兔出血症病毒镇江株的分离鉴定及VP60蛋白活性表达

试验旨在分离兔出血症病毒（rabbit hemorrhagic disease virus,RHDV）镇江株,分析其遗传进化变异,并表达具有良好活性的重组VP60蛋白。通过排除细菌感染、血凝（HA）和血凝抑制（HI）检测、动物攻毒试验与病毒传代、LD₅₀测定等方法,自江苏省镇江市某兔场发病死亡动物肝脏组织样品中分离病原并鉴定;RT-PCR方法获得VP60基因,通过分析VP60基因核苷酸及氨基酸序列研究其遗传进化;将VP60基因与pCold-Sumo载体连接,构建低温诱导、融合Sumo标签原核表达载体,15℃、IPTG诱导表达重组VP60蛋白并对表达产物进行反应原性鉴定。结果显示,分离鉴定获得RHDV ZJ2015毒株,该毒株能凝集人"O"型红血球,HA效价为11log2,其血凝性能被RHDV （AV33）抗血清抑制,该毒株的LD₅₀为10^-6.38/mL,具有较强的毒力;RT-PCR扩增得到大小约为1 740 bp的特异性条带,系统进化树分析显示,该毒株属于RHDV1抗原遗传变异株（RHDVa）,与RHDV1和RHDV2 VP60基因核苷酸序列同源性分别为89.4%~97.6%和81.1%~81.5%,氨基酸序列同源性分别为93.8%~98.3%和87.4%~87.6%。构建的低温原核融合表达质粒pCold-VP60在大肠杆菌Rosetta （DE3）中成功表达,通过SDS-PAGE及Western blotting分析,在分子质量74 ku处有特异性表达蛋白条带,且与抗血清发生特异性抗原抗体结合反应,说明重组蛋白具有良好的反应原性。本研究为开展兔病毒性出血症流行病学研究、开发新型重组疫苗与诊断试剂提供参考依据。     

兔出血症病毒CD株衣壳蛋白(VP60)基因的克隆、核酸及氨基酸序列比较分析

以兔出血症病毒 CD株人工感染家兔 ,发病死亡后 ,取肝脏匀浆 ,用 Trizol试剂从匀浆上清中提取病毒 RNA。根据 Gen Bank已发表序列保守区设计并合成引物 ,采用 RT- PCR方法扩增了 RHDV衣壳蛋白 VP6 0基因。将所扩增片段克隆到 p MD18- T载体上 ,并进行了序列测定 ,测序结果表明 ,CD株 VP6 0基因全长 174 0 bp。该序列与已发表的其他分布于世界各地的 14株 RHDV序列进行了比较和基因进化树分析。无毒株与各强毒株之间的同源性为 85 .3%～86 .5 % ,强毒株间的同源性较高 ,为 92 .1%～ 10 0 %。根据进化树可把强毒株分为 2大群 ,一群为 CD株、Iowa2 0 0 0株、0 0 - 139株、99- 0 5株 ,其余的为另一群。进一步细分 ,还可以分为 4个亚群和 7个组。但毒株之间的地域性差异并不明显。同时根据 15株 RHDV VP6 0基因的核苷酸序列推导了其所编码的氨基酸序列 ,并进行氨基酸序列的比较和亲水性、柔性区、抗原性和表面结构的比较分析。结果显示 ,各毒株氨基酸之间的的同源性在 90 .5 %～ 10 0 %之间 ,其中无毒株与各强毒株之间的同源性为 90 .5 %～ 91.9% ,强毒株间的同源性在 95 %～ 10 0 %之间 ;氨基酸变异分析未发现突变造成的 VP6 0蛋白高级结构的根本性变化     

