Interspecies Transmission of Swine Influenza A Viruses and Human Seasonal Vaccine-Mediated Protection Investigated in Ferret Model.

We investigated the infection dynamics of 2 influenza A(H1N1) virus isolates from the swine 1A.3.3.2 (pandemic 2009) and 1C (Eurasian, avian-like) lineages. The 1C-lineage virus, A/Pavia/65/2016, although phylogenetically related to swine-origin viruses, was isolated from a human clinical case. This strain infected ferrets, a human influenza model species, and could be transmitted by direct contact and, less efficiently, by airborne exposure. Infecting ferrets and pigs (the natural host) resulted in mild or inapparent clinical signs comparable to those observed with 1A.3.3.2-lineage swine-origin viruses. Both H1N1 viruses could infect pigs and were transmitted to cohoused ferrets. Ferrets vaccinated with a human 2016-17 seasonal influenza vaccine were protected against infection with the antigenically matched 1A pandemic 2009 virus but not against the swine-lineage 1C virus. Our results reaffirm the need for continuous influenza A virus surveillance in pigs and identification of candidate human vaccine viruses.

JMM Profile: Swine influenza A virus: a neglected virus with pandemic potential.

Swine influenza is an acute respiratory disease of swine caused by swine influenza A virus (SwIAV). The ability of SwIAV to spread bidirectionally from animals to humans (zoonotic), and from humans to animals (reverse zoonotic), drives coinfection that can result in gene segment exchange and elevates the risk of generating viruses with pandemic potential. Compared to human-origin influenza A viruses, current data indicate a greater diversity amongst circulating SwIAVs, with three major subtypes (classified by haemagglutinin and neuraminidase) circulating globally in swine (H1N1, H1N2 and H3N2). The lack of protection afforded by human seasonal influenza vaccines against SwIAVs exacerbates the risk associated with reassortment of human, swine and potentially avian viruses. As such, global monitoring of SwIAVs is important for both human and animal health as they represent a true 'One Health' challenge with pandemic potential.

Immunization with matrix-, nucleoprotein and neuraminidase protects against H3N2 influenza challenge in pH1N1 pre-exposed pigs.

There is an urgent need for influenza vaccines providing broader protection that may decrease the need for annual immunization of the human population. We investigated the efficacy of heterologous prime boost immunization with chimpanzee adenovirus (ChAdOx2) and modified vaccinia Ankara (MVA) vectored vaccines, expressing conserved influenza virus nucleoprotein (NP), matrix protein 1 (M1) and neuraminidase (NA) in H1N1pdm09 pre-exposed pigs. We compared the efficacy of intra-nasal, aerosol and intra-muscular vaccine delivery against H3N2 influenza challenge. Aerosol prime boost immunization induced strong local lung T cell and antibody responses and abrogated viral shedding and lung pathology following H3N2 challenge. In contrast, intramuscular immunization induced powerful systemic responses and weak local lung responses but also abolished lung pathology and reduced viral shedding. These results provide valuable insights into the development of a broadly protective influenza vaccine in a highly relevant large animal model and will inform future vaccine and clinical trial design.

Respiratory and Intramuscular Immunization With ChAdOx2-NPM1-NA Induces Distinct Immune Responses in H1N1pdm09 Pre-Exposed Pigs.

There is a critical need to develop superior influenza vaccines that provide broader protection. Influenza vaccines are traditionally tested in naive animals, although humans are exposed to influenza in the first years of their lives, but the impact of prior influenza exposure on vaccine immune responses has not been well studied. Pigs are an important natural host for influenza, are a source of pandemic viruses, and are an excellent model for human influenza. Here, we investigated the immunogenicity of the ChAdOx2 viral vectored vaccine, expressing influenza nucleoprotein, matrix protein 1, and neuraminidase in H1N1pdm09 pre-exposed pigs. We evaluated the importance of the route of administration by comparing intranasal, aerosol, and intramuscular immunizations. Aerosol delivery boosted the local lung T-cell and antibody responses, while intramuscular immunization boosted peripheral blood immunity. These results will inform how best to deliver vaccines in order to harness optimal protective immunity.

