PRRSV强弱毒感染后对PBMCs细胞嗜性和细胞因子产生情况的差异研究及HP-PRRSV T细胞表位的鉴定
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摘要
猪繁殖和呼吸障碍综合征（Porcine reproductive and respiratory syndrome, PRRS)于1987年首次在北美爆发，该病引起怀孕母猪的繁殖障碍和仔猪的呼吸道疾病，是现今世界上危害养猪业最重要的疾病之一。我国于1995年首次暴发此病，至今全国范围内均有该病的存在，并且存在很多基因型的改变。2006年暴发的高致病性猪繁殖与呼吸综合征（HP-PRRS），感染以高发病率和高死亡率为特点，从爆发之日起，迅速蔓延至全国大多省份，造成几百万头猪的死亡，严重危害了养殖业的发展。HP-PRRSV属于北美型PRRSV，在基因组nsp2区存在有30个不连续的氨基酸缺失，异于之前报道的经典PRRSV。
到现在为止，PRRS已经在世界范围内流行20多年了，然而，我们对于该病还是知之甚少，PRRSV感染后的一个典型特点就是病毒侵入机体后引起急性的病毒血症，一直到感染后14天，在这段时间内，PRRSV都可以在血清和其它易感器官中检测到。急性期过后，病毒在血清和一些器官中消失，开始在扁桃体，肺脏和一些淋巴结中持续存在。在PRRSV感染的急性期间，机体内的先天性免疫反应很弱，而且呈现不规则状态，这种对于病毒感染激发的不正常的先天性免疫抵抗可能是导致之后对抗PRRSV感染免疫应答无力的原因之一。早期细胞因子的产生是机体先天性免疫抵抗的效应细胞激活的序曲，同时，它们在协调和激活获得性免疫应答中发挥着重要的作用，保证了机体对于外来抗原的清除。然而，如果炎性细胞因子水平过高，就会引起组织损伤，甚至是机体的死亡。在细胞嗜性方面，PRRSV对具有免疫功能的单核细胞/巨噬细胞有很高的亲和性。早期的研究报道，体内实验中，PRRSV主要感染扁桃体，肺脏和其它淋巴组织中的巨噬细胞。体外实验证实，单核细胞和树突状细胞也是PRRSV潜在的靶细胞，但是，这两类细胞在体内感染过程中发挥怎样的作用尚不知晓。普遍认为，肺泡巨噬细胞（PAM）是PRRSV侵入机体后的靶细胞。
本论文的一个目的就是对于HP-PRRSV能够逃避先天性免疫应答和之后的获得性免疫应答的攻击得以在猪体内增殖的机制窥见一斑。因此，在本文的第二和第三部分，我们选取了本实验室保存的HP-PRRSV的强毒株HuN4及将其传代致弱得到的疫苗株HuN4-F112进行研究，监测HP-PRRSV强弱毒感染后体内细胞因子产生水平差异及强弱毒感染仔猪后对不同类型外周血细胞（Periperial Blood Mononuclear Cell, PBMC）细胞嗜性的差异。动物实验共有4组，每组5头仔猪，其中Group1接种疫苗株HuN4-F112；Group2首先接种疫苗株HuN4-F112，再用强毒株HuN4攻毒；Group3直接用强毒株HuN4攻毒；Group4作为空白对照。在特定时间段采集血清样品，采用实时定量的方法检测病毒载量；与对照组和疫苗免疫组相比，野毒株HuN4感染猪外周血中病毒载量呈现高水平。采用商品化ELISA试剂盒，共有6个细胞因子(IL-1β, IL-4, IL-6, IL-8,IL-10, TNF-α)进行检测。结果显示Group1，Group2，Group4外周血中病毒拷贝数相对于Group3呈现较低水平。同时强毒感染组Group3的外周血中炎性细胞因子(IL-1β, IL-4, IL-6, IL-10, TNF-α)呈现高水平，表明HP-PRRSV野毒株感染后存在较强的炎症反应。采集非抗凝血分离PBMC来检测病毒的细胞嗜性差异，研究采用双染色法上流式细胞仪检测PBMC中CD3~+，CD4~+，CD8~+T细胞，B细胞和单核细胞的感染情况。结果显示在感染后1-7天，Group1中弱毒株对各种类型PBMC的感染率要高于强毒感染组；而强毒感染组的PBMC细胞群在流式细胞仪上出现一个大的种群，初步鉴定为炎性细胞群（属于单核细胞巨噬细胞系），因此推测因为强毒株对于炎性细胞的高嗜性，导致在免疫细胞中显示了较低的感染率。本研究的结果显示PRRSV强弱毒对于PBMC细胞嗜性存在差异，而炎性细胞的大量增殖及存在是导致HP-PRRSV具有高致病性原因之一，感染后的高热反应就是一个指征。
