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Heterogeneous expression of the non-structural protein p80/p125 in cells infected with different pestiviruses
Abstract
In order to analyse the expression of the non-structural (ns) protein p80/p125 in cells infected with different pestiviruses at the protein level, radioimmunoprecipitations with the pestivirus-specific monoclonal antibody (MAb) BVD/C16 were performed. Cell lysates infected with cytopathic (cp) and non-cytopathic (ncp) bovine viral diarrhoea (BVD) virus strains and isolates, and with hog cholera (HC) virus strains were analysed. From cpBVD virus-infected cells, the MAb precipitated one or more proteins corresponding to ns p125, displaying a marked size heterogeneity. In contrast, the lower Mr ns p80 proteins from all cpBVD virus strains and isolates analysed had identical electrophoretic motility. The ncpBVD virus strains displayed either one single band or a doublet of the p125 protein and no p80 cleavage products. The p125 proteins precipitated from HC virus-infected cells showed no size heterogeneity. The possibility is discussed that multiple recombination events, including both insertions or deletions in the genomes of ncpBVD viruses, may lead to the heterogeneous expression of the ns p125 in cpBVD virus populations.
... At the molecular level, one important difference between cp BVDV and noncp BVDV is the occurrence of NS3 (p80) in cells infected with a cp virus. NS3 is colinear with the carboxyterminal part of NS2-3 (p125); the latter is present in cells infected with either cp BVDV or noncp BVDV (12,19,36,37,42,44,56). The genomes of several cp BVDV strains contain small host cell-derived insertions in the region coding for NS2-3 (34,35,55). ...
... All cp BVDV isolates can be distinguished from noncp BVDV by their ability to express NS3, a protease which cleaves at its own C terminus and which is responsible for processing of the nonstructural protein region downstream of NS3 (63). In all strains analyzed so far, NS3 has the same size (19). This observation together with data from processing studies conducted for different BVDV strains led to the conclusion that proteolytic cleavage at the amino terminus of NS3 always occurs at amino acid position 1590 or very close to it (37,55,56). ...
Objectives
To establish a nomogram for contrast-induced acute kidney injury (CI-AKI) risk assessment among patients with chronic kidney disease (CKD) undergoing coronary angiography (CAG) or percutaneous coronary intervention (PCI).
Design
Prospective observational cohort study.
Setting
Southern China.
Interventions
None.
Participants
643 consecutive patients with CKD (defined as estimated glomerular filtration rate calculated by Modification of Diet in Renal Disease formula <60 mL/min/1.73 mm ² ) were enrolled.
Outcome measures
The end point was CI-AKI defined as serum creatinine elevation ≥0.5 mg/dL or 25% from baseline within the first 48–72 hours following contrast exposure.Predictors of CI-AKI were selected by multivariable logistic regression and stepwise approach. A nomogram based on these predictors was constructed and compared with the classic Mehran Score. For validation, a bootstrap method (1000 times) was performed.
Results
The nomogram including age, weight, heart rate, hypotension, PCI and β-blocker demonstrated a better predictive value than the classic Mehran Score (area under the curve: 0.78 vs 0.71, p=0.024), as well as a well-fitted calibration curve (χ ² =12.146, p=0.145). Validation through the bootstrap method (1000 times) also indicated a good discriminative power (adjusted C-statistic: 0.76).
Conclusions
With fewer predictors and higher discriminative power, the present nomogram may be a simple and reliable tool to identify patients with CKD at risk of CI-AKI, whereas further external validations are needed.
... Antibodies. CSFV NS3/NS2-3 were detected with monoclonal antibody (MAb) C16 (36,37). The antibodies used for the detection of NS4A (GH4A1), NS4B (GL4B1), NS5A (GL5A1), and NS5B (GR5B1) were kindly provided by T. Rümenapf (University of Veterinary Medicine, Vienna, Austria) and B. Lamp (Justus-Liebig University, Giessen, Germany) (38). ...
... Titration was performed in four replicates using SK6 cells. Viral infection was detected at 72 h p.i. by IF with MAb C16 (36,37), directed against NS3/NS2-3. Mouse-specific Cy3-labeled secondary antibody (Dianova, Hamburg, Germany) was used at a dilution of 1:2,000. ...
For Flaviviridae members, the nonstructural proteins are essential for virion formation and thus exert a dual role in RNA replication and virion morphogenesis. However, it remains unclear how these proteins are functionalized for either process. In wild-type pestiviruses, the NS3/4A complex is selectively active in RNA replication, while NS2-3/4A is essential for virion formation. Mutations recently identified in BVDV-1 rendered NS3/4A capable of supporting NS2-3-independent virion morphogenesis. A comparison of NS3/4A complexes incapable/capable of supporting virion morphogenesis revealed that changes in NS3/NS4A surface interactions are decisive for the gain of function. However, so far, the role of the NS2 mutations as well as the accessory mutations additionally required in the NS2-IRES-NS3 virus variant has not been clarified. To unravel the course of genome packaging, the additional sets of mutations obtained for a second pestivirus species (CSFV) are of significant importance to develop mechanistic models for this complex process.
... Thus, cp pestiviruses represent mutants of noncp viruses which at least in most cases have been generated by recombination. For BVDV, the cp phenotype is strictly correlated with the expression of the nonstructural protein NS3, since this protein is found only in cells infected with cp viruses whereas both cp and noncp BVDV generate NS2-3 (6,11,21,22,27,38). Expression of NS3 is obviously a result of the genome rearrangements observed for cp BVDV. ...
... The cytopathogenicity of pestiviruses is correlated with the expression of the nonstructural protein NS3. For BVDV and some border disease virus strains, NS3 is expressed only by cp viruses while noncp isolates express solely NS2-3 (2,6,11,21,22,27,36,38). In the case of CSFV, noncp isolates also express a protein which is identical or at least very similar to NS3. ...
After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV defective interfering particle resulted in a chimeric defective interfering particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.
... At the molecular level, one important difference between cp BVDV and noncp BVDV is the occurrence of NS3 (p80) in cells infected with a cp virus. NS3 is colinear with the carboxyterminal part of NS2-3 (p125); the latter is present in cells infected with either cp BVDV or noncp BVDV (12,19,36,37,42,44,56). The genomes of several cp BVDV strains contain small host cell-derived insertions in the region coding for NS2-3 (34,35,55). ...
... All cp BVDV isolates can be distinguished from noncp BVDV by their ability to express NS3, a protease which cleaves at its own C terminus and which is responsible for processing of the nonstructural protein region downstream of NS3 (63). In all strains analyzed so far, NS3 has the same size (19). This observation together with data from processing studies conducted for different BVDV strains led to the conclusion that proteolytic cleavage at the amino terminus of NS3 always occurs at amino acid position 1590 or very close to it (37,55,56). ...
Molecular characterization of bovine viral diarrhea virus pair 13 revealed that isolate CP13 is composed of a cytopathogenic (cp) defective interfering particle (DI13) and a noncytopathogenic (noncp) helper virus. The DI13 genome possesses two internal deletions of 1,611 and 3,102 nucleotides. Except for a small fragment of the gene coding for glycoprotein E1, all structural protein genes are deleted together with most of the Npro gene, the region coding for nonstructural proteins p7 and NS2. While the amino terminus of NS3 seems to be strictly conserved for all other cp bovine viral diarrhea viruses, NS3 of DI13 is amino-terminally truncated and fused to 23 amino acids derived from Npro and E1. Characterization of the DI-helper virus system revealed a striking discrepancy between RNA production and generation of infectious viruses.
... Thus, cp pestiviruses represent mutants of noncp viruses which at least in most cases have been generated by recombination. For BVDV, the cp phenotype is strictly correlated with the expression of the nonstructural protein NS3, since this protein is found only in cells infected with cp viruses whereas both cp and noncp BVDV generate NS2-3 (6,11,21,22,27,38). Expression of NS3 is obviously a result of the genome rearrangements observed for cp BVDV. ...
... The cytopathogenicity of pestiviruses is correlated with the expression of the nonstructural protein NS3. For BVDV and some border disease virus strains, NS3 is expressed only by cp viruses while noncp isolates express solely NS2-3 (2,6,11,21,22,27,36,38). In the case of CSFV, noncp isolates also express a protein which is identical or at least very similar to NS3. ...
After cDNA cloning of the genome of bovine viral diarrhea virus (BVDV) isolate CP7, a full-length cDNA clone was constructed. RNA transcribed in vitro from this construct was shown to direct the generation of infectious BVDV upon transfection into bovine cells. To confirm the de novo generation of infectious BVDV from cloned cDNA a genetically tagged virus was constructed. In comparison with parental BVDV, the recombinant virus was slightly retarded in growth. The NS2 coding region of the CP7 genome contains a duplication of 27 nucleotides which is not present in the genome of its noncytopathogenic counterpart, NCP7. Exchange of a small fragment harboring this insertion against the corresponding part of the NCP7 sequence led to recovery of noncytopathogenic BVDV. Alteration of the construct by introduction of a fragment derived from a cytopathogenic BVDV defective interfering particle resulted in a chimeric defective interfering particle which exhibits a cytopathogenic phenotype. These findings confirm the hypothesis that the recombination-induced alterations in the genomes of cytopathogenic BVDV are responsible for the induction of cell lysis.
... For BVDV, cytopathogenicity is always correlated with expression of NS3, which is colinear with the carboxy-terminal part of NS2-3. While NS2-3 is expressed in both cp and noncp BVDV-infected cells, NS3 is found exclusively after infection with cp BVDV (11,13,17,21,34,35,42,43). Accordingly, NS3 is regarded as the marker protein for cp BVDV strains and is supposed to be required for the induction of cytopathic effect. ...
... It should be emphasized that the S27a* coding sequences within the genome of CP Rit are located directly upstream of the ubiquitin* coding insertion whereas the respective bovine mRNA encodes a fusion protein with the structure NH 2 -ubiquitin-S27a-COOH. For BVDV, cytopathogenicity is correlated with expression of NS3, which is not found after infection of cells with noncp BVDV (11,13,17,21,34,35,42,43,52,53). In the case of ubiquitin insertions, it has been reported that ubiquitin functions as a processing signal leading to an additional cleavage of the viral polyprotein by cellular ubiquitin C-terminal hydrolases; at least one entire ubiquitin monomer is required for processing at the C terminus of ubiquitin (51). ...