Economic evaluation of whole genome sequencing for pathogen identification and surveillance - results of case studies in Europe and the Americas 2016 to 2019.

BackgroundWhole genome sequencing (WGS) is increasingly used for pathogen identification and surveillance.AimWe evaluated costs and benefits of routine WGS through case studies at eight reference laboratories in Europe and the Americas which conduct pathogen surveillance for avian influenza (two laboratories), human influenza (one laboratory) and food-borne pathogens (five laboratories).MethodsThe evaluation focused on the institutional perspective, i.e. the 'investment case' for implementing WGS compared with conventional methods, based on costs and benefits during a defined reference period, mostly covering at least part of 2017. A break-even analysis estimated the number of cases of illness (for the example of Salmonella surveillance) that would need to be avoided through WGS in order to 'break even' on costs.ResultsOn a per-sample basis, WGS was between 1.2 and 4.3 times more expensive than routine conventional methods. However, WGS brought major benefits for pathogen identification and surveillance, substantially changing laboratory workflows, analytical processes and outbreaks detection and control. Between 0.2% and 1.1% (on average 0.7%) of reported salmonellosis cases would need to be prevented to break even with respect to the additional costs of WGS.ConclusionsEven at cost levels documented here, WGS provides a level of additional information that more than balances the additional costs if used effectively. The substantial cost differences for WGS between reference laboratories were due to economies of scale, degree of automation, sequencing technology used and institutional discounts for equipment and consumables, as well as the extent to which sequencers are used at full capacity.

Intranasal Infection of Ferrets with SARS-CoV-2 as a Model for Asymptomatic Human Infection.

Ferrets were experimentally inoculated with SARS-CoV-2 (severe acute respiratory syndrome (SARS)-related coronavirus 2) to assess infection dynamics and host response. During the resulting subclinical infection, viral RNA was monitored between 2 and 21 days post-inoculation (dpi), and reached a peak in the upper respiratory cavity between 4 and 6 dpi. Viral genomic sequence analysis in samples from three animals identified the Y453F nucleotide substitution relative to the inoculum. Viral RNA was also detected in environmental samples, specifically in swabs of ferret fur. Microscopy analysis revealed viral protein and RNA in upper respiratory tract tissues, notably in cells of the respiratory and olfactory mucosae of the nasal turbinates, including olfactory neuronal cells. Antibody responses to the spike and nucleoprotein were detected from 21 dpi, but virus-neutralizing activity was low. A second intranasal inoculation (re-exposure) of two ferrets after a 17-day interval did not produce re-initiation of viral RNA shedding, but did amplify the humoral response in one animal. Therefore, ferrets can be experimentally infected with SARS-CoV-2 to model human asymptomatic infection.

Inactivated pandemic 2009 H1N1 influenza A virus human vaccines have different efficacy after homologous challenge in the ferret model.

BACKGROUND: The 2009 pandemic H1N1 (A(H1N1)pdm09) influenza A virus (IAV) has replaced the previous seasonal H1N1 strain in humans and continues to circulate worldwide. The comparative performance of inactivated A(H1N1)pdm09 influenza vaccines remains of considerable interest. The objective of this study was to evaluate the efficacy of two licensed A(H1N1)pdm09 inactivated vaccines (AS03B adjuvanted split virion Pandemrix from GlaxoSmithKline and referred here as (V1) and non-adjuvanted whole virion Celvapan from Baxter and referred here as (V2)) in ferrets as a pre-clinical model for human disease intervention. METHODS: Naïve ferrets were divided into two groups (V1 and V2) and immunised intramuscularly with two different A/California/07/2009-derived inactivated vaccines, V1 administered in a single dose and V2 administered in 2 doses separated by 21 days. Six weeks after the first immunisation, vaccinated animals and a non-vaccinated control (NVC) group were intra-nasally challenged with 106.5 TCID50 of the isolate A/England/195/2009 A(H1N1)pdm09 with 99.1% amino acid identity to the vaccine strain. Clinical signs, lung histopathology, viral quantification and antibody responses were evaluated. RESULTS AND CONCLUSIONS: Results revealed important qualitative differences in the performance of both inactivated vaccines in relation to protection against challenge with a comparable virus in a naive animal (ferret) model of human disease. Vaccine V1 limited and controlled viral shedding and reduced lower respiratory tract infection. In contrast, vaccine V2 did not control infection and animals showed sustained viral shedding and delayed lower respiratory infection, resulting in pulmonary lesions, suggesting lower efficacy of V2 vaccine.

