Identification of classical swine fever virus protein E2 as a target for cytotoxic T cells by using mRNA-transfected antigen-presenting cells.

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.

A recombinant classical swine fever virus with a marker insertion in the internal ribosome entry site.

Based on an infectious cDNA clone of classical swine fever virus (CSFV) strain Alfort/187 (Ruggli et al., J Virol 70, 3478-3487, 1996) a full-length cDNA was constructed harbouring a nonviral 44 base insertion in the internal ribosome entry site (IRES) within the 5' nontranslated region (5'NTR) of the genome. Genome size RNA transcribed in vitro served as a positive control in routine RT-PCR used to detect CSFV RNA in diagnostic material. Unexpectedly this RNA proved to be infectious upon transfection into susceptible cells. The replication kinetics of the resulting virus vA187-Ins44 were characterized and found to be indistinguishable from its parent virus. However, a deletion mutant with 29 of the 44 inserted bases missing was detected after multiple cell culture passages. RNA secondary structure analysis of the 5'NTR showed that the 44 base insertion destroyed a stem-loop structure and a pseudoknot previously described to be essential for virus replication, demonstrating that insertions within this functionally essential IRES element are tolerated by CSFV.

Analysis of classical swine fever virus replication kinetics allows differentiation of highly virulent from avirulent strains.

To study the replication of classical swine fever virus (CSFV) in cell culture, kinetics of viral plus-strand RNA synthesis, of viral structural and non-structural protein expression as well as of secreted and cell-associated infectious virus were determined. Highly virulent, moderately virulent and avirulent strains that were tested in standardized animal experiments to confirm their virulence were used to search for in vitro parameters allowing the differentiation of strains according to their virulence. No significant qualitative or quantitative differences were found between the strains studied when either RNA replication or protein synthesis were investigated. However, the ratio of cell-associated virus versus secreted virus proved to be considerably lower for the highly virulent strains when compared to avirulent or moderately virulent strains. These data suggest that highly virulent strains of CSFV can be distinguished in cell culture from strains with reduced virulence.

Cytopathogenic and noncytopathogenic RNA replicons of classical swine fever virus.

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.

Serotyping of foot-and-mouth disease virus by antigen capture reverse transcriptase/polymerase chain reaction.

The technique of capturing of foot-and-mouth disease virus (FMDV) from clinical material in microcentrifuge tubes coated with type-specific antibodies and amplifying the viral sequences by RT/PCR in the same tube, promoted the detection and serotyping of FMDV with high sensitivity and specificity. The efficiency of antigen capturing and shelf life of the coated tubes was improved by glutaraldehyde fixation of antibodies to the tubes. Virus in infected tissues, even after storage for 25-30 years at 70 degrees C, could be successfully typed by this method. Conserved sequences flanking the variable region of immunoreactive VP1 gene of FMDV were used as primers in the assay and hence the nucleotide sequence analysis of the product could reveal the strain variation. The test has been found to be at least 125-fold more sensitive than type specific ELISA and of comparable sensitivity as other protocols for detection of FMDV by RT/PCR.

Capsid protein-encoding (P1) sequence of foot and mouth disease virus type Asia 1 ind 63/72.

Variations in the amino acid sequence of Foot-and-Mouth Disease Virus (FMDV) structural proteins are the basis for the antigenic diversity of the virus. Majority of antigenic sites for the virus neutralization are present on VP1, the major immunogenic protein. However, a few conformational epitopes are present on the structural proteins VP2 and VP3. The nucleotide sequence encoding all the four structural proteins (P1 region) of FMDV type Asia 1 Ind 63/72 was determined. The nucleotide and the deduced amino acid sequence of P1 of Asia 1 of Indian strain was compared with that of Asia 1 Israel strain. Differences were observed at 284 (14%) nucleotide positions resulting in 69 (10%) amino acid changes. The variation in the derived amino acid sequence is the highest in VP1 (14.4%) followed by VP2 (10%), VP3 (6.4%) and VP4 (3%). Deletion of two amino acids, which was observed in the case of Indian strain as well as in Israel strain indicated that these deletions are specific for type Asia 1. The P1 sequence was also compared with the corresponding region of the other serotypes O1K, A12, Cl and SAT-1. The sequence has been submitted to EMBL data bank, under accession number Y09949.

Classical swine fever virus leader proteinase Npro is not required for viral replication in cell culture.

