瘟病毒致细胞病变的遗传机制及其非结构蛋白的功能

牛病毒性腹泻—黏膜病病毒(bovine viral diarrhea—mucosal disease virus，BVDV)、边界病病毒(border disease virus，BDV)以及猪瘟病毒(classical swine fever virus，CSFV)在黄病毒家族中构成了瘟病毒属(Pestivirus)。瘟病毒是能够引起畜牧业严重损失的重要病原。这些具有囊膜的病毒粒子内包含长为9．5-12．3kb的单股正链RNA，其基因组包含有长的开放阅读框架，在病毒或细胞蛋白酶的作用下，通过共翻译或翻译后修饰途径产生成熟的病毒蛋白^[1]。从其基因组5’→3’方向依次编码N^pro、C、E^ms、E1、E2、P7(NS1)、NS2—3、NS4A、NS4B、NS5A和NS5B蛋白，其中C、E^ms、E1、E2为结构蛋白，其余为非结构蛋白。     

日本脑炎病毒非结构蛋白功能的研究进展

日本脑炎病毒(Japanese encephalitis virus, JEV)为单股正链RNA病毒,可通过蚊虫为媒介引起人畜共患的急性中枢神经系统传染病,目前暂无特效治疗药物。JEV基因全长11 kb可编码3个结构蛋白(C、PrM、E)和7个非结构蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B、NS5)。7个非结构蛋白占JEV编码蛋白质的大半,对于JEV发挥生物学功能有着重要意义。本文就JEV非结构蛋白功能进行了总结论述,以期为JEV分子生物学研究和防控提供参考。     

鹅细小病毒非结构蛋白和结构蛋白B细胞线性抗原表位的鉴定

鹅细小病毒是小鹅瘟的病原体,其基因组含有2个主要开放阅读框（ORF）,分别编码非结构蛋白（NS）和结构蛋白（VP）。为了对这2种蛋白进行抗原表位作图,设计了34个覆盖非结构蛋白NS和结构蛋白VP的50-60个氨基酸残基的重叠短肽,并进行了融合表达。用攻毒10周龄鹅血清对这34个融合蛋白进行蛋白质印迹分析,结果鉴定出NS蛋白线性抗原表位位于C末端的453-627氨基酸区域;VP蛋白线性抗原表位位于35—198、423—491、531—595、616—669和678—732氨基酸区域。     

A型流感病毒NS1蛋白研究进展

随着对A型流感病毒研究的逐渐深入，人们已经从对A型流感病毒结构蛋白的研究转移到对非结构蛋白即NS1蛋白的研究上来，目前人们已对不同亚型NS1蛋白进行了更为详细的分类，同时对NS1蛋白功能也有了更深入的认识，如抑制宿主细胞蛋白合成、诱导细胞凋亡，以及拮抗干扰素的作用等。而且，在鉴别诊断自然感染流感病毒动物与人工免疫流感疫苗动物方面有着重要的应用价值。     

牛病毒性腹泻病毒基因组结构与蛋白功能研究进展

牛病毒性腹泻病毒为黄病毒科(Flaviviridae)瘟病毒属(Pestivirus)的代表种,这个属的成员中还包括羊边界病毒(BDV)和猪瘟病毒(CSFV)。病毒基因组为单股正链RNA。论文就牛病毒性腹泻病毒已定位的4种结构蛋白、8种非结构蛋白以及3′和5′非翻译区的基因结构特征及其编码蛋白的合成与功能研究进行了综述,为牛病毒性腹泻的防控和疫苗研制提供参考。     

猪瘟病毒非结构蛋白NS4B与宿主蛋白相互作用的研究进展

猪瘟病毒(CSFV)是一种可感染猪只并引起典型或慢性猪瘟(CSF)的单链RNA包膜病毒,严重影响动物的健康和养猪业的经济发展。CSFV自身编码的结构蛋白和非结构蛋白可与宿主蛋白相互作用,促进病毒自身的复制、翻译和增殖,并影响病毒毒力和免疫反应等。CSFV的非结构蛋白NS4B在病毒生命周期中发挥重要作用,其自身结构上含有多种特定区域靶点,可与多种蛋白发生相互作用;其作为CSFV复制体碳价结构最重要的组成部分,还可被非结构蛋白NS3切割,在细胞内与CSFV自身蛋白或其他宿主蛋白,如核糖体蛋白侧柄亚基P1(RPLP1)、铁蛋白重链(FHC)、Rab蛋白22a(Rab22a)、脂肪酸合成酶(FASN)、线粒体抗病毒信号蛋白(MAVS)、肿瘤易感基因101(Tsg101)蛋白和猪指环蛋白114(pRNF114)等相互作用。本文对CSFV非结构蛋白NS4B的结构功能以及与宿主蛋白发生相互作用的研究进展进行概述,以期为CSFV致病机理研究和感染防控提供理论帮助。     

