Characterisation of vaccine-induced, broadly cross-reactive IFN-γ secreting T cell responses that correlate with rapid protection against classical swine fever virus.

Live attenuated C-strain classical swine fever viruses (CSFV) provide a rapid onset of protection, but the lack of a serological test that can differentiate vaccinated from infected animals limits their application in CSF outbreaks. Since immunity may precede antibody responses, we examined the kinetics and specificity of peripheral blood T cell responses from pigs vaccinated with a C-strain vaccine and challenged after five days with a genotypically divergent CSFV isolate. Vaccinated animals displayed virus-specific IFN-γ responses from day 3 post-challenge, whereas, unvaccinated challenge control animals failed to mount a detectable response. Both CD4(+) and cytotoxic CD8(+) T cells were identified as the cellular source of IFN-γ. IFN-γ responses showed extensive cross-reactivity when T cells were stimulated with CSFV isolates spanning the major genotypes. To determine the specificity of these responses, T cells were stimulated with recombinant CSFV proteins and a proteome-wide peptide library from a related virus, BVDV. Major cross-reactive peptides were mapped on the E2 and NS3 proteins. Finally, IFN-γ was shown to exert potent antiviral effects on CSFV in vitro. These data support the involvement of broadly cross-reactive T cell IFN-γ responses in the rapid protection conferred by the C-strain vaccine and this information should aid the development of the next generation of CSFV vaccines.

Challenge of pigs with classical swine fever viruses after C-strain vaccination reveals remarkably rapid protection and insights into early immunity.

Pre-emptive culling is becoming increasingly questioned as a means of controlling animal diseases, including classical swine fever (CSF). This has prompted discussions on the use of emergency vaccination to control future CSF outbreaks in domestic pigs. Despite a long history of safe use in endemic areas, there is a paucity of data on aspects important to emergency strategies, such as how rapidly CSFV vaccines would protect against transmission, and if this protection is equivalent for all viral genotypes, including highly divergent genotype 3 strains. To evaluate these questions, pigs were vaccinated with the Riemser® C-strain vaccine at 1, 3 and 5 days prior to challenge with genotype 2.1 and 3.3 challenge strains. The vaccine provided equivalent protection against clinical disease caused by for the two challenge strains and, as expected, protection was complete at 5 days post-vaccination. Substantial protection was achieved after 3 days, which was sufficient to prevent transmission of the 3.3 strain to animals in direct contact. Even by one day post-vaccination approximately half the animals were partially protected, and were able to control the infection, indicating that a reduction of the infectious potential is achieved very rapidly after vaccination. There was a close temporal correlation between T cell IFN-γ responses and protection. Interestingly, compared to responses of animals challenged 5 days after vaccination, challenge of animals 3 or 1 days post-vaccination resulted in impaired vaccine-induced T cell responses. This, together with the failure to detect a T cell IFN-γ response in unprotected and unvaccinated animals, indicates that virulent CSFV can inhibit the potent antiviral host defences primed by C-strain in the early period post vaccination.

A generic real-time TaqMan assay for specific detection of lapinized Chinese vaccines against classical swine fever.

Classical swine fever (CSF) is a highly contagious disease, causing severe economic losses in the pig industry worldwide. Vaccination of pigs with lapinized Chinese vaccines is still practised in some regions of the world, where the virus is enzootic, in order to prevent and control the disease. However, a single real-time assay that can detect all lapinized Chinese vaccines used widely, namely, Lapinized Philippines Coronel (LPC), Hog Cholera Lapinized virus (HCLV) and the Riems C-strain is still lacking. This study describes a real-time RT-PCR assay, targeting the N(pro) gene region, for specific detection of these lapinized vaccine strains. The assay is highly sensitive, with a detection limit of 10 genome copies per reaction for HCLV and Riems C-strain and highly specific, as more than 100 strains of wild type CSFV representing all major genotypes were not detected. The assay is also highly repeatable: the coefficient of variation of Ct values in three runs was 2.77% for the detection of 10 copies of the vaccine viral RNA. This study provides a potentially useful tool for specific detection of the lapinized Chinese vaccines, HCLV and C-strain, and the differentiation of these vaccines from wild type CSFV.

Evaluation of a primer-probe energy transfer real-time PCR assay for detection of classical swine fever virus.

This study describes evaluation of a real-time PCR assay based on primer-probe energy transfer (PriProET) technology for detection of classical swine fever virus (CSFV). The PriProET technology allows melting curve analysis following PCR amplification and thus provides a higher specificity. The assay was compared with a TaqMan assay by testing a total of 203 samples including 175 clinical specimens and 28 batches of Hog Cholera Lapinized Virus (HCLV) vaccine. The two assays gave the same results for 184 (91%) samples. Compared with the TaqMan assay, 19 additional samples were found to be positive for CSFV using the PriProET assay. In an RNA mixture of both wild type CSFV and C-strain vaccine, the melting curves displayed only one curve: either a wild type-like or a vaccine-like depending on the dominating RNA. The PriProET assay can be a routine molecular tool or a confirmative tool for diagnosis of classical swine fever (CSF), especially in the case of samples that yield an inconclusive result by the TaqMan assay.

Escape of classical swine fever C-strain vaccine virus from detection by C-strain specific real-time RT-PCR caused by a point mutation in the primer-binding site.

Classical swine fever (CSF) is one of the most important diseases of pigs. Vaccination in the European Union is limited to emergency situations. Currently, vaccination for the purpose of disease control is carried out in wild boar populations. Wild boar are in most cases vaccinated using an oral bait vaccine based on the live modified vaccine virus C-strain "Riems". A real-time reverse transcription polymerase chain reaction (RT-PCR) protocol for differentiation of C-strain "Riems" vaccine virus from CSF virus (CSFV) field isolates was published previously. In this real-time RT-PCR system differentiation is based on two nucleotide difference one at the 3' end of each of the primer-binding sites in the E(RNS) encoding genome region. During extensive diagnostic use of this protocol in an outbreak of CSF in wild boar in Germany, some C-strain positive field samples were found to give negative results in the C-strain "Riems" specific real-time RT-PCR, but positive results in a pan-CSFV real-time RT-PCR system. Moreover, sequencing of C-strain "Riems" vaccine batches for intramuscular use revealed differences in the E(RNS) encoding region. This led to the assumption that mutations in the corresponding primer-binding site of the C-strain specific system had appeared in the field, and possibly also during manufacturing of different vaccine batches. To test this hypothesis and restore sensitivity, a new primer set for detection of the possible C-strain virus quasi species was designed and tested.