The sequence encoding the viral leader proteinase Npro was replaced by the murine ubiquitin gene in a full-length cDNA clone of the classical swine fever virus (CSFV) strain Alfort/187. The recombinant virus vA187-Ubi showed growth characteristics similar to those of the parent vA187-1 virus. At two occasions cells infected with vA187-Ubi exhibited a cytopathic effect and were found to contain a subgenomic viral RNA. This RNA lacked the same viral genes as the subgenomic RNA which has been found in all cytopathogenic CSFV strains analyzed so far, but it maintained the ubiquitin sequence.

A recombinant classical swine fever virus stably expresses a marker gene.

The gene coding for bacterial chloramphenicol acetyltransferase (CAT) was inserted in frame into the viral Npro gene of the full-length cDNA clone pA187-1 of the classical swine fever virus (CSFV) strain Alfort/187. RNA transcribed in vitro from the resulting plasmid was transfected into SK-6 porcine kidney cells. Infectious progeny virus vA187-CAT recovered from transfected cells had growth characteristics indistinguishable from those of parental virus vA187-1. In cells infected with vA187-CAT the predicted fusion protein, CAT-Npro, was detected, and it retained the enzymatic activities of both CAT and Npro. The CAT gene remained stably inserted in the viral genome after 10 virus passages. Thus, marker virus vA187-CAT represents a useful tool for quantitative analysis of viral replication and gene expression.

Porcine cells persistently infected with classical swine fever virus protected from pestivirus-induced cytopathic effect.

Cytopathogenicity of classical swine fever virus (CSFV) depends on the presence of defective particles containing a subgenomic (sg) RNA with a defined deletion. In a previous report we described the spontaneous generation of this sg RNA and therefore of cytopathogenic (cp) CSFV in porcine kidney cell cultures persistently infected with CSFV. Frequently, some cells survived the CPE and could be further propagated. They remained positive for viral antigen and continued to shed complete virus and in most cases also defective virus particles. SK-6 cells that had survived the CPE (CPE(surv)cells) were used to investigate these findings further. In contrast to persistently infected cells that had not experienced a CPE, CPE(surv) cells were protected from the CPE when superinfected with cp CSFV or with cp bovine viral diarrhoea virus. Similarly, cells which were rescued and further propagated after acute infection with cp CSFV also proved to be protected from the CSFV-induced CPE. When either virus obtained from CPE(surv) cells that had spontaneously lost the sg RNA or virus from which defective particles had been removed was used to establish persistently infected cells, these cells were also protected from the CPE after superinfection with cp CSFV. These findings suggest that the virus contained in CPE(surv) cells confers on the host cell the ability to resist the CSFV-induced CPE. However, when naive cells were infected with supernatants from CPE(surv)cells that contained defective virus particles, the CPE reappeared within three to five virus passages, indicating that the sg RNA retained its cytopathogenic potential.

An indirect ELISA for the detection of antibodies against porcine reproductive and respiratory syndrome virus using recombinant nucleocapsid protein as antigen.

An indirect enzyme-linked immunosorbent assay termed rnPRRS ELISA using baculovirus-expressed and affinity-purified viral nucleocapsid protein (rNC) antigen was developed for detecting antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) in swine sera. Sera (1395) originating from different European countries were used for the validation of this assay. The rnPRRS ELISA was capable of detecting antibodies in all sera known to contain anti-PRRSV antibodies, resulting in 100% sensitivity. The specificity was 95.8%. The rnPRRS ELISA was more sensitive compared to the most widely used tests for the diagnosis of porcine reproductive and respiratory syndrome (PRRS) (i) a commercially available ELISA; (ii) the indirect immunofluorescence assay (IIFA); and (iii) the immunoperoxidase monolayer assay (IPMA). The main advantage of the rnPRRS ELISA compared to an ELISA using whole virus antigen is the use of a single immunogenic protein instead of infectious PRRSV. The rnPRRS ELISA is suitable for routine diagnosis of PRRS and also for epidemiological surveys since it allows highly reliable testing of a large number of sera within a short period of time.

Generation of cytopathogenic subgenomic RNA of classical swine fever virus in persistently infected porcine cell lines.

Two biological clones (A.1 and B.2) of the classical swine fever virus strain Alfort/187 and the recombinant virus vA187-1, derived from a cDNA clone of Alfort/187, were used to establish persistently infected cultures of the swine kidney cell lines SK-6 and PK-41. It was found that 100% of the cells in the passaged cultures were positive for viral antigen throughout the course of the experiment. Additionally, supernatants collected upon passaging of the cells continuously contained high titers of infectious virus. In six separate cultures persistently infected with either the biological clones or the recombinant virus, a cytopathic effect occurred spontaneously between passage 8 and 94. The cytopathogenic agent in the supernatants of these cultures could be passaged repeatedly, suggesting the generation of a mutant virus. Analysis of RNA from such cultures revealed the presence of a subgenomic viral RNA of approximately 8 kilobases (kb). In all six cases, this RNA had an identical internal deletion of 4764 nucleotides, including the region coding for all structural proteins. The subgenomic RNA replicated and was packaged in the presence of wild-type virus. Cells infected with cytopathogenic virus contained increased amounts of the viral protein NS3 thought to be involved in pestivirus cytopathogenicity.