兔IL-6基因真核表达载体的构建及其对pcDNA-VP60DNA疫苗佐剂效应研究

为研究兔白介素6（interleukin6,IL-6）真核表达质粒（pcDNA-IL-6）对核酸疫苗免疫效果的影响,本文构建了真核表达质粒pcDNA-IL-6,并将其与核酸疫苗pcDNA-VP60联合免疫家兔,以pcDNA-VP60和质粒载体pcDNA3．1（＋）作对照,用血凝抑制试验检测试验兔体内特异性抗体水平。结果表明：真核重组质粒pcDNA-IL-6对重组质粒pcDNA-VP60均具有免疫增强作用,从免疫后7d到70d,pcDNA-VP60／pcDNA-IL6联合免疫组抗体水平均高于pcDNA-VP60免疫组,差异具有显著统计学意义。     

Single dose adenovirus vectored vaccine induces a potent and long-lasting immune response against rabbit hemorrhagic disease virus after parenteral or mucosal administration

Rabbit hemorrhagic disease virus (RHDV) is the etiological agent of a lethal and contagious disease of rabbits that remains as a serious problem worldwide. As this virus does not replicate in cell culture systems, the capsid protein gene has been expressed in heterologous hosts or inserted in replication-competent viruses in order to obtain non-conventional RHDV vaccines. However, due to technological or safety issues, current RHDV vaccines are still prepared from organs of infected rabbits. In this work, two human type 5 derived replication-defective adenoviruses encoding the rabbit hemorrhagic disease virus VP60 capsid protein were constructed. The recombinant protein was expressed as a multimer in mouse and rabbit cell lines at levels that ranged from approximately 120 to 160 mg/L of culture. Mice intravenously or subcutaneously inoculated with a single 10(8) gene transfer units (GTU) dose of the AdVP60 vector (designed for VP60 intracellular expression) seroconverted at days 7 and 14 post-immunization, respectively. This vector generated a stronger response than that obtained with a second vector (AdVP60sec) designed for VP60 secretion. Rabbits were then immunized by parenteral or mucosal routes with a single 10(9)GTU dose of the AdVP60 and the antibody response was evaluated using a competition ELISA specific for RHDV or RHDVa. Protective hemagglutination inhibition (HI) titers were also promptly detected and IgG antibodies corresponding with inhibition percentages over 85% persisted up to one year in all rabbits, independently of the immunization route employed. These levels were similar to those elicited with inactivated RHDV or with VP60 obtained from yeast or insect cells. IgA specific antibodies were only found in saliva of rabbits immunized by intranasal instillation. The feasibility of VP60 production and vaccination of rabbits with replication-defective adenoviral vectors was demonstrated.     

Protective immune responses of recombinant VP2 subunit antigen of infectious bursal disease virus in chickens

Infectious bursal disease virus (IBDV) is the causative agent of Gumboro disease and poses a huge threat to poultry industry. The risks associated with conventional attenuated viral vaccines make it indispensable to probe into the development of novel and rationally designed subunit vaccines which are safer as well as effective. VP2 is the major host-protective antigen found in IBDV capsid. It encompasses different independent epitopes responsible for the induction of neutralizing antibody. Here, we report the efficacy of the immunodominant fragment of VP2 which induces both humoral and cellular immunity against infectious bursal disease. A 366bp fragment (52-417bp) of the VP2 gene from an IBDV field isolate was amplified and expressed in Escherichia coli as a 21kDa recombinant protein. The efficacy of rVP2(52-417) antigen was compared with two commercial IBDV whole virus vaccine strains. The rVP2(52-417) induced significantly high antibody titres in chicken compared to commercial vaccines and the anti-rVP2(52-417) sera showed reactivity with viral antigens from both commercial strains (P<0.0001) and field isolates. Also, the chicken splenocytes from rVP2(52-417) immunized group showed a significantly high proliferation (P<0.01) compared to other groups, which implies that the rVP2(52-417) fragment contains immunogenic epitopes capable of eliciting both B and T cell responses. Further, rVP2(52-417) conferred 100% protection against vIBDV challenge in the immunized chickens which was significantly higher (P<0.001) compared to 55-60% protection by commercial vaccine strains. Hence, the study confirms the efficacy of the immunodominant VP2 fragment that could be used as a potent vaccine against IBDV infection in chicken.     

