牛病毒性腹泻病毒P125基因重要区的比较与分析

牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)为黄病毒科瘟病毒属中的成员。根据在细胞培养物中是否产生病变,可将BVDV分为致细胞病变(CP)和非致细胞病变     

牛病毒性腹泻病毒生物型的遗传基础

牛病毒性腹泻病毒(BVDV)是黄病毒科、瘟病毒属成员,主要引起牛免疫抑制、腹泻及繁殖障碍,是影响全球养牛业的重要致病原。根据BVDV在体内对细胞的致病性,目前普遍将其分为2种生物型。不同生物型的基因结构及分子致病学机制明显不同,病毒基因变异可能产生新的生物型致病。我们简要综述BVDV生物型与基因结构及疾病发生之间的关系。     

牛病毒性腹泻病毒的初步鉴定

近年来,我们在内蒙羔羊下痢病流行区采集到一株牛病毒性腹泻病毒。83年到85年间,对内蒙三盟,二市,六旗(县),二个良种场的229份病粪样做了病毒检测,其阳性率达59％。该病毒粒子为直径40—80nm,具有囊膜的球形颗料。且与BVDV(牛病毒性腹泻病毒)的NADL株及Danish株有共同抗源。该病毒感染羔羊已传至22代,经补体结合反应测定,其效价提高了6倍。病羔羊的解剖病变观察,说明有病毒性肠炎的病变。病羊的恢复期血清测定为阳性。根据上述结果,我们初步鉴定该病毒为BVDV的一个株,暂命为BVDV的1nL株。     

牛BVDV C_(24)V株P80蛋白在大肠杆菌中的高效表达

根据GenBank中已发表的牛病毒性腹泻-黏膜病病毒(BVDV)P80基因序列,自行设计并合成引物,经过反转录-聚合酶链反应(RT-PCR)从BVDV C_(24)V株细胞培养液中扩增出P80基因,大小为560bp。利用酶切方法把目的基因克隆到大肠杆菌IPTG诱导表达载体PET32a(+)中,在大肠杆菌中得到了高效表达,这为建立ELISA方法检测BVDV抗体奠定了基础。     

计算机在确定牛病毒性腹泻病毒基因组常用限制性内切酶位点中的应用

应用计算机测定71种限制性内切酶在牛病毒性腹泻病毒NADL株(BVDV NADL)基因组的cDNA核苷酸序列中酶切位点,结果表明其中S8种酶的酶切位点所在的核苷酸序列数,也表明了其中13种限制性内切酶在核苷酸序列中无酶切位点。这些结果有助于进一步研究BVDV InL株的cDNA基因库。     

系统发生分析发现牛病毒性腹泻病病毒新基因亚型

本研究对我国首次分离获得的牛源牛病毒性腹泻病毒(BVDV)毒株Changchun 184(CC-184)和猪源牛病毒性腹泻病毒ZM-95进行了遗传衍化关系研究.选择主要抗原E2基因为研究对象,首先应用RT-PCR及套式PCR克隆得到CC-184和ZM-95的E2片段,通过序列测定发现CC-184和ZM-95 E2基因长度分别为1,122bp和1,125bp,各自编码374和375个氨基酸残基.核酸序列同源性比较和系统发生分析表明2株病毒均属于BVDV-1,CC-184与Osloss亲缘关系最近,都属于已有的b基因亚型,其E2基因同源性达91.8%.而ZM-95的E2基因有一个特征性的变异区,包含一个密码子序列插入,这一变异区编码了一段有别于其他瘟病毒的五肽氨基酸序列HYKKK.结果还表明ZM-95与BVDV-1现有的5个基因亚型的亲缘关系均较远,E2基因同源性最高(与Oregonc24v)只有72.4%.而BVDV 1亚型内毒株间的同源性大于85%,亚型间的同源性在69%～75%之间,充分说明ZM-95是BVDV-1中一个新发现的基因亚型.通常认为猪源BVDV来源于牛,应该与牛源BVDV有十分近的遗传关系,但是本研究发现ZM-95与其他已知牛源BVDV较低的基因同源性说明猪源BVDV还具有独立的遗传衍化与传播来源的可能性.     

