrane was not accessible to the respective monoclonal antibodies. However, CSFV particles isolated from culture supernatant were able to bind both monoclonal anti-E(rns) and anti-E2 antibodies. For CSFV and BVDV, binding of anti-E(rns) antibodies to the virions was more pronounced than that of anti-E2. This finding was unexpected since E2 is considered to be the immunodominant glycoprotein.

Monoclonal antibodies to the GP5 of porcine reproductive and respiratory syndrome virus are more effective in virus neutralization than monoclonal antibodies to the GP4.

The arterivirus porcine reproductive and respiratory syndrome virus (PRRSV) contains six structural proteins the roles of which are not completely understood. In a preceding study, immunization with the dutch isolate I10 of PRRSV had led to the development of MAbs against four structural proteins [Wieczorek-Krohmer, M., 1994. Herstellung und Charakterisierung von monoklonalen Antikörpern gegen das Virus des Porzinen Reproduktiven und Respiratorischen Syndroms (PRRSV). Inaugural-Dissertation, Ludwig-Maximilians-Universität, München] here finally identified by reaction with individual plasmid-expressed PRRSV proteins as products of ORFs 3 (GP3), 4 (GP4), 5 (GP5) and 7 (N). Surprisingly, the MAbs against GP5 revealed the presence of two antigenically distinct virus populations in the isolate I10, the population PRRSV-'PPV', isolated from plaques and the PRRSV-'EPV', gained by end point dilution. MAbs against GP3, GP4 and N reacted with both I10 populations as well as with natural PRRSV isolates. However, the anti-GP5 MAbs exclusively recognized PRRSV-'PPV'. In this study immunization of mice with both separated I10 populations confirmed that solely PRRSV-'PPV' possesses the property to induce an immune response ultimately leading to the establishment of MAbs against GP5. Whereas the 15 anti-GP5 MAbs (derived from four independent fusions) reacted exclusively with PRRSV-'PPV' of the isolate I10, anti-GP4 MAbs detected their target antigen on various isolates of European origin and were able to neutralize them. As indicated by competition assays and selection of neutralization-resistant virus mutants, all GP5 MAbs are directed against a single antigenic site on the ORF 5 protein. Both groups of neutralizing antibodies bound to the surface of purified virions demonstrating that the recognized epitopes represent surface structures of the virion envelope. However, anti-GP5 MAbs mediated the binding of more gold granules than anti-GP4 MAbs. Comparison of the neutralizing effect of anti-GP4 and anti-GP5 MAbs revealed the anti-GP5 MAbs as the more efficient antibodies. For the complete neutralization of about 100 ID50 of PRRSV-'PPV' anti-GP5 culture supernatant was effective up to a dilution of 1:1280 whereas the most effective anti-GP4 antibodies exhibited a comparable effect only up to 1:64. These results indicate that PRRSV GP5 in principle is a major target for neutralizing antibodies, as is found for other arteriviruses, but that in nature 'ORF 5 escape mutants' may develop as easily as in vitro.

Arterivirus PRRSV. Experimental studies on the pathogenesis of respiratory disease.

Pigs were infected with the porcine respiratory and reproductive syndrome virus (PRRSV) by the oronasal route. We studied the development of histological lesions, sites of virus infection and of inflammatory infiltrates by quantitative evaluation of reactive cells. The animals developed a multifocal interstitial pneumonia. Clinical signs of pneumonia were observed from day 7 to 21. In the first stage, an acute alveolitis was found, which was characterised by a hyperplasia of type II pneumocytes within the septa and an accumulation of macrophages in the alveolar spaces. Within 2-4 days p.i., virus infected cells were prominent in lymphatic organs, but their number declined rapidly during the following days. In the following period, the number of virus antigen positive cells increased in the lung. An interesting discrepancy existed between the relatively small number of virus specific cells and the degree of intensive pneumonia. As a first step to analyse mechanisms leading to the induction of pneumonia, we studied transcriptional expression of cytokines and other immunomodulatory molecules by semiquantitative RT-PCR.

Characterization of a bovine viral diarrhea virus isolated from roe deer in Germany.

The 5' untranslated region (5' UTR) of cytopathogenic pestiviruses isolated from two seronegative roe deer (Capreolus capreolus) in northern Germany was partially sequenced and compared with those of 28 other pestiviruses. Due to the occurrence within a narrow location and the complete identity of the sequenced fragments from both roe deer isolates (SH9 and SH11) they seem to belong to the same bovine virus diarrhea virus (BVDV) strain called SH9/11. This strain is highly homologous (up to 93% identity) to "classical" BVDV strains. However, SH9/11 has characteristic variations in its 5' UTR distinct from all other pestiviruses analyzed in this study. Strain SH9/11 is more similar to BVDV group I than to group II, although it is clearly separated from all other cattle isolates tested. In monoclonal antibody (mAb)-typing studies, isolate SH9 reacted with one pestivirus-specific mAb (C16), with two BVDV specific mAbs (N2B12 and D5), and with one mAb (f48) raised against the E2 protein of classical swine fever virus out of a panel of 13 mAbs. The separate position of strain SH9/11 again was demonstrated by the unique reaction pattern of isolate SH9 when compared with other mAb f48-positive BVDV and BDV strains. All these results indicate that indicate that distinct BVDV strains might exist among freeranging roe deer in Germany.