无论如何，控制及清除PRRS是研究的最终目标，然而，由于PRRSV感染后激发的免疫应答的复杂特性，包括感染后的免疫保护延迟，想要最终清除PRRSV非常困难。中和性抗体的重要作用得到广泛报道，然而也有文章报道PRRSV可以在含有中和性抗体的猪体内分离出来。早期研究表明实验感染PRRSV的康复猪体内有高水平的淋巴细胞增殖应答反应，然而这种细胞免疫应答反应在PRRSV感染早期是没有的，因此它的延迟是免疫保护无力的一个因素，因此对于抗原递呈细胞的激活是特异性免疫反应的关键步骤，是PRRSV感染后产生有效免疫保护必需的步骤。所以T细胞表位的鉴定对于新型疫苗的设计和现有疫苗的改进具有重要作用。现阶段，IFN-γELISPOT技术是检测抗原特异性T细胞的最快，最敏感的技术之一。
本论文的第四部分,采用ELISPOT技术和重叠多肽的方法对HP-PRRSV HuN4株M蛋白T细胞表位的位置进行了初步鉴定。共有全覆盖M蛋白的30条重叠多肽根据它们激发PBMC产生IFN-γ的能力进行了筛选，PBMC从HuN4-F112免疫后再用HuN4攻毒的猪体内分离得到。通过3轮的ELISPOT筛选，发现有4条短肽M3, M6, M8和M12可以单独刺激PBMC细胞产生较高表达水品的IFN-γ。为了进一步验证这4条短肽的能力，我们又采用了real-time PCR方法检测每条肽段各自刺激PBMC后的IFN-γ的表达水平，结果显示显著的高于对照组的其它短肽（P<0.05）。最后我们对这4条含有潜在T细胞表位的短肽的保守性进行了分析，表明这四个短肽具有很高的保守性。其中M6在42条序列中完全保守, M3，M12均存在两个氨基酸的变异, M8则存在3个氨基酸的变异。
综上所述，本论文一方面的结果表明将HP-PRRSV野毒株HuN4传代致弱得到疫苗株HuN4-F112改变了野毒株本身对PBMC的细胞嗜性，感染猪体后炎性细胞因子以及炎性细胞的大量浸润均不会产生，即疫苗株相比野毒株没有了严重的炎症反应。另一方面我们鉴定出了HP-PRRSV M蛋白上4条含有潜在T细胞表位的多肽，为疫苗的设计以及PRRSV免疫机理的研究奠定了基础，同时对PRRSV感染后细胞免疫应答的理解提供了研究依据。我们的结果为更好的解释及了解HP-PRRSV感染后的免疫反应提供了依据。
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.
到现在为止，PRRS已经在世界范围内流行20多年了，然而，我们对于该病还是知之甚少，PRRSV感染后的一个典型特点就是病毒侵入机体后引起急性的病毒血症，一直到感染后14天，在这段时间内，PRRSV都可以在血清和其它易感器官中检测到。急性期过后，病毒在血清和一些器官中消失，开始在扁桃体，肺脏和一些淋巴结中持续存在。在PRRSV感染的急性期间，机体内的先天性免疫反应很弱，而且呈现不规则状态，这种对于病毒感染激发的不正常的先天性免疫抵抗可能是导致之后对抗PRRSV感染免疫应答无力的原因之一。早期细胞因子的产生是机体先天性免疫抵抗的效应细胞激活的序曲，同时，它们在协调和激活获得性免疫应答中发挥着重要的作用，保证了机体对于外来抗原的清除。然而，如果炎性细胞因子水平过高，就会引起组织损伤，甚至是机体的死亡。在细胞嗜性方面，PRRSV对具有免疫功能的单核细胞/巨噬细胞有很高的亲和性。早期的研究报道，体内实验中，PRRSV主要感染扁桃体，肺脏和其它淋巴组织中的巨噬细胞。体外实验证实，单核细胞和树突状细胞也是PRRSV潜在的靶细胞，但是，这两类细胞在体内感染过程中发挥怎样的作用尚不知晓。普遍认为，肺泡巨噬细胞（PAM）是PRRSV侵入机体后的靶细胞。