Vaccines That Reduce Viral Shedding Do Not Prevent Transmission of H1N1 Pandemic 2009 Swine Influenza A Virus Infection to Unvaccinated Pigs.

Swine influenza A virus (swIAV) infection causes substantial economic loss and disease burden in humans and animals. The 2009 pandemic H1N1 (pH1N1) influenza A virus is now endemic in both populations. In this study, we evaluated the efficacy of different vaccines in reducing nasal shedding in pigs following pH1N1 virus challenge. We also assessed transmission from immunized and challenged pigs to naive, directly in-contact pigs. Pigs were immunized with either adjuvanted, whole inactivated virus (WIV) vaccines or virus-vectored (ChAdOx1 and MVA) vaccines expressing either the homologous or heterologous influenza A virus hemagglutinin (HA) glycoprotein, as well as an influenza virus pseudotype (S-FLU) vaccine expressing heterologous HA. Only two vaccines containing homologous HA, which also induced high hemagglutination inhibitory antibody titers, significantly reduced virus shedding in challenged animals. Nevertheless, virus transmission from challenged to naive, in-contact animals occurred in all groups, although it was delayed in groups of vaccinated animals with reduced virus shedding.IMPORTANCE This study was designed to determine whether vaccination of pigs with conventional WIV or virus-vectored vaccines reduces pH1N1 swine influenza A virus shedding following challenge and can prevent transmission to naive in-contact animals. Even when viral shedding was significantly reduced following challenge, infection was transmissible to susceptible cohoused recipients. This knowledge is important to inform disease surveillance and control strategies and to determine the vaccine coverage required in a population, thereby defining disease moderation or herd protection. WIV or virus-vectored vaccines homologous to the challenge strain significantly reduced virus shedding from directly infected pigs, but vaccination did not completely prevent transmission to cohoused naive pigs.

Transmission dynamics between infected waterfowl and terrestrial poultry: Differences between the transmission and tropism of H5N8 highly pathogenic avian influenza virus (clade 2.3.4.4a) among ducks, chickens and turkeys.

H5N8 highly-pathogenic avian influenza viruses (HPAIVs, clade 2.3.4.4) have spread globally via migratory waterfowl. Pekin ducks infected with a UK virus (H5N8-2014) served as the donors of infection in three separate cohousing experiments to attempt onward transmission chains to sequentially introduced groups of contact ducks, chickens and turkeys. Efficient transmission occurred among ducks and turkeys up to the third contact stage, with all (100%) birds becoming infected. Introduction of an additional fourth contact group of ducks to the turkey transmission chain demonstrated retention of H5N8-2014's waterfowl-competent adaptation. However, onward transmission ceased in chickens at the second contact stage where only 13% became infected. Analysis of viral progeny at this contact stage revealed no emergent polymorphisms in the intra-species (duck) transmission chain, but both terrestrial species included changes in the polymerase and accessory genes. Typical HPAIV pathogenesis and mortality occurred in infected chickens and turkeys, contrasting with 5% mortality among ducks.

Interspecies Transmission of Reassortant Swine Influenza A Virus Containing Genes from Swine Influenza A(H1N1)pdm09 and A(H1N2) Viruses.

Influenza A(H1N1)pdm09 (pH1N1) virus has become established in swine in the United Kingdom and currently co-circulates with previously enzootic swine influenza A virus (IAV) strains, including avian-like H1N1 and human-like H1N2 viruses. During 2010, a swine influenza A reassortant virus, H1N2r, which caused mild clinical disease in pigs in the United Kingdom, was isolated. This reassortant virus has a novel gene constellation, incorporating the internal gene cassette of pH1N1-origin viruses and hemagglutinin and neuraminidase genes of swine IAV H1N2 origin. We investigated the pathogenesis and infection dynamics of the H1N2r isolate in pigs (the natural host) and in ferrets, which represent a human model of infection. Clinical and virologic parameters were mild in both species and both intraspecies and interspecies transmission was observed when initiated from either infected pigs or infected ferrets. This novel reassortant virus has zoonotic and reverse zoonotic potential, but no apparent increased virulence or transmissibility, in comparison to pH1N1 viruses.

