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Recent reemergence of classical swine fever (CSF) in previous CSF-free areas reminds the veterinary community of this old disease [...]

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.

Emergency vaccination with live marker vaccines represents a promising control strategy for future classical swine fever (CSF) outbreaks, and the first live marker vaccine is available in Europe. Successful implementation is dependent on a reliable accompanying diagnostic assay that allows differentiation of infected from vaccinated animals (DIVA). As induction of a protective immune response relies on virus-neutralizing antibodies against E2 protein of CSF virus (CSFV), the most promising DIVA strategy is based on detection of Erns -specific antibodies in infected swine. The aim of this study was to develop and to evaluate a novel Erns -specific prototype ELISA (pigtype CSFV Erns Ab), which may be used for CSF diagnosis including application as an accompanying discriminatory test for CSFV marker vaccines. The concept of a double-antigen ELISA was shown to be a solid strategy to detect Erns -specific antibodies against CSFV isolates of different genotypes (sensitivity: 93.5%; specificity: 99.7%). Furthermore, detection of early seroconversion is advantageous compared with a frequently used CSFV E2 antibody ELISA. Clear differences in reactivity between sera taken from infected animals and animals vaccinated with various marker vaccines were observed. In combination with the marker vaccine CP7_E2alf, the novel ELISA represents a sensitivity of 90.2% and a specificity of 93.8%. However, cross-reactivity with antibodies against ruminant pestiviruses was observed. Interestingly, the majority of samples tested false-positive in other Erns -based antibody ELISAs were identified correctly by the novel prototype Erns ELISA and vice versa. In conclusion, the pigtype CSFV Erns Ab ELISA can contribute to an improvement in routine CSFV antibody screening, particularly for analysis of sera taken at an early time point after infection and is applicable as a DIVA assay. An additional Erns antibody assay is recommended for identification of false-positive results in a pig herd immunized with the licensed CP7_E2alf marker vaccine.

In Taiwan, the prevalent CSFV population has shifted from the historical genotype 3.4 (94.4 strain) to the newly invading genotype 2.1 (TD/96 strain) since 1996. This study analyzed the competition between these two virus genotypes in dual infection pigs with equal and different virus populations and with maternally derived neutralizing antibodies induced by a third genotype of modified live vaccine (MLV), to simulate that occurring in natural situations in the field. Experimentally, under various dual infection conditions, with or without the presence of maternal antibodies, with various specimens from blood, oral and fecal swabs, and internal organs at various time points, the TD/96 had consistently 1.51&minus, 3.08 log higher loads than those of 94.4. A second passage of competition in the same animals further widened the lead of TD/96 as indicated by viral loads. The maternally derived antibodies provided partial protection to both wild type CSFVs and was correlated with lower clinical scores, febrile reaction, and animal mortality. In the presence of maternal antibodies, pigs could be infected by both wild type CSFVs, with TD/96 dominating. These findings partially explain the CSFV shift observed, furthering our understanding of CSFV pathogenesis in the field, and are helpful for the control of CSF.

Classical swine fever (CSF) is an acute viral disease that has high contagion and high mortality in domestic pigs and wild boars. In Taiwan, classical swine fever virus (CSFV) of subgroup 3.4 (Native strain; 94.4/IL/94/TWN) was almost 100% isolated before 1993. In 1994, Subgroup 2.1 (Exotic strain; TD/96/TWN) was first detected, and since a severe outbreak in 1995, it has dominated the field. There is a concern that the Lapinized Philippines Coronel (LPC) of genotype 1.1, widely used in Taiwan, may not completely protect animals from the currently prevalent genotype 2.1 CSFV in the field. In this study, pigs were immunized by LPC vaccine and E2 glycoprotein expressed from cloned genes of LPC, TD/96, and 94.4 viruses, respectively before challenge with genotypically homologous viruses. The viral distributions and intensity after challenge were analyzed by using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry (IHC). Both LPC vaccine and E2 glycoproteins provided significant protection potency from CSFV. However, the LPC vaccine antigen was noted, although to a significantly milder degree (p < 0.05) than in the positive control, in lymphoid follicles of the lymph node and in various organs after the vaccinated animals were challenged with virulent ALD strain. Tissues from all experimental groups were tested negative by RT-PCR. In conclusion, the LPC attenuated virus and E2 glycoproteins offer protection against their genotypically homologous viruses. A change of vaccine seed to genotype 2.1 may be an option to combat the currently prevalent genotype 2.1 CSFVs in the field.