牦牛源BVDV非结构蛋白NS5A的原核表达及抗原表位分析

【目的】利用原核表达系统体外表达牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)NS5A基因,获得非结构蛋白NS5A,对其进行核苷酸、氨基酸序列分析,以解析BVDV非结构蛋白NS5A的功能。【方法】参考BVDV-1型毒株V006的NS5A基因序列(GenBank登录号:KX170647)设计并合成1对特异性引物,以分离到的牦牛BVDV GSTZ毒株cDNA为模板,PCR扩增NS5A基因片段,并克隆至表达载体pET-28a (+)中,构建重组原核表达载体pET28a-NS5A。经酶切初步鉴定及测序鉴定正确后,转化大肠杆菌BL21(DE3)感受态细胞,然后利用IPTG诱导表达。经10% SDS-PAGE电泳及Western blotting分析鉴定重组蛋白的表达,并根据NS5A基因的序列构建遗传发育进化树,利用DNAStar软件预测NS5A蛋白的亲水性、表面可塑性和抗原性等特性,并结合二级结构的预测对NS5A蛋白的B细胞抗原表位进行预测。【结果】PCR扩增NS5A目的基因片段为1 488 bp,双酶切和测序鉴定结果证明,重组质粒pET28a-NS5A构建成功。经10% SDS-PAGE电泳及Western blotting鉴定重组蛋白,表达出了大小为55 ku的目的蛋白,大小与预期结果相符。通过对不同BVDV毒株NS5A基因序列构建遗传发育进化树,显示GSTZ毒株NS5A在遗传进化特征上属于BVDV-1型。NS5A蛋白的亲水性主要位于12—21、32—69、75—113、120—135、143—147、152—163、165—180、215—230、265—274、296—340、348—378、389—447、455—463、469—495位氨基酸处,表面可塑性主要位于14—18、37—42、76—81、86—109、154—160、169—178、218—228、297—309、348—358、365—373、414—442、430—437、454—460位氨基酸处,柔性区域较多,主要位于14—21、37—43、67—82、86—93、97—110、152—158、169—179、218—231、240—255、296—310、313—328、344—359、364—373、413—422和472—483位氨基酸处。NS5A蛋白的B细胞抗原表位主要位于15—18、76—81、154—158、169—178、218—228、297—309、348—358、365—373和414—422位氨基酸处。【结论】成功表达并鉴定了牦牛源BVDV的非结构蛋白NS5A,系统发育进化树表明BVDV GSTZ株基因型属于BVDV-1型,NS5A蛋白具有良好的抗原性,为深入解析BVDV非结构蛋白NS5A的自身结构功能、免疫学特性以及进一步研究非结构蛋白对病毒复制的影响提供参考。     

禽流感分离株GD/1/96(H5N1)非结构蛋白结构建模

为预测H5N1亚型禽流感病毒(AIV)NS1蛋白全长结构,本研究以A/Goose/Guangdong/1/96(H5N1)株NS1蛋白氨基酸序列为目标序列,通过同源建模以及蛋白质结构叠合预测出NS1蛋白全长单体以及二聚体初始结构,采用约束分子动力学方法对初始结构进行优化,并利用立体化学、折叠可靠性、残基包装质量等评判方法对优化后的模型进行评估。结果表明所建立的NS1蛋白全长单体及二聚体结构模型具有很高的可信度,可用于基于结构的功能研究及虚拟筛选等工作。     

蓝舌病毒结构与组装机制研究进展

蓝舌病毒(Bluetongue virus,BTV)是一种以媒介昆虫为传播媒介侵染野生反刍动物和家畜的世界范围流行的病原微生物。BTV颗粒是由10个基因片段组成的双股RNA病毒,分别编码7个结构蛋白(VP1-VP7)和至少4个非结构蛋白(NS1、NS2、NS3/3a和NS4)组成的连续蛋白质层的复杂结构,BTV已被作为大型无囊膜dsRNA病毒研究的模型系统。近年来,通过反向遗传学(RG)、低温电子显微镜(Cryo-EM)、蛋白晶体结构解析等研究分析方法,为BTV病毒蛋白结构、病毒蛋白之间功能关系、病毒组装/拆卸等方面取得了相当大的研究进展,该文对BTV病毒衣壳蛋白结构、核心蛋白的组装、基因组RNA组装等方面机制进行了综述。     