Molecular characterization of cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strain CP Rit, a temperature-sensitive strain widely used for vaccination, revealed that the viral genomic RNA is about 15.2 kb long, which is about 2.9 kb longer than the one of noncytopathogenic (noncp) BVDV strains. Molecular cloning and nucleotide sequencing of parts of the genome resulted in the identification of a duplication of the genomic region encoding nonstructural proteins NS3, NS4A, and part of NS4B. In addition, a nonviral sequence was found directly upstream of the second copy of the NS3 gene. The 3' part of this inserted sequence encodes an N-terminally truncated ubiquitin monomer. This is remarkable since all described cp BVDV strains with ubiquitin coding sequences contain at least one complete ubiquitin monomer. The 5' region of the nonviral sequence did not show any homology to cellular sequences identified thus far in cp BVDV strains. Databank searches revealed that this second cellular insertion encodes part of ribosomal protein S27a. Further analyses included molecular cloning and nucleotide sequencing of the cellular recombination partner. Sequence comparisons strongly suggest that the S27a and the ubiquitin coding sequences found in the genome of CP Rit were both derived from a bovine mRNA encoding a hybrid protein with the structure NH2-ubiquitin-S27a-COOH. Polyprotein processing in the genomic region encoding the N-terminal part of NS4B, the two cellular insertions, and NS3 was studied by a transient-expression assay. The respective analyses showed that the S27a-derived polypeptide, together with the truncated ubiquitin, served as processing signal to yield NS3, whereas the truncated ubiquitin alone was not capable of mediating the cleavage. Since the expression of NS3 is strictly correlated with the cp phenotype of BVDV, the altered genome organization leading to expression of NS3 most probably represents the genetic basis of cytopathogenicity of CP Rit.
... The mAbs WS381 and WS384 were derived from the BDV strain 87/6 of genotype 1.b [45,46]. The mAb BVD/C16 (Institute of Virology, University of Veterinary Medicine Hannover, Germany [47]) was generated against the BVDV strain NADL. This mAb was used as an infection control confirming that the cell layer was 70-90% infected by the corresponding viruses listed in Table 1. ...
Classical swine fever virus (CSFV) shares high structural and antigenic homology with bovine viral diarrhea virus (BVDV) and border disease virus (BDV). Because all three viruses can infect swine and elicit cross-reactive antibodies, it is necessary to differentiate among them with regard to serological diagnosis of classical swine fever. To understand the mechanism of cross-reactivity, it is important to define common or specific epitopes of these viruses. For this purpose, epitope mapping of six monoclonal antibodies (mAbs) was performed using recombinant expressed antigenic domains of CSFV and BDV E2 proteins. One CSFV-specific conformational epitope and one CSFV and BDV common epitope within domain B/C of E2 were identified. Site-directed mutagenesis confirmed that residues G725 and V738/I738 of the CSFV-specific epitope and P709/L709 and E713 of the second epitope are important for mAbs binding. Infection of CSFV in porcine cells was significantly reduced after pre-incubation of the cells with the domain B/C of E2 or after pre-incubation of CSFV with the mAbs detecting domain B/C. 3D structural modeling suggested that both epitopes are exposed on the surface of E2. Based on this, the identified epitopes represent a potential target for virus neutralization and might be involved in the early steps of CSFV infection.
... CSFV neutralizing Ab titres higher than 1/32 can be protective, preventing viral excretion and transmission (Terpstra and Wensvoort, 1988). The antibodies induced in infected pigs recognize E rns , E2 and NS3 proteins (Graham et al., 2012b;Greiser-Wilke et al., 1992;König et al., 1995;Weiland et al., 1992). Anti-NS3 antibodies cross-react with NS3 of different pestiviruses and are not neutralizing (Weiland et al., 1992). ...
Classical swine fever (CSF) is among the most relevant viral epizootic diseases of swine. Due to its severe economic impact, CSF is notifiable to the world organization for animal health. Strict control policies, including systematic stamping out of infected herds with and without vaccination, have permitted regional virus eradication. Nevertheless, CSF virus (CSFV) persists in certain areas of the world and has re-emerged regularly. This review summarizes the basic established knowledge in the field and provides a comprehensive and updated overview of the recent advances in fundamental CSFV research, diagnostics and vaccine development. It covers the latest discoveries on the genetic diversity of pestiviruses, with implications for taxonomy, the progress in understanding disease pathogenesis, immunity against acute and persistent infections, and the recent findings in virus-host interactions and virulence determinants. We also review the progress and pitfalls in the improvement of diagnostic tools and the challenges in the development of modern and efficacious marker vaccines compatible with serological tests for disease surveillance. Finally, we highlight the gaps that require research efforts in the future.
... Viral infection with BVDV or BDV was detected 72 hpi by IF using MAb 8.12.7 directed against NS3/NS2-3, kindly provided by E. J. Dubovi (Cornell University, Ithaca, NY) (90). Infection of SK6 cells with CSFV isolates was detected by indirect IF with MAb C16 directed against CSFV NS2-3/NS3 (104,108). ...
Only noncp pestivirus strains are capable of establishing life-long persistent infections to generate the virus reservoir in the field. The molecular basis for this biotype is only partially understood and only investigated in depth for BVDV-1 strains. Temporal control of viral RNA replication correlates with the noncp biotype and is mediated by limiting amounts of cellular DNAJC14 that activate the viral NS2 protease to catalyze the release of the essential replicase component NS3. Here, we demonstrate that several species of noncp pestiviruses depend on DNAJC14 for their RNA replication. Moreover, all cp pestiviruses, in sharp contrast to their noncp counterparts, replicate independently of DNAJC14. The generation of a cp BVDV in the persistently infected animal is causative for onset of mucosal disease. Therefore, the observed strict biotype-specific difference in DNAJC14 dependency should be further examined for its role in cell type/tissue tropism and the pathogenesis of this lethal disease.
... By a variety of mechanisms, such as cellular RNA insertions, cp BVDV arises by RNA recombination of ncp BVDV (Qi et al., 1992;Ridpath and Bolin, 1995;Tautz et al., 1994). Unlike in cp BVDV, the NS2/NS3 region is not cleaved in ncp BVDV (Greiser-Wilke et al., 1992). The soluble form of the envelope glycoprotein E rns of pestivirus degrades immunostimulatory viral single-and double-stranded RNA and inhibits ss-and dsRNA-induced type I IFN synthesis (Lussi and Schweizer, 2016). ...
Bovine viral diarrhea virus (BVDV) and bovine respiratory syncytial virus (BRSV) infections contribute to the bovine respiratory disease complex (BRDC), which is a multi-factorial disorder involving co-infections of viruses and bacteria including mycoplasma. BRDC causes great economic losses to the United States feedlot industry. BVDV infection induces immunosuppression in infected animals. BVDV Npro binds and degrades the transcription factor interferon regulatory factor-3 (IRF-3) and effectively blocks type I interferon (type I IFN) expression in host cells. BRSV nonstructural proteins, NS1 and NS2, block activation of IRF-3. In calves, concurrent infection with BVDV and BRSV resulted in more severe clinical signs of disease and extensive lung lesions than infection with either virus alone. The objective of this study was to extend the understanding of the role of the Npro of noncytopathic BVDV-2 (pestivirus B) on type I IFN pathway signaling in bovine turbinate (BT) cells during single and co-infection with BRSV. Based on real-time quantitative-reverse transcription-polymerase chain reaction, the BVDV-2 mutant with dysfunctional Npro (BVDV2-E) significantly up-regulated protein kinase R (PKR), TANK-Binding Kinase 1, IRF-3, IRF-7, and interferon-β (IFN-β) mRNAs compared to BVDV-2 wild-type (BVDV2-wt) and BRSV in single and co-infected BT cells. BRSV-infected cells expressed significantly up-regulated PKR, IRF-3, IRF-7, and IFN-β mRNAs, whereas BVDV2-wt, but not BVDV2-E, abolished this up-regulation in co-infection. No significant differences were observed in mitochondrial antiviral signaling, Nuclear Factor-κB (NF-κB), and NIMA-Interacting 1 mRNAs. A dual-luciferase reporter assay showed that BVDV2-wt significantly increased NF-κB activity compared to BVDV2-E, while BVDV2-E significantly increased IFN-β activity compared to BVDV2-wt. BT cells infected with BVDV2-E produced more IRF-3 protein compared to cells infected with BRSV or BVDV2-wt. The BRSV titer and RNA levels significantly increased in cells co-infected with BRSV/BVDV2-wt compared to cells co-infected with BRSV/BVDV2-E or infected with BRSV alone. BVDV2-E single and co-infected cells synthesized type I IFN significantly higher than BVDV2-wt single and co-infected BT cells. These findings are useful in defining the role of the intact BVDV-2 Npro on type I IFN pathway signaling and support the understanding of the mechanism underlying the synergistic action of BVDV2-wt and BRSV inhibition of type I IFN. The inhibition of BRSV-induced signals by BVDV augments BRSV infection. Advisor: Clayton L. Kelling, Ph.D.
... In addition, E rns and NS3 also induce detectable antibodies [30][31][32]. E2 is under purifying selection pressure [33], likely due to the immune response, this is most probably also the case for NS3, although perhaps to a lesser degree, as it is a nonstructural protein and does not elicit neutralizing antibodies [34], however it is a strong activator of cytotoxic T lymphocytes [35][36][37]. In principle, these apparent SNPs could be due to errors by the reverse transcriptase; however, this is unlikely as most of the SNPs were detected in multiple independent cDNAs and also in previously published sequences. ...
Background:
Direct molecular cloning of full-length cDNAs derived from viral RNA is an approach to identify the individual viral genomes within a virus population. This enables characterization of distinct viral haplotypes present during infection.
Results:
In this study, we recover individual genomes of classical swine fever virus (CSFV), present in a pig infected with vKos that was rescued from a cDNA clone corresponding to the highly virulent CSFV Koslov strain. Full-length cDNA amplicons (ca. 12.3 kb) were made by long RT-PCR, using RNA extracted from serum, and inserted directly into a cloning vector prior to detailed characterization of the individual viral genome sequences. The amplicons used for cloning were deep sequenced, which revealed low level sequence variation (< 5%) scattered across the genome consistent with the clone-derived origin of vKos. Numerous full-length cDNA clones were generated using these amplicons and full-genome sequencing of individual cDNA clones revealed insights into the virus diversity and the haplotypes present during infection. Most cDNA clones were unique, containing several single-nucleotide polymorphisms, and phylogenetic reconstruction revealed a low degree of order.
Conclusions:
This optimized methodology enables highly efficient construction of full-length cDNA clones corresponding to individual viral genomes present within RNA virus populations.