Detection of antibodies against classical swine fever virus in swine sera by indirect ELISA using recombinant envelope glycoprotein E2.

Recombinant envelope protein E2 (gp55) of classical swine fever virus (CSFV) strain Alfort/187 was evaluated as an alternative to whole virus as ELISA antigen for the detection of antibodies against CSFV. A glycosylated and a non-glycosylated form of E2 was expressed in the baculovirus system. Six histidine residues added at the carboxy terminus of each of the recombinant proteins allowed purification by nickel-chelate affinity chromatography. Comparison of the antigenic properties of the two proteins in indirect and blocking ELISAs revealed that the glycosylated form resulted in both higher sensitivity and specificity. The indirect ELISA, using glycosylated E2, either derived from crude cell extract or affinity-purified, was validated by testing a total of 2719 porcine sera. Its final version proved to be as sensitive (98.3%) as the virus neutralization test when sera from infected pig herds were examined, and highly specific (99.6%) when applied to test negative sera. It is therefore suitable for large scale monitoring of classical swine fever.

Nucleotide sequence of classical swine fever virus strain Alfort/187 and transcription of infectious RNA from stably cloned full-length cDNA.

The complete nucleotide sequence of the genome of classical swine fever virus (CSFV) strain Alfort/187 was determined from three cDNA libraries constructed by cloning of DNA fragments obtained from independent sets of reverse transcription and PCR. The cDNA fragments were then assembled and inserted downstream of a T7 promoter in a P15A-derived plasmid vector to obtain the full-length cDNA clone pA187-1. The first nucleotide of the CSFV genome was positioned at the transcription start site of the T7 promoter. Cleavage at an SrfI restriction site introduced at the exact 3' end of the cloned viral cDNA allowed the in vitro synthesis of full-length viral RNA by runoff transcription. This RNA proved to be infectious after transfection into porcine kidney cells. Infectivity was not increased after capping of the synthetic RNA. Virus recovered from transfected cells was titrated in porcine kidney cells by endpoint dilution using indirect immunofluorescence and a CSFV-specific monoclonal antibody. RNA transcripts generated from plasmid DNA isolated from bacteria which had been cultured and cloned 10 times remained infectious, indicating that the full-length clone is stable in bacterial cells. A silent point mutation introduced at position 11842 of the genome was retained in the recombinant virus recovered from transfected cells. An infectious chimeric construct was obtained by replacing a 696-bp fragment in pA187-1 with the corresponding cDNA fragment from the CSFV strain CAP. The stably cloned full-length CSFV cDNA allows site-specific mutagenesis of the viral genome and thus will be useful for detailed molecular characterization of the virus as well as for studies of viral pathogenesis.

Rapid detection and characterization of foot-and-mouth disease virus by restriction enzyme and nucleotide sequence analysis of PCR products.

Reverse transcription coupled with PCR was used for the detection of foot-and-mouth disease virus serotypes A, C, and O in organ extracts from experimentally infected cattle. Primers were selected from conserved sequences flanking the genome region coding for the major antigenic site of the capsid located in the C-terminal part of viral protein 1 (VP1). Because this region of the capsid is highly variable its coding sequence is considered to be the most appropriate for the characterization of virus isolates and, therefore, for the determination of the epidemiological relationships between viruses of the same serotype. For differentiation between serotypes and for detailed characterization of individual virus isolates restriction enzyme cleavage and nucleotide sequence analysis of the respective PCR products were carried out. In order to minimize the time required for sample preparation from clinical material, viral RNA was released from particles by heating the sample for 5 min at 90 degrees C. Finally, an air thermocycler was used, which allows performance of a PCR of 30 cycles in approximately 20 min. The results show that reverse transcription PCR followed by restriction enzyme analysis and/or nucleotide sequence analysis of the PCR products is useful for the rapid detection and differentiation of foot-and-mouth disease virus.

Baculovirus expression and affinity purification of protein E2 of classical swine fever virus strain Alfort/187.