Statistical modelling of data showing pandemic H1N1 2009 swine influenza a virus infection kinetics in vaccinated pigs.

A swine influenza A pandemic 2009 H1N1 (pH1N1) virus was used in a pig challenge model to investigate the efficacy of whole inactivated vaccines homologous or heterologous to the challenge virus as well as a commercial vaccine. Nasal shedding of viral RNA was monitored daily by real-time, quantitative RT-PCR (RRT-qPCR) as detailed (1). Here we report the statistical modelling of the viral RNA shedding kinetics.

Head Start Immunity: Characterizing the Early Protection of C Strain Vaccine Against Subsequent Classical Swine Fever Virus Infection.

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.

Vaccine-mediated protection of pigs against infection with pandemic H1N1 2009 swine influenza A virus requires a close antigenic match between the vaccine antigen and challenge virus.

Swine influenza A virus (SwIV) infection has considerable economic and animal welfare consequences and, because of the zoonotic potential, can also have public health implications. The 2009 pandemic H1N1 'swine-origin' infection is now endemic in both pigs and humans. In Europe, avian-like H1avN1, human-like H1huN2, human-like swine H3N2 and, since 2009, pandemic H1N1 (pH1N1) lineage viruses and reassortants, constitute the dominant subtypes. In this study, we used a swine pH1N1 challenge virus to investigate the efficacy of whole inactivated virus vaccines homologous or heterologous to the challenge virus as well as a commercial vaccine. We found that vaccine-mediated protection was most effective when vaccine antigen and challenge virus were homologous and correlated with the specific production of neutralising antibodies and a cellular response to the challenge virus. We conclude that a conventional whole inactivated SwIV vaccine must be antigenically matched to the challenge strain to be an effective control measure.

CD1- and CD1+ porcine blood dendritic cells are enriched for the orthologues of the two major mammalian conventional subsets.

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.

Aerosol Delivery of a Candidate Universal Influenza Vaccine Reduces Viral Load in Pigs Challenged with Pandemic H1N1 Virus.

Influenza A viruses are a major health threat to livestock and humans, causing considerable mortality, morbidity, and economic loss. Current inactivated influenza vaccines are strain specific and new vaccines need to be produced at frequent intervals to combat newly arising influenza virus strains, so that a universal vaccine is highly desirable. We show that pandemic H1N1 influenza virus in which the hemagglutinin signal sequence has been suppressed (S-FLU), when administered to pigs by aerosol can induce CD4 and CD8 T cell immune responses in blood, bronchoalveolar lavage (BAL), and tracheobronchial lymph nodes. Neutralizing Ab was not produced. Detection of a BAL response correlated with a reduction in viral titer in nasal swabs and lungs, following challenge with H1N1 pandemic virus. Intratracheal immunization with a higher dose of a heterologous H5N1 S-FLU vaccine induced weaker BAL and stronger tracheobronchial lymph node responses and a lesser reduction in viral titer. We conclude that local cellular immune responses are important for protection against influenza A virus infection, that these can be most efficiently induced by aerosol immunization targeting the lower respiratory tract, and that S-FLU is a promising universal influenza vaccine candidate.

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.

Differential detection of classical swine fever virus challenge strains in C-strain vaccinated pigs.

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.

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.

Proteome-wide screening reveals immunodominance in the CD8 T cell response against classical swine fever virus with antigen-specificity dependent on MHC class I haplotype expression.

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: core241₋255 PESRKKLEKALLAWA and NS31902₋1912 VEYSFIFLDEY, or minimal length antigenic peptides: E2996₋1003 YEPRDSYF, NS21223₋1230 STVTGIFL and NS5A3070₋3078 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 NS1223₋1230 STVTGIFL and NS31902₋1912 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.

Assessment of the phenotype and functionality of porcine CD8 T cell responses following vaccination with live attenuated classical swine fever virus (CSFV) and virulent CSFV challenge.

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.