猪瘟病毒非结构蛋白NS5A与Beclin1相互作用并促进病毒增殖

为探究宿主蛋白Beclin1在猪瘟病毒(classical swine fever virus, CSFV)非结构蛋白NS5A激活细胞自噬反应过程中的作用及具体分子机制,本研究在感染CSFV及表达NS5A蛋白的ST细胞中,利用qRT-PCR方法检测Beclin1、PI3K/Akt通路相关因子表达变化情况;利用激光共聚焦、Co-IP及GST-pulldown等方法研究Beclin1与NS5A相互作用关系;通过在ST细胞中过表达或敲低Beclin1,研究其对CSFV复制的影响。结果表明,ST细胞感染猪瘟病毒或外源表达NS5A蛋白,Beclin1转录和蛋白表达水平均显著升高,且PI3K/Akt通路相关因子表达水平与之呈正相关。此外,CSFV NS5A蛋白与Beclin1蛋白在细胞中存在共定位且具有相互作用。最后,作者发现在细胞中过表达Beclin1,对CSFV复制起到明显促进作用;反之,利用siRNA敲低Beclin1后,抑制PI3K/Akt通路活化,CSFV增殖表现出明显抑制效应。以上结果表明,Beclin1蛋白对CSFV复制具有促进作用,其机制是通过与NS5A的相互作用调控PI3K/Ak...     

Preliminary Study on a Novel Atypical Congenital Tremor of Piglets and Its Etiology

To investigate the infection of atypical porcine pestivirus (APPV) in China, autopsywas carried out for clinical suspected piglet of congenital tremor, viscera pathologic changes were observed. Two pairs of primersfor detection of the NS2-3 and NS5B genes by RT-PCR were designed according to the genome of APPV. The positive product was sequenced using DNAStar and Mega 7.0 softwares,and drawing the molecular evolution trees. The results showed that the lesions were similar to classical swine fever, but without infarction of the spleen and peripheral hemorrhage of the mandibular lymph node.Epidemic materials amplified products were consistent with the expected size, the sequences were APPV fragments by BLAST. APPV and other pestivirus belonged to two branch with the homologies about 70%. The homologies of NS2-3 and NS5B genes among the isolates and the APPV reference strains were about 76.9% to 98.8%, and the homologies of NS2-3 gene of 7 isolates were 89.7% to 94.8%,the homologies of NS5B gene of 12 isolates were 82.2% to 100.0%. This was the first report for the infection of APPV in piglets in China.     

一种新型仔猪先天性震颤及其病原的初步研究

为了解国内猪群是否存在新型仔猪先天性震颤及其病原,本试验对临床疑似仔猪先天性震颤病例进行剖检,观察组织脏器的病理变化;根据GenBank上登录的瘟病毒基因组序列设计2对引物,以典型震颤仔猪的病料RNA为模板,进行RT-PCR,并对阳性产物进行测序;用DNAStar和Mega 7.0软件对所测序列进行分析,绘制系统进化树。结果表明,疑似仔猪先天性震颤病猪的组织脏器病理变化与猪瘟的病理变化相似,但无明显的脾脏梗死和淋巴结周边出血;从采集的病料中均扩增到与预期大小基本一致的条带;所测序列经BLAST比对,均为仔猪先天性震颤瘟病毒（APPV）的相应基因序列片段;系统进化树结果显示,哺乳动物瘟病毒可分为两大进化分支,APPV为单独一分支,其他瘟病毒为另一分支,两分支的同源性低于70%;所测的NS2-3和NS5B基因序列与GenBank上登录的APPV相应序列同源性在76.9%~98.8%之间,表明本研究所测定的序列是APPV的基因组序列;测定的7条NS2-3基因序列之间的同源性在89.7%~94.8%之间,12条NS5B基因序列的同源性在82.2%~100.0%之间。本试验首次证实国内猪群中存在新型仔猪先天性震颤病及其病原（APPV）。     

Ovine pestiviruses: their taxonomic status revealed by palindromic nucleotide substitutions

We examined previously identified border disease virus (BDV) strains by using a newly proposed genotyping procedure based on palindromic nucleotide substitutions (PNS) in the 5'-untranslated region (UTR), and found 22 (41.5%) out of 53 strains of BDV in the nucleotide sequence databases are not of BDV. All the 22 ovine pestivirus strains were allocated to the BVDV species according to the PNS, and were compared with reference strains of pestivirus 1 (BVDV-Ia,-Ib, and-Ic genovars), pestivirus 2 (BVDV-II genovar), pestivirus 3 (BDV) and pestivirus 4 (CSFV), respectively. Ten strains (Weybridge, A553, B1056, D771/1, D861, D1120/1, D1432/P, Q1161/1, Q1161/2, 114817) showed a palindromic structure in the 5'-UTR characteristic to the BVDV-Ia genovar, three strains (7535, 7546, 7548) were characteristic to the BVDV-Ib genovar, and nine strains (BD-78, 59386, SCP, Lees, C413, 167237, 168149, 173157, 175375) belonged to the BVDV-II genovar.     