An outbreak of rabbit hemorrhagic disease (RHD) caused by Lagovirus europaeus GI.2/rabbit hemorrhagic disease virus 2 (RHDV2) in Ehime, Japan

A total of ten 1–2-year-old rabbits died within 2 weeks at a facility in Ehime prefecture in May 2019. Necropsy revealed liver discoloration and fragility, hemorrhage of some organs and blood coagulation failure. On histopathologic examination, necrotizing hepatitis was a common finding, together with fibrin thrombi in the small vessels and hemorrhage in some organs. Rabbit hemorrhagic disease (RHD) virus gene was detected in liver samples, and viral particles of approximately 32 nm in diameter were found in the cytoplasm of degenerated hepatocytes by electron microscopy. Phylogenetic analysis based on the partial VP60 gene sequence classified it as Lagovirus europaeus GI.2/RHDV2. This is the first confirmed outbreak of RHD caused by globally emerging GI.2/RHDV2 in Japan.     

兔病毒性出血症病毒“LQ”株的分离鉴定

对成都龙泉某兔场疑似兔病毒性出血症(RHD)发病兔的病料进行细菌学检查以及血凝性、特异性鉴定,并进一步进行致病性鉴定。结果表明:RHDV LQ株对人"O"型红细胞具有高度血凝性,血凝效价达10×212;RHDV抗血清可特异性抑制RHDV LQ株对人"O"型红细胞的凝集;RHDV LQ株对家兔的LD50为10-6.87/mL,是一株对家兔具有高致病力的强毒株。     

Detection of antibodies against porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs

The humoral antibody response against the nonstructural protein NS1 and the structural protein VP2 of porcine parvovirus (PPV) was evaluated by immuno-peroxidase test (IPT) and enzyme linked immuno sorbent assay (ELISA) using recombinant PPV antigens. The coding sequence for NS1 and VP2 was inserted into the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) genome resulting in two recombinant baculoviruses AcNPV-NS1 and AcNPV-VP2, respectively. Sf9 cells (Spodoptora frugidiperda) inoculated with AcNPV-NS1 producing recombinant nonstructural protein (rNS1) and AcNPV-VP2 producing recombinant virion protein (rVP2) were used in IPT and ELISA to analyse serum antibodies. Pigs vaccinated with an inactivated whole virus vaccine and experimentally infected pigs were studied. Significant titers against rVP2 were obtained in both vaccinated and infected pigs. Specific antibodies against rNS1 could only be detected in infected pigs and NS1 may in this way allow the specific detection of infected animals. Analysis of serum samples collected up to 18 days post infection (p.i.) from four pigs experimentally infected with PPV showed that antibodies against rNS1 and rVP2 could in all cases be detected on day 9 p.i. Two individual pigs were inoculated twice with PPV and the antibody response was followed 89 days after second inoculation. Serum antibodies against borth rVP2 and rNS1 could be detected for this period of time.     

Detection of RHDV strains in the Iberian hare (Lepus granatensis): earliest evidence of rabbit lagovirus cross-species infection

Rabbit hemorrhagic disease virus (RHDV) is a highly lethal Lagovirus, family Caliciviridae, that threatens European rabbits (Oryctolagus cuniculus). Although a related virus severely affects hares, cross-species infection was only recently described for new variant RHDV in Cape hares (Lepus capensis mediterraneus). We sequenced two strains from dead Iberian hares (Lepus granatensis) collected in the 1990s in Portugal. Clinical signs were compatible with a Lagovirus infection. Phylogenetic analysis of the complete capsid gene positioned them in the RHDV genogroup that circulated on the Iberian Peninsula at that time. This is the earliest evidence of RHDV affecting a species other than European rabbits.     

