杆状病毒表达系统(The Baculovirus Expression System)

·研究论文·Chinese Journal of Animal Infectious Diseases中国动物传染病学报非洲猪瘟病毒p30蛋白在杆状病毒表达系统中的表达与免疫检测摘 要：p30蛋白是非洲猪瘟病毒（ASFV ）免疫原性最强的结构蛋白之一，由CP204L 基因编码。

本研究利用Bac-to-Bac 昆虫杆状病毒真核表达系统将ASFV 中CP204L 插入pFastBac1载体下游，将形成的重组质粒pFastBac-p30以转座子的形式插入到DH10Bac 中的杆状病毒穿梭质粒（Bacmid ）中，筛选出重组Bacmid-p30后，将Bacmid-p30转染Sf9细胞，并继续传代3次，获得重组杆状病毒，命名为rBac-p30。

分别通过间接免疫荧光（IFA ）和免疫印迹（Western blot ）鉴定了ASFV 阳性血清可特异性识别Sf9细胞中表达的p30。

以此真核表达p30蛋白包被ELISA 板，可区分ASFV 阴阳性血清。

以上结果表明，本研究初步建立了检测ASFV 血清抗体的间接ELISA 方法，为后续建立非洲猪瘟抗体检测方法和p30亚单位疫苗研究奠定基础。

关键词：非洲猪瘟病毒；p30蛋白；杆状病毒表达系统中图分类号：S852.65文献标志码：A文章编号：1674-6422（2023）04-0170-06Production and Immunological Detection of African Swine Fever Virus p30 byBaculovirus Expression SystemLIAO Xinxin 1,2, ZHONG Qiuping 2, SHI Xinjin 2, WEI Changqing 2, SUN Haiwei 2, LIU Yingnan 2,AO Qingying 2, XIE Zhenhua 2, WU Jing 1, CHEN Hongjun 2(1. Hunan Engineering Research Center of Livestock and Poultry Health Care, Colleges of V eterinary Medicine, Hunan Agricultural University,Changsha 410128, China; 2. Shanghai V eterinary Research Institute, CAAS, Shanghai 200241, China)收稿日期：2021-11-01基金项目：十三五国家重点研发专项资助项目（2018YFC08400400，2017YFD0502300）；自然科学基金联合基金项目重点支持项目-区域创新发展联合基金项目（U19A2039）作者简介：廖欣欣，女，硕士研究生，临床兽医学专业通信作者：邬静，E-mail：****************.cn；陈鸿军，E-mail：***************.cn Abstract: The p30 protein encoded by CP204L gene is one of the most immunogenic structural proteins of African swine fever virus (ASFV). In this study, the Bac-to-Bac insect baculovirus eukaryotic expression system was used to insert CP204L in ASFV into the downstream of pFastBac1 vector, and the resulting recombinant plasmid pFastBac-p30 was inserted into Baculovirus shuttle plasmid (Bacmid) in DH10Bac as a transposon. After screening the recombinant Bacmid-P30 was transfected into Sf9 cells, and the recombinant Baculovirus rBac-p30 was obtained expression of p30 protein. Indirect immunofl uorescence (IFA) and Western blot were used to identify p30 expression in Sf9 cells by ASFV positive serum specifi city. The recombinant p30 protein was used to coat ELISA plates to distinguish positive and negative serum samples of ASFV . The development of an indirect ELISA method laid the foundation for further establishment of an ASFV antibody detection method and p30 subunit vaccine research.Key words: African Swine fever virus; p30 protein; baculovirus expression system2023,31(4):170-175廖欣欣1,2，钟秋萍2，史馨瑾2，魏常青2，孙海伟2，刘英楠2，敖清莹2，谢振华2，邬 静1，陈鸿军2（1.湖南农业大学动物医学院 畜禽保健湖南省工程研究中心，长沙410128；2.中国农业科学院上海兽医研究所，上海200241· 171 ·廖欣欣等：非洲猪瘟病毒p30蛋白在杆状病毒表达系统中的表达与免疫检测第31卷第4期非洲猪瘟（African swine fever, ASF）是由非洲猪瘟病毒（African swine fever virus, ASFV）引起的猪的一种热性、急性、高度接触性传染病[1]。

山西农业科学2022，50（5）：714-719Journal of Shanxi Agricultural Sciences doi:10.3969/j.issn.1002-2481.2022.05.15doi应用杆状病毒表达系统有效表达PEDV纤突蛋白郝建伟1，薛春宜2，曹永长2（1.晋中学院生物科学与技术系，山西晋中030600；2.中山大学生命科学学院/有害生物控制与资源利用国家重点实验室，广东广州510006）摘要：为了解决猪流行性腹泻病毒（Porcine epidemic diarrhea virus，PEDV）变异毒株纤突蛋白（S蛋白）分子质量大、难以表达的问题，研究采用Signal4.0软件预测S蛋白信号肽，构建表达替换信号肽S蛋白的重组质粒，鉴定正确后重组质粒转化DH10Bac感受态细胞并进行蓝白斑筛选，挑取白斑进行菌落PCR检测，检测无误后提取Bacmid DNA转染Sf9细胞，制备重组杆状病毒。