牛病毒性腹泻病毒的分子生物学研究进展

牛病毒性腹泻病毒（Bovineviraldiarrheavirus,BVDV）,也称牛病毒性腹泻一粘膜病病毒（Bovinevlraldi。h。一mucosaldi。。virus,BVD一MDV）,在分类学上属黄病毒科（Flaviviri－d。）,瘟病毒属（Pestiv＿）［l］。鉴于其基因结构及分子生物学特征近来也有人提议新设一瘟病毒科［’,’l。BVDV是瘟病毒属的代表病毒,与属内的猪瘟病毒（HOgCh。le。Vi。,HCV;或称CI。icalswinefevervi。s,CSFV）及羊边界病病毒（Borderdi。asevirus,BDV）,在血清学上有交叉反应［‘］,BVDV感染牛可引起多种临床症状,如：高热、流…     

我国牛病毒性腹泻流行现状与防控策略

我国牛病毒性腹泻病(Bovine viral diarrhea,BVD)的流行比较复杂,其病原BVDV (BVDV-1和BVDV-2)不仅仅局限于已知易感动物牛群感染,其他动物种群中感染BVDV-1和BVDV-2的现象也值得注意,如猪群中BVDV感染很大程度上混淆了猪瘟等病原的监测,从而加剧病程发展。牛病毒性腹泻病毒(Bovine viral diarrhea virus,BVDV)可致持续感染(Persistent infection,PI),这一特性导致该病的净化面临巨大困难,对整个养殖场的健康发展形成了严峻威胁。BVDV抗原变异速率非常快,目前BVDV-1已有22个亚型,BVDV-2有4个亚型,鉴于病原在自然界的适应和演进特性,对该病的防控措施迟后其病原的变异速度。因此,定期摸清BVDV-1和BVDV-2在我国的流行现状是实施疫病净化的第一步和关键步骤,进一步借鉴国外BVD净化成功经验,综合考虑我国国情,采取适宜的防控策略,逐步净化该病原感染,有助于促进国内养殖业的健康发展。     

囊泡相关膜蛋白A调控牛病毒性腹泻病毒复制

[背景]牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)是致犊牛腹泻的重要病原之一,而目前BVDV与宿主因子互作机理研究较少,成为限制BVDV防控的重要原因。[目的]探明囊泡相关膜蛋白A (vesicle-associated membrane protein A,VAPA)对BVDV复制的影响。[方法]根据GenBank中VAPA基因,使用Benchling和CHOPCHOP等平台设计靶向VAPA的向导RNA(small guide RNA,sgRNA),融合后克隆至慢病毒lentiCRISPR v2载体中,包装慢病毒后感染牛肾细胞(Madin-Darby bovine kidney,MDBK),使用嘌呤霉素连续筛选5代,使用Western Blot检测VAPA蛋白敲除(knockout,KO)情况;BVDV感染VAPA KO细胞不同时间后,收集细胞提取总RNA,并将等质量的RNA反转录成cDNA,使用实时荧光定量PCR (real-time quantitative PCR,RT-qPCR)和免疫荧光分析(immunofluorescence a...     

