共查询到20条相似文献，搜索用时 203 毫秒
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A K Cheung 《American journal of veterinary research》1990,51(2):222-226
Pseudorabies virus (PRV) immediate-early (IE) protein is a nonglycosylated polypeptide localized in the nuclei of infected cells. The IE protein is a regulatory protein that is only synthesized during viral replication and is presented to the immune system of PRV-infected swine. Antibodies to the IE protein were demonstrated in swine with induced or naturally acquired infection. However, antiserum raised against purified IE protein could not neutralize PRV in vitro. 相似文献
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T Murata X Xuan H Otsuka 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》1999,61(5):453-457
Bovine herpesvirus-1 (BHV-1) has been used as a vector of live recombinant vaccines for cattle which express the genes of other pathogens. Because of the importance of the choice of the promoter which allows the efficient expression of the foreign genes in the BHV-1 vector, we compared the relative efficacy of various promoters integrated in the BHV-1 genome. The promoter sequences of the BHV-1 thymidine kinase (tk), gB, gC, SV40 early, and pseudorabies virus (PRV) immediate early (IE) genes were placed at the upstream of the open reading frame of the chloramphenycol acetyl transferase (CAT) gene and the promoter-CAT sequences were integrated into the tk gene of BHV-1 by homologous recombination. The promoter activity was assayed by measuring the CAT activity in the extracts of Madin Darby bovine kidney (MDBK) cells infected with the recombinant BHV-1. The PRV IE promoter was activated earlier and maintained at a higher level activity than the BHV-1 gB or gC promoters throughout the most of the growth phase of BHV-1. At the late phase, however, the activities of the BHV-1 gB and gC promoters reached the higher level. The BHV-1 tk promoter activity was low and the SV40 early promoter was hardly activated when integrated into the BHV-1 genome. promoter, recombinant BHV-1. 相似文献
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伪狂犬病病毒(Pseudorabies virus,PRV)变异株(JS-2012株)在Vero细胞上40℃传代培养120代后,获得了1株弱毒株(JS-2012-F120株)。该毒株UL46基因3'端有29个碱基插入,导致了130个氨基酸突变。为了研究UL46基因移码突变对PRV生物学特性的影响,本研究用JS-2012-F120的ΔUL46基因替换了JS-2012的UL46基因,构建了重组病毒JS-2012-ΔUL46,并对该重组病毒、高温传代毒株(JS-2012-F120)及其亲本强毒株(JS-2012)在细胞上的生长特性和对小鼠的毒力进行了比较分析。结果显示,与亲本毒株(JS-2012)相比,传代毒株(JS-2012-F120)病毒滴度明显提高,对小鼠的毒力明显减弱;重组病毒(JS-2012-ΔUL46)在Vero细胞上的生长趋势没有明显改变,对小鼠的毒力减弱。因此,UL46基因移码突变对PRV在Vero细胞上的复制没有明显影响,但减弱了PRV对小鼠的毒力。 相似文献
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G Iglesias J W Harkness 《Comparative immunology, microbiology and infectious diseases》1989,12(3):47-56