Pseudorabies virus glycoprotein L is necessary for virus infectivity but dispensable for virion localization of glycoprotein H.

Herpesviruses contain a number of envelope glycoproteins which play important roles in the interaction between virions and target cells. Although several glycoproteins are not present in all herpesviruses, others, including glycoproteins H and L (gH and gL), are conserved throughout the Herpesviridae. To elucidate common properties and differences in herpesvirus glycoprotein function, corresponding virus mutants must be constructed and analyzed in different herpesvirus backgrounds. Analysis of gH- mutants of herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PrV) showed that in both viruses gH is essential for penetration and cell-to-cell spread and that its presence is required for virion localization of gL. Since gH homologs are found complexed with gL, it was of interest to assess the phenotype of gL- mutant viruses. By using this approach, HSV-1 gL has been shown to be required for entry and for virion localization of gH (C. Roop, L. Hutchinson, and D. Johnson, J. Virol. 67:2285-2297, 1993). To examine whether a similar phenotype is associated with lack of gL in another alphaherpesvirus, PrV, we constructed two independent gL- PrV mutants by insertion and deletion-insertion mutagenesis. The salient findings are as follows: (i) PrV gL is required for penetration of virions and cell-to-cell spread; (ii) unlike HSV-1, PrV gH is incorporated into the virion in the absence of gL; (iii) virion localization of gH in the absence of gL is not sufficient for infectivity; (iv) in the absence of gL, N-glycans on PrV gH are processed to a greater extent than in the presence of gL, indicating masking of N-glycans by association with gL; and (v) an anti-gL polyclonal antiserum is able to neutralize virion infectivity but did not inhibit cell-to-cell spread. Thus, whereas PrV gL is essential for virus replication, as is HSV-1 gL, gL- PrV mutants exhibit properties strikingly different from those of HSV-1. In conclusion, our data show an important functional role for PrV gL in the viral entry process, which is not explained by a chaperone-type mechanism in gH maturation and processing.

Porcine reproductive and respiratory syndrome virus (PRRSV): monoclonal antibodies detect common epitopes on two viral proteins of European and U.S. isolates.

Sixteen hybridoma cell lines secreting monoclonal antibodies (mAbs) directed against two dutch isolates of the causative virus of Porcine Reproductive and Respiratory Syndrome (PRRSV) were produced. The hybridoma cells resulted from fusions of SP2/0 myeloma cells with splenocytes of STU mice immunized with purified PRRSV after induction of immunotolerance against host cell constituents. Screening of supernatant fluids was performed by an indirect immunofluorescence assay on PRRSV-infected porcine alveolar macrophages. Immunoblotting studies revealed that the mAbs had different protein specificities. One mAb reacted with a viral 15 kD protein, eleven were directed against a 40-50 kD protein, and four against a 30-40 kD protein. The mAb against the 15 kD putative nucleocapsid protein as well as five mAbs against the 40-50 kD protein recognized epitopes on these proteins which are conserved in various European and U.S. isolates of PRRSV.

Differentiation between MHC-restricted and non-MHC-restricted porcine cytolytic T lymphocytes.

The immune system of swine is unique in that the expression of CD4 and CD8 antigens defines four subpopulations of resting extrathymic T lymphocytes. Beyond phenotypic differences to other species, porcine T lymphocytes, particularly when derived from infected animals, are known to show high non-specific cytolytic in vitro activity. Here we describe the putative porcine CD6 antigen (workshop CD6; wCD6) which enables a phenotypic separation of T lymphocytes responsible for major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxicity. The putative porcine CD6 analogue, wCD6, a protein with a molecular mass of 110,000, shows high specificity for T lymphocytes and is neither expressed on B lymphocytes nor on cells of the myeloid lineage. In the extrathymic T-lymphocyte compartment wCD6 characterizes two T-lymphocyte fractions: wCD6+ T lymphocytes including both CD4+ T-helper cell subpopulations (CD4+CD8- and CD4+CD8+) and within the CD4-CD8+ fraction cells with high CD8 antigen density. In contrast the CD4-CD8- gamma/delta T-cell receptor (TCR) subset and CD4-CD8+ cells with low CD8 antigen density are included in the wCD6- T-lymphocyte fraction. Functional studies with separated wCD6 fractions revealed that the wCD6- cells can be characterized by spontaneous and non-MHC restricted cytolytic activity, whereas the wCD6+ T lymphocytes are responsible for MHC-restricted T-cell functions. Thus, the porcine wCD6 is an important antigen to discriminate between MHC-restricted and non-MHC-restricted cytotoxicity.

