广西猪瘟病毒E0和E2基因的克隆及序列分析

通过分析目前广西猪瘟病毒(CSFV)的E0和E2基因特征,为了解广西地区CSFV的分子流行病学、遗传变异及综合防控提供科学依据。试验采用RT-PCR方法,对阳性猪瘟样品进行CSFV的E0及E2基因的扩增,经克隆、测序后,利用DNAStar软件对序列进行比对分析,同时绘制系统遗传进化树。结果表明,从阳性猪瘟样品中成功扩增CSFV的E0及E2基因。序列比对分析发现,GX2毒株与参考毒株的E0基因核苷酸同源性在83.1%~94.1%,其推导氨基酸同源性在85.9%~99.6%;与参考毒株的E2基因核苷酸同源性为81.7%~93.7%,其推导氨基酸同源性为89.0%~97.0%;E0与E2基因均属于基因Ⅱ群。氨基酸变异位点分析表明,E0蛋白的RNase活性区域氨基酸基序位点没有发生变异;E2蛋白中15个位点上的半胱氨酸均未发生变异,但单抗识别位点S734R发生变异。遗传进化分析显示测定的GX2毒株与近年来广西CSFV流行毒株的变异趋势相似,与中国传统疫苗株HCLV、经典强毒株Shimen的同源性较低,亲缘关系较远,与广西近年来的流行毒株GXWZ02株的同源性较高,亲缘关系较近。     

非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位

【目的】制备非洲猪瘟病毒（African swine fever virus,ASFV）p30蛋白的单克隆抗体（monoclonal antibodies,MAbs）并初步分析其所识别的线性抗原表位,为ASFV及其抗体检测方法的建立及p30蛋白结构和功能的研究奠定基础。【方法】将原核表达并纯化的p30重组蛋白作为免疫原,免疫6—8周龄BALB/c雌鼠,每两周免疫1次,共免疫3次,首次免疫是抗原与等体积的弗氏完全佐剂乳化后免疫,第二次和第三次免疫与等体积的弗氏不完全佐剂乳化,3次免疫后1 w断尾采血,间接酶联免疫吸附试验（ELISA）检测血清抗体效价,选择血清效价最高的小鼠进行加强免疫,3 d后取小鼠脾淋巴细胞与SP2/0骨髓瘤细胞按照4﹕1的比例使用PEG进行常规细胞融合。利用重组p30蛋白作为包被抗原,间接ELISA筛选阳性杂交瘤细胞,有限稀释法进行克隆纯化,直至筛出能够稳定分泌抗体的MAbs。将ASFV接种于猪肺泡巨噬细胞,以筛选的MAbs为一抗、兔抗鼠HRP-IgG为二抗,进行间接免疫荧光试验（IFA）。将感染和未感染ASFV的细胞沉淀处理后进行 SDS-PAGE并转印至硝酸纤维素膜,分别以IFA鉴定为阳性的MAbs上清为一抗、兔抗鼠HRP-IgG为二抗,进行Western blotting分析,筛选获得p30 MAbs。根据已知序列设计引物扩增p30ab与p30bc两段截短基因,其中p30ab代表由第86—153位氨基酸残基的截短体,p30bc代表由第120—187位氨基酸残基的截短体,原核表达部分重叠的截短p30蛋白,最终获得重组蛋白GST-p30ab与重组蛋白GST-p30bc。分别以GST-p30ab和GST-p30bc融合蛋白为包被抗原,以5株MAbs为一抗,以兔抗鼠HRP-IgG为二抗, 通过间接ELISA方法初步定位p30蛋白的抗原表位。【结果】以纯化的重组蛋白为包被抗原,经间接ELISA试验筛选出25株可分泌抗重组 p30蛋白的杂交瘤细胞株。IFA结果显示,5株MAbs（8F4、1D3、1H2、6C3和8E11）与ASFV感染的猪肺泡巨噬细胞IFA 试验呈阳性;Western blotting结果显示,5株MAbs均能够与ASFV感染的细胞呈阳性反应,与未感染病毒的细胞呈阴性反应。试验构建的p30截短体重组蛋白GST-p30ab以可溶和包涵体两种形式表达,而GST-p30bc仅以包涵体形式表达,以两组截短体融合蛋白为包被抗原,通过间接ELISA检测出MAbs 8F4、1H2和6C3与两个重组蛋白均能有效结合,证明MAbs 8F4、1H2和6C3抗原识别区域为两组截短蛋白重叠区域,即第120—153位氨基酸;MAbs 8E11与1D3则只能与GST-p30ab蛋白结合, 证明MAbs 8E11与1D3抗原识别区域为两个重组蛋白的非重叠区域,即第86—119位氨基酸。【结论】本研究可溶性地表达了p30蛋白的第86—153位氨基酸截短体重组蛋白,制备了5株p30 MAbs,定位到2个p30蛋白抗原表位。结合ELISA和IFA,可建立十分可靠的ASFV及其抗体的检测手段。     

Porcine circovirus type 2 decreases the infection and replication of attenuated classical swine fever virus in porcine alveolar macrophages

Recently, it has been noted that porcine circovirus type 2 (PCV2) infection adversely affects the protective efficacy of Lapinized Philippines Coronel (LPC) vaccine, an attenuated strain of classical swine fever virus (CSFV), in pigs. In order to investigate the possible mechanisms of the PCV2-derived interference, an in vitro model was established to study the interaction of LPC virus (LPCV) and PCV2 in porcine alveolar macrophages (AMs). The results showed that PCV2 reduced the LPCV infection in AMs and the levels of PCV2-derived interference were dose-dependent. The PCV2-derived interference also reduced the replication level of LPCV in AMs. The full-length PCV2 DNA and its fragment DNA C9 CpG-ODN were involved in the reduction of LPCV infection in AMs, whereas UV-inactivated PCV2 was not. In addition, a moderate negative correlation between the LPCV antigen-containing rate and IFN-γ production was observed, and had a dose-dependent trend with the level of PCV2-inoculation. The results of the present study may partially explain how PCV2 infection interferes with the efficacy of LPC vaccine.     