Mapping of two antigenic domains on the NS3 protein of the pestivirus bovine viral diarrhea virus

The immunodominant NS3 (p80) protein of the pestivirus bovine viral diarrhea virus (BVDV) functions as a serine protease and a RNA helicase. To identify antigenic domains of the BVDV NS3, a panel of monoclonal antibodies (mAbs) was tested against fragments of the protein expressed in E. coli. Two large overlapping NS3 fragments, A (amino acids [aa] 1-434) and B (aa 368-683) which together contain all NS3 sequences, were used to screen mAbs for reactivity. Two mAbs, 21.5.8 and 1.11.3, were reactive to fragment A (in ELISA only) and one mAb, 20.10.6, was reactive to fragment B (in ELISA and Western blotting). Further mapping demonstrated that the smallest fragment mAbs 21.5.8 and 1.11.3 bound to was comprised of aa 205-369 (domain A). In Western blotting, the smallest fragment reactive with mAb 20.10.6 was comprised of aa 368-549 (domain B). However, in indirect ELISA, mAb 20.10.6 also demonstrated high reactivity to a smaller fragment comprising aa 368-512 (domain B'). This indicated that the epitope of mAb 20.10.6 was conformational and not linear. Blocking ELISAs using these mAbs and type 1 and type 2 BVDV antisera demonstrated that an immunodominant region of the NS3 protein in cattle is defined by aa 205-549.     

A multiplex RT-PCR assay for the rapid and differential diagnosis of classical swine fever and other pestivirus infections

Classical swine fever is a highly contagious viral disease causing severe economic losses in pig production almost worldwide. All pestivirus species can infect pigs, therefore accurate and rapid pestivirus detection and differentiation is of great importance to assure control measures in swine farming. Here we describe the development and evaluation of a novel multiplex, highly sensitive and specific RT-PCR for the simultaneous detection and rapid differentiation between CSFV and other pestivirus infections in swine. The universal and differential detection was based on primers designed to amplify a fragment of the 5′ non-coding genome region for the detection of pestiviruses and a fragment of the NS5B gene for the detection of classical swine fever virus. The assay proved to be specific when different pestivirus strains from swine and ruminants were evaluated. The analytical sensitivity was estimated to be as little as 0.89 TCID₅₀. The assay analysis of 30 tissue homogenate samples from naturally infected and non-CSF infected animals and 40 standard serum samples evaluated as part of two European Inter-laboratory Comparison Tests conducted by the European Community Reference Laboratory, Hanover, Germany proved that the multiplex RT-PCR method provides a rapid, highly sensitive, and cost-effective laboratory diagnosis for classical swine fever and other pestivirus infections in swine.     

Effects of feed enzymes on nutritive value of soyabean meal fed to broilers

1. The effects of two enzyme products on the nutritive value of soyabean meal (SBM) were investigated with the emphasis on changes in composition of non-starch polysaccharides (NSP) along the digestive tract. Enzyme A was a commercially available product containing mainly hemicellulase, pectinase, beta-glucanase and some protease activities and Enzyme B was an experimental product with mainly beta-galactanase activity. 2. Enzymes were added at the recommended dosage (normal) and at 5 times the recommended dosage (high) to a semi-purified diet based on maize with SBM as the sole protein source. 3. The enzymes had no effect on digesta viscosity in the jejunum or ileum. 4. Enzyme A at the high dosage significantly (P<0.05) improved AMEN, reduced excreta moisture content and improved ileal protein digestibility. The addition of the same enzyme at the recommended dosage had no effect on any of the above parameters. 5. Analysis of the monosaccharide composition revealed that Enzyme A tended to reduce the amounts of rhamnose and galactose in the soluble and insoluble NSP fractions in thejejunal and ileal digesta. The reduction was significant (P<0.05) when the same enzyme was added at the high dosage. 6. Enzyme B significantly (P<0.05) improved AMEN of the diet but not the growth or the feed conversion ratio (FCR) of the birds. Enzyme B at the high dosage significantly reduced (P<0.05) ileal protein digestibility. 7. Enzyme B significantly (P<0.05) increased the amount of free sugars in thejejunum and reduced (P<0.05) the concentration of soluble NSP in the ileum. 8. Analysis of the monosaccharide composition in the jejunal and ileal digesta showed that this enzyme was highly effective in releasing galactose from both the soluble and insoluble NSP fractions. 9. It is concluded that glycanases with galactanase and pectinase activities supplemented at appropriate dosages can improve the digestibility of the NSP in SBM and increase the metabolisable energy content of the diet containing high levels of SBM. 10. Furthermore, the addition of Enzyme B at the high dosage significantly (P<0.05) reduced protein digestibility without any measurable reduction in growth performance.     