Four Aujeszky's disease (pseudorabies) virus variants were characterized in vitro by investigation of their resistance to heat at 48 degrees C, sensitivity to trypsin and ability to replicate in pig alveolar macrophages, two of these variants (Ls-1 and Ls-2) were cloned previously from a single isolate of virus and showed differing pathogenicity for pigs; the virulent Stanley strain; and the non-virulent NIA-4 strain were included for comparison. Heat treatment produced slight decreases in infectivity but no significant differences were observed in the rates of inactivation. Both Ls-1 and Ls-2 were significantly more sensitive to trypsin treatment than the other two. The comparison of progeny virus titres after replication in alveolar macrophages allowed further differentiation among variants. Ls-1 and Ls-2 had similar titres in cultures infected with high virus input but in cultures infected with low virus input (0.1 TCID50/cell) Ls-1 produced higher titre. The difference in titres at 48 h post-infection was statistically significant (P less than 0.05). The cytopathogenicity for macrophages of the strains was correlated with their virulence for pigs, Stanley strain being the most cytopathogenic and NIA-4 the least. 相似文献
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为了研究伪狂犬病病毒（pseudorabies virus,PRV）立即早期180基因（immediate early 180,IE180）及其功能,试验采用设计的特异性引物通过PCR法对IE180基因进行扩增,并成功构建了IE180克隆载体,经序列测定后,用DNAStar软件对该序列和已发表的7个PRV参考株的IE180基因核苷酸序列及推导的氨基酸序列进行比对分析。结果表明,HB-98株IE180基因编码区大小为4425 bp,编码1474个氨基酸,与GenBank中登录的参考株的IE180基因核苷酸同源性为98.4%～99.1%,氨基酸同源性为97.8%～98.4%。系统进化树分析结果表明,该毒株与毒株TNL(登录号：AF352564.2）的亲缘关系较近。 相似文献
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为获得FMDV的衣壳蛋白前体P1和P1与3C基因的重组伪狂犬病毒,并评价两重组病毒的免疫效果,进行了本项研究。将构建的重组转移质粒pIESZP1和pUTK3CP1,用脂质体转染预先感染0.1 MOI PRV的Vero细胞,经120 h培养收获病毒后,再分别用蓝斑筛选和BrdU加压筛选重组病毒3~5次。经过挑斑纯化、扩增培养后用PCR、IFA检测筛选表达的FMDVP1蛋白。用这2株病毒分别免疫BALB/c小鼠,通过SN和ELISA方法检测免疫小鼠的抗体水平。成功地获得了2株可以正确表达目的蛋白的重组伪狂犬病毒rPRVP1和rPRV3CP1。2株重组病毒均可诱导小鼠产生特异性抗FMDV抗体,两重组病毒各项免疫指标之间没有明显的差异。 相似文献
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Field strains of Aujeszky's disease virus (ADV) were attenuated by heat treatment and serial passage at sub-optimal growth temperatures in chicken embryo fibroblasts (CEF). At chosen passage levels, virus was titrated in cell culture and in mice. For each strain, the pathogenicity was expressed as a mouse lethal index (MLI), defined as the inverse of the log10 (CCID50:LD50). MLIs determined for field strains displayed a wide range of comparatively high values. The attenuation of field strains was accompanied by a rapid fall in MLI values, particularly in the initial stages. Heat-treated ADV attenuated faster than untreated ADV, when passaged at 30 degrees C. Passage at 27 degrees C resulted in considerably accelerated attenuation compared to passage at 30 degrees C, in the case of both untreated and heat-treated ADV. MLIs were determined for attenuated ADV strains that had been tested in 6-day-old piglets. Low MLI values were found to correlate with low virulence in piglets and high MLI values with virulence. 相似文献
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Effect of temperature, relative humidity and medium on the aerosol stability of infectious bovine rhinotracheitis virus. 
