Classical Swine Fever Virus E2 Research Articles

Genetic characterization of E2 gene of classical swine fever virus by restriction fragment length polymorphism and phylogenetic analysis

An RT-nested PCR (RT-nPCR)-based restriction fragment length polymorphism (RFLP) analyses of the E2 gene were developed for genetic subtyping and differentiation of vaccinated and infected classical swine fever virus (CSFV) strains. RT-nPCR identified 96 CSFV-positive samples from 321 clinical specimens from southeastern China during 2003-2008. The PCR products of positive samples were further differentiated using MspI digestion, 23 were identified as the C-strain, 62 as field strains, and 11 as mixture of the vaccine strain and field ones. RFLP with BglI, DdeI, DraI, and PstI were then used for subtyping of the field CSFV isolates. Thirty-eight field isolates phylogenetically classified as subgroup 2.1 based on E2 were divided into 11 subtypes by this RFLP scheme. Both RFLP profiling and sequence-based phylogenetic analysis revealed genetic diversity of CSFV in the field. Three novel substitutions at amino acid positions 17, 93, and 286 were identified in the predominant subtype VI strains isolated in 2008 as compared to other strains including historical subtype VI strains. These results suggest that CSFV in China experienced gradual variations and evolutionary accumulation progress. Thus, the RFLP methods targeting on the CSFV E2 gene are suitable for epidemiological survey in endemic area where the C-strain is applied for vaccination. Combination of the RFLP schemes with sequence-based phylogenetic analysis could provide more detailed information on transmission of CSFV in the region or even its evolution.

Antigenic domains analysis of classical swine fever virus E2 glycoprotein by mutagenesis and conformation-dependent monoclonal antibodies

Glycoprotein E2 of classical swine fever virus (CSFV) is the major antigenic protein exposed on the outer surface of the virion that induces main neutralizing antibodies during infection in pigs. This study displays the differences in antigenicity of E2 between vaccine and field strains of CSFV by their variable reaction patterns between expressed proteins and monoclonal antibodies (mAbs). The D/A domains of various CSFVs were relatively conserved and recognized by all mAbs against the A domain. However, mAbs against B/C domains were able to differentiate field viruses TD/96/TWN (subgroup 2.1) and 94.4/IL/94/TWN (subgroup 3.4) from the vaccine virus LPC/AHRI (subgroup 1.1). By analysis of expressed truncated proteins, the epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779. Site-directed mutagenesis further showed that residues 693C, 737C, 771L, 772L, 773F and 774D were critical for the reactivity of E2 protein with mAbs. Thus, the B/C domains are responsible for antigen specificity among various CSFVs, and the disulfide bond and motif 771LLFD 774 are essential for the structural integrity of its conformational recognition. These data significantly increase our understanding of the antigenic structure of E2 for antibody binding.

An indirect ELISA of classical swine fever virus based on quadruple antigenic epitope peptide expressed in E.coli

In this study, a synthesized quadruple antigenic epitope gene region of the classical swine fever virus (CSFV) E2 glycoprotein was expressed in E. coli to a obtain target protein. This target protein was used as a coating antigen to establish an indirect ELISA for specifically detecting anti-CSFV antibodies in serum samples from pigs. The P/N cut-off value of this assay was 1.92 by receiver operating characteristic curve (ROC) analysis based on 30 negative sera and 80 positive samples. The test gave 97.5% sensitivity and 96.7% specificity compared with the indirect hemagglutination (IHA) test. The inter-assay and intra-assay coefficients of variation (CVs) for 16 sera were both ≤6.8%. No cross-reactivity between the coating antigen and anti-bovine viral diarrhoea virus (BVDV) antibodies was observed.

Selection of a Less Pathogenic BVDV Strain for the Construction of Avirulent Chimeric Pestivirus

To select a less pathogenic bovine viral diarrhea virus (BVDV) strain for the construction of chimeric pestivirus harboring classical swine fever virus (CSFV) E2 gene, five Korean BVDV isolates (four type 1 isolates and a type 2 isolate) were evaluated for their pathological and biological properties with respect to porcine infection. Each of five groups of 100-day-old pigs was inoculated intranasally with one of the five BVDV isolates. No clinical sign or leukopenia was observed in any pig throughout the duration of the experiment, but viruses were detected in blood, nasal discharges and postmortem samples using RT-PCR. These results indicated that although the five BVD viruses could infect pigs, they did not cause clinically apparent symptoms. Because of its proper infection dynamics shown in this preliminary animal study and its fast growth rate and quick CPE in cell culture, one isolate (KD26-1) was chosen among the five isolates to test its virulence and immunogenic properties in 40-day-old piglets. Neither clinical sign nor pathological lesion was observed in 40-day-old piglets during the course of infection of isolate KD26-1. The first neutralizing antibodies were detectable 14 days post-inoculation (PI) and increased to 1:128~1:256 28 days PI. A BVDV specific gene was detectable by RT-PCR in tonsil, spleen, inguinal lymph node and brain until 14 days PI. According to this study, it can be concluded that isolate KD26-1 has little pathological effect in pigs and is a candidate for construction of chimeric pestivirus harboring CSFV E2 gene.