... After two passages cell culture supernatant was collected and the cells were fixed by heat treatment (80°C for 3 h). For detection of viral antigen an indirect immune-peroxidase staining was performed using an NS3-specific monoclonal mouse antibody BVD/C16 ( [27], dilution 1:25 in PBS-0.01%Tween) and a polyclonal rabbit anti-mouse horseradish peroxidase conjugate (dilution 1:200 in PBS-0.01% Tween, Catalogue no.: P0260, DAKO, Denmark). ...
Translation and replication of positive stranded RNA viruses are directly initiated in the cellular cytoplasm after uncoating of the viral genome. Accordingly, infectious virus can be generated by transfection of RNA genomes into susceptible cells. In the present study, efficiency of conventional virus isolation after inoculation of cells with infectious sample material was compared to virus recovery after transfection of total RNA derived from organ samples of pigs infected with Classical swine fever virus (CSFV). Compared to the conventional method of virus isolation applied in three different porcine cell lines used in routine diagnosis of CSF, RNA transfection showed a similar efficiency for virus rescue. For two samples, recovery of infectious virus was only possible by RNA transfection, but not by the classical approach of virus isolation. Therefore, RNA transfection represents a valuable alternative to conventional virus isolation in particular when virus isolation is not possible, sample material is not suitable for virus isolation or when infectious material is not available. To estimate the potential risk of RNA prepared from sample material for infection of pigs, five domestic pigs were oronasally inoculated with RNA that was tested positive for virus rescue after RNA transfection. This exposure did not result in viral infection or clinical disease of the animals. In consequence, shipment of CSFV RNA can be regarded as a safe alternative to transportation of infectious virus and thereby facilitates the exchange of virus isolates among authorized laboratories with appropriate containment facilities. © 2015 Meyer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
... BVDV-1a and BVDV-1b seem to be the most prevalent genogroups worldwide (Fulton et al., 2003;Vilcek et al., 2004). In addition, due to their effects on permissive cells, two biotypes can be distinguished, the cytopathic (cp) and the noncytopathic (ncp) (Donis and Dubovi, 1987;Greiser-Wilke et al., 1992). Animals which become infected intrauterinely with ncp-BVDV (50 to 150 days of gestation) are epidemiologically important, as they may become tolerant to the virus. ...
Sixteen bovine viral diarrhea virus (BVDV) isolates collected in Costa Rica between 1987 and 2006 from dairy cattle were analyzed by RT-PCR and determined to belong to BVDV species 1. Furthermore, eleven of these isolates were genotyped using the nucleotide sequences of the Npro region of the viral genome. Phylogenetic analysis indicated that all samples examined clustered within the BVDV-1b subtype.
... Im Gegensatz zu nzp BVDVs wurde im nicht-zytopathogenen Biotyp des KSPV eine Prozessierung von NS2-3 zu reifem NS3 beobachtet (Donis et al., 1987;Podock et al., 1987;Greiser-Wilke et al., 1992). Zytopathogene KSP-Feldisolate wurden nur selten beschrieben. ...
The biosynthesis of Classical swine fever virus (CSFV) nonstructural proteins was
characterized with regard to hierarchy and dynamics of proteolytic maturation. A wildtypic
non-cytopathogenic isolate and two cytopathogenic CSFV constructs were analysed to allow
the comparison with data about the closely related BVDV. The following results were
achieved:
1 A GST-NS2-3 fusion protein, a C-terminal fragment of the NS3 helicase, a Cterminal
fragment of NS4B, and the entire NS5A of CSFV were expressed in E.
coli und purified by Ni2+ ion affinity chromatography. Following the immunization
of laboratory animals, monoclonal antibodies (mAbs) against these proteins were
produced and characterized.
2 Indirect immunofluorescence experiments showed a tight association of all CSFV
nonstructural proteins with membranes of the endoplasmic reticulum. Immunoblot
analyses of a cp CSFV replicon and ncp CSFV led to the identification of NS4-5
and NS2-3 precursors, NS5A/B and NS4A/B processing intermediates, and of all
mature nonstructural proteins. In addition, a novel internal processing of NS3 was
detected separating the protease and helicase domains of this molecule.
3 The nonstructural protein processing of ncp CSFV, cp CSFV-JIV, and a cp CSFV
replicon was characterized by immunoprecipitation together with pulse chase
analyses. The nonstructural protein maturation of the ncp biotype of CSFV occurs
very slowly, while an efficient generation of all nonstructural proteins takes place
in the cp biotype. Half-lives of precursors and mature proteins were determined to
directly compare the different biotypes. These analyses showed that the mature
nonstructural proteins of CSFV are stable (T1/2: 1,5-4 h). The maturation of
precursor molecules was faster (T1/2: 0,5-2,5 h) than the turnover of mature
products in the cp biotype. In contrast, a constant amount of mature proteins results
from a slow maturation of precursor molecules in the ncp biotype (T1/2: 2,5-5,5 h).
These data indicate that enhanced expression of NS3 in cp pestiviruses is
responsible for an accelerated processing of the other nonstructural proteins. In
contrast, inefficient NS2-3 cleavage in ncp pestiviruses restricts NS3 protease
activity and thereby regulates the concentration of all mature nonstructural
proteins.
... Virus isolation was carried out on PK15 or SK6 cells according to the Technical Part of the ''Diagnostic Manual'' of the European Commission (Anonymous, 2002). Viral antigen was detected after heat fixation 72 h post inoculation by indirect immuno-peroxidase staining with the pestivirus-specific monoclonal antibody (mab) C16 (Greiser-Wilke et al., 1992) or the CSFV E2-specific mab HC26, respectively. ...
Due to the tremendous socio-economic impact of classical swine fever (CSF) outbreaks, emergency vaccination scenarios are continuously under discussion. Unfortunately, all currently available vaccines show restrictions either in terms of marker capacities or immunogenicity. Recent research efforts were therefore directed at the design of new modified live marker vaccines. Among the most promising candidates the chimeric pestiviruses "CP7_E2alf" and "flc11" were identified. Within an international research project, these candidates were comparatively tested in challenge experiments after a single oral vaccination. Challenge infection was carried out with highly virulent CSF virus strain "Koslov", 14 or 21 days post vaccination (dpv), respectively. Safety, efficacy, and marker potential were addressed. All assessments were done in comparison with the conventional "gold standard" C-strain "Riems" vaccine. In addition to the challenge trials, multiple vaccinations with both candidates were performed to further assess their marker vaccine potential. All vaccines were safe and yielded full protection upon challenge 21 days post vaccination. Neither serological nor virological investigations showed major differences among the three vaccines. Whereas CP7_E2alf also provided clinical protection upon challenge at 14 days post vaccination, only 50% of animals vaccinated with flc11, and 83% vaccinated with C-strain "Riems" survived challenge at this time point. No marked differences were seen in protected animals. Despite the fact that all multiple-vaccinated animals stayed sero-negative in the accompanying marker test, the discriminatory assay remains a weak point due to delayed or inexistent detection of some of the vaccinated and subsequently infected animals. Nevertheless, the potential as live marker vaccines could be confirmed for both vaccine candidates. Future efforts will therefore be directed at the licensing of "Cp7_E2alf" as the first live marker vaccine for CSF.
... In contrast, cp CSFV strain Alfort-Jiv described in this study combines helper virus-independent replication and genetic stability during propagation in tissue culture cells and thus is well suited to compare the properties of cp and non-cp CSFV in both susceptible cells and the natural host. The cytopathogenicity of pestiviruses including BVDV, BDV, and CSFV has been shown to correlate with the expression of large amounts of NS3, the C-terminal part of NS2-3 (5,9,20,21,23,27,30,51). The genetic background and the resulting molecular mechanisms that lead to the efficient release of free NS3 are remarkably diverse among cp pestivirus genomes, including (i) genomic deletions fusing the translation start codon (51) or the autoprotease N pro gene (72) directly to the 5Ј end of the viral NS3 gene, (ii) duplicated viral sequences located in the NS2 gene (70), (iii) point mutations in the NS2 gene (37), and (iv) the insertion of cleavage sites for cellular proteases directly upstream of NS3 (6,12,16,25,71). ...
For the important livestock pathogens classical swine fever virus (CSFV) and bovine viral diarrhea virus (BVDV), cytopathogenic (cp) and non-cp viruses are distinguished according to the induction of apoptosis in infected tissue culture cells. However, it is currently unknown whether cp CSFV differs from non-cp CSFV with regard to virulence in the acutely infected host. In this study, we generated helper virus-independent CSFV Alfort-Jiv, which encompasses sequences encoding domain Jiv-90 of cellular J-domain protein interacting with viral protein (Jiv). Expanding the knowledge of BVDV, our results suggest that Jiv acts as a regulating cofactor for the nonstructural (NS) protein NS2 autoprotease of CSFV and initiates NS2-3 cleavage in trans. For Alfort-Jiv, the resulting expression of large amounts of NS3 correlated with increased viral RNA synthesis and viral cytopathogenicity. Moreover, both cp Alfort-Jiv and the parental non-cp CSFV strain Alfort-p447 efficiently replicate in cell culture. Animal experiments demonstrated that in contrast to parental non-cp Alfort-p447, infection with cp Alfort-Jiv did not cause disease in pigs but induced high levels of neutralizing antibodies, thus elucidating that cp CSFV is highly attenuated in its natural host. In contrast to virulent Alfort-p447, the attenuated CSFV strain Alfort-Jiv induces the expression of cellular Mx protein in porcine PK-15 cells. Accordingly, the remarkable difference between cp and non-cp CSFV with regard to the ability to cause classical swine fever in pigs correlates with different effects of cp and non-cp CSFV on cellular antiviral defense mechanisms.
... The rabbit polyclonal antisera against recombinant affinity-purified CSFV N pro (42), CSFV C, and porcine IRF3 were described previously (6). The viral nonstructural protein NS3 was detected using the monoclonal antibody (MAb) C16 (16), kindly provided by Irene Greiser-Wilke, Hannover Veterinary School, Hannover, Germany. The anti-FLAG M2 MAb and the anti-␣-tubulin MAb were purchased from Sigma. ...