Codon optimization of the rabbit hemorrhagic disease virus (RHDV) capsid gene leads to increased gene expression in Spodoptera frugiperda 9 (Sf9) cells

Rabbit hemorrhagic disease (RHD) is contagious and highly lethal. Commercial vaccines against RHD are produced from the livers of experimentally infected rabbits. Although several groups have reported that recombinant subunit vaccines against rabbit hemorrhagic disease virus (RHDV) are promising, application of the vaccines has been restricted due to high production costs or low yield. In the present study, we performed codon optimization of the capsid gene to increase the number of preference codons and eliminate rare codons in Spodoptera frugiperda 9 (Sf9) cells. The capsid gene was then subcloned into the pFastBac plasmid, and the recombinant baculoviruses were identified with a plaque assay. As expected, expression of the optimized capsid protein was markedly increased in the Sf9 cells, and the recombinant capsid proteins self-assembled into virus-like particles (VLPs) that were released into the cell supernatant. Rabbits inoculated with the supernatant and the purified VLPs were protected against RHDV challenge. A rapid, specific antibody response against RHDV was detected by an ELISA in all of the experimental groups. In conclusion, this strategy of producing a recombinant subunit vaccine antigen can be used to develop a low-cost, insect cell-derived recombinant subunit vaccine against RHDV.     

Adenovirus-based oral vaccine for rabbit hemorrhagic disease

Vaccine antigens for rabbit hemorrhagic disease virus (RHDV) are currently derived from inactivated RHDV obtained from the livers of experimentally infected rabbits or from several recombinant immunogens. However, the application of these vaccine antigens has been restricted because of biosecurity and immunity characteristics. In the current study, a recombinant adenovirus expressing the RHDV capsid protein (VP60) was constructed and the expression of the recombinant protein was identified through western blot analysis using RHDV-positive rabbit sera. Eighteen rabbits were immunized by injection, direct oral instillation, or using bait. They were challenged with RHDV isolate three weeks after boost immunization. In all cases, the rabbits immunized with the recombinant adenovirus developed RHDV-specific antibodies and cell immune response. The rabbits injected with the recombinant adenovirus were completely protected against RHDV challenge. The adenovirus expression system may provide a strategy for the immunization of rabbits, particularly for the control of RHDV in wild rabbits.     

鸡传染性法氏囊病病毒rVP2蛋白高效组装亚病毒颗粒的鉴定

为获得高纯度的鸡传染性法氏囊病病毒（infectious bursal disease virus,IBDV）VP2蛋白自组装的亚病毒颗粒（subviral particles,SVPs）,并对重组VP2蛋白（rVP2）SVPs的组装效率和均一性进行评价,本试验利用大肠杆菌表达IBDV rVP2,通过SDS-PAGE和Western blotting对蛋白表达情况和免疫反应性进行鉴定,通过阴离子交换层析和切向流超滤浓缩系统纯化rVP2蛋白,并通过透射电镜（TEM）、高效液相尺寸排阻色谱（HPSEC）、蔗糖密度梯度离心及粒径和表面电位（Zeta potential）等多种检测技术对单个SVP组装形态和整体组装情况进行分析检测,初步建立了IBDV rVP2 SVPs组装效率的系统性检测方法。结果显示,在大肠杆菌中实现IBDV rVP2的大部分可溶性表达,且能与鸡IBDV阳性血清有明显的免疫反应性,纯化后rVP2纯度提高66.9倍,回收率达到93.3%。TEM观察rVP2自组装成直径约25 nm的SVPs,视野内SVPs大小均一,均匀分散。HPSEC检测结果显示,rVP2 SVPs的分子质量约为2 482 ku,与20个VP2三聚体形成的T=1二十面体SVPs分子质量一致。蔗糖密度梯度离心检测结果显示,rVP2 SVPs体积分布均一,组装效率高。粒径和Zeta电位检测结果表明,rVP2 SVPs颗粒分散度较好、体系稳定,有利于rVP2 SVPs的长期保存。本试验通过多种检测技术对rVP2 SVPs的组装情况进行检测分析,实现了rVP2 SVPs的高效组装,为IBDV SVPs疫苗的质量控制提供了有力支撑。     

