RESUMO
The aim of this study was to analyze the immunogenic response elicited in swine by two synthetic peptides derived from GP5 to understand the role of lineal B epitopes in the humoral and B-cell-mediated response against the porcine reproductive and respiratory syndrome virus (PRRSV). For inoculation, twenty-one-day-old pigs were allocated into six groups: control, vehicle, vaccinated (Ingelvac-PRRSV, MLV®), non-vaccinated and naturally infected, GP5-B and GP5-B3. At 2 days post-immunization (dpi), the GP5-B3 peptide increased the serum concentrations of cytokines associated with activate adaptive cellular immunity, IL-1ß (1.15 ± 1.15 to 10.17 ± 0.94 pg/mL) and IL-12 (323.8 ± 23.3 to 778.5 ± 58.11 pg/mL), compared to the control group. The concentration of IgGs anti-GP5-B increased in both cases at 21 and 42 dpi compared to that at 0 days (128.3 ± 8.34 ng/mL to 231.9 ± 17.82 and 331 ± 14.86 ng/mL), while IgGs anti-GP5-B3 increased at 21 dpi (105.1 ± 19.06 to 178 ± 15.09 ng/mL) and remained at the same level until 42 dpi. Also, antibody-forming/Plasma B cells (CD2+/CD21-) increased in both cases (9.85 ± 0.7% to 13.67 ± 0.44 for GP5-B and 15.72 ± 1.27% for GP5-B3). Furthermore, primed B cells (CD2-/CD21+) from immunized pigs showed an increase in both cases (9.62 ± 1.5% to 24.51 ± 1.3 for GP5-B and 34 ± 2.39% for GP5-B3) at 42 dpi. Conversely the naïve B cells from immunized pigs decreased compared with the control group (8.84 ± 0.63% to 6.25 ± 0.66 for GP5-B and 5.78 ± 0.48% for GP5-B3). Importantly, both GP5-B and GP5-B3 peptides exhibited immunoreactivity against serum antibodies from the vaccinated group, as well as the non-vaccinated and naturally infected group. In conclusion, GP5-B and GP5-B3 peptides elicited immunogenicity mediated by antigen-specific IgGs and B cell activation.
RESUMO
We analyzed the T-cell responses induced by lineal epitopes of glycoprotein 5 (GP5) from PRRSV to explore the role of this protein in the immunological protection mediated by T-cells. The GP5 peptides were conjugated with a carrier protein for primary immunization and booster doses. Twenty-one-day-old pigs were allocated into four groups (seven pigs per group): control (PBS), vehicle (carrier), PTC1, and PTC2. Cytokine levels were measured at 2 days post-immunization (DPI) from serum samples. Cytotoxic T-lymphocytes (CTLs, CD8+) from peripheral blood were quantified via flow cytometry at 42 DPI. The cytotoxicity was evaluated by co-culturing primed lymphocytes with PRRSV derived from an infectious clone. The PTC2 peptide increased the serum concentrations of pro-inflammatory cytokines (i.e., TNF-α, IL-1ß, IL-8) and cytokines that activate the adaptive cellular immunity associated with T-lymphocytes (i.e., IL-4, IL-6, IL-10, and IL-12). The concentration of CTLs (CD8+) was significantly higher in groups immunized with the peptides, which suggests a proliferative response in this cell population. Primed CTLs from immunized pigs showed cytolytic activity in PRRSV-infected cells in vitro. PTC1 and PTC2 peptides induced a protective T-cell-mediated response in pigs immunized against PRRSV, due to the presence of T epitopes in their sequences.
Assuntos
Síndrome Respiratória e Reprodutiva Suína , Vírus da Síndrome Respiratória e Reprodutiva Suína , Vacinas Virais , Suínos , Animais , Vírus da Síndrome Respiratória e Reprodutiva Suína/metabolismo , Síndrome Respiratória e Reprodutiva Suína/prevenção & controle , Anticorpos Antivirais , Citocinas/metabolismo , Fator de Necrose Tumoral alfa , EpitoposRESUMO
BACKGROUND: Worldwide, Porcine Reproductive and Respiratory Syndrome (PRRS) is among the diseases that cause the highest economic impact in modern pig production. PRRS was first detected in Costa Rica in 1996 and has since then severely affected the local swine industry. Studies of the molecular characterization of circulating strains, correlation with clinical records, and associations with pathogens associated with Porcine Respiratory Disease Complex (PRDC) have not been done in Costa Rica. RESULTS: Sequencing and phylogenetic analysis of ORF5 proved that PRRSV-2 was the only species detected in all locations analyzed. These sequences were grouped into three clusters. When comparing samples from San Jose, Alejuela, and Puntarenas to historical isolates of the previously described lineages (1 to 9), it has been shown that these were closely related to each other and belonged to Lineage 5, along with the samples from Heredia. Intriguingly, samples from Cartago clustered in a separate clade, phylogenetically related to Lineage 1. Epitope analysis conducted on the GP5 sequence of field isolates from Costa Rica revealed seven peptides with at least 80% amino acid sequence identity with previously described and experimentally validated immunogenic regions. Previously described epitopes A, B, and C, were detected in the Santa Barbara-Heredia isolate. CONCLUSIONS: Our data suggest that the virus has three distinct origins or introductions to the country. Future studies will elucidate how recently introduced vaccines will shape the evolutionary change of circulating field strains.
