RESUMEN
BACKGROUND: Infectious diseases of farmed and wild animals pose a recurrent threat to food security and human health. The macrophage, a key component of the innate immune system, is the first line of defence against many infectious agents and plays a major role in shaping the adaptive immune response. However, this phagocyte is a target and host for many pathogens. Understanding the molecular basis of interactions between macrophages and pathogens is therefore crucial for the development of effective strategies to combat important infectious diseases. RESULTS: We explored how porcine pluripotent stem cells (PSCs) can provide a limitless in vitro supply of genetically and experimentally tractable macrophages. Porcine PSC-derived macrophages (PSCdMs) exhibited molecular and functional characteristics of ex vivo primary macrophages and were productively infected by pig pathogens, including porcine reproductive and respiratory syndrome virus (PRRSV) and African swine fever virus (ASFV), two of the most economically important and devastating viruses in pig farming. Moreover, porcine PSCdMs were readily amenable to genetic modification by CRISPR/Cas9 gene editing applied either in parental stem cells or directly in the macrophages by lentiviral vector transduction. CONCLUSIONS: We show that porcine PSCdMs exhibit key macrophage characteristics, including infection by a range of commercially relevant pig pathogens. In addition, genetic engineering of PSCs and PSCdMs affords new opportunities for functional analysis of macrophage biology in an important livestock species. PSCs and differentiated derivatives should therefore represent a useful and ethical experimental platform to investigate the genetic and molecular basis of host-pathogen interactions in pigs, and also have wider applications in livestock.
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Virus de la Fiebre Porcina Africana , Enfermedades Transmisibles , Virus de la Fiebre Porcina Africana/genética , Animales , Interacciones Huésped-Patógeno/genética , Macrófagos , Células Madre , PorcinosRESUMEN
Classical swine fever (CSF) is a highly contagious disease caused by the classical swine fever virus (CSFV). The live attenuated C-strain vaccine is highly efficacious, initiating protection within several days of delivery. The vaccine strain is detected in the tonsil early after inoculation, yet little is known of the role that tonsillar immune cells might play in initiating protection. Comparing the C-strain vaccine with the pathogenic CSFV Alfort-187 strain, changes in the myeloid cell compartment of the tonsil were observed. CSFV infection led to the emergence of an additional CD163+CD14+ cell population, which showed the highest levels of Alfort-187 and C-strain infection. There was also an increase in both the frequency and activation status (as shown by increased MHC-II expression) of the tonsillar conventional dendritic cells 1 (cDC1) in pigs inoculated with the C-strain. Notably, the activation of cDC1 cells coincided in time with the induction of a local CSFV-specific IFN-γ+ CD8 T cell response in C-strain vaccinated pigs, but not in pigs that received Alfort-187. Moreover, the frequency of CSFV-specific IFN-γ+ CD8 T cells was inversely correlated to the viral load in the tonsils of individual animals. Accordingly, we hypothesise that the activation of cDC1 is key in initiating local CSFV-specific CD8 T cell responses which curtail early virus replication and dissemination.
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Linfocitos T CD8-positivos/metabolismo , Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Tonsila Palatina/inmunología , Vacunas Virales/administración & dosificación , Animales , Antígenos CD/metabolismo , Antígenos de Diferenciación Mielomonocítica/metabolismo , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/fisiología , Células Dendríticas/metabolismo , Interferón gamma/metabolismo , Receptores de Lipopolisacáridos/metabolismo , Células Mieloides/metabolismo , Tonsila Palatina/citología , Tonsila Palatina/virología , Receptores de Superficie Celular/metabolismo , Porcinos , Vacunas Atenuadas/administración & dosificación , Vacunas Atenuadas/inmunología , Carga Viral , Vacunas Virales/inmunologíaRESUMEN
BACKGROUND: Control of classical swine fever (CSF) by vaccination ideally requires that field strain infection can be detected irrespective of the vaccination status of the herd. To inform on the usefulness of molecular tests compatible with genetic Differentiation of Infected from Vaccinated Animals (DIVA) principles when using live-attenuated vaccines, tonsil homogenates from a vaccination-challenge experiment were analyzed using a differential real-time qRT-PCR for the C-strain vaccine or real-time qRT-PCR assays developed to specifically detect the challenge strains used. RESULTS: In animals with high or moderate levels of blood viraemia, which were not, or not fully, protected by vaccination, challenge virus RNA was readily detected in tonsil homogenates. In three out of the seven vaccinated animals that had high or moderate viraemia, the vaccine strain RNA also could be detected but at lower levels. Lower but varying levels of challenge and/or vaccine virus RNA were detected in tonsil homogenate samples from animals with no or low-level viraemia, and in groups solely consisting of such animals, no transmission of infection to naïve in-contact animals occurred. In one group of animals that were vaccinated 3 days prior to challenge, viraemia levels varied from high to absent and transmission of challenge virus to naïve in-contact animals occurred. The DIVA assay revealed challenge virus in all tonsil homogenates from this group, even in those animals that did not have viraemia and were protected from clinical disease by vaccination. Such animals, particularly in a low biosecurity/informal farm setting, could constitute a risk for disease control in the field. CONCLUSIONS: Genetic DIVA testing is useful for detecting the presence of field virus infection especially in non-viraemic animals without overt clinical signs but which are incompletely protected by vaccination. Such tests could particularly be useful to inform decisions prior to and during cessation of a control strategy that employs vaccination.