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下载免费PDF全文 Aerosols of infectious bovine rhinotracheitis virus were generated with a Devilbiss 40 nebulizer from Eagle's minimum essential medium, nasal secretion from a noninfected calf and nasal secretion from a calf artificially infected with infectious bovine rhinotracheitis virus and aged in a rotating drum at temperatures of 6 degrees C or 32 degrees C and relative humidities of 30% or 90%. The aerosols were sampled at seven minutes after start of spraying, one hour, two hours and three hours with an all glass impinger (AGI-30) and titrated for infectivity in cell cultures. Physical decay was determined by a rhodamine B tracer technique. During spraying (seven minutes from start of spraying), the virus was usually more stable in aerosols of nasal secretion from a noninfected calf and at 90% relative humidity. In nasal secretion from a noninfected calf the virus survived best at 90% relative humidity when the temperature was 6 degrees C and best at 30% relative humidity when the temperature was 32 degrees C. During aging, biological decay was greater at the higher temperature, and at 6 degrees C, the highest decay rates occurred at 30% relative humidity in Eagle's minimum essential medium and at 90% relative humidity in nasal secretion from a noninfected calf. The stability of infectious bovine rhinotracheitis virus infected nasal secretion was not widely different from that in noninfected nasal secretion, although under certain conditions greater survival occurred in the noninfected secretion. 相似文献
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为检测伪狂犬病病毒(PRV)在体外细胞中糖蛋白H(gH)的表达情况,本研究构建原核表达重组质粒pET-gHN660,并在大肠杆菌中诱导表达重组蛋白,纯化的gH重组蛋白免疫实验动物制备抗PRV gH抗体,经western blot和IFA检测到病毒感染细胞中gM蛋白的表达,病毒感染细胞后表达的gH蛋白大小为95 ku,定位于细胞浆中,gH蛋白在病毒感染细胞4 h可以检出,随PRV的复制gH蛋白表达增加,gH蛋白可以作为PRV复制的指示蛋白.本研究利用制备的抗体分析感染细胞中gH蛋白的表达情况,初步探讨感染细胞中PRV的复制,为PRV和宿主相互作用的研究奠定基础. 相似文献
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伪狂犬病病毒Bartha株TK-/LacZ+突变株的构建及其生物学特性研究 总被引：3，自引：1，他引：2
本研究将伪狂犬病病毒Bartha株基因组与含有LacZ标志基因的TK基因转移质料pUEKPZ共转染猪肾传代细胞PK－5，细胞出现病变后，反复冻融3次收毒，按1：5稀释接种于IBRS－2细胞。在X－gal存在下挑取蓝斑，蓝斑筛选3次，再进行空斑试验，同时用PCR扩增LacZ基因，经3次空斑纯化，随机挑取的空斑均能扩增出LacZ基因，证实所获得的重组病毒为伪狂犬病病毒Bartha株TK^-/LacZ^ 突变株。TCID50试验表明，TK失活对Bartha株在细胞上增殖无影响；Balb/C小鼠试验表明，该突变对Balb/C小鼠的安全性明显高于Bartha亲本毒株。 相似文献
