RESUMO
Nervous necrosis virus (NNV) is one of the greatest threats to Mediterranean aquaculture, infecting more than 170 fish species and causing mortalities up to 100% in larvae and juveniles of susceptible species. Intensive aquaculture implies stressed conditions that affect the welfare of fish and their ability to fight against infections. In fact, a higher susceptibility to NNV has been related to poor welfare conditions. In order to analyze the physiological link between stressed conditions and increased susceptibility to NNV, as well as its possible role in the pathogenesis of this disease, we reared shi drum (Umbrina cirrosa) juveniles (30.7 ± 3.10 g body weight), which are expected to be asymptomatic upon NNV infection, at three stocking densities (2, 15, and 30 kg/m3) for 27 days and subsequently challenged them with NNV. We firstly characterized the stressed conditions of the specimens before and after infection and recorded the mortalities, demonstrating that stressed specimens reared at 30 kg/m3 suffered mortalities. However, the viral loads in different tissues were similar in all experimental groups, allowing horizontal and vertical transmission of the virus from asymptomatic specimens. All of these data suggest that shi drum tolerates wide ranges of culture densities, although high densities might be a setback for controlling NNV outbreaks in this species. In an attempt to understand the molecular pathways orchestrating this susceptibility change in stressed conditions, we performed a transcriptomic analysis of four tissues under mock- and NNV-infected conditions. In addition to the modification of the exceptive pathways such as cell adhesion, leukocyte migration, cytokine interaction, cell proliferation and survival, and autophagy, we also observed a heavy alteration of the neuroactive ligand-receptor pathway in three of the four tissues analyzed. Our data also point to some of the receptors of this pathway as potential candidates for future pharmacological treatment to avoid the exacerbated immune response that could trigger fish mortalities upon NNV infection.
Assuntos
Doenças dos Peixes , Nodaviridae , Infecções por Vírus de RNA , Animais , Nodaviridae/fisiologia , Doenças dos Peixes/virologia , Doenças dos Peixes/imunologia , Infecções por Vírus de RNA/imunologia , Infecções por Vírus de RNA/veterinária , Suscetibilidade a Doenças , Aquicultura , Carga ViralRESUMO
Investigations of population structuring in wild species are fundamental to complete the bigger picture defining their ecological and biological roles in the marine realm, to estimate their recovery capacity triggered by human disturbance and implement more efficient management strategies for fishery resources. The Blackspot Seabream (Pagellus bogaraveo, Brünnich 1768) is a commercially valuable deep-water fish highly exploited over past decades. Considering its exploitation status, deepening the knowledge of intraspecific variability, genetic diversity, and differentiation using high-performing molecular markers is considered an important step for a more effective stock assessment and fishery management. With one of the largest efforts conceived of and completed by countries overlooking the Atlantic and Mediterranean coasts in recent years, a total of 320 individuals were collected from different fishing grounds in the Mediterranean Sea and Atlantic Ocean and analysed using 29 microsatellite loci. We applied multiple statistical approaches to investigate the species' connectivity and population structure across most of its described distribution area. Considering the incomplete knowledge regarding the migratory behaviour of adults, here we suggest the importance of egg and larval dispersal in sustaining the observed genetic connectivity on such a large geographical scale.
RESUMO
Infectious diseases are a burden for aquaculture. Antigen processing and presentation (APP) to the immune effector cells that fight pathogens is key in the adaptive immune response. At the core of the adaptive immunity that appeared in lower vertebrates during evolution are the variable genes encoding the major histocompatibility complex (MHC). MHC class I molecules mainly present peptides processed in the cytosol by the proteasome and transported to the cell surface of all cells through secretory compartments. Professional antigen-presenting cells (pAPC) also express MHC class II molecules, which normally present peptides processed from exogenous antigens through lysosomal pathways. Autophagy is an intracellular self-degradation process that is conserved in all eukaryotes and is induced by starvation to contribute to cellular homeostasis. Self-digestion during autophagy mainly occurs by the fusion of autophagosomes, which engulf portions of cytosol and fuse with lysosomes (macroautophagy) or assisted by chaperones (chaperone-mediated autophagy, CMA) that deliver proteins to lysosomes. Thus, during self-degradation, antigens can be processed to be presented by the MHC to immune effector cells, thus, linking autophagy to APP. This review is focused on the essential components of the APP that are conserved in teleost fish and the increasing evidence related to the modulation of APP and autophagy during pathogen infection.