Classical swine fever (CSF), an economically important and highly contagious disease of pigs, is caused by classical swine fever virus (CSFV). In Taiwan, CSFVs from field outbreaks belong to two distinct genotypes. The historical genotype 3.4 dominated from the 1920s to 1996, and since 1996, the newly invading genotype 2.1 has dominated. To explain the phenomenon of this virus shift in the field, representative viruses belonging to genotypes 2.1 and 3.4 were either inoculated alone (single infection) or co-inoculated (co-infection), both in vivo and in vitro, to compare the virus replication and pathogenesis. In pigs co-infected with the genotype 2.1 TD/96/TWN strain and the genotype 3.4 94.4/IL/94/TWN strain, the newly invading genotype 2.1 was detected earlier in the blood, oral fluid, and feces, and the viral loads were consistently and significantly higher than that of the historical genotype 3.4. In cell cultures, the ratio of secreted virus to cell-associated virus of the genotype 2.1 strain was higher than that of the genotype 3.4 strain. This study is the first to demonstrate a possible explanation of virus shift in the field, wherein the newly invading genotype 2.1 replicates more efficiently than did genotype 3.4 and outcompetes the replication and pathogenicity of genotype 3.4 in pigs in the field.

Classical swine fever (CSF) is an economically important, highly contagious disease of swine worldwide. CSF is caused by classical swine fever virus (CSFV), and domestic pigs and wild boars are its only natural hosts. The two main strategies used to control CSF epidemic are systematic prophylactic vaccination and a non-vaccination stamping-out policy. This review compares the protective efficacy of the routinely used modified live vaccine (MLV) and E2 subunit vaccines and summarizes the factors that influence the efficacy of the vaccines and the challenges that both vaccines face to CSF control. Although MLV provide earlier and more complete protection than E2 subunit vaccines, it has the drawback of not allowing differentiation between infected and vaccinated animals (DIVA). The marker vaccine of E2 protein with companion discriminatory test to detect antibodies against E rns allows DIVA and is a promising strategy for future control and eradication of CSF. Maternal derived antibody (MDA) is the critical factor in impairing the efficacy of both MLV and E2 subunit vaccines, so the well-designed vaccination programs of sows and piglets should be considered together. Because of the antigen variation among various genotypes of CSFV, antibodies raised by either MLV or subunit vaccine neutralize genotypically homologous strains better than heterologous ones. However, although this is not a major concern for MLV as the induced immune responses can protect pigs against the challenge of various genotypes of CSFVs, it is critical for E2 subunit vaccines. It is thus necessary to evaluate whether the E2 subunit vaccine can completely protect against the current prevalent strains in the field. An ideal new generation of vaccine should be able to maintain the high protective efficiency of MLV and overcome the problem of antigenic variations while allowing for DIVA.

Recently, it has been noted that porcine circovirus type 2 (PCV2) infection adversely affects the protective efficacy of Lapinized Philippines Coronel (LPC) vaccine, an attenuated strain of classical swine fever virus (CSFV), in pigs. In order to investigate the possible mechanisms of the PCV2-derived interference, an in vitro model was established to study the interaction of LPC virus (LPCV) and PCV2 in porcine alveolar macrophages (AMs). The results showed that PCV2 reduced the LPCV infection in AMs and the levels of PCV2-derived interference were dose-dependent. The PCV2-derived interference also reduced the replication level of LPCV in AMs. The full-length PCV2 DNA and its fragment DNA C9 CpG-ODN were involved in the reduction of LPCV infection in AMs, whereas UV-inactivated PCV2 was not. In addition, a moderate negative correlation between the LPCV antigen-containing rate and IFN-γ production was observed, and had a dose-dependent trend with the level of PCV2-inoculation. The results of the present study may partially explain how PCV2 infection interferes with the efficacy of LPC vaccine.