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【目的】 研究他莫昔芬（Tamoxifen）在PK15细胞模型上对猪伪狂犬病病毒（Pseudorabies virus,PRV）感染的抗病毒作用。【方法】 以PK15细胞为模型,采用CCK-8细胞计数法检测他莫昔芬对细胞活力的影响;利用ANNEXIN V-FITC/PI凋亡试剂盒检测他莫昔芬对细胞周期和凋亡的影响;利用CytoFLEX流式细胞仪和荧光显微镜检测他莫昔芬处理细胞感染PRV-GFP后病毒增殖的差异;利用实时荧光定量PCR和Western blotting方法分别检测他莫昔芬处理细胞感染PRV-QXX后PRV gB基因mRNA和蛋白表达水平的变化;利用病毒滴度测定法检测他莫昔芬处理细胞感染PRV-QXX后对病毒的抑制情况。【结果】 他莫昔芬用药浓度在6 μmol/L时对细胞活力无影响;在6 μmol/L以下时,与空白组相比,他莫昔芬处理组对细胞周期与凋亡没有显著影响（P>0.05）。在同一时间点,他莫昔芬处理组PRV-GFP在PK15细胞中的增殖速度极显著低于对照组（P<0.01）;实时荧光定量PCR结果表明,他莫昔芬处理后极显著抑制了PRV gB基因mRNA在PK15细胞中的表达（P<0.01）;Western blotting结果显示,不同给药浓度同一时间点,他莫昔芬都显著或极显著抑制了PRV gB蛋白的表达（P<0.05;P<0.01）;病毒滴度测定结果表明,在PK15细胞中,同一时间点他莫昔芬处理组的PRV-QXX子代病毒的复制速度显著或极显著低于对照组（P<0.05;P<0.01）。【结论】 他莫昔芬能显著抑制PRV在PK15细胞中的增殖。 相似文献
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【目的】 构建混合谱系激酶结构域样(mixed lineage kinase domain-like，MLKL)基因敲除的PK-15细胞株(PK-15 MLKL-KO)，研究敲除MLKL基因对猪伪狂犬病病毒(Pseudorabies virus，PRV)复制的影响。【方法】 根据MLKL序列设计特异性编辑位点，利用CRISPR/Cas9基因编辑技术构建MLKL-sgRNA编辑载体，转染至PK-15细胞，经嘌呤霉素药物筛选获得多克隆细胞系，通过有限稀释法获得PK-15 MLKL-KO单克隆细胞株。通过靶基因组PCR、测序和Western blotting验证MLKL基因在PK-15细胞上的敲除水平;采用Reed-Muench法检测病毒增殖水平;采用PI染色和荧光显微镜观察细胞坏死情况。【结果】 试验成功构建MLKL-sgRNA载体，筛选出1株MLKL基因缺失647 bp的PK-15细胞株，Western blotting未检测到MLKL蛋白的表达。与PK-15细胞相比，PK-15 MLKL-KO细胞极显著或显著提高了PRV GD-WH(感染后36 h除外)和PRV Bartha-K61的病毒滴度(P<0.01;P<0.05)。PRV GD-WH和PRV Bartha-K61感染PK-15 MLKL-KO细胞后，坏死细胞明显减少。【结论】 本研究构建了MLKL基因敲除的PK-15细胞株，与PK-15细胞相比，PK-15 MLKL-KO细胞显著提高了PRV GD-WH和PRV Bartha-K61的复制和存活能力，为PRV Bartha-K61疫苗生产过程中提高病毒产量提供了一种可行性策略。 相似文献
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为了解神经肽S（NPS）及其受体（NPSR）在伪狂犬病病毒（PRV）感染过程中的作用,本试验采用细胞培养、RT-PCR、实时荧光定量PCR及shRNA技术研究NPS和NPSR在PRV体外感染过程中的表达变化,以及NPS和NPSR对病毒基因和相关细胞因子的变化。RT-PCR和实时荧光定量PCR结果显示,PRV感染培养的小鼠胚胎成纤维细胞系（3T3细胞）后12和18 h,NPS和NPSR mRNA的表达水平极显著上升（P<0.01）;通过shRNA技术稳定干扰3T3细胞中NPSR表达后,PRV gE基因的表达极显著下调（P<0.01）;添加外源性NPS可显著增强PRV感染的3T3细胞PRV gE基因及细胞因子白介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的表达水平（P<0.05）;添加NPSR抑制剂后,则极显著抑制细胞中PRV gE基因和细胞因子IL-6和TNF-α的表达水平（P<0.01）,该作用效果与NPS的作用效果相反。上述结果表明,降低NPSR的表达能有效抑制PRV在3T3细胞上的复制,说明NPSR是PRV有效感染3T3细胞所必需的重要因子;外源性NPS可通过调节炎症细胞因子的表达而加剧PRV感染诱导的炎症反应。本研究结果为深入探索NPS和NPSR在PRV感染动物体内的调节作用奠定了基础。 相似文献
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为建立猪伪狂犬病病毒(PRV)gE基因缺失疫苗和野毒感染的快速鉴别诊断方法,本研究根据猪伪狂犬病野毒具有gD表面抗原基因和gE毒力基因,而基因缺失疫苗只有gD基因无gE基因的特性,针对gD/gE基因的5'端核苷酸序列自行设计引物,建立鉴别PRV gE基因缺失疫苗和野毒感染的二重PCR诊断方法,并进行了特异性、敏感性和重复性试验;利用所建立的检测方法对临床疑似样品进行了检测.结果表明,成功建立了鉴别PRV gE基因缺失疫苗和野毒感染的二重PCR诊断方法,该方法灵敏度高,最低检出限为100拷贝/μL;重复性好;特异性强,可特异性地扩增出PRV细胞毒中的gD和gE基因及gE基因缺失疫苗毒中的gD基因,但对PK-15细胞和猪流行性腹泻病毒等其他8种病原扩增不出任何条带;自835份临床疑似PRV感染病料中共检测出PRV gD和gE基因双阳性样品即野毒感染阳性样品267份,PRV gD基因单阳性样品28份,选取该方法检测出的26份PRV野毒感染阳性样品用于病原分离培养,两种方法的符合率为96.1%.说明本试验建立的二重PCR鉴别诊断方法快速、灵敏、特异,对于临床上疫苗毒和野毒感染的快速鉴别诊断具有重要意义. 相似文献