Effect of food enzymes on utilisation of lupin carbohydrates by broilers

1. The effects of 3 commercial enzyme products on the nutritive value of 2 lupin species were investigated with the emphasis on changes in composition of non-starch polysaccharides (NSPs) along the digestive tract. Enzyme A contained primarily cellulase, beta-glucanase and xylanase activities, enzyme B primarily hemicellulase, pentosanase and xylanase activities, and enzyme C primarily hemicellulase, pectinase and beta-glucanase activities. 2. The enzymes were added to semi-purified diets based on sorghum and casein containing 35% whole seed lupins (Lupinus angustifolius cv Gungurru or Lupinus albus cv Kiev mutant). Control diets contained no lupins. 3. Food conversion ratio (FCR), excreta moisture content and apparent metabolisable energy (AME) were affected by lupin species but not by enzyme supplementation. 4. In diets with L. angustifolius, enzyme C significantly increased digesta viscosity and increased the concentration of soluble NSPs in all sections of the intestine. 5. Digestibility of protein and NSPs in the ileum and microbial fermentation in the ileum and caeca were not affected by adding enzymes to diets containing L. angustifolius. 6. Enzyme addition to diets with L. albus did not affect digesta viscosity nor concentration of soluble NSPs but caused a significantly (P<0.05) reduced concentration of insoluble NSP in the ileum. 7. Enzyme addition to L. albus significantly (P<0.05) increased NSP digestibility in the ileum but had no effects on protein digestibility and fermentation in the ileum and caeca.     

Characterization of NS3, NS5A and NS5B of classical swine fever virus through mutation and complementation analysis

In order to get further insight into the organization of the pestiviral replication machinery, characterization of NS3, NS5A and NS5B of classical swine fever virus (CSFV) through mutation and complementation analysis was performed. Mutation analysis in genomic replicons and subgenomic replicons indicated importance of the GDD motif in NS5B, the DEYH motif in NS3 and the conserved sequence C2717-C2740-C2742-C2767 in the NS5A for CSFV recover and viral RNA synthesis. Complementation experiments were performed between subgenomic replicons, between RNA replicons or between RNA replicon and expressed nonstructural protein. Rescue of virus and recover of viral RNA synthesis were examined in these complementation experiments. Results showed that mutations within NS5A, neither NS5B nor NS3, can be trans-complemented, strongly suggesting that NS5B and NS3 function in cis mode for regulation of replication. We assumed that the necessary membrane association of CSFV NS5B and NS3 could occur only when they are being translated and originated from an identical translation template, with the exception of NS5A whose membrane association might occur post-translationally.     

猪繁殖与呼吸综合征病毒蛋白质研究进展

猪繁殖与呼吸综合征病毒(PRRSV)是一种折叠的正义RNA病毒,属于动脉炎病毒科(Arteriviridae),尼多病毒目(Nidovirales)。除了动脉炎病毒科,尼多病毒目还包括冠状病毒科(Coronaviruses)。15.1~15.2 kb长的PRRSV基因组通过互相重叠的开放性阅读框编码蛋白质。ORF1a翻译过程中产生pp1a多聚蛋白。ORF1b表达产生了 pp1ab多聚蛋白。pp1a和pp1ab经病毒蛋白酶处理后又生成了14个非结构蛋白。一些非结构蛋白为蛋白酶(NSP1、NSP1β、NSP2和NSP4)、RNA-依赖性RNA聚合酶(NSP9)、解旋酶(NSP10)和核酸内切酶(NSP11)。ORFs2-5编码GP2-GP5,ORF6编码M,ORF7编码N蛋白。ORF2b完全包括在ORF2内,表达小的非糖基化E或2b蛋白。相当一部分的囊膜蛋白是nidoviruses所特有的,所有的结构蛋白都是感染所必需的。