牛病毒性腹泻病毒Erns蛋白激活NOD样受体热蛋白结构域相关蛋白3炎症小体诱发细胞焦亡的研究

【目的】牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)是引起牛病毒性腹泻-黏膜病的关键病毒。BVDV的结构蛋白Erns可在病毒感染的初期削弱宿主的免疫防御,引发牛群炎症反应。核苷酸寡聚化结构域样受体(nucleotide-binding oligomerization domain, NOD)热蛋白结构域相关蛋白3 (NLRP3)炎症小体是NOD样受体(NOD-like receptor, NLRs)家族重要成员,调控炎症性疾病的发生发展,同时激活的NLRP3炎症小体能够引起宿主细胞焦亡,进而诱发级联放大的炎症反应。但BVDV Erns蛋白在BVDV感染诱发炎症反应的分子机制尚不清楚。【方法】为进一步探索Erns蛋白对BVDV感染激活NLRP3炎症小体诱发细胞焦亡的影响,构建了BVDV Erns蛋白的真核表达质粒pCMV-HA-Erns,过表达BVDV Erns蛋白,检测BVDV感染细胞中NLRP3炎症小体组分[半胱氨酸蛋白酶(caspase-1)、凋亡相关斑点样蛋白(apoptosis-associated speck-like protein, ASC)和NLRP3]、IL-1β的mRNA转录水平和蛋白表达水平,以及细胞死亡调节蛋白(gasdermin D, GSDMD)的基因表达和蛋白剪切情况,并通过扫描电镜观察牛睾丸(bovine testis, BT)细胞膜成孔及BT细胞内容物释放情况,以分析Erns蛋白诱导BT细胞产生细胞焦亡。【结果】Erns蛋白能够显著引起NLRP3炎症小体活化进而激活caspase-1,活化的caspase-1一方面切割GSDMD,形成有活性的GSDMD-N端并在BT细胞膜形成孔洞,释放内容物,诱导BT细胞发生细胞焦亡;另一方面活化的caspase-1切割pro-IL-1β,形成有活性的IL-1β,并释放到BT细胞外,引起BT细胞上清中IL-1β水平上升。【结论】系统解析了BVDV Erns蛋白激活NLRP3炎症小体介导细胞焦亡的产生,对疫苗及治疗药物的研制具有重要指导意义。     

Bovine viral diarrhea virus: its effects on estradiol, progesterone and prostaglandin secretion in the cow

Bovine viral diarrhea virus (BVDV) is a major cattle pathogen responsible for a spectrum of symptoms, including reproductive failure. This study was designed to establish the effects of BVDV infection on estradiol, progesterone and PGF2alpha secretion in the cow. Seven BVDV-free cows were challenged with non-cytopathogenic BVDV (strain Pe 515: 5x10(6) tissue culture infected dose50) so that peak viremia occurred during the initial phase of luteal development in a synchronized estrous cycle. Ovulation was also synchronized in 7 sham-infected animals. Within 2 wk of inoculation, viremia, leukopenia and serum neutralizing antibodies were recorded in all of the BVDV-infected cows but not the sham-infected animals. Between Day 4 and Day 9 post estrus the BVDV-infected cows had significantly (P<0.01) lower plasma estradiol levels than the sham-infected animals. However, the BVDV infection did not alter rectal temperatures, plasma progesterone concentrations or PGF2alpha secretion 17, 18 and 19 d post estrus. These data highlight a potential causal link between BVDV viremia, endocrine dysfunction and poor fertility in the cow.     

Evidence of a humoral immune response against the prokaryotic expressed N-terminal autoprotease (N<Superscript>pro</Superscript>) protein of bovine viral diarrhoea virus

Bovine viral diarrhoea virus (BVDV) is an economically important pathogen of cattle and sheep belonging to the genus Pestivirus of the family Flaviviridae. Although the BVDV non-structural N-terminal protease (N^pro) acts as an interferon antagonist and subverts the host innate immunity, little is known about its immunogenicity. Hence, we expressed a recombinant BVDV N^pro-His fusion protein (28 kDa) in E. coli and determined the humoral immune response generated by it in rabbits. The antigenicity of the N^pro protein was confirmed by western blot using anti-BVDV hyperimmune cattle, sheep and goat serum, and anti-N^pro rabbit serum. When rabbits were immunized with the N^pro protein, a humoral immune response was evident by 4 weeks and persisted till 10 weeks post immunization as detected by ELISA and western blot. Despite N^pro-specific antibodies remaining undetectable in 80 serum samples from BVDV-infected sheep and goats, BVDV hyperimmune sera along with some of the field cattle, sheep and goat sera with high BVDV neutralizing antibody titres were found positive for N^pro antibodies. Our results provide evidence that despite the low immunogenicity of the BVDV N^pro protein, a humoral immune response is induced in cattle, sheep and goats only with repeated BVDV exposure.     