Assuntos
Apresentação de Antígeno , Autofagia , Imunidade Adaptativa , Animais , Antígenos/metabolismo , Autofagia/genética , Antígenos de Histocompatibilidade/metabolismo , Antígenos de Histocompatibilidade Classe II/metabolismo , Lisossomos/metabolismo , Peptídeos/metabolismoRESUMO
The production of the aquaculture industry has increased to be equal to that of the world fisheries in recent years. However, aquaculture production faces threats such as infectious diseases. Betanodaviruses induce a neurological disease that affects fish species worldwide and is caused by nervous necrosis virus (NNV). NNV has a nude capsid protecting a bipartite RNA genome that consists of molecules RNA1 and RNA2. Four NNV strains distributed worldwide are discriminated according to sequence homology of the capsid protein encoded by RNA2. Since its first description over 30 years ago, the virus has expanded and reassortant strains have appeared. Preventive treatments prioritize the RGNNV (red-spotted grouper nervous necrosis virus) strain that has the highest optimum temperature for replication and the broadest range of susceptible species. There is strong concern about the spreading of NNV in the mariculture industry through contaminated diet. To surveil natural reservoirs of NNV in the western Mediterranean Sea, we collected invertebrate species in 2015 in the Alboran Sea. We report the detection of the RGNNV strain in two species of cephalopod mollusks (Alloteuthis media and Abralia veranyi), and in one decapod crustacean (Plesionika heterocarpus). According to RNA2 sequences obtained from invertebrate species and reported to date in the Mediterranean Sea, the strain RGNNV is predominant in this semienclosed sea. Neither an ecosystem- nor host-driven distribution of RGNNV were observed in the Mediterranean basin.
Assuntos
Decapodiformes/virologia , Reservatórios de Doenças/veterinária , Nodaviridae/isolamento & purificação , Pandalidae/virologia , Animais , Reservatórios de Doenças/virologia , Peixes/classificação , Peixes/virologia , Genoma Viral/genética , Mar Mediterrâneo , Nodaviridae/classificação , Nodaviridae/genética , Filogenia , RNA Viral/genética , Frutos do Mar/classificação , Frutos do Mar/virologiaRESUMO
The highly migratory Atlantic bluefin tuna (ABFT) is currently managed as two distinct stocks, in accordance with natal homing behavior and population structuring despite the absence of barriers to gene flow. Larval fish are valuable biological material for tuna molecular ecology. However, they have hardly been used to decipher the ABFT population structure, although providing the genetic signal from successful breeders. For the first time, cooperative field collection of tuna larvae during 2014 in the main spawning area for each stock, the Gulf of Mexico (GOM) and the Mediterranean Sea (MED), enabled us to assess the ABFT genetic structure in a precise temporal and spatial frame exclusively through larvae. Partitioning of genetic diversity at nuclear microsatellite loci and in the mitochondrial control region in larvae spawned contemporarily resulted in low significant fixation indices supporting connectivity between spawners in the main reproduction area for each population. No structuring was detected within the GOM after segregating nuclear diversity in larvae spawned in two hydrographically distinct regions, the eastern GOM (eGOM) and the western GOM (wGOM), with the larvae from eGOM being more similar to those collected in the MED than the larvae from wGOM. We performed clustering of genetically characterized ABFT larvae through Bayesian analysis and by Discriminant Analysis of Principal Components (DAPC) supporting the existence of favorable areas for mixing of ABFT spawners from Western and Eastern stocks, leading to gene flow and apparent connectivity between weakly structured populations. Our findings suggest that the eastern GOM is more prone for the mixing of breeders from the two ABFT populations. Conservation of this valuable resource exploited for centuries calls for intensification of tuna ichthyoplankton research and standardization of genetic tools for monitoring population dynamics.