Classical swine fever virus: Antigenic architecture of the E2 glycoprotein elucidated by epitope mapping using synthetic peptides

Detailed epitope characterization of the major envelope glycoprotein E2 of the classical swine fever virus (CSFV) is important for our understanding of interactions between the virus and the host immune system, as well as for the development of CSFV-specific diagnostic assays and epitope- or peptide-based marker vaccines. As was shown previously by competitive binding assay, monoclonal antibodies obtained by our group recognize eight epitopes on the E2 protein. Here, we report mapping of five linear, nonoverlapping B-cell epitopes that use a set of synthetic peptides, which encompass the full sequence of the CSFV E2 protein (Shimen strain). Two of the epitopes are located in the antigenic domain A of the E2, while another three belong to a highly structured region of this glycoprotein. The alignment of the identified gene sequences was performed for 12 CSFV strains, three strains of bovine viral diarrhea virus (BVDV), and two strains of the border disease virus (BDV). The data obtained could be used to improve CSFV diagnostic assays, as well as to investigate the effects of aminoacid substitutions in E2 on its antigenic properties.

Immune responses induced by a BacMam virus expressing the E2 protein of classical swine fever virus in mice

Non-replicating baculovirus-mediated gene transfer into mammalian cells has been developed as a vaccine strategy against a number of diseases in several animal models. In the present study, the BacMam vector, a baculovirus pseudotyped with the glycoprotein from vesicular stomatitis virus, was used as a recombinant vector to express classical swine fever virus (CSFV) E2 protein under the control of the immediate early 1 (ie1) promoter from shrimp white spot syndrome virus. The E2 gene was efficiently expressed in both insect and mammalian cells. Intramuscular injection of mice with the recombinant baculovirus resulted in the production of high-titers of CSFV-specific neutralizing antibodies. Specific lymphoproliferative responses to CSFV stimulation were detected in the splenocytes of the immunized mice as demonstrated by CFSE staining assay and WST-8 assay. This study demonstrates that the BacMam virus vector can efficiently express the E2 protein and effectively induce immune responses against CSFV. This is a first step in the demonstration that the pseudotyped baculovirus-delivered CSFV E2 gene can be a potential non-replicating vaccine against CSFV infections.

Generation and efficacy evaluation of a recombinant adenovirus expressing the E2 protein of classical swine fever virus

Classical swine fever virus (CSFV) is the causative agent of classical swine fever (CSF), which causes significant economic losses to the pig industry worldwide. The E2 glycoprotein of CSFV is the main target for neutralizing antibodies. This study was aimed to develop a recombinant human adenovirus type 5 expressing the CSFV E2 gene (rAdV-E2) and evaluate its efficacy in rabbits and pigs. The results showed that the rabbits and the pigs immunized with the rAdV-E2 developed high-level CSFV-specific neutralizing antibodies. The rAdV-E2-immunized rabbits were protected from fever induced by infection with C-strain, which is pathogenic to the rabbit, and the rAdV-E2-immunized pigs were protected from lethal challenge with highly virulent Shimen strain. This indicates that the recombinant adenovirus can be an attractive candidate vaccine for preventing CSF.

Antigenic differentiation of classical swine fever viruses in China by monoclonal antibodies

