Differential Virus Torpism For PBMCs And Production Of Early Cytokines In Pigs Inoculated With High And Low Virulent PRRSV And Identiifcation Of T Cell Epitopes Of HP-PRRSV

Porcine reproductive and respiratory syndrome (PRRS), first detected in1987in North America, ischaracterized with either reproductive failure in pregnant sows or respiratory disease in young pigs, andis recognized to be one of the most important diseases to swine industry worldwide. Classical PRRSwas first confirmed in China in1995. The virus soon spread all over the country and considerablegenetic variation has been identified. In early2006, a highly pathogenic PRRSV (HP-PRRSV) emergedin China. Infections were characterized by high morbidity and mortality. Since its initial outbreaks, thedisease has spread to most provinces in China, resulting in millions of pig deaths and severely affectedthe swine industry. HP-PRRSV belongs to the North American genotype with a genomic marker ofdiscontinued30amino acid deletion in Nsp2region, which is different from other PRRSV strainsreported previously.To date, PRRS is endemic in the global swine population, which is more than20years after theemergence of PRRS. Unfortunately, our understanding of this disease is still far from clear. A typicalepisode of PRRSV infection is that the virus causes an acute viraemic phase up to14days postinoculation (DPI) during which the virus can be detected in serum and all susceptible organs. This acutephase is followed by virus elimination from serum and most organs and by persistent replication intonsils, lungs and some lymph nodes. Previous studies reported that in the acute viraemic phase ofPRRSV infection pigs developed weak and atypical innate immune responses. This inadequaterecognition of virus infection by the innate defence mechanisms could be responsible for the initiallycrippled immune response. Production of early cytokines is a prelude to activation of the effector cellsof innate resistance. Proinflammatory cytokines are believed to play an important role in porcinerespiratory disease by coordinating and activating the adaptive immune response, which enables thehost to eliminate offending pathogens. However, if cytokine levels become excessive, tissue damageand even death of the host can occur. PRRSV has a highly specific tropism for cells of the monocyte/macrophage lineage, which are essential for immune function. Previous studies in pigs showed thatPRRSV mainly infected a subpopulation of differentiated macrophages that were present in tonsils,lungs and other lymphoid tissues. Besides macrophages, in vitro analysis of susceptible cells identifiedcultivated monocytes and dendritic cells as potential targets but their roles during PRRSV infection inpigs remained unknown. Lung pathogenesis represents another feature of PRRSV infection. Porcinealveolar macrophages (PAMs) are generally considered to be a major target for PRRSV.One objective of the present study was to gain insight into the mechanisms by which HP-PRRSVcould evade innate immunity and later adaptive response. So in the second and third part of the article,HP-PRRSV strain HuN4and its attenuated live vaccine strain HuN4-F112were used in the presentstudy to illustrtae the correlation of altered virus tropism for different types of peripheral bloodmononuclear cells (PBMCs) and production of early cytokines with pathogenicity in piglets. There arefour groups, five piglets each. The piglets in Group1were vaccinated with the vaccine virus HuN4-F112strain, piglets in Group2were vaccinated with the vaccine virus HuN4-F112strain and28days later challenged with virulent HuN4strain along with Group3piglets. Meanwhile,piglets inGroup4were mock-vaccinated control. Blood samples were collected from day1through day28postinoculation (DPI) from these piglets to detect serum virus loads by quantitative real-time PCR. Lowervirus loads were detected in serum samples from vaccinated piglets prior to challenge as compared withchallenged vaccinates and controls. The levels of6cytokines (IL-1β, IL-4, IL-6, IL-8, IL-10and TNF-α)in serum samples were also examined by commercial ELISA kits. Data showed that HP-PRRSV strainHuN4induced earlier occurrence and higher levels of inflammatory cytokines in infected piglets.PBMCs were isolated from EDTA-treated blood samples and tested for their susceptibility to PRRSVby dual-color flow cytometry. During the first7DPI, higher positive rates of CD3, CD4and CD8Tlymphocytes, B lymphocytes and monocytes were demonstrated in vaccinated alone piglets than allchallenged piglets. Results obtained from the present study indicate that alteration of virus tropsim forPBMCs and modification of host cytokine production may correlate with pathogenicity of HP-PRRSVin piglets.Any way, to control and eliminate PRRS is the ultimate goal. However, due to the variable featuresof immune response to PRRSV, which includes limited protection from the infection, it is difficult.PRRS is one of the most challenging research topics in veterinary viral immunology. The importantroles of neutralizing antibodies were reported, nerveless, experiment showed that PRRSV could also beisolated from blood of pigs with neutralizing antibodies. Previous studies showed that pigs recoveredfrom experimental PRRSV infection had strong lymphocyte proliferative responses. And this CMIresponse determined by lymphocyte blastogenesis and adaptive cytokine production was delayed. Innateand adaptive immune responses are required for effective PRRS protection. And the activation of APCsis a key step in the induction of antigen-specific immunity. Currently, the localization of T-cell epitopesis important for the development of new vaccines and the improvement of existing vaccines. And IFN-γELIspot assay is one of the fastest and most sensitive ways to detect antigen-spcific T cells.In the fourth part of the article, ELIspot analysis with overlapping peptides was used to confirmand characterize T-cell epitopes of membrane protein in highly pathogenic PRRSV strain HuN4. Thirtypentadecapeptides spanned in membrane protein were screened on the basis of their capability to elicitinterferon-γ (IFN-γ) responses in the culture of peripheral blood mononuclear cells (PBMCs) obtainedfrom pigs immunized with PRRSV HuN4-F112strain and challenged with HP-PRRSV strain HuN4.After three rounds of screening, individual peptide M3, M6, M8or M12could elicit high expression ofIFN-γ. In order to further explore the relationship between these peptides and PRRSV specificcell-mediated immune response and identify T-cell epitopes, real-time PCR was used to confirm theinduction of IFN-γ transcription with high quality in cells stimulated by these peptides. The screening ofPRRSV isolates indicated that M6was fully conserved, M3and M12were relatively conserved withvariation of two amino acids, and M8was conserved with variation of three amino acids.In summary, the present study has demonstrated that HP-PRRSV has altered its tropism forPBMCs after extensive cell culture passages and reduced its capability to induce severe inflammation and cell infiltration in infected piglets by lower production of cytokines. And we have identified fourT-cell epitopes in membrane protein of HP-PRRSV, which will benefit the vaccination studies to primeantigen-specific T cells as well as to provide a tool for analyzing immunopathogenesis of PRRSV. Ourstudy will contribute to the understanding of the cell-medicated immunity against PRRSV. Therefore,our current investigations will provide a reasonable explanation and enhanced understanding forHP-PRRSV immunology.