RESUMO
The spawning grounds of the Atlantic bluefin tuna (Thunnus thynnus) are traditionally considered to be the Gulf of Mexico (Gulf of Mexico) and the Mediterranean Sea (Mediterranean Sea). However, for the western Atlantic, unequivocal evidence of bluefin spawning outside the Gulf of Mexico has been shown. In this study we present the first records of genetically confirmed bluefin larvae in the southern Bay of Biscay (eastern Atlantic). These findings provide evidence of bluefin spawning activity outside the Mediterranean Sea, in the north-eastern Atlantic. However, our results suggest that the bluefin spawning in the Bay of Biscay is a sporadic phenomenon.
Assuntos
Baías , Atum , Animais , Golfo do México , Mar MediterrâneoRESUMO
The diversity of protists was researched in the Alboran Sea (SW Mediterranean Sea) by means of high-throughput sequencing technologies based on the amplification of the V9 region of 18S rRNA. Samples were collected at different depths in seven stations following an environmental gradient from a coastal upwelling zone to the core of an oligotrophic anticyclonic gyre (AG). Sampling was performed during summer, when the water column was stratified. The superphyla Alveolata, Stramenopila and Rhizaria accounted for 84% of the total operational taxonomic units (OTUs). The most diverse groups were Dinophyceae (21% of OTUs), Marine Alveolates-II (MALV-II; 20%), Ciliophora (9%) and MALV-I (6%). In terms of read abundance, the predominant groups were Dinophyceae (29%), Bacillariophyta (14%), MALV-II (11%) and Ciliophora (11%). Samples were clustered into three groups according to the sampling depth and position. The shallow community in coastal stations presented distinguishable patterns of diatoms and ciliates compared with AG stations. These results indicate that there was a strong horizontal coupling between phytoplankton and ciliate communities. Abundance of Radiolaria and Syndiniales increased with depth. Our analyses demonstrate that the stratification disruption produced by the AG caused shifts in the trophic ecology of the plankton assemblages inducing a transition from bottom-up to top-down control.
Assuntos
Alveolados , Rhizaria , Alveolados/genética , Biodiversidade , Mar Mediterrâneo , RNA Ribossômico 18S/genética , Rhizaria/genéticaRESUMO
The cytotoxic T-lymphocyte-mediated killing of virus-infected cells requires previous recognition of short viral antigenic peptides bound to human leukocyte antigen class I molecules that are exposed on the surface of infected cells. The cytotoxic T-lymphocyte response is critical for the clearance of human respiratory syncytial virus infection. In this study, naturally processed viral human leukocyte antigen class I ligands were identified with mass spectrometry analysis of complex human leukocyte antigen-bound peptide pools isolated from large amounts of human respiratory syncytial virus-infected cells. Acute antiviral T-cell response characterization showed that viral transcription determines both the immunoprevalence and immunodominance of the human leukocyte antigen class I response to human respiratory syncytial virus. These findings have clear implications for antiviral vaccine design.
Assuntos
Antígenos de Histocompatibilidade Classe I/imunologia , Imunidade Celular/imunologia , Vírus Sincicial Respiratório Humano/genética , Vírus Sincicial Respiratório Humano/imunologia , Transcrição Gênica , Sequência de Aminoácidos , Animais , Apresentação de Antígeno/imunologia , Extratos Celulares , Linhagem Celular , Humanos , Epitopos Imunodominantes/imunologia , Ligantes , Camundongos Transgênicos , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/imunologia , Proteoma/metabolismo , Vírus Sincicial Respiratório Humano/química , Linfócitos T/imunologia , Espectrometria de Massas em TandemRESUMO
CD8(+) T cells identify and kill infected cells through the specific recognition of short viral antigens bound to human major histocompatibility complex (HLA) class I molecules. The colossal number of polymorphisms in HLA molecules makes it essential to characterize the antigen-presenting properties common to large HLA families or supertypes. In this context, the HLA-B*27 family comprising at least 100 different alleles, some of them widely distributed in the human population, is involved in the cellular immune response against pathogens and also associated to autoimmune spondyloarthritis being thus a relevant target of study. To this end, HLA binding assays performed using nine HLA-B*2705-restricted ligands endogenously processed and presented in virus-infected cells revealed a common minimal peptide motif for efficient binding to the HLA-B*27 family. The motif was independently confirmed using four unrelated peptides. This experimental approach, which could be easily transferred to other HLA class I families and supertypes, has implications for the validation of new bioinformatics tools in the functional clustering of HLA molecules, for the identification of antiviral cytotoxic T lymphocyte responses, and for future vaccine development.