Assuntos
Fases de Leitura Aberta/genética , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Sequência de Aminoácidos , Animais , Costa Rica/epidemiologia , Epitopos/análise , Filogenia , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Síndrome Respiratória e Reprodutiva Suína/virologia , SuínosRESUMO
Equine Arteritis Virus (EAV) has been shown to induce apoptosis in vitro but the induction of this mechanism has not been previously associated with any viral gene product. In this work, we found a cytotoxicity effect of the EAV gP5 protein on baculovirus-insect cells and a low yield of protein recovery. Besides, different morphological features by electron transmission microscopy, DNA fragmentation in agarose gel, TUNEL analysis and caspase 3 activity were found. All these findings indicate that the EAV gP5 protein induces apoptosis in insect cells.
Assuntos
Antígenos Virais/metabolismo , Apoptose , Equartevirus/fisiologia , Proteínas do Envelope Viral/metabolismo , Fatores de Virulência/metabolismo , Animais , Antígenos Virais/genética , Baculoviridae/genética , Equartevirus/patogenicidade , Expressão Gênica , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Células Sf9 , Spodoptera , Proteínas do Envelope Viral/genética , Fatores de Virulência/genéticaRESUMO
Equine viral arteritis (EVA) is a contagious viral disease that frequently causes mild or subclinical infections in adult horses. Only one EAV serotype has been described. However, there are differences in antigenicity, pathogenicity and neutralization characteristics of virus field strains. The interaction of two viral proteins, GP5 and M, is critical for infectivity and amino acid changes in the GP5 sequences have an effect on the neutralizing phenotype, regardless the effects of other viral proteins. The objective of the present study was to evaluate the neutralization phenotypes of the 5 unique Argentine EAV strains reported and to compare them with the neutralization phenotypes of the EAV-UCD reference strain, with special emphasis on the analysis of M and GP5 proteins. The strains had a similar neutralization phenotype pattern when anti-EAV serum, derived from EAV seropositive horses, was used in the analysis. Meanwhile, low titers were observed when equine polyclonal anti-EAV reference sera were used in the assay. Argentine strains have almost the same amino acid substitutions, with the exception of LP01 strain, that mainly involves the first variable region V1, especially in neutralization sites B and C. However, they are fairly different from the EAV-UCD strain. Nevertheless, the nucleotide and amino acid differences observed among the Argentine strains LP02/R, LP02/C, LP02/P and LP-LT-ARG did not show any variations in the neutralization phenotype.
La arteritis viral equina (AVE) ocasiona infecciones, en su mayoría subclínicas, pero puede causar abortos y enfermedad respiratoria. Si bien se ha descrito un solo serotipo de AVE, existen diferencias en cuanto a la antigenicidad, patogenicidad y patrones de neutralización en las cepas de campo. Los ORF5 y ORF6 del virus codifican las proteínas de envoltura GP5 y M; la interacción entre estas proteínas es crítica para la infectividad. Los cambios en las secuencias de aminoácidos en la proteína GP5, especialmente en la región V1, afectan el fenotipo neutralizante, sin tener en cuenta variaciones aminoacídicas de otras proteínas virales. En este estudio evaluamos los fenotipos neutralizantes de las 5 únicas cepas de arteritis viral equina aisladas en Argentina y los comparamos con los de la cepa de referencia EAV-UCD por virus neutralización cruzada y análisis de secuencias aminoacídicas de las proteínas M y GP5. Las cepas argentinas presentaron un patrón de neutralización similar cuando se utilizaron sueros positivos del banco de sueros, mientras que fueron neutralizadas en menor medida por los sueros policlonales de referencia anti-AVE. A excepción de la cepa LP01, las cepas argentinas tienen casi las mismas sustituciones aminoacídicas en la primera región variable V1 de la proteína GP5, específicamente en los sitios neutralizantes B y C, pero difieren en gran medida respecto de la cepa de referencia EAV-UCD. Las diferencias encontradas en los aislamientos LP02/R, LP02/C, LP02/P y LT-LP-ARG no se reflejaron en variaciones en el fenotipo neutralizante.