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表达H3N2亚型猪流感病毒HA基因重组伪狂犬病病毒的构建 总被引：4，自引：1，他引：4
将SV40启动子控制下的LacZ基因表达盒和CMV启动子控制下的H3N2亚型猪流感病毒（SIV H3N2）的HA基因插入到伪狂犬病病毒（PRV）通用转移载体pBdTK-Uni中，获得转移载体pLTK-HA。将该载体与PRV Bartha-K61株基因组DNA通过脂质体法共转染Vero细胞，经过10代蓝斑筛选、纯化和PCR鉴定获得了一株插入SIV HA基因的重组伪狂犬病病毒，命名为rPRV-HA。Western blotting和间接免疫荧光试验证实HA基因在重组病毒感染的细胞中获得了表达。用不同的细胞（PK-15、IBRS-2、Vero和鸡胚成纤维细胞）对该重组病毒与亲本病毒的增殖滴度和致细胞病变进行比较，未见显著差异，对第30代重组病毒的HA基因进行序列分析，表明该重组病毒遗传性状稳定。 相似文献
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Pregnant sows, immune against pseudorabies after vaccination, were inoculated at 70 days of gestation either with autologous blood mononuclear cells that had been infected in vitro with pseudorabies virus (PRV) or with cell-free PRV. The infected cells or cell-free PRV were inoculated surgically into the arteria uterina. Eight sows (A to H) had been vaccinated with an inactivated vaccine. The titer of seroneutralizing antibodies in their serum varied between 12 and 48. Five sows (A to E) were inoculated with autologous mononuclear cells, infected either with a Belgian PRV field strain or with the Northern Ireland PRV strain NIA3. These 5 sows aborted their fetuses: 2 of them (B and C) 3 days after inoculation, and the other 3 (A, D, and E) 10, 11, and 12 days after inoculation, respectively. Sows F, G, and H were inoculated with a cell-free PRV field strain. They farrowed healthy litters after normal gestation. Neutralizing antibodies were absent against PRV in the sera of the newborn pigs, which were obtained prior to the uptake of colostrum. The 23 fetuses that were aborted in sows B and C 3 days after the inoculation were homogeneous in appearance and size. Foci of necrosis were not detected in the liver. Viral antigens were located by immunofluorescence in individual cells in lungs, liver, and spleen of 15 fetuses. Virus was isolated from the liver, lungs, or body fluids of 12 fetuses. The 39 fetuses that were aborted in sows A, D, and E between 10 and 12 days after inoculation were of 2 types: 17 were mummified and 22 were normal-appearing.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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伪狂犬病病毒HS株tk基因的PCR扩增与克隆 总被引：5，自引：0，他引：5
参照伪狂犬病病毒（Ｐｓｅｕｄｏｒａｂｉｅｓｖｉｒｕｓ,ＰＲＶ）ＮＩＡ－３株ｔｋ基因序列,设计并合成１对长２２ｍｅｒ的引物,引物间距１．５ｋｂ,其内包含完整的ＰＲＶｔｋ基因。以ＢＨＫ２１细胞繁殖的ＰＲＶ湖北地方强毒株（ＨＳ－９３０４）基因组为模板进行ＰＣＲ扩增。琼脂糖凝胶电泳检测显示扩增主带清晰,长约１．５ｋｂ,符合设计要求。扩增片段克隆至由ｐＵＣ１８质粒改制而成的Ｔ载体中,限制性内切酶ＢａｍＨＩ、ＳｍａⅠ、ＸｈｏⅠ、ＨｉｎｄⅢ酶切分析证实,扩增片段的酶切位点与ｔｋ基因一致,说明扩增和克隆片段包含ＰＲＶｔｋ基因。 相似文献