The phylogenetic diversity of classical swine fever virus (CSFV) in China has been extensively studied previously, with the report of the classification of Chinese CSFVs into four subgroups within two of the established genotypes, but the antigenic differences amongst Chinese CSF viruses still remain unknown. To address this issue, 21 CSFV field strains isolated in China between 1996 and 2006 were grown in cell culture and characterized in comparison with two Chinese reference strains: a virulent strain Shimen and a vaccine strain CSF lapinized virus (hog cholera lapinized virus in China, HCLV), by indirect immunofluorescence assay (IFA) with a panel of 28 monoclonal antibodies (mAbs) against four pestiviruses, CSFV, bovine viral diarrhoea virus-1 (BVDV-1), bovine viral diarrhoea virus-2 (BVDV-2) and border disease virus (BDV). All 23 CSFV strains reacted only with CSFV-specific mAbs, not with those raised against BVDV-1, BVDV-2 and BDV. Of the former mAbs, those directed against CSFV E2 protein recognized more isolates than those directed against E rns and NS2/3. Of nine CSFV E2-specific mAbs used, WH303 and WH302 reacted with all 23 strains, confirming their value in differentiating CSFV from other pestiviruses. Furthermore, different strains had different patterns of reactivity with CSFV-specific mAbs, and mAbs other than WH303 and WH302 did not recognize all strains. This study provides the first evidence for the existence of antigenic differences among Chinese CSFVs.

Assessment of the cell-mediated immunity induced by alphavirus replicon-vectored DNA vaccines against classical swine fever in a mouse model

We have previously shown that an alphavirus replicon-vectored DNA vaccine (pSFV1CS-E2) encoding the E2 glycoprotein of classical swine fever virus (CSFV) completely protected the immunized pigs from lethal challenge. These animals developed only low or moderate level viral-specific antibody titers before challenge, implying that cell-mediated immunity (CMI) probably played an important role in the protective immunity against CSFV conferred by the DNA vaccine. In this study, the CMI induced by pSFV1CS-E2 and its derivative pSFV1CS-E2-UL49 encoding a fusion protein of CSFV E2 and pseudorabies virus (PRV) VP22 was evaluated in a mouse model by lymphoproliferation assays based on CFSE or WST-8, intracellular cytokine staining, and cytokine ELISA. The results showed that both vaccines induced CSFV-specific lymphoproliferative responses and cytokine production, and pSFV1CS-E2-UL49 induced stronger lymphoproliferative responses and higher cytokine levels than pSFV1CS-E2. These findings suggest that the alphavirus replicon-delivered DNA vaccines are capable of inducing CMI, and PRV VP22 is able to enhance the immunogenicity of the co-delivered antigen.

Classical swine fever virus E2 glycoprotein antigen produced in adenovirally transduced PK-15 cells confers complete protection in pigs upon viral challenge

E2 is the major envelope glycoprotein present on the outer surface of the classical swine fever virus (CSFV). It is exposed as a homodimer originated by disulfide linkage and represents an important target for the induction of neutralizing immune responses against the viral infection. The E2his glycoprotein nucleotide sequence used in this work contains the CSFV E2 extracellular domain preceded by the tissue plasminogen signal peptide and a hexa-histidine tag in the 3' terminus. The recombinant antigen was produced at a range of 120-150 microg/mL in the culture media of epithelial kidney pig cells, transduced with a replication defective adenoviral vector (Ad-E2his) generated by means of cloning the E2his sequence in the vector genome. The glycoprotein was obtained from clarified culture media as a homodimer of 110 kDa with purity over 95% after a single affinity chromatography step in Ni-NTA Agarose column. The E2his characterization by lectin-specific binding assay showed the presence of N-linked oligosaccharides of both hybrid and complex types. The protective capacity of E2his was demonstrated in two immunization and challenge experiments in pigs using doses of 15 or 30 microg of the glycoprotein, emulsified in Freund's adjuvant. The intramuscular immunization followed by a unique boost three weeks later, elicited high titers of neutralizing antibodies between the second and the fourth week after the primary vaccination. The immunized animals were fully protected from the viral infection after challenge with 10(5) PLD(50) of homologous CSFV "Margarita" strain administered by intramuscular injection. Consequently, no clinical signs of the disease or viral isolation from lymphocytes were detected in the vaccinated pigs. These results suggest that the E2his antigen produced in mammalian cells may be a feasible vaccine candidate for CSF prevention.

The selection pressure analysis of classical swine fever virus envelope protein genes Erns and E2

Classical swine fever virus, one member of the family Flaviviridae, is the pathogen of CSF, an economically important and highly contagious disease of pigs. Knowledge of virus genes under positive selection pressure can help identify molecular determinants of virulence or pathogenesis without prior knowledge of the mechanisms governing virulence and pathogenesis and clarify the driving force of classical swine fever virus evolution. The positive selection pressure acting on envelope protein genes Erns, E1 and E2 of classical swine fever virus were assessed and a site-by-site analysis of the dN/dS ratio was performed, to identify specific codons undergoing diversifying positive selection. Whilst no significant evidence for positive selection was observed in E1, four positively selected sites (208 in Erns and 72, 75, and 200 in E2) were identified. The positively selected site (208) of Erns corresponds to one of the amino acid substitutions (Ser to Arg) found in an HS-binding CSFV variant. The mutant at the positively selected site (75) is located within an O-glycosylation motif and altered the predicted glycosylation pattern. In addition, Thr at the positively selective site 200 are directly involved with mAb WH308 with which CS vaccine strain does not react, unlike most of the virulent CSFV strains.