Impact of BVDV infection of white-tailed deer during second and third trimesters of pregnancy

While it has been demonstrated that persistent bovine viral diarrhea virus (BVDV) infections can be established in white-tailed deer (Odocoileus virginianus) following in utero exposure in the first trimester of gestation, there is little to no information regarding the outcome of infection in later stages of pregnancy in deer. Our goal was to observe the impact of infection of white-tailed deer in the second and third trimesters of pregnancy. Five white-tailed deer in the second trimester of pregnancy and four in the third trimester were infected with a BVDV type 2 virus previously isolated from a BVDV-infected deer harvested from the wild. Infection of deer in the second trimester of pregnancy resulted in loss of the pregnancy in three of five deer. Fawns born to the two remaining deer appeared normal and were born BVDV antigen-negative with neutralizing serum antibodies against BVDV. Infection of does in the third trimester of pregnancy did not result in fetal death or persistent infection and all does gave birth to live, healthy fawns that were BVDV antigen-negative and born with antibodies against BVDV. These results, combined with those previously reported regarding BVDV infection in the first trimester of pregnancy, suggest that the impact of BVDV infection of pregnant white-tailed deer is very similar to that observed in pregnant cattle.     

Characterization of cross-reacting antigens on the Epstein-Barr virus envelope and plasma membranes of producer cells.

A rabbit antiserum has been prepared against the B95-8 transforming strain of EBV. The antiserum has a high virus neutralizing titer (approximately 1:1000) against both the marmoset B95-8 EBV and the human P3HR-1 EBV. The neutralizing antibodies may be absorbed completely with EBV producer cell lines, but not with nonproducer cell lines or producer cell lines treated with phosphonoacetic acid (PAA) so as to be nonproducer. After repeated absorption with PAA-treated B95-8, the serum remains reactive with the membranes of producer cell lines as judged by immunofluorescence or the 125I--Staphylococcal protein A radioimmunoassay. Thus the neutralizing antigens are expressed on the membranes of producer cell lines and may be purified from this source using the serum and 125I--Staph A binding as an assay. The ability of the serum to differentiate between producer and nonproducer cells by means of cell surface determinants has been exploited to achieve a separation of these two populations from the same culture. Immunoprecipitation by the protein A technique shows that the serum recognizes two polypeptides from producer cells of approximate molecular weights 150,000 and 75,000.     

抗禽流感病毒H5N1亚型单克隆抗体制备初报

目的制备禽流感H5N1亚型病毒的单克隆抗体,为相关研究提供工具。方法以禽流感H5N1亚型病毒免疫BALBc小鼠,取其脾细胞和SP20细胞融合,用血凝抑制试验(HI)和酶联免疫反应(ELISA)检测培养上清,并将阳性融合细胞稀释克隆化3次直至100%孔均为阳性,筛选阳性克隆株,运用免疫荧光法评估单克隆抗体检测病毒感染的犬肾细胞(MDCK)。结果得到三株稳定分泌抗体的细胞并命名为F8、F9、G11,抗体亚型鉴定结果分别为IgG1、IgG2a和IgG2b;在免疫荧光法单克隆抗体能够检测出感染MDCK细胞的病毒。结论建立了3株抗禽流感H5N1亚型病毒的单克隆抗体细胞株,其产生的一株高特异性的McAbG11能够用于H5N1亚型禽流感病毒感染诊断,并可能应用于禽流感病毒H5N1亚型感染的防治。     