本论文的一个目的就是对于HP-PRRSV能够逃避先天性免疫应答和之后的获得性免疫应答的攻击得以在猪体内增殖的机制窥见一斑。因此，在本文的第二和第三部分，我们选取了本实验室保存的HP-PRRSV的强毒株HuN4及将其传代致弱得到的疫苗株HuN4-F112进行研究，监测HP-PRRSV强弱毒感染后体内细胞因子产生水平差异及强弱毒感染仔猪后对不同类型外周血细胞（Periperial Blood Mononuclear Cell, PBMC）细胞嗜性的差异。动物实验共有4组，每组5头仔猪，其中Group1接种疫苗株HuN4-F112；Group2首先接种疫苗株HuN4-F112，再用强毒株HuN4攻毒；Group3直接用强毒株HuN4攻毒；Group4作为空白对照。在特定时间段采集血清样品，采用实时定量的方法检测病毒载量；与对照组和疫苗免疫组相比，野毒株HuN4感染猪外周血中病毒载量呈现高水平。采用商品化ELISA试剂盒，共有6个细胞因子(IL-1β, IL-4, IL-6, IL-8,IL-10, TNF-α)进行检测。结果显示Group1，Group2，Group4外周血中病毒拷贝数相对于Group3呈现较低水平。同时强毒感染组Group3的外周血中炎性细胞因子(IL-1β, IL-4, IL-6, IL-10, TNF-α)呈现高水平，表明HP-PRRSV野毒株感染后存在较强的炎症反应。采集非抗凝血分离PBMC来检测病毒的细胞嗜性差异，研究采用双染色法上流式细胞仪检测PBMC中CD3~+，CD4~+，CD8~+T细胞，B细胞和单核细胞的感染情况。结果显示在感染后1-7天，Group1中弱毒株对各种类型PBMC的感染率要高于强毒感染组；而强毒感染组的PBMC细胞群在流式细胞仪上出现一个大的种群，初步鉴定为炎性细胞群（属于单核细胞巨噬细胞系），因此推测因为强毒株对于炎性细胞的高嗜性，导致在免疫细胞中显示了较低的感染率。本研究的结果显示PRRSV强弱毒对于PBMC细胞嗜性存在差异，而炎性细胞的大量增殖及存在是导致HP-PRRSV具有高致病性原因之一，感染后的高热反应就是一个指征。
无论如何，控制及清除PRRS是研究的最终目标，然而，由于PRRSV感染后激发的免疫应答的复杂特性，包括感染后的免疫保护延迟，想要最终清除PRRSV非常困难。中和性抗体的重要作用得到广泛报道，然而也有文章报道PRRSV可以在含有中和性抗体的猪体内分离出来。早期研究表明实验感染PRRSV的康复猪体内有高水平的淋巴细胞增殖应答反应，然而这种细胞免疫应答反应在PRRSV感染早期是没有的，因此它的延迟是免疫保护无力的一个因素，因此对于抗原递呈细胞的激活是特异性免疫反应的关键步骤，是PRRSV感染后产生有效免疫保护必需的步骤。所以T细胞表位的鉴定对于新型疫苗的设计和现有疫苗的改进具有重要作用。现阶段，IFN-γELISPOT技术是检测抗原特异性T细胞的最快，最敏感的技术之一。
本论文的第四部分,采用ELISPOT技术和重叠多肽的方法对HP-PRRSV HuN4株M蛋白T细胞表位的位置进行了初步鉴定。共有全覆盖M蛋白的30条重叠多肽根据它们激发PBMC产生IFN-γ的能力进行了筛选，PBMC从HuN4-F112免疫后再用HuN4攻毒的猪体内分离得到。通过3轮的ELISPOT筛选，发现有4条短肽M3, M6, M8和M12可以单独刺激PBMC细胞产生较高表达水品的IFN-γ。为了进一步验证这4条短肽的能力，我们又采用了real-time PCR方法检测每条肽段各自刺激PBMC后的IFN-γ的表达水平，结果显示显著的高于对照组的其它短肽（P<0.05）。最后我们对这4条含有潜在T细胞表位的短肽的保守性进行了分析，表明这四个短肽具有很高的保守性。其中M6在42条序列中完全保守, M3，M12均存在两个氨基酸的变异, M8则存在3个氨基酸的变异。
综上所述，本论文一方面的结果表明将HP-PRRSV野毒株HuN4传代致弱得到疫苗株HuN4-F112改变了野毒株本身对PBMC的细胞嗜性，感染猪体后炎性细胞因子以及炎性细胞的大量浸润均不会产生，即疫苗株相比野毒株没有了严重的炎症反应。另一方面我们鉴定出了HP-PRRSV M蛋白上4条含有潜在T细胞表位的多肽，为疫苗的设计以及PRRSV免疫机理的研究奠定了基础，同时对PRRSV感染后细胞免疫应答的理解提供了研究依据。我们的结果为更好的解释及了解HP-PRRSV感染后的免疫反应提供了依据。
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.
引文
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