基因重组抗原酶联免疫法检测兔出血症病毒抗体及其标准化

以重组兔出血症病毒(RHDV)VP60蛋白为抗原，建立了RHDV抗体间接ELISA检测方法。优化的试验反应务件为：重组VP60的包被质量浓度为1．0mg／L，用10％牛血清封闭，以大肠杆菌提取物稀释被检血清以消除非特异性反应。将所建立的ELISA与现行血凝抑制(HI)试验比较发现，不同免疫状态的兔血清的RHDV ELISA抗体与HI抗体均呈正相关。对11个RHD免疫兔场1130份血清样品的抗体检测表明，各免疫兔群血清RHDV抗体水平不完全一致，D值在1．09～1．76之间，显著高于非免疫兔(0．05)及SPF兔(0．02)，低于高免兔(2．34)。在此基础上，研制了RHDV抗体酶联免疫检测试剂盒，测定了其主要指标，制定了各成分的质量控制标准，为兔群进行免疫学监测及评价疫苗的免疫效果提供了便利。     

中国株兔出血症病毒衣壳蛋白VP60基因在大肠杆菌中的表达及其免疫原性鉴定

应用RT—PCR技术扩增编码兔出血症病毒(rabbit hemorrhagic disease virus,RHDV)WX84株衣壳蛋白VP60基因，将PCR产物按相应的阅读框架克隆到表达性载体pGEX-6P-1中谷胱甘肽转移酶(GST)基因的下游。将重组质粒转化入大肠杆菌BL21株，在1．0mmol／L 1PTG和37C的条件下诱导，VP60—GST基因融合蛋白获了高效表达。经聚丙烯酰胺凝胶电泳和western-blotting试验证实所表达的融合蛋白产物相对分子质量与预期的87000相符。将表达产物经电泳切胶回收目的条带后免疫小鼠，所得抗血清应用间接ELISA检测，与RHDV病毒粒子呈阳性反应。试验结果表明，大肠杆菌中表达的RHDVVP60融合蛋白在抗原性上与天然衣壳蛋白具有一定的相似性。     

Efficacy of VP2 protein expressed in E. coli for protection against highly virulent infectious bursal disease virus

The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specific-pathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.     

用抗原免疫构建分泌兔出血症病毒抗独特型抗体的杂交瘤细胞

用纯化的兔出血症病毒（ｒａｂｂｉｔｈａｅｍｏｒｈａｇｉｃｄｉｓｅａｓｅｖｉｒｕｓ,简称,ＲＨＤＶ）免疫Ｂａｌｂ／ｃ小鼠。正如网络学说所指出的,从免疫鼠的杂交瘤细胞克隆中,不仅筛选出能分泌抗ＲＨＤＶ抗体（Ａｂ１）的细胞克隆,同时也筛选出能分泌抗ＲＨＤＶ独特型抗体（ａｎｔｉ－ｉｄｉｏｔｙｐｉｃａｎｔｉｂｏｄｉｅｓ,Ａｂ２）的杂交瘤细胞,用抗原而不用单克隆抗体（Ａｂ１）免疫,诱导产生抗独特型抗体（Ａｂ２）是一种具有广阔的应用前景的新技术     

兔出血症病毒VP60蛋白真核表达及其免疫原性研究

将兔出血症病毒(Rabbit hemorrhagic disease virus,RHDV)衣壳蛋白VP60基因,克隆到真核表达载体pcDNA3.1(+)中,构建pcDNA-VP60,并将其转染Vero细胞.间接免疫荧光(indirect immunofluorescence assay,IFA)检测pcDNA-VP60在细胞中的表达情况,同时将pcDNA-VP60免疫实验兔,观察血清中特异性抗体变化情况.试验结果表明,本文构建的pcDNA-VP60不仅能在Vero细胞中表达VP60蛋白,而且能够诱导机体产生免疫应答.     