RNase of classical swine fever virus: biochemical characterization and inhibition by virus-neutralizing monoclonal antibodies.

The structural glycoprotein E0 of classical swine fever virus (CSFV) possesses an intrinsic RNase activity. Here we present the first comprehensive biochemical characterization of E0, using a recombinant glycoprotein expressed in insect cells. We were able to show that the presence of neither carbohydrate moieties nor disulfide bonds is a prerequisite for RNase activity. In addition, virus-neutralizing and nonneutralizing anti-E0 monoclonal antibodies were tested for their ability to influence RNase activity. In these experiments, the antibodies which effectively blocked the infection of STE cells also exerted a high degree of E0 RNase inhibition. This correlation suggests that the RNase activity of CSFV E0 plays a role in the viral life cycle.

Differentiation of classical swine fever virus (CSFV) strains using monoclonal antibodies against structural glycoproteins.

Two panels of monoclonal antibodies (mAbs) against the classical swine fever virus (CSFV) envelope glycoproteins E2 (12 mAbs) and E0 (11 mAbs) were established and tested by immunoperoxidase binding assay against 135 pestivirus strains and isolates. Variability of the binding pattern was demonstrated for CSFV and also for bovine viral diarrhea virus (BVDV) strains and isolates. The panels of mAbs against E2 and E0 led to very different reactivity patterns. Particular mAbs against E2 reacted with (i) all CSFV isolates, (ii) only 4 out of 126 CSFV isolates, or (iii) about 90% of the tested CSFV isolates and 78% of ruminant pestivirus isolates. Anti CSFV E0 mAbs allowed the detection of a greater variability among the CSFV strains and isolates than the anti E2 mAbs. None of the 11 anti E0 mAbs recognized an epitope conserved for CSFV or showed crossreactivity with ruminant pestiviruses. The use of both panels of mAbs against two CSFV structural glycoproteins led to the discrimination of 21 antigenic types of CSFV strains and isolates. The described panels can be used to trace the origin of CSFV after outbreaks of the disease.

Discrimination between two subsets of porcine CD8+ cytolytic T lymphocytes by the expression of CD5 antigen.

Previous work has revealed striking differences in the peripheral T-lymphocyte compartments of swine compared to other species. The key difference is the existence of four T-lymphocyte subpopulations defined by their CD4/CD8 expression. Besides this difference in the CD4/CD8 antigen expression, we report here another unusual antigen distribution on porcine extrathymic T lymphocytes: the expression of the 63,000 MW CD5 antigen. In porcine thymus the CD5 antigen shows a biphasic antigen density, whereas the majority of thymocytes are characterized by an intermediate CD5 expression. In the extrathymic T-lymphocyte compartment, CD5 also shows a heterogeneous antigen distribution as defined by three subsets: CD5high, CD5dim and CD5- T lymphocytes. Analyses of the CD5 expression on the four CD4/CD8-defined peripheral T-cell subpopulations revealed that all CD4+ T lymphocytes, CD4+ CD8+ as well as CD4+ CD8- T lymphocytes, belonged to the subset with high CD5 antigen density. The CD4- CD8- subpopulation, containing in the majority T-cell receptor (TcR) gamma delta T lymphocytes, was characterized by dim CD5 expression. The most notable difference was the division of the CD4- CD8+ cytolytic T-lymphocyte subpopulation into two CD5-defined subsets: CD4- CD5- CD8+ lymphocytes with spontaneous cytolytic activity against tumour cells, and CD4- CD5+ CD8+ T lymphocytes with major histocompatibility complex (MHC)-restricted cytolytic function. Thus, the porcine CD5 antigen is an important marker to discriminate between CD5- CD8+ natural killer (NK) cells and CD5+ CD8+ progenitors of MHC-restricted cytolytic T lymphocytes.

Proteins encoded in the 5' region of the pestivirus genome--considerations concerning taxonomy.

The first protein encoded within the pestivirus open reading frame is a nonstructural protein which removes itself from the polyprotein by autoproteolytic cleavage. The following nucleocapsid protein ends just before a putative signal sequence preceding three glycosylated proteins. All three glycoproteins are part of the viral envelope and exist in the form of disulfide-linked dimers. Pestiviruses have recently been reclassified as members of the family Flaviviridae which now comprises three genera, namely flavivirus, hepatitis C virus group and pestivirus. All members of the family have certain characteristics in common like the overall genome organization and the strategy of gene expression. Major differences exist, however, between the genera; the most obvious ones concern proteins encoded in the 5' region of the respective genomes.