Assuntos
Antígenos Virais/metabolismo , Antígeno HLA-B27/metabolismo , Proteínas Virais/metabolismo , Motivos de Aminoácidos , Animais , Apresentação de Antígeno , Antígenos Virais/imunologia , Linhagem Celular , Antígeno HLA-B27/química , Humanos , Ligantes , Camundongos , Ligação Proteica , Estabilidade Proteica , Vírus Sincicial Respiratório Humano/imunologia , Proteínas Virais/imunologiaRESUMO
In the classical human leukocyte antigen (HLA) class I antigen processing and presentation pathway, the antigenic peptides are generated from viral proteins by multiple proteolytic cleavages of the proteasome (and in some cases other cytosolic proteases) and transported to the endoplasmic reticulum (ER) lumen where they are exposed to aminopeptidase activity. In human cells, two different ER-resident enzymes, ERAP1 and ERAP2, can trim the N-terminally extended residues of peptide precursors. In this study, the possible cooperative effect of generating five naturally processed HLA-B27 ligands by both proteases was analyzed. We identified differences in the products obtained with increased detection of natural HLA-B27 ligands by comparing double versus single enzyme digestions by mass spectrometry analysis. These in vitro data suggest that each enzyme can use the degradation products of the other as a substrate for new N-terminal trimming, indicating concerted aminoproteolytic activity of ERAP 1 and ERAP2.
Assuntos
Aminopeptidases/metabolismo , Antígeno HLA-B27/metabolismo , Proteólise , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Aminopeptidases/química , Humanos , Ligantes , Antígenos de Histocompatibilidade Menor , Dados de Sequência Molecular , Multimerização Proteica , Estrutura Quaternária de Proteína , Vírus Sincicial Respiratório Humano/metabolismo , Proteínas Virais/químicaRESUMO
The cytotoxic CD8(+) T lymphocyte-mediated cellular response is important for the elimination of virus-infected cells and requires the prior recognition of short viral peptide antigens previously translocated to the endoplasmic reticulum by the transporter associated with antigen processing (TAP). However, individuals with nonfunctional TAP complexes or infected cells with TAP molecules blocked by specific viral proteins, such as the cowpoxvirus, a component of the first source of early empirical vaccination against smallpox, are still able to present several HLA class I ligands generated by the TAP-independent antigen processing pathways to specific cytotoxic CD8(+) T lymphocytes. Currently, bioterrorism and emerging infectious diseases have renewed interest in poxviruses. Recent works that have identified HLA class I ligands and epitopes in virus-infected TAP-deficient cells have implications for the study of both the effectiveness of early empirical vaccination and the analysis of HLA class I antigen processing in TAP-deficient subjects.
Assuntos
Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Apresentação de Antígeno , Linfócitos T CD8-Positivos/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Redes e Vias Metabólicas , Vacina Antivariólica/imunologia , Vacinação/métodos , Transportadores de Cassetes de Ligação de ATP/deficiência , Transportadores de Cassetes de Ligação de ATP/metabolismo , Epitopos de Linfócito T/imunologia , Humanos , Vacina Antivariólica/administração & dosagemRESUMO
Respiratory syncytial virus causes lower respiratory tract infections in infancy and old age, affecting also immunocompromised patients. The viral fusion protein is an important vaccine candidate eliciting antibody and cell-mediated immune responses. CD8(+) cytotoxic T lymphocytes (CTLs) are known to have a role in both lung pathology and viral clearance. In BALB/c mice, the fusion protein epitope F249-258 is presented to CTLs by the murine major histocompatibility complex (MHC) class I molecule K(d). In cells infected with recombinant vaccinia viruses encoding the fusion protein, F249-258 is presented by MHC class I molecules through pathways that are independent of the transporters associated with antigen processing (TAP). We have now found that F249-258 can be generated from non-infectious virus from an exogenous source. Antigen processing follows a lysosomal pathway that appears to require autophagy. As a practical consequence, inactivated virus suffices for in vivo priming of virus-specific CTLs.
