Factors affecting the infectivity of tissues from pigs with classical swine fever: thermal inactivation rates and oral infectious dose.

Outbreaks of classical swine fever are often associated with ingestion of pig meat or products derived from infected pigs. Assessment of the disease risks associated with material of porcine origin requires knowledge on the likely amount of virus in the original material, how long the virus may remain viable within the resulting product and how much of that product would need to be ingested to result in infection. Using material from pigs infected with CSFV, we determined the viable virus concentrations in tissues that comprise the majority of pork products. Decimal reduction values (D values), the time required to reduce the viable virus load by 90% (or 1 log10), were determined at temperatures of relevance for chilling, cooking, composting and ambient storage. The rate of CSFV inactivation varied in different tissues. At lower temperatures, virus remained viable for substantially longer in muscle and serum compared to lymphoid and fat tissues. To enable estimation of the temperature dependence of inactivation, the temperature change required to change the D values by 90% (Z values) were determined as 13 °C, 14 °C, 12 °C and 10 °C for lymph node, fat, muscle and serum, respectively. The amount of virus required to infect 50% of pigs by ingestion was determined by feeding groups of animals with moderately and highly virulent CSFV. Interestingly, the virulent virus did not initiate infection at a lower dose than the moderately virulent strain. Although higher than for intranasal inoculation, the amount of virus required for infection via ingestion is present in only a few grams of tissue from infected animals.

Partial Activation of natural killer and γδ T cells by classical swine fever viruses is associated with type I interferon elicited from plasmacytoid dendritic cells.

Vaccination with live attenuated classical swine fever virus (CSFV) vaccines can rapidly confer protection in the absence of neutralizing antibodies. With an aim of providing information on the cellular mechanisms that may mediate this protection, we explored the interaction of porcine natural killer (NK) cells and γδ T cells with CSFV. Both NK and γδ T cells were refractory to infection with attenuated or virulent CSFV, and no stimulatory effects, as assessed by the expression of major histocompatibility complex (MHC) class II (MHC-II), perforin, and gamma interferon (IFN-γ), were observed when the cells were cultured in the presence of CSFV. Coculture with CSFV and myeloid dendritic cells (mDCs) or plasmacytoid dendritic cells (pDCs) showed that pDCs led to a partial activation of both NK and γδ T cells, with upregulation of MHC-II being observed. An analysis of cytokine expression by infected DC subsets suggested that this effect was due to IFN-α secreted by infected pDCs. These results were supported by ex vivo analyses of NK and γδ T cells in the tonsils and retropharyngeal lymph nodes from pigs that had been vaccinated with live attenuated CSFV and/or virulent CSFV. At 5 days postchallenge, there was evidence of significant upregulation of MHC-II but not perforin on NK and γδ T cells, which was observed only following a challenge of the unvaccinated pigs and correlated with increased CSFV replication and IFN-α expression in both the tonsils and serum. Together, these data suggest that it is unlikely that NK or γδ T cells contribute to the cellular effector mechanisms induced by live attenuated CSFV.

Development and validation of a multiplex, real-time RT PCR assay for the simultaneous detection of classical and African swine fever viruses.

A single-step, multiplex, real-time polymerase chain reaction (RT-PCR) was developed for the simultaneous and differential laboratory diagnosis of Classical swine fever virus (CSFV) and African swine fever virus (ASFV) alongside an exogenous internal control RNA (IC-RNA). Combining a single extraction methodology and primer and probe sets for detection of the three target nucleic acids CSFV, ASFV and IC-RNA, had no effect on the analytical sensitivity of the assay and the new triplex RT-PCR was comparable to standard PCR techniques for CSFV and ASFV diagnosis. After optimisation the assay had a detection limit of 5 CSFV genome copies and 22 ASFV genome copies. Analytical specificity of the triplex assay was validated using a panel of viruses representing 9 of the 11 CSFV subgenotypes, at least 8 of the 22 ASFV genotypes as well as non-CSFV pestiviruses. Positive and negative clinical samples from animals infected experimentally, due to field exposure or collected from the UK which is free from both swine diseases, were used to evaluate the diagnostic sensitivity and specificity for detection of both viruses. The diagnostic sensitivity was 100% for both viruses whilst diagnostic specificity estimates were 100% for CSFV detection and 97.3% for ASFV detection. The inclusion of a heterologous internal control allowed identification of false negative results, which occurred at a higher level than expected. The triplex assay described here offers a valuable new tool for the differential detection of the causative viruses of two clinically indistinguishable porcine diseases, whose geographical occurrence is increasingly overlapping.

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.

Characterisation of virus-specific peripheral blood cell cytokine responses following vaccination or infection with classical swine fever viruses.

Existing live attenuated classical swine fever virus (CSFV) vaccines provide a rapid onset of complete protection but pose problems in discriminating infected amongst vaccinated animals. With a view to providing additional information on the cellular mechanisms that may contribute to protection, which in turn may aid the development of the next generation of CSFV vaccines, we explored the kinetics of the cytokine responses from peripheral blood cells of pigs vaccinated with an attenuated C-strain vaccine strain and/or infected with a recent CSFV isolate. Peripheral blood cells were isolated over the course of vaccination/infection and stimulated in vitro with C-strain or UK2000/7.1 viruses. Virus-specific responses of peripheral blood cells isolated from C-strain vaccinated pigs were dominated by the production of IFN-gamma. IFN-gamma production in response to the C-strain virus was first detected in vaccinates 9 days post-vaccination and was sustained over the period of observation. In contrast, cells from challenge control animals did not secrete IFN-gamma in response to stimulation with C-strain or UK2000/7.1 viruses. Supernatants from UK2000/7.1 infected animals contained significant levels of pro-inflammatory cytokines from day 8 post-infection and these cytokines were present in both virus and mock stimulated cultures. The results suggest that the C-strain virus is a potent inducer of a type-1 T cell response, which may play a role in the protection afforded by such vaccines, whereas the pro-inflammatory cytokine responses observed in cultures from infected pigs may reflect a pathological pro-inflammatory cascade initiated in vivo following the replication and spread of CSFV.