The genome region encoding the major envelope glycoprotein E2 (gp55) of the classical swine fever virus (CSFV) strain Alfort/187 was cloned and sequenced. The E2 gene, either with or without additional authentic 5'-terminal sequences coding for two variants of a putative signal sequence, was used to construct recombinant baculoviruses expressing the respective glycosylated and nonglycosylated E2 protein in insect cells. The signal sequences mediated glycosylation in insect cells, but no efficient secretion of the protein into the cell culture supernatant was observed. Six histidine residues introduced at the carboxy terminus of E2 allowed purification of E2 protein by Ni(2+)-chelate affinity chromatography. The proteins obtained were characterized and their immunological properties were compared by western blot analysis.

[Diagnosis of contagious diseases in animals using PCR]. / Tierseuchendiagnostik mittels PCR.

The PCR is used for diagnostic purposes as it allows to detect infections agents within a much shorter time than by cultural isolation. In addition, it can detect non-infectious viruses and bacteria in clinical samples. These advantages are important factors in the diagnosis of highly contagious animal diseases (mainly caused by viruses) since a rapid laboratory diagnosis will allow to take immediate disease control actions. PCR is routinely used at the Institute of African and classical swine fever virus, foot and mouth disease virus, Aujeszky's disease virus, porcine reproductive and respiratory syndrome virus, as well as Newcastle disease virus. The isolate can be further characterized by direct nucleotide sequencing of the amplified DNA. Since reliability of the results as well as as prevention of contaminations are vital to PCR, this method should be carried out by appropriately trained personnel. In addition, it requires a high level of technical infrastructure.

Detection of hog cholera virus and differentiation from other pestiviruses by polymerase chain reaction.

Reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection and differentiation of pestiviruses. For this purpose, one primer pair was selected from a highly conserved region of the genome of pestiviruses. Using these primers (PEST 1-PEST 2), DNA fragments of between 72 and 74 bp could be amplified from all pestivirus isolates tested. In order to differentiate hog cholera virus (HCV) from bovine viral diarrhea virus (BVDV) and border disease virus (BDV), we selected a primer pair from a conserved region in the genome of HCV strains that differed from that sequenced in the genome of BVDV strains. By using these primers (HCV 1-HCV 2), a DNA fragment of 478 bp could be specifically amplified from HCV isolates. By these means, viral RNA was detected in extracts of lymph node, spleen, tonsil, and lung. Such extracts were used directly for RT-PCR without prior RNA isolation. We also performed multiplex PCR by using both the PEST 1-PEST 2 and HCV 1-HCV 2 primer pairs in a single reaction. This allowed the differentiation of HCV from BVDV and BDV in one step. To assess the sensitivity of the method, RT-PCR was compared with virus propagation in tissue culture and subsequent detection by immunofluorescence staining. The results show that RT-PCR is useful for the rapid detection and differentiation of pestiviruses.

Approaches to the identification of non-essential genes of African swine fever virus.

It is poorly understood why vaccines could not be developed for the control and prevention of African swine fever (ASF) virus infection. The aim of our study was to identify genes non-essential for ASF virus replication because there were indications that certain viral gene products, which apparently are non-essential for viral replication, conferred protection from death due to ASF. A cosmid library representing the genome of ASF virus strain France 64 was established and characterized. Then, in order to inactivate viral genes by insertion, the beta-galactosidase (beta-gal) gene was introduced either randomly or at specific locations of selected cloned DNA fragments. These constructions were transfected into cells which had been previously infected with a cell-culture-adapted viral strain in order to allow the generation of recombinant progeny virus. Viable recombinant progeny was identified by at least one of the following means: (1) expression of beta-gal; (2) detection of beta-gal specific DNA by plaque hybridization, and (3) absence of a functional product of the inactivated gene. Presently, we are characterizing a recombinant virus with an insertionally inactivated thymidine kinase gene.

Specific diagnosis of parvovirus enteritis in dogs and cats by in situ hybridization.

In a retrospective study, the fixed intestines of 10 dogs and 10 cats with intestinal lesions characteristic of parvovirus infection were assayed for the presence of parvovirus by in situ hybridization and immunohistochemistry. Parvoviral nucleic acid was localized by in situ hybridization in intestinal tissue in all 10 dogs and in nine of the 10 cats, whereas antigen was detected only in seven of 10 canine and eight of 10 feline intestines by immunohistochemistry. We conclude that an aetiological diagnosis can be established with a high degree of certainty by routine histology. Demonstration of the infectious agent by in situ hybridization, however, proves to be a valuable specific tool which allows an exact cellular localization of parvovirus in formalin-fixed, paraffin wax-embedded tissue sections.