Identification of a novel virulence determinant within the E2 structural glycoprotein of classical swine fever virus

Classical swine fever virus (CSFV) E2 glycoprotein contains a discrete epitope (TAVSPTTLR, residues 829–837 of CSFV polyprotein) recognized by monoclonal antibody (mAb) WH303, used to differentiate CSFV from related ruminant pestiviruses, Bovine Viral Diarrhea Virus (BVDV) and Border Disease Virus (BDV), that infect swine without causing disease. Progressive mutations were introduced into mAb WH303 epitope in CSFV virulent strain Brescia (BICv) to obtain the homologous amino acid sequence of BVDV strain NADL E2 (T SFNMDTL A). In vitro growth of mutants T1v (T SFSPTTLR), T2v (T SFNPTTLR), T3v (T SFNMTTLR) was similar to parental BICv, while mutants T4v (T SFNMDTLR) and T5v (T SFNMDTL A) exhibited a 10-fold decrease in virus yield and reduced plaque size. In vivo, T1v, T2v or T3v induced lethal disease, T4v induced mild and transient disease and T5v induced mild clinical signs. Protection against BICv challenge was observed at 3 and 21 days post-T5v infection. These results indicate that E2 residues TAVSPTTLR play a significant role in CSFV virulence.

In planta production of two peptides of the Classical Swine Fever Virus (CSFV) E2 glycoprotein fused to the coat protein of potato virus X

BackgroundClassical Swine Fever (CSFV) is one of the most important viral infectious diseases affecting wild boars and domestic pigs. The etiological agent of the disease is the CSF virus, a single stranded RNA virus belonging to the family Flaviviridae.All preventive measures in domestic pigs have been focused in interrupting the chain of infection and in avoiding the spread of CSFV within wild boars as well as interrupting transmission from wild boars to domestic pigs. The use of plant based vaccine against CSFV would be advantageous as plant organs can be distributed without the need of particular treatments such as refrigeration and therefore large areas, populated by wild animals, could be easily covered.ResultsWe report the in planta production of peptides of the classical swine fever (CSF) E2 glycoprotein fused to the coat protein of potato virus X. RT-PCR studies demonstrated that the peptide encoding sequences are correctly retained in the PVX construct after three sequential passage in Nicotiana benthamiana plants. Sequence analysis of RT-PCR products confirmed that the epitope coding sequences are replicated with high fidelity during PVX infection. Partially purified virions were able to induce an immune response in rabbits.ConclusionPrevious reports have demonstrated that E2 synthetic peptides can efficiently induce an immunoprotective response in immunogenized animals. In this work we have showed that E2 peptides can be expressed in planta by using a modified PVX vector. These results are particularly promising for designing strategies for disease containment in areas inhabited by wild boars.

The protective immune response induced by B cell epitope of classical swine fever virus glycoprotein E2

Classical swine fever virus (CSFV) envelope glycoprotein E2 is a major protective immunogen responsible for eliciting neutralizing antibodies and conferring protective immunity against the virus. Based on the core sequence (TAVSPTTLR, 829–837 aa) of the B cell linear epitope of the CSFV E2 protein identified by Lin et al. [Lin, M., Lin, F., Mallory, M., Alfonso, C., 2004. Deletions of structural glycoprotein E2 of classical swine fever virus strain Alfort/187Resolve a lineal epitope of monoclonal antibody WH303 and the minimal N-terminal domain essential for binding immunoglobulin G antibodies of a pig hyperimmune serum. J. Virol. 74 (24), 11619–11625], two oligonucleotides MF and MR were synthesized and used to construct by PCR a gene cassette encoding a 15 amino acid polypeptide M (CTAVSPTTLRTEVVK), which spans 828–842 amino acids of E2. The gene cassette was fused in-frame to 3′ terminal of glutathione S transferase gene (GST) of the prokaryotic expression vector pGEX-6p-1, resulting in the recombinant plasmid pGEX-M. After transformation into Escherichia coli BL21 a soluble fusion protein GST-M with expected size of 28 kDa was expressed after inducing with isopropyl-β- d-thiogalactoside (IPTG). Enzyme-linked immunosorbent assay (ELISA) and Western blot analysis showed that the purified GST-M had good reactivity with swine anti-CSFV serum and rabbit anti-CSFV E2 serum. Further vaccination trials showed that the fusion protein GST-M could elicit effectively immune response protecting rabbits and pigs from virulent challenge. This study showed a possibility for developing epitope-based vaccines against CSFV.

