Effects Of PRRSV Specific Peptides On CD4~+?CD8~+ And CD4~+CD8~+T Cell Phenotype In Porcine Bone Marrow,Spleen,Thymus And Hilar Lymph Node

Porcine reproductive and respiratory syndrome virus causes serious damage to animal husbandry in China every year.The main feature of PRRS is the destruction of the reproductive system and the respiratory system.PRRSV can induce B and T lymphocytes apoptosis and a long-lasting immunosuppression,resulting in an increase in infection with other viruses and bacteria and in much higher mortality.However,the immunosuppression machnism of PRRS remains unclear.PRRSV is a single strand positive enveloped RNA virus,in which N and GP5 are structural proteins of PRRSV.Several studies have suggested that the two proteins play important parts in stimulating T cell responses in PRRSV infected pigs.In the present paper,we investigated the effects of specific peptide,PRRSV-GP5(GP5aa96-104)and PRRSV-N(Naa49-57)on the phenotype expression of T cells in the bone marrow,spleen,thymus and hilar lymph nodes from PRRS immunized pigs.In this experiment,we designed and commercially synthesized two PRRSV specific peptides PRRSV-GP5(GP5aa96-104)and PRRSV-N(Naa49-57).Immunization with CSF and PRRS was performed according to routine immunization procedures.The antibody levels of CSF and PRRS in peripheral blood were detected by indirect ELISA and blocking ELISA at 28 days of immunization.We collected porcine bone marrow,spleen,thymus and hilar lymph nodes,and isolated the lymphocytes from them.When the cells were cultured in RPMR-1640 medium with 10% bovine serum for 24 h and 48 h,the cells were co-cultured with one of the two PRRS specific peptides(0.15ug/ml).At 24 h co-culture,respectively,the mixtures were centrifuged at 1500 rpm for 15 min,discarded the supernatant.Then the cells were washed with phosphate-bufferedsaline(PBS)for four times and re-suspended in PBS(PH=7.4)with the concentration adjusted to approximately 1106cells/m L.Cells were stained with PE-CD3 ?PE-cy5.5-CD4 and Alexa-flour647-CD8 antibodies for 30 min at 4 °C.After the cells were rinsed in PBS three times,Flow cytometry was performed to detect the changes of T lymphocyte phenotype expression.The results show that: For two kinds of PRRS specific peptides to stimulate T lymphocytes,thymus is the most lasting organ in the transformation of the four kinds of tissues.During this period,CD4~+CD8~+T lymphocytes play a major role in the thymus.We also find that PRRSV-N specific peptide causes a transient decrease of CD4~+T lymphocytes in the thymus and hilar lymph nodes.Interestingly,PRRSV-N specific peptide had significant inhibitory effect on T lymphocytes in bone marrow at 24 h,and PRRSV-GP5 specific peptide had the effect on 48 h.In addition,CD4~+T lymphocytes in the bone marrow showed a response to PRRSV-N specific peptide stimulation in response to other T cell phenotypes,such as PRRSV-GP5 specific peptides for CD8~+T lymphocytes.It can be found that two kinds of specific peptides play a role in the changes of T lymphocyte phenotype in bone marrow,spleen,thymus and hilar lymph nodes.The research aimed to provide the material for the study of the immune mechanism of PRRSV and provide the theoretical basis for the development of subunit vaccine.However,the specific mechanism needs further study.