牛病毒性腹泻病毒Erns蛋白的中华仓鼠卵巢细胞表达及免疫原性分析

本研究利用中华仓鼠卵巢(Chinese hamster ovary,CHO)细胞表达系统制备牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV) E^rns蛋白,并分析其免疫原性。以BVDV-1 NADL标准毒株基因序列为基础,构建BVDV E^rns蛋白重组真核表达质粒pcDNA3.1-BVDV-E^rns,转染悬浮培养的CHO细胞,进行上清分泌表达。SDS-PAGE分析E^rns蛋白的表达和纯化,并用抗His单克隆抗体和BVDV阳性血清进行Western blotting鉴定纯化蛋白;进一步使用纯化的E^rns蛋白免疫新西兰大白兔,通过间接酶联免疫吸附试验(enzyme linked immunosorbent assay,ELISA)和细胞间接免疫荧光(indirect immunofluorescence,IFA)实验检测血清抗体水平及其免疫反应活性,用病毒中和实验测定免疫兔血清的中和抗体滴度。BCA蛋白定量试剂盒检测纯化的E^rns     

抗胆汁螺杆菌单克隆抗体的制备

目的制备抗胆汁螺杆菌单克隆抗体（McAbs）。方法用胆汁螺杆菌B2m株皮下免疫BALB/c小鼠,采用杂交瘤技术进行融合。以酶联免疫吸附实验（ELISA）筛选抗胆汁螺杆菌单克隆杂交瘤细胞株并初步鉴定其特异性;免疫印迹试验测定单抗所结合的抗原表位;免疫双向扩散试验确定IgG亚类;腹腔接种法、辛酸-硫酸铵盐析法大量制备、纯化单克隆抗体。结果经过ELISA筛选获得11个阳性杂交瘤细胞株,其效价最高达1：4×10^5以上,并与实验动物常见的15种病原菌呈阴性反应;IgG亚类为IgG2a和IgG2b;免疫印迹试验显示,6株（A-F）与胆汁螺杆菌大约相对分子质量（172、0、21、30、52、66）×10^3的抗原特异结合,5株（G-K）皆与胆汁螺杆菌、幽门螺杆菌等三种螺杆菌大约相对分子质量（52、82）×10^3的抗原呈阳性反应,表明A-F株针对的是胆汁螺杆菌特异性抗原,G-K株可能具有属特异性。结论筛选的单克隆抗体具有较高的特异性和敏感性,所结合的抗原为胆汁螺杆菌或螺杆菌的免疫优势抗原,为进一步的种、属生物学特性研究、菌株分型及血清学检测方法建立等奠定了基础。     

人和猴T淋巴细胞表面TRBC受体和E受体的比例研究

In 1985, rosette formation of human and macaque pan-T lymphocytes with tree shrew red blood cells (TRBC) (TRBC rosette) was first found by Ben K et al, showing different physico-chemical properties from that of rosette formation with sheep red blood cells (E-rosette). In order to approach the correlation between TRBC receptor, E receptor (CD2) and other differentiation antigens (CDs) on T lymphocytes, rosette inhibition assay and antigenic modulation or co-modulation were performed with monoclonal antibodies (McAbs) to CDs, and the distribution of TRBC receptor in other peripheral immunocytes, cell lines was also examined. TRBC rosette appeared in 88.8% of E rosette positive peripheral blood lymphocytes (E(+)-PBL) and in 4.16% of E(-)-PBL. TRBC receptor was also found on all T cell lines tested (CEM, H33 HJ-JA 1, Jurkat, MLA-144, Molt-3, Molt-4, Molt-4 clone 8, PEER) and some myeloid lines (U 937 and HL 60), but not on human granulocytes, B cell lines (Daudi, Raji and Reh) and myeloid line K 562. The modulation or co-modulation of CD 3, TCR, CD 5, CD 6 and CD 7 with McAbs OKT 3, T 108 (F 1), T 136 (F 101-15), T 149 (M-T 604) and T 152 (7 G 5) did not affect TRBC rosette formation of PBL. TRBC rosette of human and rhesus monkey PBL was not inhibited by T 11.1 McAb OKT 11 (CD 2 McAb), in contrast human and rhesus monkey E rosette formations were obviously blocked at inhibition rates of 77.9% and 49.3%, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)