Viruses are detected by different classes of pattern recognition receptors that lead to the activation of interferon regulatory
factors (IRF) and consequently to the induction of alpha/beta interferon (IFN-α/β). In turn, efficient viral strategies to
escape the type I IFN-induced antiviral mechanisms have evolved. Previous studies established that pestivirus Npro antagonizes the early innate immune response by targeting the transcription factor IRF3 for proteasomal degradation. Here,
we report that Npro of classical swine fever virus (CSFV) interacts also with IRF7, another mediator of type I IFN induction. We demonstrate
that the Zn-binding domain of Npro is essential for the interaction of Npro with IRF7. For IRF3 and IRF7, the DNA-binding domain, the central region, and most of the regulatory domain are required
for the interaction with Npro. Importantly, the induction of IRF7-dependent type I IFN responses in plasmacytoid dendritic cells (pDC) is reduced after
wild-type CSFV infection compared with infection with virus mutants unable to interact with IRF7. This is associated with
lower levels of IRF7 in pDC. Consequently, wild-type but not Npro mutant CSFV-infected pDC show reduced responses to other stimuli. Taken together, the results of this study show that CSFV
Npro is capable of manipulating the function of IRF7 in pDC and provides the virus with an additional strategy to circumvent the
innate defense.
... Neutralizing antibodies specifically recognize the structural E rns and E2 proteins [8,9] and their presence has been associated with protection [10]. Antibodies against the NS3 protein are also induced upon infection but, despite their ability to recognize different pestiviruses, they do not neutralize the virus [11]. E2 envelope glycoprotein is the major target for neutralizing antibo-dies during natural CSFV infection [10,12,13] and it has been the main component in the design of CSFV DIVA vaccines [1,3,7]. ...
... Briefly, cells were detached from culture dishes by trypsin treatment, fixed with 4% paraformaldehyde in PBS and permeabilized with 0.1% saponin in PBS. Viral NS3 expression was detected with the monoclonal antibody C16 [45] kindly provided by Irene Greiser-Wilke, Hannover Veterinary School, Hannover. To this end, the fixed and permeabilized cells were incubated in presence of C16 diluted with PBS/0.3% ...
Virus replicon particles (VRP) are genetically engineered infectious virions incapable of generating progeny virus due to partial or complete deletion of at least one structural gene. VRP fulfil the criteria of a safe vaccine and gene delivery system. With VRP derived from classical swine fever virus (CSF-VRP), a single intradermal vaccination protects from disease. Spreading of the challenge virus in the host is however not completely abolished. Parameters that are critical for immunogenicity of CSF-VRP are not well characterized. Considering the importance of type I interferon (IFN-α/β) to immune defence development, we generated IFN-α/β-inducing VRP to determine how this would influence vaccine efficacy. We also evaluated the effect of co-expressing granulocyte macrophage colony-stimulating factor (GM-CSF) in the vaccine context. The VRP were capable of long-term replication in cell culture despite the presence of IFN-α/β. In vivo, RNA replication was essential for the induction of an immune response. IFN-α/β-inducing and GM-CSF-expressing CSF-VRP were similar to unmodified VRP in terms of antibody and peripheral T-cell responses, and in reducing the blood levels of challenge virus RNA. Importantly, the IFN-α/β-inducing VRP did show increased efficacy over the unmodified VRP in terms of B-cell and T-cell responses, when tested with secondary immune responses by in vitro restimulation assay.
... A common characteristic of cp BVDV strains is the efficient production of NS3 throughout infection, which represents a major difference from ncp BVDV (15,18,35,40). Cytopathogenic BVDV strains can use various strategies for expression of NS3, including processing of the precursor protein NS2-3 (1, 9, 24, 32-34, 36, 37, 42, 49). ...
For Bovine viral diarrhea virus (BVDV), the type species of the genus Pestivirus in the family Flaviviridae, cytopathogenic (cp) and noncytopathogenic (ncp) viruses are distinguished according to their effect on cultured cells. It has been established that cytopathogenicity of BVDV correlates with efficient production of viral nonstructural protein NS3 and with enhanced viral RNA synthesis. Here, we describe generation and characterization of a temperature-sensitive (ts) mutant of cp BVDV strain CP7, termed TS2.7. Infection of bovine cells with TS2.7 and the parent CP7 at 33 degrees C resulted in efficient viral replication and a cytopathic effect. In contrast, the ability of TS2.7 to cause cytopathogenicity at 39.5 degrees C was drastically reduced despite production of high titers of infectious virus. Further experiments, including nucleotide sequencing of the TS2.7 genome and reverse genetics, showed that a Y1338H substitution at residue 193 of NS2 resulted in the temperature-dependent attenuation of cytopathogenicity despite high levels of infectious virus production. Interestingly, TS2.7 and the reconstructed mutant CP7-Y1338H produced NS3 in addition to NS2-3 throughout infection. Compared to the parent CP7, NS2-3 processing was slightly decreased at both temperatures. Quantification of viral RNAs that were accumulated at 10 h postinfection demonstrated that attenuation of the cytopathogenicity of the ts mutants at 39.5 degrees C correlated with reduced amounts of viral RNA, while the efficiency of viral RNA synthesis at 33 degrees C was not affected. Taken together, the results of this study show that a mutation in BVDV NS2 attenuates viral RNA replication and suppresses viral cytopathogenicity at high temperature without altering NS3 expression and infectious virus production in a temperature-dependent manner.
... The other important change at the protein level distinguishing cpBVDV from noncpBVDV is the expression of ~80 in addition to ~125. While the above-mentioned fusion proteins show a high degree of variability concerning nature and size, not only the mere presence of ~80 in cpBVDV-infected cells but also its size is strictly conserved (Purchio et al., 1984;Donis and Dubovi, 1987a;Greiser-Wilke et al., 1992;Meyers et a/., unpublished). A prerequisite for generation of ~80 is the introduction of a protease cleavage site at the aminoterminus of this protein. ...
Two cytopathogenic isolates of bovine viral diarrhea virus (cpBVDV) have been analyzed. For both viruses two regions of their genomic RNAs were found to be duplicated and rearranged. The viral genomes contain a small duplicated element (SD) derived from the genomic 5' end far downstream of its original context. This sequence is followed by a larger duplication which encompasses the region coding for the protein p80(LD), a molecular marker for cpBVDV. The SD element codes for the viral protease p20. In the case of the viruses analyzed here the aminoterminus of p80 is generated by autoproteolytic removal of the preceding SD-encoded protease. For one of the cpBVDV isolates a specific fusion protein (p28) could be identified which is composed of p20 and part of p10, another viral protein. Molecular characterization of the respective noncytopathogenic counterpart revealed that duplication and rearrangement of sequences as well as the expression of p28 and p80 are specific for the cytopathogenic virus.
... CSFV vA187-1 was derived from cloned cDNA as described before (Ruggli et al., 1996). Detection of replicon and/or virus replication was carried out by immunoperoxidase staining of the cells for viral proteins E2 or NS3 using monoclonal antibody (Mab) HC/TC26 (Greiser-Wilke et al., 1990) or Mab C16 (Greiser-Wilke et al., 1992), respectively (Moser et al., 1999). ...
Bicistronic genomes of the classical swine fever virus (CSFV) strain Alfort/187 (A187) were established by insertion of a second cistron consisting of an internal ribosome entry site of the encephalomyocarditis virus and a coding sequence in the 3' untranslated region of the genome. Introduction of the selectable marker gene for neomycin phosphotransferase into the second cistron of the CSFV replicon A187 Delta E2-CAT allowed the establishment of porcine SK-6 cell lines constitutively expressing the respective bicistronic replicon RNA. In cells transfected with RNA representing the full-length viral genome and containing the gene coding for bacterial enhanced green fluorescence protein (EGFP) in the second cistron infectious bicistronic virus was synthesized. Expression of EGFP in cells infected with this virus indicated the potential of CSFV as a viral vector. Finally, after insertion of the sequence encoding the signal peptide of the CSFV E2 protein followed either by the E2 or the E2-p7 sequence into the replicon A187 Delta E2 which carries an in frame deletion of 465 nucleotides in the E2 gene, infectious viruses vA187 Delta E2-IRES-sigE2 and vA187 Delta E2-IRES-sigE2p7, respectively, were obtained. This shows that E2 deletion mutants can be complemented by expression of E2 from a separate cistron.
... Electroporation of the respective RNA (10 g each) into SK-6 cells was performed exactly as described above for DNA (chapter 2.2). One tenth of the electroporated cells was seeded into two wells of a 24-well plate for staining by IPA [29] with either NS3-specific mAb C16 [30] or with mAb HC/TC26 recognizing the CSFV E2 protein [28]. The specific infectivity of the RNA was determined similarly as previously described for in vitro synthesized full-length CSFV RNA [24] in an infectious centre assay [31] except that no agarose overlay was used. ...
A cDNA clone of the classical swine fever virus (CSFV) strain Alfort/187 [Ruggli N, Tratschin JD, Mittelholzer C, Hofmann MA. Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA. J Virol 1996;70(6):3478-87] was used to construct two E2 deletion mutants lacking either the complete E2 gene or, alternatively, a stretch of 204 nucleotides encoding 68 amino acids located in the C-terminal region of the E2 glycoprotein. The respective in vitro synthesized mutant RNAs replicated in SK-6 cells but no infectious virus was generated. Both replicons could be packaged into virus particles in SK-6 cells constitutively expressing E2 of CSFV. For the resulting CSF virus replicon particles (CSF-VRP) A187-E2del373 and A187-E2del68 titers of 10(6) and 10(7) TCID(50)/ml, respectively, were obtained. Oronasal vaccination with 10(7) TCID(50) of either of the two CSF-VRP protected pigs against a challenge with a lethal dose of CSFV strain Eystrup. In contrast, after intradermal vaccination VRP A187-E2del68 but not VRP A187-E2del373 lacking the complete E2 gene induced a protective immune response. We conclude that E2-complemented CSF-VRP have the potential to be used as live-attenuated non-transmissible oral vaccines for pigs. In addition, our data suggest that E2 of CSFV is dispensable for the induction of mucosal but not of parenteral immunity.
... For CSFV-specific protein expression , transfected or infected cells were washed twice in PBS, fixed in 4 % (w/v) paraformaldehyde for 15 min and washed again. Then, the cells were stained for 20 min at 4 uC with the anti-E2 mAb HC26 (Greiser-Wilke et al., 1990) (kindly provided by Bommeli AG) or the anti-NS3 mAb C16 (Greiser-Wilke et al., 1992) (kindly provided by Dr Greiser-Wilke, Hannover, Germany), diluted in 0?3 % (w/v) saponin (S4521; Sigma-Aldrich) permeabilization solution . After washing with PBS containing 0?1 % (w/v) saponin, Alexa-488 or Alexa-546 fluorochrome-labelled anti-mouse secondary antibody (Molecular Probes) – diluted in the permeabilizing solution – was added. ...