Assuntos
Apresentação de Antígeno , Epitopos de Linfócito T/imunologia , Lisossomos/metabolismo , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sinciciais Respiratórios/imunologia , Linfócitos T Citotóxicos/imunologia , Vacinas Virais/imunologia , Idoso , Animais , Antígenos Ly/metabolismo , Antígenos Virais/genética , Antígenos Virais/imunologia , Autofagia , Linhagem Celular , Epitopos de Linfócito T/genética , Antígenos de Histocompatibilidade Classe I/imunologia , Humanos , Hospedeiro Imunocomprometido , Lactente , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/imunologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologia , Vírus Sinciciais Respiratórios/metabolismo , Linfócitos T Citotóxicos/virologiaRESUMO
Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. In addition, HRSV poses a serious health risk in immunocompromised individuals and the elderly. It has been reported that this virus can infect mouse antigen-presenting cells, including B lymphocytes. In these B cells, HRSV infection upregulates the expression of activation markers, including MHC class II and CD86, but not MHC class I molecules. Here, we report that HRSV infection of spleen B lymphocytes downregulated TLR4. Either blocking with anti-TLR4 antibody or genetic deletion, but not functional deficiency of TLR4, moderately reduced the infectivity of HRSV in B lymphocytes. HRSV-infected B lymphocytes with deleted TLR4 upregulated MHC class II and CD86 molecules to the same levels as TLR4(+) wild type B cells. Since the activation of monocytes and macrophages by HRSV was previously reported to depend on TLR4, the current study indicates that these cells and B lymphocytes respond to HRSV infection with different activation pathways.
Assuntos
Linfócitos B/imunologia , Ativação Linfocitária/imunologia , Vírus Sincicial Respiratório Humano/imunologia , Receptor 4 Toll-Like/imunologia , Animais , Linfócitos B/metabolismo , Linfócitos B/virologia , Antígeno B7-2/imunologia , Antígeno B7-2/metabolismo , Separação Celular , Células Cultivadas , Feminino , Citometria de Fluxo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Camundongos , Camundongos Knockout , Vírus Sincicial Respiratório Humano/fisiologia , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismo , Regulação para CimaRESUMO
Human respiratory syncytial virus (HRSV) is the most common cause of severe respiratory infections in infants and young children, often leading to hospitalization. Although human airway epithelial cells are the main target of HRSV, it has been reported that this virus can also infect professional antigen-presenting cells such as macrophages and dendritic cells, promoting upregulation of maturation markers. Here, we report that mouse spleen B220(+) B lymphocytes were susceptible to HRSV infection in vitro, probably involving a glycosaminoglycan-dependent mechanism. In contrast, neither CD4(+) nor CD8(+) T lymphocytes were infected. In B lymphocytes, HRSV infection upregulated major histocompatibility complex (MHC) class II but not MHC class I molecules and induced the expression of the activation marker CD86.
Assuntos
Linfócitos B/metabolismo , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Infecções por Vírus Respiratório Sincicial/imunologia , Vírus Sincicial Respiratório Humano/fisiologia , Animais , Linfócitos B/imunologia , Linfócitos B/virologia , Biomarcadores/metabolismo , Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Glicosaminoglicanos/imunologia , Glicosaminoglicanos/metabolismo , Antígenos de Histocompatibilidade Classe I/imunologia , Antígenos de Histocompatibilidade Classe I/metabolismo , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Infecções por Vírus Respiratório Sincicial/virologiaRESUMO
Antigen processing of respiratory syncytial virus (RSV) fusion (F) protein epitopes F85-93 and F249-258 presented to cytotoxic T-lymphocytes (CTLs) by the murine major histocompatibility complex (MHC) class I molecule Kd was studied in different viral contexts. Epitope F85-93 was presented through a classical endogenous pathway dependent on the transporters associated with antigen processing (TAP) when the F protein was expressed from either RSV or recombinant vaccinia virus (rVACV). At least in cells infected with rVACV encoding either natural or cytosolic F protein, the proteasome was required for epitope processing. In cells infected with rVACV encoding the natural F protein, an additional endogenous TAP-independent presentation pathway was found for F85-93. In contrast, epitope F249-258 was presented only through TAP-independent pathways, but presentation was brefeldin A sensitive when the F protein was expressed from RSV, or mostly resistant when expressed from rVACV. Therefore, antigen-processing pathways with different mechanisms and subcellular localizations are accessible to individual epitopes presented by the same MHC class I molecule and processed from the same protein but in different viral contexts. This underscores both the diversity of pathways available and the influence of virus infection on presentation of epitopes to CTLs.
