湖南柑橘衰退病调查分析及弱毒系筛选

 The investigation showed that stem-pitting Citrus tristeza virus (CTV)occurred commonly in citrus production areas in several varieties of Hunan Province. Accurate detection of CTV strains was performed by p23/PCR method, PCR and the results indicated that the most samples were infected with several CTV isolates. Three mild strains were isolated and their pathogenicity was identified by biological identification, it indicated that p23/PCR groups had uniformity with the pathogenicity of CTV isolates. Furthermore, three mild isolates were tested in the cross protection by analysis of biological symptoms and composition of p23 gene. Different protecting effects were observed among these strains and W17 mild isolate was effective.     

小西葫芦绿斑驳花叶病毒生物学特性及传播方式

小西葫芦绿斑驳花叶病毒(Zucchini green mottle mosaic virus,ZGMMV)是我国新报道的侵染葫芦科作物的烟草花叶病毒属病毒.从感染病毒的葫芦叶片中分离出ZGMMV,摩擦接种西瓜(Citrullus lanatus)、葫芦(Lagenaria siceraria)、西葫芦(Cucurbit...     

猫泛白细胞减少症病毒北京株NS1基因克隆与生物信息学分析

为分析猫泛白细胞减少症病毒（FPV）北京株（FPV-BJ 05株）NS1基因的分子特征及其编码蛋白的生物学功能,本研究对FPV-BJ 05株NS1基因进行PCR扩增、克隆及序列测定。应用生物信息学软件分析FPV-BJ 05株与GenBank上登录的11株FPV参考株NS1基因的同源性,并预测NS1蛋白理化性质、信号肽、跨膜结构、B细胞抗原表位、磷酸化位点、亚细胞定位及蛋白结构与功能、高级结构等。结果显示,NS1基因全长2 007 bp,编码668个氨基酸,且与其他FPV分离株NS1基因核苷酸序列同源性为98.8%~99.3%,氨基酸序列同源性为97.9%~98.8%。系统进化树分析结果显示,FPV-BJ 05株NS1蛋白与GenBank上登录的3株FPV参考株处于同一大分支,但由于氨基酸的突变导致其属于独立的一小分支。NS1蛋白既无信号肽也无跨膜结构域,属于非分泌型的疏水性蛋白。B细胞抗原表位预测结果显示,NS1蛋白柔韧性较好,抗原性及表面可及性较高,预测该蛋白含有13个优势抗原位点。修饰结构预测表明,NS1蛋白含有62个潜在磷酸化位点,27个O-糖基化位点和2个N-糖基化位点。亚细胞定位结果显示,NS1蛋白在细胞质和细胞核的概率较高,分别为69.6%和17.4%。NS1蛋白二级结构中α-螺旋、延伸链、无规则卷曲和β-转角分别占39.37%、15.42%、39.37%和5.84%。应用在线软件SWISS-Modle对NS1蛋白进行建模,预测其三级结构,结果发现NS1蛋白主要以α-螺旋为主。本试验结果将为北京地区FPV免疫诊断及核酸疫苗研究提供理论依据。     

一种基于RPA的番茄褪绿病毒检测方法

番茄褪绿病毒Tomato chlorosis virus(ToCV)引起番茄褪绿病毒病,给番茄生产造成严重危害。开发快速准确的检测方法对该病害的防控具有重要意义。利用番茄褪绿病毒外壳蛋白(CP)基因序列,设计特异性引物,建立了ToCV的重组酶聚合酶等温扩增(recombinase polymerase amplification, RPA)检测方法,同时分析了该方法的灵敏度和特异性。结果表明,建立的ToCV-RPA方法在38℃恒温下40 min可从ToCV阳性的番茄样品中扩增出246 bp的特异性条带。扩增时间短,对设备要求低,且与番茄其他病毒无交叉反应,特异性好,灵敏度可达到PCR方法的10倍,适用于ToCV的快速检测。     

禽腺联病毒全基因组的克隆及感染性病毒的拯救

为了克隆禽腺联病毒(Avian adeno-associated virus,AAAV)全基因组用于构建基因转移载体研究,以鸡胚致死孤儿病毒(CELO)作为辅助病毒与AAAV共接种SPF鸡胚进行AAAV的增殖,将AAAV约4.7kb双链基因组DNA与pCR2.1载体连接,构建了含AAAV全基因组的重组质粒pAAAV并进行了测序。序列分析表明,AAAV YZ-1株的基因组为4684bp,两端具有141bp的末端倒置重复序列和Rep蛋白结合位点特征序列,与GenBank中收录的AAAV DA-1株和VR-865株的核苷酸序列同源性分别为95.0%和92.2%。将pAAAV质粒转染CELO病毒感染的鸡胚肝细胞系,获得了感染性AAAV病毒粒子,结果证明克隆的AAAV基因组中存在与病毒复制和包装相关的正确关键序列,可用于重组AAAV载体的构建。     

Study on Synergistic Effect of VA5 Immunopotentiator on Pigeon NDV Inactivated Vaccine