Vaccination of pigs against Classical swine fever virus (CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3-NS4A were used to identify viral genes encoding CTL epitopes. Monocyte-derived dendritic cells (DCs) and fibrocytes served as the APCs. In vitro translation of the mRNA and microscopic analysis of transfected cells demonstrated that E2 and NS3-NS4A could be identified. APCs transfected with either of the mRNA molecules restimulated CSFV-specific T cells to produce gamma interferon and specific cytotoxic activity against CSFV-infected target cells. The presence of CTL epitopes on E2 was confirmed by using d/d-haplotype MAX cells expressing E2 constitutively as target cells in d/d-haplotype CTL assays. A potent CTL activity against E2 was detected early (1-3 weeks) after CSFV challenge. This work corroborates the existence of CTL epitopes within the non-structural protein domain NS3-NS4A of CSFV. Furthermore, epitopes on the E2 protein can also now be classified as targets for CTLs, having important implications for vaccine design, especially subunit vaccines. As for the use of mRNA-transfected APCs, this represents a simple and efficient method to identify viral genes encoding CTL epitopes in outbred populations.
Classical swine fever virus (CSFV) shares high antigenic homology with other members of the genus Pestivirus. Because several pestivirus species can also infect swine, eliciting cross-reactive antibodies, it is important to define CSFV-specific epitopes for the differential diagnosis of classical swine fever (CSF) by serology. For this purpose, epitope mapping of seven monoclonal antibodies (mAbs), recognizing sites on the D/A domain of glycoprotein E2, was performed using recombinant expressed antigenic domains and mutants of E2, as well as an overlapping peptide library. Three CSFV-specific epitopes, i.e., 780-IEEMGDDFGFGLCPF-794, 810-NGSAFYLVCPIGWTG-824, and 846-REKPF-850, were identified within the D/A domain of E2. Site-directed mutagenesis further confirmed that residues 783-MGD-785, 789-FGLCPF-794, 813-AFYLVCPIGWTG-824, and 846-REK-848 were critical residues in these regions. In addition, a F789S difference within the epitope 780-IEEMGDDFGFGLCPF-794 was responsible for the absence of binding of two mAbs to the E2 protein of the live attenuated CSFV vaccine strain Riems. Structural modeling revealed that, the three epitopes are located near each other, suggesting that they may form a more complex conformational epitope on the D/A domain in vivo. Six of the mAbs neutralized viruses of diverse genotypes, indicating that the target epitopes are involved in virus interaction with cells. The binding of CSFV to cells was significantly reduced after pre-incubation with either truncated E2 proteins comprising the D/A domain or with the CSFV-specific mAbs targeting the domain D/A. These epitopes identified on the D/A domain are important targets for virus neutralization that might be involved in the early steps of CSFV infection. These findings reveal potential candidates for improving the differential diagnosis of pestiviruses by serology.
Cytopathogenic (cp) pestiviruses frequently emerge in cattle that are persistently infected with the bovine viral diarrhea virus (BVDV) as a consequence of RNA recombination and mutation. They induce apoptosis in infected tissue cultures, are highly attenuated in the immunocompetent host, and unable to establish persistent infections after diaplacental infections. Cp strains of BVDV have been used as naturally attenuated live vaccines and for species-specific plaque reduction tests for the indirect serological detection of BVDV. Here, we present a genetically engineered cp strain of the classical swine fever virus (CSFV). Cytopathogenicity of the strain was induced by the insertion of ubiquitin embedded in a large NS3 to NS4B duplication. The CSFV RNA genome was stabilized by the inactivation of the NS2 autoprotease, hindering the deletion of the insertion and the reversion to a wild-type genome. Additional insertion of a mCherry gene at the 5′-end of the E2 gene allowed fluorescence-verified plaque reduction assays for CSFV, thus providing a novel, cost-efficient diagnostic tool. This genetically stabilized cp CSFV strain could be further used as a basis for potential new modified live vaccines. Taken together, we applied reverse genetics to rationally fixate a typical cp NS3 duplication in a CSFV genome.
IntroductionDirect Antigen DetectionVirus IsolationAntibody DetectionReverse Transcription Polymerase Chain Reaction (RT-PCR) AssaysMilk As A Diagnostic SampleScreening For Persistently Infected (PI) AnimalsSummary And Conclusions
Functional disruption of dendritic cells (DCs) is an important strategy for viral pathogens to evade host defences. Monocytotropic viruses such as classical swine fever virus (CSFV) could employ such a mechanism, since the virus can suppress immune responses and induce apoptosis without infecting lymphocytes. Here, CSFV was shown to infect and efficiently replicate in monocyte- and in bone marrow-derived DCs. Interestingly, the infected DCs displayed neither modulated MHC nor CD80/86 expression. Stimulation of DCs with IFN-α/TNF-α or polyinosinic-polycytidylic acid (pIC) induced phenotypic maturation with increased MHC and CD80/86 expression, both with mock-treated and infected DCs. In addition, the T cell stimulatory capacity of CSFV-infected DCs was maintained both in a polyclonal T cell stimulation and in specific antigen-presentation assays, requiring antigen uptake and processing. Interestingly, similar to macrophages, CSFV did not induce IFN-α responses in these DCs and even suppressed pIC-induced IFN-α induction. Other cytokines including interleukin (IL)-6, IL-10, IL-12 and TNF-α were not modulated. Taken together, these results demonstrated that CSFV can replicate in DCs and control IFN type I responses, without interfering with the immune reactivity. These results are interesting considering that DC infection with RNA viruses usually results in DC activation.
Author Summary
Plasmacytoid dendritic cells (pDC) represent the most potent producers of interferon type I and are therefore of major importance in antiviral defences. A TLR7-dependent induction of interferon-α in pDC by infected cells in the absence of virions has been demonstrated for hepatitis C virus. Here, we show that this pathway is also very efficient for classical swine fever virus, a pestivirus that is also a member of the Flaviviridae. Our data indicate a transfer of RNA from the donor cell to pDC in a cell-contact-dependent manner requiring intact lipid rafts and cytoskeleton of the donor cell. Importantly, we demonstrate that the enigmatic viral Erns protein unique to pestiviruses efficiently prevents this pathway of pDC activation. This novel function of Erns is dependent on its RNase activity within intracellular compartments. The present study underlines the importance of pDC activation by infected cells and identifies a novel pathway of virus escaping the interferon system. Considering that Erns is required for pestiviruses to establish persistent infection of foetuses after transplacental virus transmission resulting in the development of immunotolerant animals, this report also points on a possible role of pDC in preventing immunotolerance after viral infection of foetuses.
Chimeric pestivirus CP7_E2alf is a promising live marker vaccine candidate against classical swine fever. Prior to a possible application in the field, several safety aspects have to be addressed. Due to the fact that CP7_E2alf is based on a bovine viral diarrhea virus backbone, its behavior in ruminants is of particular interest. In the framework of this study, its innocuousness in non-target species was addressed by inoculation of calves, young goats, lambs, and rabbits. To this means, high titres of CP7_E2alf were applied orally to three animals of each species. Additional animals were left as unvaccinated contact controls. During the study, all animals remained clinically healthy, and neither fever nor leukopenia were observed. Virus could not be isolated from purified white blood cells or from nasal or faecal excretions. Moreover, none of the animals (inoculated or contact control) seroconverted. In the target species, innocuousness, shedding and transmission of vaccine virus was addressed in different animal trials that were carried out primarily for the purpose of efficacy, potency or duration of immunity studies. In all experiments, CP7_E2alf proved to be completely safe for the vaccinees and unvaccinated contact controls. Furthermore, no shedding or transmission was detected in any of the experiments. Even after parental vaccination, vaccine virus genome was barely detectable in blood or organ samples of vaccinated animals. Thus, CP7_E2alf can be regarded as completely safe for both target and non-target species.
Bovine viral diarrhea virus (BVDV), strain NADL, was originally isolated from an animal with fatal mucosal disease. This isolate is cytopathic in cell culture and produces two forms of NS3-containing proteins: uncleaved NS2-3 and mature NS3. For BVDV NADL, the production of NS3, a characteristic of cytopathic BVDV strains, is believed to be a consequence of an in-frame insertion of a 270-nucleotide cellular mRNA sequence (called cIns) in the NS2 coding region. In this study, we constructed a stable full-length cDNA copy of BVDV NADL in a low-copy-number plasmid vector. As assayed by transfection of MDBK cells, uncapped RNAs transcribed from this template were highly infectious (>10(5) PFU/microg). The recovered virus was similar in plaque morphology, growth properties, polyprotein processing, and cytopathogenicity to the BVDV NADL parent. Deletion of cIns abolished processing at the NS2/NS3 site and produced a virus that was no longer cytopathic for MDBK cells. This deletion did not affect the efficiency of infectious virus production or viral protein production, but it reduced the level of virus-specific RNA synthesis and accumulation. Thus, cIns not only modulates NS3 production but also upregulates RNA replication relative to an isogenic noncytopathic derivative lacking the insert. These results raise the possibility of a linkage between enhanced BVDV NADL RNA replication and virus-induced cytopathogenicity.
Proteolytic processing of polyproteins is considered a crucial step in the life cycle of most positive-strand RNA viruses. An enhancement of NS2-3 processing has been described as a major difference between the noncytopathogenic (non-CP) and the cytopathogenic (CP) biotypes of pestiviruses. The effects of accelerated versus delayed NS2-3 processing on the maturation of the other nonstructural proteins (NSP) have never been compared. In this study, we analyzed the proteolytic processing of NSP in Classical swine fever virus (CSFV). Key to the investigation was a panel of newly developed monoclonal antibodies (MAbs) that facilitated monitoring of all nonstructural proteins involved in virus replication (NS2, NS3, NS4A, NS5A, and NS5B). Applying these MAbs in Western blotting and radioimmunoprecipitation allowed an unambiguous identification of the mature proteins and precursors in non-CP CSFV-infected cells. Furthermore, the kinetics of processing were determined by pulse-chase analyses for non-CP CSFV, CP CSFV, and a CP CSFV replicon. A slow but constant processing of NS4A/B-5A/B occurred in non-CP CSFV-infected cells, leading to balanced low-level concentrations of mature NSP. In contrast, the turnover of the polyprotein precursors was three times faster in CP CSFV-infected cells and in cells transfected with a CP CSFV replicon, causing a substantial increase of mature NSP concentrations. We conclude that a delayed processing not only of NS3 but further of all NSP represents a hallmark of regulation in non-CP pestiviruses.
Hannover, Tierärztliche Hochsch., Diss., 2000 (Nicht für den Austausch).
