猪繁殖与呼吸综合征病毒蛋白免疫调节作用与免疫逃逸机制

Two decades after its discovery, porcine reproductive and respiratory syndrome virus (PRRSV) is still one of the most globally devastating viruses threatening the swine industry. PRRSV has developed diverse mechanisms to evade porcine antiviral immune responses. It can negatively modulate the host immune responses, resulting in persistent infection and secondary immunodeficiency. Although several studies have been carried out to elucidate the PRRSV protein in regulation of host immune response and the viral evasion strategies, there are several aspects that still remain unclear. This proposal wants to identify the viral proteins that up-regulate the regulatory T lymphocytes and IL-10 expression and the amino acid in the viral proteins possess the immune evasion properties under the pressure of antibodies against PRRSV. The expression of inflammatory cytokines will be first comparatively determined in the peripheral blood mononuclear cells (PBMCs) and monocyte-derived DCs infected with different PRRSV strains having different pathogenic in pigs. And some viral proteins possible having this characteristic will be expressed and purified and the expression of inflammatory cytokines will be detected in the DC treated with the recombinant proteins. Thirdly, some recombinant PRRSV will be constructed by using infectious cDNA cloning techniques and the viral proteins possible having this characteristic will be valuated in in vitro and in vovo. Meanwhile, some PRRSV isolates will be cultured and passaged in Marc-145 cells and porcine alveolar macrophages with the mediums containing antibodies to PRRSV, and the acquired passaged viruses having the different antigenic and pathogenic characterization will be selected. Based on the sequencing results of these viruses, a panel of mutants with deletions/mutations at the viral ORFs will be constructed by using infectious cDNA cloning techniques, and the amino acids associated with the antigenic and pathogenic changes will be determined. The putative results will be very useful for elucidating the mechanisms of PRRSV immunology and developing new vaccines for preventing and controlling this disease in the future.

猪繁殖与呼吸综合征病毒（PRRSV）被发现至今已有20多年，但依然是目前造成全球养猪业严重经济损失的最重要病毒之一。现有研究表明，PRRSV具有多种免疫逃逸机制，感染猪体可引起免疫抑制，出现持续感染和二次感染，但其免疫调节和免疫逃逸机制尚不十分清楚。本项目拟采用不同生物学特性的PRRSV分离株，比较其诱导宿主IL-10和调节性T细胞（Treg）免疫功能，通过病毒编码蛋白表达和感染性cDNA克隆技术，构建基因嵌合体或突变体重组病毒，经体外树突细胞及猪体感染试验，探索PRRSV诱导IL-10表达或诱生Tregs的功能蛋白及其关键氨基酸基元；同时，通过PRRSV在抗体环境中在Marc-145和PAM细胞连续培养传代，观察不同代次病毒抗原和毒力变异生物学表型和基因特征，采用病毒拯救技术，揭示病毒抗原和毒力变异关键基因位点，从而丰富PRRSV免疫机制理论基础，为该病毒新型疫苗研究奠定基础。

猪繁殖与呼吸综合征病毒(PRRSV)是影响世界养猪业的最重要病原之一。PRRSV具有多种免疫逃逸机制，感染猪体可引起免疫抑制，但其免疫调节和免疫逃逸机制尚不十分清楚。本项目通过PRRSV促进IL10分泌的功能蛋白、诱导产生调节性T细胞（Tregs）的功能蛋白及在抗体压力下免疫逃逸分子机制三个方面的研究，取得如下重要进展和成果：（1）阐明高致病性PRRSV（HP-PRRSV）诱导IL10表达能力较强。成功构建IL10启动子活性检测系统，解析了PRRSV促进IL10分泌的功能蛋白，明确了N蛋白的N-N非共价结合区在IL-10的诱导产生中扮演了重要作用。（2）阐明了HP-PRRSV具有较强的诱生调节性Treg T细胞的能力,明确了N蛋白15N和46R残基是诱导Treg细胞的关键位点。（3）发现PRRSV感染能够上调MoDCs分泌型sCD83表达，阐明PRRSV N和nsp10蛋白通过Sp1及NF-κB信号通路调节CD83表达,发现PRRSV N蛋白非共价区43-44aa及nsp10的第192-196aa和G214-216aa是上调sCD83表达的关键氨基酸位点。（4）发现PRRSV nsp1通过促进sCD83分泌抑制树突细胞抗原提呈作用，并抑制树突细胞介导的T细胞增殖作用，揭示了PRRSV nsp1锌指结构域是其上调sCD83表达的关键氨基酸或关键区域。（5）制备获得中和抗体环境压力下的PRRSV基因变异毒株，首次阐明HP-PRRSV GP5 第102和104、M蛋白第70位氨基酸残基在该病毒逃避中和抗体作用方面有重要作用。(6)阐明了临床免疫压力下，PRRSV基因变异和重组多样性，明确了我国该病毒分子流行病学特征，为该病毒抗原变异监测提供了重要方法。（7）明确了Nsp2的第323-433aa和628-747aa是PRRSV诱导炎性因子产生的关键区域。（8）发现了Poly（I:C）通过IFIT3介导MAVS信号通路抑制PRRSV复制。(9)克隆表达了干扰素下游信号通路中的Mx2、Vipeirn和胆固醇-25-羟化酶等重要蛋白基因，明确了其抗病毒及其分子机制，为PRRSV抗病毒药物研究奠定了基础。（10）成功构建了PRRSV反向遗传操作系统，拯救获得了上述关键功能域基因突变重组PRRSV，明确了其免疫抑制作用显著降低，为PRRSV新型疫苗研究奠定了重要基础。