P.217 High rate of spontaneous viral clearance in patients with symptomatic acute hepatitis C

To evaluate the effects of the Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-beta (TRIF) on immune responses induced by DNA vaccines, mice were immunized with the eukaryotic expression plasmid pcDNA/E2 encoding classical swine fever virus (CSFV) E2 alone or in combination with the TRIF genetic adjuvant. Immune responses were examined in immunized mice. Our data demonstrates that co-delivery of the DNA vaccine pcDNA/E2 with the TRIF adjuvant augmented specific humoral and cellular immune responses in a mouse model. Vaccination of pigs confirmed that the pcDNA/E2 in combination with TRIF conferred total protection against lethal challenge with highly virulent CSFV. We conclude that TRIF enhances the effects of the DNA vaccine against CSFV infection and could be used as a potential genetic adjuvant for DNA vaccines in large animal species.

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

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

The E2 Glycoprotein of Classical Swine Fever Virus Is a Virulence Determinant in Swine

To identify genetic determinants of classical swine fever virus (CSFV) virulence and host range, chimeras of the highly pathogenic Brescia strain and the attenuated vaccine strain CS were constructed and evaluated for viral virulence in swine. Upon initial screening, only chimeras 138.8v and 337.14v, the only chimeras containing the E2 glycoprotein of CS, were attenuated in swine despite exhibiting unaltered growth characteristics in primary porcine macrophage cell cultures. Additional viral chimeras were constructed to confirm the role of E2 in virulence. Chimeric virus 319.1v, which contained only the CS E2 glycoprotein in the Brescia background, was markedly attenuated in pigs, exhibiting significantly decreased virus replication in tonsils, a transient viremia, limited generalization of infection, and decreased virus shedding. Chimeras encoding all Brescia structural proteins in a CS genetic background remained attenuated, indicating that additional mutations outside the structural region are important for CS vaccine virus attenuation. These results demonstrate that CS E2 alone is sufficient for attenuating Brescia, indicating a significant role for the CSFV E2 glycoprotein in swine virulence.

Oronasal vaccination with classical swine fever virus (CSFV) replicon particles with either partial or complete deletion of the E2 gene induces partial protection against lethal challenge with highly virulent CSFV

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.

Spontaneous excision of BAC vector sequences from bacmid-derived baculovirus expression vectors upon passage in insect cells.

Repeated baculovirus infections in cultured insect cells lead to the generation of defective interfering viruses (DIs), which accumulate at the expense of the intact helper virus and compromise heterologous protein expression. In particular, Autographa californica multicapsid nucleopolyhedovirus (AcMNPV) DIs are enriched in an origin of viral DNA replication (ori) not associated with the homologous regions (hrs). This non-hr ori is located within the coding sequence of the non-essential p94 gene. We investigated the effect of a deletion of the AcMNPV non-hr ori on the heterologous protein expression levels following serial passage in Sf21 insect cells. Using homologous ET recombination in E. coli, deletions within the p94 gene were made in a bacterial artificial chromosome (BAC) containing the entire AcMNPV genome (bacmid). All bacmids were equipped with an expression cassette containing the green fluorescent protein gene and a gene encoding the classical swine fever virus E2 glycoprotein (CSFV-E2). For the parental (intact) bacmid only, a strong accumulation of DIs with reiterated non-hr oris was observed. This was not observed for the mutants, indicating that removal of the non-hr ori enhanced the genetic stability of the viral genome upon passaging. However, for all passaged viruses it was found that the entire BAC vector including the expression cassette was spontaneously deleted from the viral genome, leading to a rapid decrease in GFP and CSFV-E2 production. The rationale for the (intrinsic) genetic instability of the BAC vector in insect cells and the implications with respect to large-scale production of proteins with bacmid-derived baculoviruses are discussed.

Establishment and application of bicistronic classical swine fever virus genomes for foreign gene expression and complementation of E2 deletion mutants

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 A187ΔE2-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 A187ΔE2 which carries an in frame deletion of 465 nucleotides in the E2 gene, infectious viruses vA187ΔE2-IRES-sigE2 and vA187ΔE2-IRES-sigE2p7, respectively, were obtained. This shows that E2 deletion mutants can be complemented by expression of E2 from a separate cistron.