Due to the vast economic consequences of classical swine fever (CSF) outbreaks, emergency vaccination plans are under discussion in European Union Member States. However, animals vaccinated with the conventional C-strain vaccine are subject to trade restrictions. To ease these restrictions, potent marker vaccines are required. One promising candidate is the chimeric pestivirus CP7_E2alf. For emergency vaccination in a CSF outbreak scenario, early onset of immunity is required. Here, the studies performed with a CP7_E2alf virus stock produced under good manufacturing conditions (GMP) are reported. In challenge experiments, CP7_E2alf induced full clinical protection 1 week after intramuscular vaccination, and 2 weeks after oral immunization. Furthermore, even after application of diluted vaccine preparations complete protection could be achieved if challenge infection was carried out 4 weeks after vaccination. In conclusion, GMP-produced CP7_E2alf proved to be a suitable marker vaccine candidate - also for emergency vaccination - both after intramuscular and oral application.
Classical swine fever (CSF) is one of the most important diseases of pigs. Although prophylactic vaccination is banned within the European Union, emergency vaccination, allowing differentiation of vaccinated from infected animals, is an option for disease control. Up to now, these strategies are based on antibody detection. In this context, conventional modified live vaccines are not suitable. A promising perspective could be genetic differentiation of vaccinated from infected animals where field virus strains are differentiated from vaccine viruses by sequence differences. This concept could also be used with marker vaccines. To this end, a set of real-time reverse transcription-polymerase chain reaction (RT-PCR) assays was developed and validated. Specific primers and probes were designed for detection of the C-strain "Riems" vaccine virus or the chimeric marker vaccine candidate CP7_E2alf. A heterologous internal positive control was also included. The assays were then multiplexed to detect simultaneously either CSF field virus, C-strain "Riems", and the internal control or CSF field virus, CP7_E2alf, and the internal control. To validate both systems, samples from vaccination/challenge trials were tested. Only samples from vaccinated animals were found to be positive, while all samples from wild type virus-infected animals and a broad test panel of different pestiviruses were negative. Field application of the "C-strain Riems" specific assay was proven with wild boar samples from surveillance programs in Germany and France. In conclusion, ready-to-use RT-PCR sets are presented as reliable tools for genetic differentiation of vaccinated from infected animals for CSFV eradication strategies.
An antigen capture enzyme immunoassay (EIA) for the detection of bovine viral diarrhoea (BVD) viral antigen in peripheral blood lymphocytes of cattle was used for the screening of 241 animals. The test used a monoclonal antibody directed against a conserved antigenic domain of a nonstructural protein (p125/p80) of pestiviruses for antigen capture. Bound antigen was detected with a pestivirus-specific polyclonal peroxidase conjugate. In parallel the samples were analysed by routine virus isolation procedures based on cell culture. Virus isolation and antigen capture EIA were positive in 54 cases. The latter test scored one additional sample.
Genomic RNA of noncytopathic (NCP) bovine viral diarrhea virus (BVDV) strain SD-1 was extracted directly from serum obtained from a persistently infected animal. cDNA was synthesized and amplified by polymerase chain reaction (PCR) before cloning. The complete genomic nucleotide sequence was determined by sequencing at least two different clones from independent PCR reactions. The 5' and 3' end sequences of the SD-1 genome was determined from 5'-3' ligation clones. The complete genome sequence was comprised of 12,308 nucleotides containing one large open reading frame which encodes an amino acid sequence of 3898 residues with a calculated molecular weight of 438 kDa. In contrast to cytopathic (CP) BVDV strain NADL, which contains a cellular RNA insert of 270 nucleotides and CP BVDV strain Osloss, which has an inserted ubiquitin RNA sequence of 228 nucleotides, the NCP strain SD-1 had no insertion along the genome. Sequence comparison with other pestiviruses revealed that the overall nucleotide sequence homologies of SD-1 are 88.6% with NADL, 78.3% with Osloss, 67.1% with HoCV Alfort, and 67.2% with HoCV Brescia. The overall deduced amino acid sequence homologies of SD-1 are 92.7% with NADL, 86.2% with Osloss, 72.5% with HoCV Alfort, and 71.2% with HoCV Brescia. The most conserved nucleotide and amino acid sequences are located in the 5' untranslated region (5'UTR) and nonstructural protein p80 region, respectively. The viral glycoproteins, particularly gp53, and nonstructural proteins p54 and p58 have the lowest homology comparing both nucleotide and amino acid sequences between SD-1 and other pestiviruses. Extensive analyses of amino acid sequences for the viral structural proteins and nonstructural protein p54 regions from five pestiviruses led to the identification of four conserved domains (designated as C1, C2, C3, C4) and three highly variable domains (designated as V1, V2, V3) within this region. The C1, C2, and C3 domains are located in the capsid protein p14, glycoprotein gp48, and gp25, respectively. The C4 domain is located in the junction between gp53 and p54. Interestingly, out of three variable domains, two (V1, V2) are located in the same glycoprotein gp53. The third variable domain is located in the nonstructural protein p54.
After molecular RNA cloning of the Alfort strain (Alfort/LCRV) of hog cholera virus (HCV), the nucleotide sequence of about 70% of the total genome was determined. This sequence was compared with homologous parts of previously published pestivirus genomes. The average homology with another clone of the Alfort strain (Alfort/FRC) was found to be lower (86.1%) than with Brescia strain of HCV (94.3%), while, compared with NADL, Osloss and SD-1 (3 different strains of bovine viral diarrhea virus, BVDV), the average homology was 67%. Although the amino-acid sequences show a higher degree of conservation, they had a similar degree of homology (92.7, 96.7, 69, 68.2 and 69%, respectively). Partial sequence comparison also revealed that Alfort/LCRV strain was more closely related to Alfort 187 (98.6% for the nucleotides and amino acids) and Weybridge (97.3% for the nucleotides and 96.1% for the amino acids) strains of HCV than it was to Alfort/FRC. These results may indicate that the Alfort/FRC strain has undergone more genomic variations during its historical passage. Genomic relationships among the pestiviruses are discussed.
BVDV isolates exist as two biotypes differentiated at the molecular level by production of a p80 polypeptide. Insertions consisting of host cell sequences and/or duplicated and rearranged viral sequences have been observed in the portion of the genome coding for the p80 polypeptide in some, but not all, cytopathic BVDV. The significance of these insertions to biotypic expression has yet to be demonstrated. It has been hypothesized that recombination results in the production of the p80 polypeptide by introduction of a cleavage site into a precursor polypeptide or the introduction of a second copy of the p80 gene. Because inserts have not been identified in all cytopathic BVDV examined, it appears that recombination may not be the only mechanism involved in biotypic determination.
Porcine bone marrow stroma cell (BMSC) cultures producing cells of granulocyte-lineage were established. Hog cholera (HC) virus ALD and Alfort strains replicated in the porcine BMSC cultures showing distinct cytopathic effect (CPE). The differentiation of granulocyte-lineage cells in the cultures ceased after infection with HC virus. Polyclonal antibody against the ALD strain inhibited completely the development of CPE of the both ALD and Alfort strains. Monoclonal antibodies (mAbs) specific to the ALD strain inhibited CPE of the ALD strain, while CPE of the Alfort strain was not affected by those mAbs, suggesting that CPE induced in the BMSC cultures is due to HC virus.
Cytopathogenic (cp) bovine viral diarrhea virus (BVDV) strains are generated in cattle persistently infected with noncytopathogenic (noncp) BVDV.cp BVDV strains are considered crucial for the development of fatal mucosal disease. Comparative analysis of cp and noncp BVDV strains isolated from one animal suffering from mucosal disease revealed that the genomes of the cp BVDV strain (CP7) and the corresponding noncp BVDV strain (NCP7) are highly homologous. However, only the genome of CP7 contains an insertion of 27 nucleotides in the NS2 coding region. The inserted sequence represents a duplication of bases 4064 to 4090 of the viral genome, located between the formerly neighboring nucleotides 4353 and 4354. Parts of the viral polyproteins of CP7 and NCP7 were expressed in the T7 vaccinia virus system. These studies revealed that the insertion identified in the CP7 genome is necessary and sufficient for the induction of NS2-3 cleavage. Since the expression of NS3 is strictly correlated to cp BVDV, the insertion identified in the genome of BVDV CP7 represents most likely the relevant mutation leading to the evolvement of CP7 from NCP7.
This chapter provides an overview of various aspects of pestiviruses, including diseases they cause and their molecular biology. General diseases caused by the pestiviruses include bovine viral diarrhea and mucosal disease, border disease, and classical swine fever. Molecular cloning and sequencing of pestiviral genomes, as well as expression of defined parts of their polyproteins, allowed crucial conclusions concerning—in particular, the strategy of gene expression, genome organization, composition of virions, relationships of the three species at the molecular level. One remarkable property of pestiviruses is the existence of two biotypes that were recognized according to morphological changes they cause during growth in tissue culture cells. Noncytopathogenic (noncp) pestiviruses replicate without clearly visible effects, while cytopathogenic (cp) viruses lead to lysis of appropriate target cells. The molecular basis for this distinction is the subject of current investigations and is the major focus of this chapter, which arise by nonhomologous RNA recombination.
Vaccination with live cytopathogenic (cp) bovine viral diarrhoea virus (BVDV) is often used for control of this disease. In animals which are persistently infected with noncytopathogenic (ncp) BVDV this can lead to the outbreak of mucosal disease (MD). To simulate vaccination of such animals and to monitor the clinical-virological course after superinfection, nine clinically healthy calves which were persistently viremic were superinfected with different cp BVDV strains. One animal succumbed to early onset MD within three weeks after superinfection. During the observation period of 18 months four animals developed severe clinical signs. While two animals developed late onset MD, the other two had to be euthanized due to clinical signs which could not be related to the superinfecting BVDV. These results indicated that after superinfection or vaccination of persistently infected calves with cp BVDV the probability of developing early and/or late onset MD is significantly increased. The risks arising from uncritical vaccination of herds with unknown virological status in relation with the control of BVDV conforming to the actual official guidelines are discussed.
To determine the minimal requirements for autonomous RNA replication of classical swine fever virus (CSFV), genomes having in-frame deletions within the genes for structural and flanking nonstructural proteins were constructed, based on an infectious cDNA clone of CSFV Alfort/187. RNA was transcribed in vitro from the respective plasmids and transfected into SK-6 swine kidney cells. The replication competence of the RNA was determined by immunostaining transfected cells for CSFV NS3 protein and by analysis of cell extracts for viral RNA, as well as protein synthesis at different times after transfection. The genes encoding N(pro), C, E(rns), E1, E2, p7, and NS2 proved to be dispensable for RNA replication, but the efficiency of replication varied strongly between individual constructs. RNA replicons containing the complete NS2-NS3 gene persisted in transfected cells and continued to replicate without causing any obvious morphological or functional damage to the cells, whereas genomes lacking the NS2 gene replicated more efficiently and induced a cytopathic effect. These findings suggest that NS2, although it is not essential for pestivirus RNA replication, has a regulatory function therein. Both cytopathogenic and noncytopathogenic replicons were packaged into virus particles provided in trans by a cotransfected full-length helper virus genome.