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Peste Porcina Clásica/diagnóstico , Vacunas Virales/inmunología , Animales , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Peste Porcina Clásica/virología , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/inmunología , Tonsila Palatina/virología , Reacción en Cadena en Tiempo Real de la Polimerasa , Porcinos/inmunología , Porcinos/virología , Vacunas Virales/uso terapéutico , Viremia/inmunología , Viremia/veterinaria , Viremia/virologíaRESUMEN
The last outbreak of classical swine fever (CSF) in the UK occurred in 2000. A total of 16 domestic pig holdings in the East Anglia region were confirmed as infected over a 3-month period. Obtaining viral genome sequences has since become easier and more cost-effective and has accordingly been applied to trace viral transmission events for a variety of viruses. The rate of genetic evolution varies for different viruses and is influenced by different transmission events, which will vary according to the epidemiology of an outbreak. To examine if genetic changes over the course of any future CSF outbreak would occur to supplement epidemiological investigations and help to track virus movements, the E2 gene and full genome of the virus present in archived tonsil samples from 14 of these infected premises were sequenced. Insufficient changes occurred in the full E2 gene to discriminate between the viruses from the different premises. In contrast, between 5 and 14 nucleotide changes were detected between the genome sequence of the virus from the presumed index case and the sequences from the other 13 infected premises. Phylogenetic analysis of these full CSFV genome sequences identified clusters of closely related viruses that allowed to corroborate some of the transmission pathways inferred by epidemiological investigations at the time. However, other sequences were more distinct and raised questions about the virus transmission routes previously implicated. We are thus confident that in future outbreaks, real-time monitoring of the outbreak via full genome sequencing will be beneficial.
RESUMEN
The understanding of the pathogenic mechanisms and the clinicopathological forms caused by currently circulating African swine fever virus (ASFV) isolates is incomplete. So far, most of the studies have been focused on isolates classified within genotypes I and II, the only genotypes that have circulated outside of Africa. However, less is known about the clinical presentations and lesions induced by isolates belonging to the other twenty-two genotypes. Therefore, the early clinicopathological identification of disease outbreaks caused by isolates belonging to, as yet, not well-characterised ASFV genotypes may be compromised, which might cause a delay in the implementation of control measures to halt the virus spread. To improve the pathological characterisation of disease caused by diverse isolates, we have refined the macroscopic and histopathological evaluation protocols to standardise the scoring of lesions. Domestic pigs were inoculated intranasally with different doses (high, medium and low) of ASFV isolate Ken05/Tk1 (genotype X). To complement previous studies, the distribution and severity of macroscopic and histopathological lesions, along with the amount and distribution of viral antigen in tissues, were characterised by applying the new scoring protocols. The intranasal inoculation of domestic pigs with high doses of the Ken05/Tk1 isolate induced acute forms of ASF in most of the animals. Inoculation with medium doses mainly induced acute forms of disease. A less severe but longer clinical course, typical of subacute forms, characterised by the presence of more widespread and severe haemorrhages and oedema, was observed in one pig inoculated with the medium dose. The severity of vascular lesions (haemorrhages and oedema) induced by high and medium doses was not associated with the amount of virus antigen detected in tissues, therefore these might be attributed to indirect mechanisms not evaluated in the present study. The absence of clinical signs, lesions and detectable levels of virus genome or antigen in blood from the animals inoculated with the lowest dose ruled out the existence of possible asymptomatic carriers or persistently infected pigs, at least for the 21 days period of the study. The results corroborate the moderate virulence of the Ken05/Tk1 isolate, as well as its capacity to induce both the acute and, occasionally, subacute forms of ASF when high and medium doses were administered intranasally.