Drivers and risk factors for circulating African swine fever virus in Uganda, 2012–2013

We explored observed risk factors and drivers of infection possibly associated with African swine fever (ASF) epidemiology in Uganda. Representative sub-populations of pig farms and statistics were used in a case-control model. Indiscriminate disposal of pig viscera and waste materials after slaughter, including on open refuse dumps, farm-gate buyers collecting pigs and pig products from within a farm, and retention of survivor pigs were plausible risk factors. Wire mesh-protected windows in pig houses were found to be protective against ASF infection. Sighting engorged ticks on pigs, the presence of a lock for each pig pen and/or a gate at the farm entrance were significantly associated with infection/non-infection; possible explanations were offered. Strict adherence to planned within-farm and community-based biosecurity, and avoidance of identified risk factors is recommended to reduce infection. Training for small-scale and emerging farmers should involve multidimensional and multidisciplinary approaches to reduce human-related risky behaviours driving infection.     

Comparison of two real-time RT-PCR assays for differentiation of C-strain vaccinated from classical swine fever infected pigs and wild boars

Classical swine fever is one of the most important infectious diseases for the pig industry worldwide due to its economic impact. Vaccination is an effective means to control disease, however within the EU its regular use is banned owing to the inability to differentiate infected and vaccinated animals, the so called DIVA principle. This inability complicates monitoring of disease and stops international trade thereby limiting use of the vaccine in many regions. The C-strain vaccine is safe to use and gives good protection. It is licensed for emergency vaccination in the EU in event of an outbreak. Two genetic assays that can distinguish between wild type virus and C-strain vaccines have recently been developed. Here the results from a comparison of these two real-time RT-PCR assays in an interlaboratory exercise are presented. Both assays showed similar performance.     

Up-regulated expression of PD-1 and its ligands during acute Classical Swine Fever virus infection in swine

In order to investigate the relationship between the PD-1 pathway and impairment of immune responses with the CSFV infection, the mRNA expression of PD-1 and its ligands were evaluated by quantitative polymerase chain reaction (qPCR) during artificial CSFV infection. Simultaneously, expression of IL-2 and IL-10 mRNA were detected. The T cell proliferation and CSFV load in plasma were also measured. Results showed that the expression of PD-1 and its ligands mRNA were significantly increased (p < 0.01) in PBMC from 3 to 7 days post infection (dpi). Meanwhile the level of IL-10 was up-regulated (p < 0.01). The IL-2 mRNA was not obviously changed but it is significantly increased from 14 dpi. The T cell proliferation was notably decreased at 7 dpi. The CSFV load was also increased in plasma. Overall, our results suggest that the expression of PD-1 and its ligands were up-regulated and probably correlated with immune inhibition during acute CSFV infection.     

某猪场猪瘟免疫程序的调整及免疫效果评价

为了更好地预防和控制猪瘟,找出适合四川某猪场的猪瘟免疫程序,笔者根据实际情况对免疫程序进行了调整。然后用ELISA和IHA法对免疫程序调整前后的不同日龄猪血清进行了抗体检测,结果显示:调整前用ELISA法检测到3日龄、25日龄、50日龄、90日龄和120日龄猪只的猪瘟抗体阳性率分别为80%、54.54%、77.78%、80%和95%,用IHA法测得的阳性率分别为100%、63.64%、77.78%、90%和95%;调整后用ELISA法测得的猪瘟抗体阳性率分别为100%、80%、77.27%、77.78%和95%,用正向IHA法测得的抗体阳性率分别为97.73%、94.44%、96.15%、100%和100%。调整前用ELISA和IHA测得的平均阳性率为77.46%、85.28%,调整后则变为86.01%、97.66%,虽然均符合农业部颁布的猪群猪瘟抗体阳性率应不低于70%的标准,但免疫程序调整后的抗体阳性率明显高于调整前,可见调整后的免疫程序更适合于该猪场。     

黄芪多糖对猪瘟活疫苗免疫应答的佐剂作用

为研究黄芪多糖对猪瘟活疫苗的佐剂作用,分别用0.5％黄芪多糖、疫苗专用稀释液、生理盐水（对照）稀释猪瘟耐热保护剂活疫苗免疫猪,测定免疫后不同时间段内血清中 IgG 及其亚型IgG1、IgG2、IgG3、IgG4和细胞因子IL 2、IL 6的含量。结果显示,与对照组相比,黄芪多糖作为疫苗稀释液可以显著提高血清中IgG及其亚型和细胞因子IL 2、IL 6的含量。与疫苗专用稀释液组相比,在免疫后第7、14、28天,黄芪多糖组IgG含量均提高,分别提高1.6％（P＞0.05）、6.4％（P＜0.05）、7.1％（P＜0.05）,IL 2含量分别提高12.1％（P＜0.05）、2.9％（P＞0.05）、15.3％（P＜0.05）,IL 6含量差异均不显著。表明,黄芪多糖作为疫苗稀释液对猪瘟活疫苗具有良好的佐剂作用。