Virulent classical swine fever (CSF) represents an immunomodulatory viral infection that perturbs immune functions. Circulatory and immunopathological disorders include leukopenia, immunosuppression and haemorrhage. Monocytic cells - targets for CSF virus (CSFV) infection - could play critical roles in the immunopathology, owing to their production of immunomodulatory and vasoactive factors. Monocytes and macrophages (Mphi) are susceptible to virus infection, as a consequence of which prostaglandin E2 (PGE2) production is enhanced. The presence of PGE2 in serum from CSFV-infected pigs correlated with elevated PGE2 productivity by the peripheral blood mononuclear cells from these same animals. It was noted that these PGE2-containing preparations did not inhibit, but actually enhanced, lymphocyte proliferation. The proinflammatory cytokines tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-6 were not involved, although elevated IL-1 production could relate to lymphocyte activation. Nevertheless, IL-1 was not the sole element: infected Mphi produced lympho-stimulatory activity but little IL-1. This release of immunomodulatory factors, following CSFV infection of monocytic cells, was compared with other characteristics of the disease. Therein, PGE2 and IL-1 production was noted to coincide with the onset of fever and the coagulation disorders typical of CSF. Consequently, these factors are of greater relevance to the haemorrhagic disturbances, such as petechia and infarction, rather than the leukopenia found in CSF.
Tissue alterations and distribution of BVDV antigen were examined in nine cattle with early onset and five cattle with late onset mucosal disease (MD) to evaluate the possibility to differentiate between the two disease entities. MD was induced by inoculation of persistently viremic cattle with different strains of cytopathogenic BVDV. Animals which developed early onset MD became moribund approximately 2 weeks post-inoculation (pi); animals with late onset MD 42-115 days pi. All animals were euthanized and necropsied when moribund. Macroscopic lesions were found in the upper and lower digestive tract of cattle with early and late onset MD. In cattle with late onset MD, lesions in the oral cavity were generally milder and in the intestinal tract they were not only associated with GALT, but frequently affected the mucosa outside. Histologically, the abrupt changes between hyperplastic and atrophic areas of mucosa were striking in the cattle with late onset MD. This corresponded with the multifocal distribution of areas of mucosa in which intense staining for BVD-virus antigen could be demonstrated. In both courses of MD, a severe depletion of Peyer's patches was noted, but only in late onset MD, there was a complete loss of architecture. The most distinctive difference was the presence of vascular lesions which were observed in all five cattle with late onset MD, but in none of the animals with early onset MD. The vasculopathy was characterized by segmental necrosis of vascular walls and lymphohistiocytic perivasculitis in arterioles and small arteries in the submucosa of the intestine.
When compared to noncytopathic (ncp) bovine viral diarrhoea virus (BVDV), some cytopathic (cp) BVDV contain additional sequences in the NS2-3 genomic region. One of these insertions, which is 270 nucleotides long and of host origin (cINS), was first described for strain NADL. To find out how frequently this type of insertion occurs in other cp BVDV, 32 cp BVDV field isolates and the BVDV reference cp strain Indiana were screened using RT-PCR which detected cINS in NADL. For most cp viruses an RT-PCR product of 402bp indicated the presence of NS2-3 genes without insertions. In addition, one or two DNA fragments, around 600-850bp in size, were amplified from the genomes of 13 cp viruses indicating the presence of insertions. Sequencing of the PCR products, i.e. 402bp DNA fragment (with no insertion) and longer fragments (with insertion) revealed the location of the insertions in the NS2-3 coding region of eight cp BVDV genomes. All of the insertions were confirmed to be of the cINS type and were located in a very similar position to that found previously in the NADL genome. They were in the same reading frame as the viral polypeptide and they encoded 90-140 amino acids. The 5' and 3' ends of the insertions were different in most of the cp isolates studied. Interestingly, a 14-amino-acid stretch at the 5'-end of the insertion in the cp 5569 isolate as well as 15 amino acids at the 3'-end of the insertion in the cp 5.19516 isolate were not homologous to the cINS sequence. No significant matches for these stretches were found in the EMBL and Swissprot databases.
An assay for the bovine viral diarrhoea virus (BVDV) replicase was developed using extracts from BVDV-infected cells. The replicase activity was maximal approximately 8 h post-infection as measured by the generation of a genomic length radiolabelled RNA. Using a semi-denaturing gel system, three virus-specific in vitro radiolabelled nascent RNA species were identified. A fast-migrating RNA was demonstrated to be the double-stranded replicative form (RF). A second form was shown to be a partially single-stranded/partially double-stranded RNA, characteristic of the replicative intermediate (RI). A third form, which was often undetectable, migrated between the RF and RI and was probably genomic viral RNA. The optimal replicase activity was dependent on 5-10 mM Mg2+ and although it was also active in 1-2 mM Mn2+ it was inhibited at higher concentrations. The optimum KCl concentration for labelling of the RI and RF were different, suggestive of at least two distinct replicase activities. These results are supportive of a semi-conservative model of BVDV RNA replication.
Lipopolysaccharide (LPS) impairs classical swine fever virus (CSFV) replication in monocytic cells, which are primary targets for CSFV and mediators of virus-induced immunomodulation. Although soluble antiviral factors including interferons (IFN) were not detected, IFN-alpha and IFN-beta mRNA were induced. The serine threonine protein kinase inhibitor 2-aminopurine, impeded this antiviral activity. These results indicate that the LPS-induced antiviral state employs signaling pathways, in which the double-stranded RNA-dependent protein kinase (PKR) is actively involved.
Classical swine fever virus (CSFV) replicates efficiently in cell lines and monocytic cells, including macrophages (MΦ), without
causing a cytopathic effect or inducing interferon (IFN) secretion. In the present study, the capacity of CSFV to interfere
with cellular antiviral activity was investigated. When the porcine kidney cell line SK-6 was infected with CSFV, there was
a 100-fold increased capacity to resist to apoptosis induced by polyinosinic-polycytidylic acid [poly(IC)], a synthetic double-stranded
RNA. In MΦ, the virus infection inhibited poly(IC)-induced alpha/beta IFN (type I IFN) synthesis. This interference with cellular
antiviral defense correlated with the presence of the viral Npro gene. Mutants lacking the Npro gene (ΔNpro CSFV) did not protect SK-6 cells from poly(IC)-induced apoptosis, despite growth properties and protein expression levels
similar to those of the wild-type virus. Furthermore, ΔNpro CSFV did not prevent poly(IC)-induced type I IFN production in MΦ but rather induced type I IFN in the absence of poly(IC)
in both MΦ and the porcine kidney cell line PK-15, but not in SK-6 cells. With MΦ and PK-15, an impaired replication of the
ΔNpro CSFV compared with wild-type virus was noted. In addition, ΔNpro CSFV, but not wild-type CSFV, could interfere with vesicular stomatitis virus replication in PK-15 cells. Taken together,
these results provide evidence for a novel function associated with CSFV Npro with respect to the inhibition of the cellular innate immune system.
The RNA genomes of cytopathogenic bovine viral diarrhea virus (BVDV) isolates contain insertions highly homologous to cellular sequences. For two of them the insert was identified as ubiquitin coding sequence. The genome of BVDV Osloss contains exactly one ubiquitin gene monomer. In the case of BVDV CP1 the cellular insertion comprises one complete ubiquitin gene and part of a second monomer. The host cell-derived element in the CP1 genome is embedded in a large duplication of about 2.4 kb of viral sequences. Cellular insertion and duplication were not found in the genome of NCP1, the noncytopathogenic counterpart of CP1. These results strongly suggest that recombination between viral and cellular RNA is responsible for development of the cytopathogenic viruses, which is linked to pathogenesis of a lethal disease in cattle.
Cytopathic and noncytopathic reference strains as well as Canadian field isolates of bovine viral diarrhea virus were analyzed by neutralization and immunofluorescence tests using a bovine viral diarrhea virus-specific neutralizing monoclonal antibody. Results on reference strains indicated three major antigenic groups: I) NADL-like, II) New York 1-like and III) Oregon C24V-like. Field isolates could be segregated into groups I and II and none could be typed into the group III. It appears that most bovine viral diarrhea virus strains share a common antigen which carries a major neutralization epitope. These characteristics would make this monoclonal antibody a useful reagent for taxonomic and epizootiological studies.
Variation of the intracellular polypeptides induced in calf testis cells by 5 cloned isolates of bovine virus diarrhoea virus (BVDV) was examined. Three of the isolates were cytopathic (NADL, C 2415 and Pe 515 c) and two were non-cytopathic (C 1226 and Pe 515 nc) in these cells. The isolates Pe 515 c and Pe 515 nc were both isolated from an animal with clinical signs of mucosal disease. In cells infected with NADL, 8 virus specific proteins (vp 1 to vp 8) with molecular weights ranging from 120,000 (vp 1) to 23,000 (vp 8) were detected. Isolates C 2415 and Pe 515 c gave a similar array of polypeptides to NADL, but the 3 cytopathic isolates could be distinguished by the variation in the molecular weights of some of the proteins. The non-cytopathic isolates could also be distinguished from each other by this type of molecular variation; however, one feature that characterised these strains, when compared to the cytopathic isolates, was the absence of vp 2. Comparison of the polypeptides induced by Pe 515 c and Pe 515 nc showed that apart from the lack of vp 2 in the Pe 515 nc virus profile, the molecular weights of the other viral proteins were similar. This supports serological evidence that for mucosal disease to occur the pair of cytopathic and non-cytopathic viruses must be closely related. Four of the polypeptides induced by Pe 515 c were shown to be glycoproteins.
Three outbreaks of mucosal disease were investigated. Careful examination of 47 cattle that were persistently viraemic with bovine virus diarrhoea virus (BVDV) revealed no clinical disease, no or low levels of BVDV antibody and only non-cytopathic virus in their blood. The four animals with mucosal disease all showed clinical disease and both cytopathic and non-cytopathic virus in their blood. Following post mortem examination, there were particularly high levels of cytopathic virus in gut tissue. A hypothesis for the induction of mucosal disease is suggested. It states that animals become persistently infected with non-cytopathic virus following in utero infection and when, in post natal life, they become superinfected with a cytopathic virus, then mucosal disease ensues. The experimental reproduction of mucosal disease in support of this hypothesis is described.