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Classical swine fever (CSF) is among the most relevant viral epizootic diseases of swine. Due to its severe economic impact, CSF is notifiable to the world organisation for animal health. Strict control policies, including systematic stamping out of infected herds with and without vaccination, have permitted regional virus eradication. Nevertheless, CSF virus (CSFV) persists in certain areas of the world and has re-emerged regularly. This review summarizes the basic established knowledge in the field and provides a comprehensive and updated overview of the recent advances in fundamental CSFV research, diagnostics and vaccine development. It covers the latest discoveries on the genetic diversity of pestiviruses, with implications for taxonomy, the progress in understanding disease pathogenesis, immunity against acute and persistent infections, and the recent findings in virus-host interactions and virulence determinants. We also review the progress and pitfalls in the improvement of diagnostic tools and the challenges in the development of modern and efficacious marker vaccines compatible with serological tests for disease surveillance. Finally, we highlight the gaps that require research efforts in the future.
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Virus de la Fiebre Porcina Clásica/genética , Peste Porcina Clásica , Animales , Peste Porcina Clásica/diagnóstico , Peste Porcina Clásica/epidemiología , Peste Porcina Clásica/virología , PorcinosRESUMEN
African swine fever virus (ASFV) causes a lethal, haemorrhagic disease in domestic swine that threatens pig production across the globe. Unlike domestic pigs, warthogs, which are wildlife hosts of the virus, do not succumb to the lethal effects of infection. There are three amino acid differences between the sequence of the warthog and domestic pig RELA protein; a subunit of the NF-κB transcription factor that plays a key role in regulating the immune response to infections. Domestic pigs with all 3 or 2 of the amino acids from the warthog RELA orthologue have been generated by gene editing. To assess if these variations confer resilience to ASF we established an intranasal challenge model with a moderately virulent ASFV. No difference in clinical, virological or pathological parameters were observed in domestic pigs with the 2 amino acid substitution. Domestic pigs with all 3 amino acids found in warthog RELA were not resilient to ASF but a delay in onset of clinical signs and less viral DNA in blood samples and nasal secretions was observed in some animals. Inclusion of these and additional warthog genetic traits into domestic pigs may be one way to assist in combating the devastating impact of ASFV.
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Fiebre Porcina Africana/prevención & control , Ligasas/genética , FN-kappa B/genética , Fiebre Porcina Africana/genética , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/genética , Virus de la Fiebre Porcina Africana/patogenicidad , Animales , Animales Salvajes/genética , Ligasas/metabolismo , FN-kappa B/metabolismo , Ingeniería de Proteínas/métodos , Sus scrofa/genética , PorcinosRESUMEN
Classical Swine Fever Virus (CSFV) is an ongoing threat to the pig industry due to the high transmission and mortality rates associated with infection. Live attenuated vaccines such as the CSFV C strain vaccine are capable of protecting against infection within 5 days of vaccination, but the molecular mechanisms through which this early protection is mediated have yet to be established. In this study, we compared the response of pigs vaccinated with the C strain to non-vaccinated pigs both challenged with a pathogenic strain of CSFV. Analysis of transcriptomic data from the tonsils of these animals during the early stages after vaccination and challenge reveals a set of regulated genes that appear throughout the analysis. Many of these are linked to the ISG15 antiviral pathway suggesting it may play a role in the rapid and early protection conferred by C strain vaccination.