This study was designed to evaluate the synergistic effect of VA5 immunopotentiator on pigeon ND4416 inactivated vaccine.160 healthy pigeons were randomly divided into four groups,including ND4416 strain inactivated vaccine group (NDV group),ND4416 strain inactivated vaccine and immunopotentiator (VA5) mixed group (NDV+VA5 group),La Sota inactivated vaccine group (La Sota group) and normal saline as the control group (C group),to assess their vaccine efficacy against virulent pigeon NDV by serological analysis and animal testing.Pigeons sera were collected at different time points after immunization and measured the HI antibody titer of each group.The results showed that VA5 immunopotentiator significantly improved the serum antibody level of pigeons immunized with pigeon Newcastle disease inactivated vaccine (P<0.05).In addition,comparative test of spleen lymphocyte transformation was conducted at various time points after immunization.The results indicated that VA5 effectively stimulated the lymphocyte transformation of immune pigeon.Pigeons in each groups were challenged with ND4416 strain at the 30th,90th and 180th d after immunization.The results presented that the NDV+VA5 group had 100% protection rate and higher than La Sota group.The duration of immunization test showed that the antibody titer of NDV+VA5 group reached peak 11.20log₂ at the 21st d,remained 7.50log₂ at 180th d,and the protection rate remained 100% at 180th d.It indicated that VA5 immunopotentiator sustained the immune duration of pigeon NDV vaccine up to 180 d.Moreover,the in vitro detoxification test results suggested that VA5 immunopotentiator reduced the in vitro detoxification cycle of pigeons after challenge.Overall,this study suggested VA5 immunopotentiator could significantly improve the immune efficacy of pigeon Newcastle disease inactivated vaccine,which provided a basis for further enhancing the efficacy of Newcastle disease vaccine,as well increased experimental data for the application of immunopotentiators.     

Coxiella burnetii: Serological reactions and bacterial shedding in primiparous dairy cows in an endemically infected herd—impact on milk yield and fertility

Coxiella burnetii (C. burnetii) is the causative agent of Q fever both in humans and animals. The objectives of this study were to investigate seropositivity and bacterial shedding in heifers and primiparous cows in an endemically infected herd and to assess the effects on post‐partum diseases, fertility and milk production. At the age of 9 months, 96 Holstein heifers were included. Sampling was performed reproduction‐orientated: at the beginning of the study, at detection of first pregnancy, 3 weeks before expected calving date (blood serum), at parturition and after 21, 42, 100 and 150 days in milk (DIM) (blood serum, vaginal swabs and milk). Serum samples were investigated by a commercial ELISA for the presence of specific antibodies and vaginal swabs and milk samples by PCR to detect C. burnetii DNA. Individual animal data (calving ease, stillbirth, retained foetal membranes, puerperal metritis, endometritis after 42 DIM, presence of corpus luteum after 42 DIM, interval calving‐first service, interval calving‐conception, number of inseminations until 150 DIM, proportion of pregnant cows until 100 and 150 DIM, proportion of pregnant cows after first service and data of the dairy herd improvement test) were documented. All heifers were seronegative at the age of 9 months and 3 weeks before the expected calving date. Subsequently, the proportion of seropositive animals and the antibody score increased significantly towards 42 and 100 DIM, respectively. Vaginal C. burnetii shedding was highest at parturition (30.9%), while the most positive milk samples were detected after 100 DIM (15.3%). Coxiella burnetii seropositivity and shedding had no impact on parameters of reproduction. However, milk fat yield was declined in puerperal vaginal shedders and cows which seroconverted during their first 42 DIM, respectively.     

牛病毒性腹泻病毒P125基因重要区的比较与分析

根据牛病毒性腹泻病毒（ＢＶＤＶ）ＮＡＤＬ株的序列，以计算机辅助设计，化学合成１对引物（Ｗ１／Ｗ２）；应用ＲＴ－ＰＣＲ对国内不同地区分离的４株ＢＶＤＶ的Ｐ１２５基因外源序列插入区进行了扩增，并将扩增的片段进行克隆和序列测定，同时以ＤＮＡＳＩＳ和ＰＲＯＳＩＳ计算机软件将测定的核苷酸序列及推导的氨基酸序列进行比较分析。结果证实，国内分离的４株ＢＶＤＶ在这一区域中既没有外源序列的插入，也没有基因重组、基因重排或基因缺失，但存在某些核苷酸和氨基酸的替换，表明病毒的致细胞病变作用除与外源序列的插入、基因重组、基因重排或基因缺失有关外，可能还存在其他机制。核苷酸序列的同源性及系统树分析的结果表明，国内的ＢＶＤＶ毒株存在Ｉａ和Ｉｂ２个基因亚型，它们均属基因Ｉ型ＢＶＤＶ     

2007-2010年江苏地区猪繁殖与呼吸综合征病毒RT-PCR检测及部分Nsp2基因序列分析

本研究参考GenBank已发表的猪繁殖与呼吸综合征病毒（Porcine reproductive and respiratory syndrome virus,PRRSV）Nsp2基因序列,在高致病性病毒Nsp2基因缺失区的两端保守区设计并合成了一对引物,建立并优化了能够区分经典PRRSV和高致病性PRRSV的RT-PCR诊断方法,并利用该方法对2007～2010年间江苏地区的45份可疑临床病料进行了检测。结果表明临床病料阳性率为40％,所有毒株均属于高致病性毒株。对PRRSV阳性病毒的Nsp2基因序列分析表明,所有18株PRRSV均属于美洲型毒株,与中国高致病性PRRSV代表毒株JXA1、WUH1的氨基酸同源性分别在82．2％～97．6％、80．4％～95.3％。此外,18株病毒的Nsp2共同存在不连续的30个氨基酸缺失,缺失位置与同期中国高致病性PRRSV具有相同的特征。通过本研究掌握了江苏地区2007～2010年间PRRSV流行情况及Nsp2基因变异特征,为地方猪繁殖与呼吸综合征的临床诊断和防治提供参考依据。