兔瘟病毒VP60蛋白原核表达及其应用

为获得活性良好的兔出血症病毒基因工程抗原,本研究对兔出血症病毒ZB分离株的VP60基因进行了原核表达与初步应用。参照ZB株分离病毒VP60基因序列,设计合成一对特异性引物,PCR扩增长876bp的VP60基因片段。将目的片段定向克隆至pET30a表达载体中,经鉴定正确后,重组质粒转化BL21表达菌,经IPTG诱导后获得了以包涵体形式表达的重组蛋白,重组蛋白纯化后,Western blot检测表明具有良好的抗原性与特异性,以该蛋白作为诊断抗原,初步建立了检测兔瘟病毒抗体的间接ELISA诊断方法。本研究为RHDV分子流行病学调查提供了参考,为VP60蛋白结构与功能研究、RHDV抗体检测试剂盒及新型疫苗的研制奠定基础。     

Development and characterization of a potential diagnostic monoclonal antibody against capsid protein VP1 of the chicken anemia virus

Chicken anemia virus (CAV) is an important viral pathogen that causes anemia and severe immunodeficiency syndrome in chickens worldwide. In this study, a potential diagnostic monoclonal antibody against the CAV VP1 protein was developed which can precisely recognize the CAV antigen for diagnostic and virus recovery purposes. The VP1 gene of CAV encoding the N-terminus-deleted VP1 protein, VP1Nd129, was cloned into an Escherichia (E.) coli expression vector. After isopropyl-β-D-thiogalactopyronoside induction, VP1Nd129 protein was shown to be successfully expressed in the E. coli. By performing an enzyme-linked immunoabsorbent assay using two coating antigens, purified VP1Nd129 and CAV-infected liver tissue lysate, E3 monoclonal antibody (mAb) was found to have higher reactivity against VP1 protein than the other positive clones according to the result of limiting dilution method from 64 clones. Using immunohistochemistry, the presence of the VP1-specific mAb, E3, was confirmed using CAV-infected liver and thymus tissues as positive-infected samples. Additionally, CAV particle purification was also performed using an immunoaffinity column containing E3 mAb. The monoclonal E3 mAb developed in this study will not only be very useful for detecting CAV infection and performing histopathology studies of infected chickens, but may also be used to purify CAV particles in the future.     

兔出血症病毒VP60基因真核表达质粒的构建及初步评价

利用pMD18-T-VP60质粒（包含RHDVTP株VP60基因）PCR扩增并酶切回收VP60基因片段,插入到真核表达载体pcDNA3．1（＋）中,构建真核表达质粒pcDNA—VP60,对重组质粒进行酶切和PCR鉴定;将重组质粒转染RK13细胞,进行间接免疫荧光和Western-blot检测;将构建好的重组质粒作为核酸疫苗,腿部肌肉注射免疫小鼠,采用间接ELISA法检测抗体水平;采用淋巴细胞增殖试验（MTT法）和流式细胞术（FACS）检测细胞免疫情况;采用PCR方法检测重组质粒在小鼠体内的生物安全性。结果显示,成功构建pcDNA—VP60真核表达质粒,并在RK13细胞中获得表达。重组质粒作为核酸疫苗能够诱导小鼠体内产生特异性抗体而且抗体水平随免疫次数增加而逐渐增高,在免疫后49d时达到峰值;脾淋巴细胞刺激指数和CD4＋、CD8＋淋巴细胞亚群数量百分率明显增高,且与对照组存在明显差异。经PCR鉴定VP60基因未整合到小鼠主要脏器染色体上。结果表明,构建的pcDNA—VP60真核表达质粒能够诱导小鼠产生较强的体液免疫和细胞免疫应答,重组质粒对小鼠是安全的。