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Peste Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Transcriptoma/inmunología , Vacunas Virales/inmunología , Animales , Virus de la Fiebre Porcina Clásica , Porcinos , Vacunas Atenuadas/inmunologíaRESUMEN
The palatine tonsil is the portal of entry for food and air and is continuously subjected to environmental challenges, including pathogens, which use the tonsil and pharynx as a primary site of replication. In pigs, this includes the viruses causing porcine respiratory and reproductive syndrome, and classical and African swine fever; diseases that have impacted the pig production industry globally. Despite the importance of tonsils in host defense, little is known regarding the phenotype of the myeloid cells resident in the porcine tonsil. Here, we have characterized five myeloid cell populations that align to orthologous populations defined in other mammalian species: a CD4+ plasmacytoid dendritic cell (DC) defined by expression of the conserved markers E2.2 and IRF-7, a conventional dendritic cell (cDC1) population expressing CADM1highCD172alow and high levels of XCR1 able to activate allogeneic CD4 and CD8 T cells; a cDC2 population of CADM1dim cells expressing FLT3, IRF4, and CSF1R with an ability to activate allogeneic CD4 T cells; CD163+ macrophages (MÏ´s) defined by high levels of endocytosis and responsiveness to LPS and finally a CD14+ population likely derived from the myelomonocytic lineage, which showed the highest levels of endocytosis, a capacity for activation of CD4+ memory T cells, combined with lower relative expression of FLT3. Increased knowledge regarding the phenotypic and functional properties of myeloid cells resident in porcine tonsil will enable these cells to be targeted for future vaccination strategies to current and emerging porcine viruses.
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Células Mieloides/metabolismo , Tonsila Palatina/citología , Fenotipo , Animales , Presentación de Antígeno/inmunología , Biomarcadores , Células Cultivadas , Citocinas/metabolismo , Expresión Génica , Antígenos de Histocompatibilidad Clase II/genética , Antígenos de Histocompatibilidad Clase II/inmunología , Inmunofenotipificación , Linfocitos/inmunología , Linfocitos/metabolismo , Porcinos , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismoRESUMEN
Conventional dendritic cells (cDC) are professional antigen-presenting cells that induce immune activation or tolerance. Two functionally specialised populations, termed cDC1 and cDC2, have been described in humans, mice, ruminants and recently in pigs. Pigs are an important biomedical model species and a key source of animal protein; therefore further understanding of their immune system will help underpin the development of disease prevention strategies. To characterise cDC populations in porcine blood, DC were enriched from PBMC by CD14 depletion and CD172a enrichment then stained with lineage mAbs (Lin; CD3, CD8α, CD14 and CD21) and mAbs specific for CD172a, CD1 and CD4. Two distinct porcine cDC subpopulations were FACSorted CD1- cDC (Lin-CD172+ CD1-CD4-) and CD1+ cDC (Lin-CD172a+ CD1+ CD4-), and characterised by phenotypic and functional analyses. CD1+ cDC were distinct from CD1- cDC, expressing higher levels of CD172a, MHC class II and CD11b. Following TLR stimulation, CD1+ cDC produced IL-8 and IL-10 while CD1- cDC secreted IFN-α, IL-12 and TNF-α. CD1- cDC were superior in stimulating allogeneic T cell responses and in cross-presenting viral antigens to CD8 T cells. Comparison of transcriptional profiles further suggested that the CD1- and CD1+ populations were enriched for the orthologues of cDC1 and cDC2 subsets respectively.
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Antígenos CD1/análisis , Células Sanguíneas/química , Células Sanguíneas/inmunología , Células Dendríticas/química , Células Dendríticas/inmunología , Animales , Antígenos de Superficie/análisis , Células Sanguíneas/clasificación , Citocinas/metabolismo , Células Dendríticas/clasificación , Citometría de Flujo , Perfilación de la Expresión Génica , Porcinos , Enfermedades de los PorcinosRESUMEN
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.
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Virus de la Fiebre Porcina Clásica/aislamiento & purificación , Peste Porcina Clásica/virología , Carne/virología , Animales , Peste Porcina Clásica/transmisión , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/patogenicidad , Genotipo , Masculino , Músculos/virología , Porcinos , Temperatura , Carga Viral/veterinaria , Inactivación de VirusRESUMEN
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.
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Virus de la Fiebre Porcina Clásica/inmunología , Células Dendríticas/inmunología , Células Dendríticas/virología , Interferón Tipo I/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Linfocitos T/inmunología , Animales , Células Cultivadas , Técnicas de Cocultivo , Antígenos de Histocompatibilidad Clase II/análisis , Interferón Tipo I/metabolismo , Células Asesinas Naturales/efectos de los fármacos , Perforina/análisis , Porcinos , Linfocitos T/efectos de los fármacos , Factores de Tiempo , Regulación hacia ArribaRESUMEN
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.