Assuntos
Antígenos Virais/metabolismo , Vírus Sinciciais Respiratórios/imunologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/virologia , Membro 2 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Membro 3 da Subfamília B de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Apresentação de Antígeno , Antígenos Virais/genética , Linhagem Celular , Epitopos de Linfócito T/genética , Epitopos de Linfócito T/metabolismo , Antígenos H-2/genética , Antígenos H-2/metabolismo , Humanos , Camundongos , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Vírus Sinciciais Respiratórios/genética , Vírus Sinciciais Respiratórios/patogenicidade , Transfecção , Vaccinia virus/genética , Vaccinia virus/imunologia , Proteínas Virais de Fusão/genética , Proteínas Virais de Fusão/imunologiaRESUMO
Cytotoxic CD8(+) T lymphocytes kill infected cells that display major histocompatibility complex (MHC) class I molecules presenting peptides processed from pathogen proteins. In general, the peptides are proteolytically processed from newly made endogenous antigens in the cytosol and require translocation to the endoplasmic reticulum (ER) for MHC class I loading. This last task is performed by the transporters associated with antigen processing (TAP). Sampling of suspicious pathogen-derived proteins reaches beyond the cytosol, and MHC class I loading can occur in other secretory or endosomal compartments besides the ER. Peptides processed from exogenous antigens can also be presented by MHC class I molecules to CD8(+) T lymphocytes, in this case requiring delivery from the extracellular medium to the processing and MHC class I loading compartments. The endogenous or exogenous antigen can be processed before or after its transport to the site of MHC class I loading. Therefore, mechanisms that allow the full-length protein or processed peptides to cross several subcellular membranes are essential. This review deals with the different intracellular pathways that allow the traffic of antigens to compartments proficient in processing and loading of MHC class I molecules for presentation to CD8(+) T lymphocytes and highlights the need to molecularly identify the transporters involved.
Assuntos
Linfócitos T CD8-Positivos/metabolismo , Peptídeos/química , Proteínas/química , Animais , Apresentação de Antígeno , Antígenos/química , Transporte Biológico , Citosol/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Sistema Imunitário , Ligantes , Complexo Principal de Histocompatibilidade , Modelos Biológicos , Estrutura Terciária de Proteína , Linfócitos T/metabolismoRESUMO
Human respiratory syncytial virus (RSV) is a major cause of respiratory infection in children and in the elderly. The RSV fusion (F) glycoprotein has long been recognized as a vaccine candidate as it elicits cytotoxic T-lymphocyte (CTL) and antibody responses. Two murine H-2K(d)-restricted CTL epitopes (F85-93 and F92-106) are known in the F protein of the A2 strain of RSV. F-specific CTL lines using BCH4 fibroblasts that are persistently infected with the Long strain of human RSV as stimulators were generated, and it was found that in this strain only the F85-93 epitope is conserved. Motif based epitope prediction programs and an F2 chain deleted F protein encoded in a recombinant vaccinia virus enabled identification of a new epitope in the Long strain, F249-258, which is presented by K(d) as a 9-mer (TYMLTNSEL) or a 10-mer (TYMLTNSELL) peptide. The results suggest that the 10-mer might be a naturally processed endogenous K(d) ligand. The CD8(+) T-lymphocyte responses to epitopes F85-93 and F249-258 present in the F protein of RSV Long were found to be strongly skewed to F85-93 in in vitro multispecific CTL lines and in vivo during a secondary response to a recombinant vaccinia virus that expresses the entire F protein. However, no hierarchy in CD8(+) T-lymphocyte responses to F85-93 and F249-258 epitopes was observed in vivo during a primary response.