We amplified and sequenced the p125 coding regions of a 'homologous' pair of BVDV biotypes, Pe515 cytopathogenic and non-cytopathogenic. The sequences were aligned with the published sequences of Osloss, NADL and the HCV Alfort strains, but no insertions of host sequence were observed in that region.
Thirty-three pestivirus strains were grown in cell culture and characterized by immunostaining with 19 monoclonal antibodies (MAbs) raised against hog cholera virus (HCV), with 42 MAbs against bovine viral diarrhoea virus (BVDV) and with 13 MAbs against border disease virus (BDV). Seven MAbs reacted with all pestivirus strains tested, eight MAbs detected only the seven HCV strains, three detected only the 16 BVDV strains. No MAb was found that was specific for BDV. BVDV and BDV strains were broadly cross-reactive with the MAbs, indicating a close relationship between these two species, whereas HCV strains were characterized as distinct from BVDV and BDV.
Eight monoclonal antibodies directed against the hog cholera virus (HCV) strain Alfort/187 and displaying broad cross-reactivity with other HCV strains were characterized. An enzyme immunoassay on fixed monolayers of porcine or bovine cells infected with 14 different strains and isolates of HCV and 12 bovine viral diarrhea viruses (BVDV), respectively, showed that all antibodies reacted with HCV only. Seven antibodies recognized all HCV tested, thus indicating that they were directed against conserved epitopes. All antibodies neutralized the homologous strain and different patterns of the other HCV tested. Radioimmunoprecipitation analysis showed that the monoclonal antibodies were directed against a doublet of 56-60 kDa, presumably representing the major envelope glycoprotein of HCV. The results of reciprocal antibody blocking assays allowed the mapping of two distinct conserved antigenic domains on this protein.
Introduction. The term ‘pestivirus’ was coined in 1973 to group together two antigenically related enveloped RNA viruses: hog cholera virus (HCV) and bovine viral diarrhoea virus (BVDV; Horzinek, 1973). A third animal pathogen, the border disease virus (BDV) of sheep, was found later to be a close relative of BVDV. Pestiviruses are among the smallest enveloped animal RNA viruses (about 40 nm in diameter) and possess a nucleocapsid of non-helical, probably icosahedral symmetry (Horzinek et al., 1967); they share these traits with the numerous flaviviruses, of which the arthropod-borne yellow fever virus is the prototype. The pestiviruses are not arthropod-borne and currently hold generic status in the family Togaviridae. Previously, flaviviruses also held generic status in this family. However, when details of flavivirus molecular structure, replication strategy and gene sequence became known in the early 1980s, the Togaviridae Study Group recognized the fundamental differences and proposed the creation of the new family Flaviviridae with Flavivirus as the only genus (Westaway et al., 1985).
The genome of bovine viral diarrhea virus (BVDV) contains a single large open reading frame capable of encoding 449 kDa of protein. Short segments from along the length of the molecularly cloned BVDV genome were engineered so as to be expressed as bacterial fusion polypeptides in Escherichia coli. These BVDV analog fusion proteins were used as immunogens to generate a panel of sequence-specific antisera. These antiserum reagents were in turn employed in immunoprecipitation analyses to identify the authentic BVDV protein to which they were directed. The results allowed for the identification and positioning along the genome of BVDV gene products accounting for approximately 83% of the coding capacity of the virus. A preliminary map of the genetic organization of BVDV is presented and discussed.
The RNA genome of the cytopathic NADL isolate of bovine viral diarrhea virus (BVDV) has been molecularly cloned and the nucleotide sequence determined. The cloned sequence was 12,573 nucleotides in length, corresponding to a molecular weight of 4.3 X 10(6), having a base composition of 32.2% A, 25.7% G, 22.1% U, and 20.0% C. However, the sequences at the 5' and 3' termini of the RNA have not been unequivocally established. A single major open reading frame extending the length of the molecule was found in the viral-sense (positive polarity) sequence. This open reading frame was capable of encoding 3988 amino acids, representing 449 kDa of protein.
Both cytopathic and noncytopathic bovine viral diarrhea virus (BVDV) were isolated from 16 of 17 bovine spleens representing 11 herds that had experienced acute BVD and from 12 of 21 bovine spleens from 1 herd affected with chronic BVD. It was concluded that isolation of cytopathic and noncytopathic BVDV from the same spleen probably indicates that an animal with a persistent, noncytopathic BVDV infection was superinfected with a cytopathic BVDV. The prevalence (greater than 70%) of 2 viruses in the spleen of cattle with acute or chronic BVD suggested that persistent infection with noncytopathic BVDV may be an important factor in the pathogenesis of BVD.
Eight healthy cattle that were persistently infected with noncytopathic bovine viral diarrhea virus (BVDV) were inoculated with cell culture fluids that contained noncytopathic or cytopathic BVDV. A severe disease occurred after inoculation with cytopathic BVDV. The clinical signs, lesions, and immune response were consistent with those of clinical BVDV infections.
Two biotypes of bovine viral diarrhea-mucosal disease virus are present in nature: one that induces cytopathology in infected bovine cells and the other that infects cells without overt cytopathology. Infections with both types of virus yield similar amounts of infectious progeny virus. Field and laboratory isolates of both biotypes of bovine viral diarrhea (BVD) virus were analyzed by radioimmunoprecipitation and polyacrylamide gel electrophoresis of infected cell extracts. The noncytopathic biotype BVD (NCB-BVD) virus isolates can be differentiated from cytopathic biotype BVD (CB-BVD) isolates on the basis of peculiar polypeptide profiles they induce in the infected cell. The most abundant polypeptide in CB-BVD infected cells is the 80K polypeptide. NCB-BVD virus-infected cells lack the 80K polypeptide and induce a predominant 118K polypeptide. D-[2-3H]Mannose labeling of cells infected with NCB-BVD indicated that at least three polypeptides are N-glycosylated: 75K, 56K-58K, and 48K. In addition the sizes and ratios of the glycoproteins induced by all virus isolates showed a marked variation. We present evidence indicating that there is remarkable heterogeneity among the field viral isolates of BVD and this methodology is of potential value for molecular epidemiology studies.
Neutralising antibody to non-cytopathogenic and cytopathogenic strains of bovine virus diarrhoea virus (BVDV) was assayed in a microtitre test in which cultures of calf testis cells were stained by the immunoperoxidase method to detect viral replication. Fourteen BVDV strains were compared in cross neutralisation tests with antisera prepared in gnotobiotic calves. Ten of the strains comprised five pairs of non-cytopathogenic and cytopathogenic BVDV. Each pair was isolated from an animal with mucosal disease. All five animals were from five separate outbreaks of the disease. Each pair of strains from the same outbreak was found to be antigenically indistinguishable. In contrast, when the coefficient of antigenic similarity was calculated 11 of 45 comparisons between the pairs and 46 of 91 comparisons between all 14 viruses gave R values that distinguished strains. The observations suggest that an antigenic spectrum within a single related group exists for BVDV strains, rather than distinct serotypes. The findings are also consistent with the suggestion that cytopathogenic strains from natural outbreaks of mucosal disease arise by mutation from non-cytopathogenic virus.
Virus-specific proteins were examined in cultured cells infected with bovine viral diarrhea virus. By using antisera obtained from virus-infected animals, three major virus-specific polypeptides with molecular weights of 115,000 (115K), 80K, and 55K were observed. Minor proteins of 45,000 and 38,000 daltons were also noted. Tryptic peptide mapping indicated that the 115K and the 80K polypeptides were structurally related. The 55K protein was glycosylated and appeared not to be related to the 115K and 80K proteins. Pulse-chase experiments failed to demonstrate any procursor-product relationship among any of these proteins, and all three polypeptides were found in purified virion preparations. The significance of these findings with respect to the replication of bovine viral diarrhea virus is discussed.
The purpose of this study was the identification of antigenic differences between cytopathic (cp) and noncytopathic (ncp) bovine viral diarrhoea viruses (BVDV). Cells infected with 19 strains of each viral biotype were analyzed for reactivity with the monoclonal antibody (mab) BVD/C38. Reactivity was examined using an enzyme immunoassay on fixed infected monolayers of fetal calf kidney cells. In the majority of cases, the mab discriminated between cells infected with each of the two viral biotypes. Three reactivity patterns could be distinguished. Most cpBVDV strains yielded monolayers where 80–100% of infected cells reacted with the mab. Most of the ncpBVDV infected cells showed either no reaction, or only single cells or foci were stained. However, about one third of either cp- or ncpBVDV strains tested yielded infected monolayers where 30–50% of the cells reacted with the antibody. Cell damage other than the typical cytopathic effect might be responsible for the BVD/C38 reactivity of cells infected with BVDV.
In addition, it was analyzed whether the antigenic marker associated with cpBVDV was expressed in cells infected with viral isolates from 21 animals with clinical mucosal disease. In 14 cases cpBVDV was isolated and the antigenic marker was found throughout. In seven cases ncpBVDV was cultivated and the antigenic marker was detected in four isolates.
In previous work, we developed a preliminary description of the genetic organization of the prototypic pestivirus bovine viral diarrhea virus (BVDV). In order to refine this genetic map and to further elucidate the gene products and expression strategy of this virus, we have generated a broad panel of sequence-specific antibody reagents. Use of these reagents not only allowed the identification of several previously undescribed viral polypeptides, but when used in in vivo pulse-chase experiments, they identified precursor polyproteins and processing intermediates. Data generated from these studies provide a more accurate and complete view of viral gene organization, as well as insight into several aspects of protein processing and the gene expression strategy employed by this pestivirus. These experiments also revealed varying stability and turnover rates for the mature BVDV proteins. These latter results have implications for the functional roles of certain gene products.
The history of pestivirus taxonomy is surprisingly consistent: almost 30 years ago it was recognized that pestiviruses are structurally akin to flaviviruses, and recent nucleotide sequence data have confirmed this resemblance at the level of genome organization. For other enveloped positive stranded RNA viruses with ikosahedral nucleocapsids e.g. equine arteritis and lactate dehydrogenase virus of mice a taxonomic dilemma is encountered; while virions resemble ("non-arthropod-borne") togaviruses, the replication via a nested set of subgenomic RNAs is corona and torovirus-like. Pestiviruses, flaviviruses and the hepatitis C virus group have been assigned generic status in the Flaviviridae family.
Detection of antibodies against hog cholera virus and bovine viral diarrhea virus in porcine serum. A comparative examination using CF, PLA and NPLA assays
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