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Virus de la Fiebre Porcina Africana/genética , Fiebre Porcina Africana/diagnóstico , Asfarviridae/genética , Infecciones por Virus ADN/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Enfermedades de los Porcinos/diagnóstico , Porcinos/virología , Fiebre Porcina Africana/virología , Virus de la Fiebre Porcina Africana/aislamiento & purificación , Animales , Asfarviridae/aislamiento & purificación , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/virología , Sensibilidad y Especificidad , Enfermedades de los Porcinos/virologíaRESUMEN
Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-γ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-γ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-γ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core241â255 PESRKKLEKALLAWA and NS31902â1912 VEYSFIFLDEY, or minimal length antigenic peptides: E2996â1003 YEPRDSYF, NS21223â1230 STVTGIFL and NS5A3070â3078 RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-α in addition to IFN-γ. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS1223â1230 STVTGIFL and NS31902â1912 VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides could be restricted by additional haplotypes. Thus the antigenic regions and epitopes identified represent attractive targets for evaluation of their vaccine potential against CSFV.
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Linfocitos T CD8-positivos/inmunología , Virus de la Fiebre Porcina Clásica/inmunología , Genes MHC Clase I/inmunología , Epítopos Inmunodominantes/genética , Sus scrofa/inmunología , Secuencia de Aminoácidos , Análisis de Varianza , Animales , Secuencia de Bases , Cartilla de ADN/genética , Ensayo de Immunospot Ligado a Enzimas , Epítopos , Epítopos de Linfocito T/genética , Citometría de Flujo , Datos de Secuencia Molecular , Proteómica/métodos , Análisis de Secuencia de ADN , Vacunación/métodosRESUMEN
Vaccination with live attenuated classical swine fever virus (CSFV) induces solid protection after only 5 days, which has been associated with virus-specific T cell gamma interferon (IFN-γ) responses. In this study, we employed flow cytometry to characterize T cell responses following vaccination and subsequent challenge infections with virulent CSFV. The CD3(+) CD4(-) CD8(hi) T cell population was the first and major source of CSFV-specific IFN-γ. A proportion of these cells showed evidence for cytotoxicity, as evidenced by CD107a mobilization, and coexpressed tumor necrosis factor alpha (TNF-α). To assess the durability and recall of these responses, a second experiment was conducted where vaccinated animals were challenged with virulent CSFV after 5 days and again after a further 28 days. While virus-specific CD4 T cell (CD3(+) CD4(+) CD8α(+)) responses were detected, the dominant response was again from the CD8 T cell population, with the highest numbers of these cells being detected 14 and 7 days after the primary and secondary challenges, respectively. These CD8 T cells were further characterized as CD44(hi) CD62L(-) and expressed variable levels of CD25 and CD27, indicative of a mixed effector and effector memory phenotype. The majority of virus-specific IFN-γ(+) CD8 T cells isolated at the peaks of the response after each challenge displayed CD107a on their surface, and subpopulations that coexpressed TNF-α and interleukin 2 (IL-2) were identified. While it is hoped that these data will aid the rational design and/or evaluation of next-generation marker CSFV vaccines, the novel flow cytometric panels developed should also be of value in the study of porcine T cell responses to other pathogens/vaccines.
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Linfocitos T CD8-positivos/inmunología , Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Vacunas Virales/inmunología , Animales , Antígenos CD/análisis , Peste Porcina Clásica/inmunología , Citometría de Flujo , Inmunofenotipificación , Porcinos , Subgrupos de Linfocitos T/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
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.
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Virus de la Fiebre Porcina Clásica/inmunología , Porcinos/inmunología , Porcinos/virología , Vacunación/métodos , Animales , Anticuerpos Neutralizantes/inmunología , Peste Porcina Clásica/prevención & control , Peste Porcina Clásica/transmisión , Virus de la Fiebre Porcina Clásica/patogenicidad , Interferón gamma/metabolismo , Masculino , Linfocitos T/inmunología , Linfocitos T/virología , Factores de TiempoRESUMEN
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.
Asunto(s)
Virus de la Fiebre Porcina Clásica/inmunología , Peste Porcina Clásica/inmunología , Peste Porcina Clásica/prevención & control , Interferón gamma/metabolismo , Linfocitos T/inmunología , Vacunas Virales/inmunología , Secuencia de Aminoácidos , Animales , Reacciones Cruzadas/inmunología , Epítopos/química , Epítopos/inmunología , Interferón gamma/inmunología , Masculino , Datos de Secuencia Molecular , Porcinos , Vacunas Atenuadas , Proteínas Virales/química , Proteínas Virales/inmunologíaRESUMEN
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.