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. The conformational epitope(s) on B/C domains were mapped to the N-terminal 90 residues of E2 between amino acids 690 and 779 (Chang et al., 2010a). To mimic the conformational epitopes, a set of synthetic cyclized peptides spanning the B/C domains of E2 were used to react with monoclonal antibodies (mAbs) against E2 and with swine anti-CSFV polyclonal sera. All antibodies recognized a highest common element, (753)RYLASLHKKALPTSV(767), on the double-looped peptides. This epitope region has not been revealed previously in the literature. Both substitution-scanning of residues (753)RYLASLHKKALPTSV(767) on a double-looped peptide and site-directed mutagenesis of expressed E2 demonstrated that residues (761)K, (763)L and (764)P were critical for the reactivity with mAbs. In addition, the up- and downstream residues (753)R, (754)Y, (755)L and (765)T were also crucial. Alignment showed that this stretch of amino acids was relatively conserved among various CSFVs. Thus, we identified a motif (753)RYLASLHKKALPT(765), which may be part of group-specific antigen and important for the structural integrity of conformational epitope recognition.

The Lapinized Philippines Coronel (LPC) vaccine, an attenuated strain of classical swine fever virus (CSFV), is an important tool for the prevention and control of CSFV infection and is widely and routinely used in most CSF endemic areas, including Taiwan. The aim of this study was to investigate whether PCV2 infection affects the efficacy of the LPC vaccine. Eighteen 6-week-old, cesarean-derived and colostrum-deprived (CDCD), crossbred pigs were randomly assigned to four groups. A total of 105.3 TCID50 of PCV2 was experimentally inoculated into pigs through both intranasal and intramuscular routes at 0 days post-inoculation (dpi) followed by LPC vaccination 12 days later. All the animals were challenged with wild-type CSFV (ALD stain) at 27 dpi and euthanized at 45 dpi. Following CSFV challenge, the LPC-vaccinated pigs pre-inoculated with PCV2 showed transient fever, viremia, and viral shedding in the saliva and feces. The number of IgM+, CD4+CD8-CD25+, CD4+CD8+CD25+, and CD4-CD8+CD25+ lymphocyte subsets and the level of neutralizing antibodies against CSFV were significantly higher in the animals with LPC vaccination alone than in the pigs with PCV2 inoculation/LPC vaccination. In addition, PCV2-derived inhibition of the CSFV-specific cell proliferative response of peripheral blood mononuclear cells (PBMCs) was demonstrated in an ex vivo experiment. These findings indicate that PCV2 infection decreases the efficacy of the LPC vaccine. This PCV2-derived interference may not only allow the invasion of wild-type CSFV in pig farms but also increases the difficulty of CSF prevention and control in CSF endemic areas.

Envelope glycoprotein E2 of classical swine fever virus (CSFV) is the major antigen that induces neutralizing antibodies in infected pigs. Our previous study revealed that N-terminal 90 residues (domains B/C) of E2 play key roles in differentiating vaccine strain LPC/AHRI (subgroup 1.1) from the two field strains TD/96/TWN (subgroup 2.1) and 94.4/IL/94/TWN (subgroup 3.4) (Chang et al., 2010). This study further analyzed the reaction patterns between monoclonal antibodies (mAbs) and expressed hybrid N-terminal of E2 of the above-mentioned viruses, revealing that mAbs T33 and C2, mAbs V8 and T23, and mAbs L7 and L150 required binding sites specifically at residues 690-714 in domain B, residues 715-740 in domain C, and residues 741-765 in domain C, respectively. Site-directed mutagenesis further demonstrated that residues (713)E and (729)D were critical for antigenic specificity of field strain (94.4/IL/94/TWN), while residues (705)D and (761)K were specific for vaccine strain (LPC/AHRI). These specific residues likely mediated in determining the topography of mAb binding sites of E2 to allow for differentiation between strains based on the premise that the structural integrity of the conformational epitope is maintained.

By analyzing the E2 sequences of classical swine fever virus from field outbreaks in Taiwan during 1993–2001, three virus populations with distinct genotypes were determined including one historical (subgroup 3.4) and two exotic (subgroup 2.1) strains. The first subgroup 2.1 virus was isolated in 1994 and further sporadic outbreaks occurred after 1996. Phylogenetic analysis using the E2 region has segregated the Taiwanese strains of 2.1virus into two different genotypes (termed 2.1a and 2.1b). The 2.1b viruses were only isolated in 2001 and shared approximately 94.8% nucleotide identities to the 2.1a viruses in the total genomic sequences. The results suggest that the 2.1a and 2.1b viruses may be introduced from different origins.