采用杆状病毒表达系统表达目的蛋白，采用免疫印迹法（West⁃ern blot）和串联式飞行时间质谱（MALDI-TOF-TOF）技术鉴定表达蛋白，采用SDS-PAGE半定量方法测定蛋白含量；以Balb/c小鼠为材料进行免疫试验，应用中和试验、ELISPOT试验测定小鼠抗体滴度及细胞因子水平。

结果表明，S蛋白氨基端20个氨基酸为蛋白信号肽，重组质粒经双酶切鉴定构建成功，经菌体PCR检测验证表达骨架构建完成，转染Sf9细胞后获得重组病毒rB-PEDV-S；重组病毒表达蛋白经Western-blot和MALDI-TOF-TOF鉴定为目的蛋白，蛋白质量浓度可达0.0086μg/μL；替换原有信号肽可有效提高S蛋白产量，以0.86μg/只剂量免疫小鼠后，可刺激小鼠产生IL-4，但血清抗体水平与PBS组无明显差异。

替换信号肽有助于S蛋白表达，利用表达蛋白免疫小鼠可有效刺激IL-4产生。

关键词：猪流行性腹泻病毒（PEDV）；杆状病毒；免疫印迹法；串联式飞行时间质谱；蛋白表达；疫苗中图分类号：S858.28文献标识码：A文章编号：1002‒2481（2022）05‒0714‒06Effective Expression of Spike Protein of PEDV with Expression System of Bac-to-BacHAO Jianwei1，XUE Chunyi2，CAO Yongchang2（1.Department of Biological Science and Technology，Jinzhong University，Jinzhong030600，China；2.School of Life Sciences，State Key Laboratory of Biocontrol，Sun Yat-sen University，Guangzhou510006，China）Abstract：In order to solve the problem of large molecular weight and difficult expression of spike protein(S protein)of the mutant strain of porcine epidemic diarrhea virus(PEDV),in this study,the S protein signal peptide was predicted by Signal software(Version4.0),and the recombinant plasmid expressing the replacement of signal peptide S protein was constructed. Then the recombinant plasmid was transfected into DH10bac competent cells after identification,the white-blue plaque selection tests were conducted,and white spots were selected for further colony PCR detection.After the detection was confirmed,the Bacmid DNA was extracted and transfected into Sf9cells to prepare recombinant baculovirus.The target protein was expressed by baculovirus expression system and the expressed protein was identified by Western blot and MALDI-TOF-TOF methods The protein content was determined by SDS-PAGE semi quantitative method.Balb/c mice were used for immune experiment. The neutralization experiment and ELISPOT experiment were to determine the antibody titer and cytokine level.The results showed that the20amino acids in the amino terminal of S protein were protein signal peptides.The recombinant plasmid was successfully constructed through identification by double enzyme digestion.The bacteria PCR detection confirmed that the construction of the expression skeleton was completed,then,the recombinant virus rB-PEDV-S was obtained after transfected into Sf9cells.The protein expressed by the recombinant virus was identified as the target protein by verification of Western blot and MALDI-TOF-TOF,and the protein concentration was0.0086μg/μL,which showed that replacing the original signal peptide would effectively increase the yield of S protein.Immunization of mice by the S protein with the dosage of0.86μg/mice could stimulate mice to produce IL-4,but there was no significant differences between serum antibody level and that in PBS groups.The results proved that replacement of signal peptide contributed to the expression of S protein,and immunization of mice with the expressed protein could effectively stimulate production of IL-4.Key words：Porcine epidemic diarrhea（PEDV）;baculovirus;Western-blot;MALDI-TOF-TOF;expression of protein;收稿日期：2021-12-15基金项目：“十三五”重点研发计划项目（2016YFD0500101）作者简介：郝建伟（1983-），男，山西大同人，高级兽医师，博士，主要从事动物病毒学研究工作。

IntroductionThe Baculovirus Expression Vector System (BEVS) is a widely used tool for the production and expression of baculoviruses and recombinant proteins. The Sf9 and Sf21 insect cell lines, derived from the ovaries of the fall armyworm Spodoptera frugiperda, have been the cell lines of choice when using the BEVS technology. Recently, Protein Sciences Corporation1developed a new cell line derived from Spodoptera frugiperda, which is morphologically and genetically different from the Sf9 cell line2. The cell line, expres SF+®, was developed using a series of stringent selection steps in serum-free medium with added insulin2. The cell line was designed to possess characteristics that favor the commercial production of baculoviruses and recombinant proteins.EX-CELL TM420 Serum-Free Medium for nsect Cells is a serum-free, protein-free medium designed and optimized for suspension culture of Spodopteran cell lines. Previous studies with this medium have shown that Sf9 and Sf21 cells adapt easily to EX-CELL TM420, and that EX-CELL TM420 supports high cell densities, baculovirus production and secreted and intracellular recombinant protein expression3,4. Experiments were designed to assess the ease with which expres SF+®cells adapt to EX-CELL TM420, to evaluate cell growth and viability in shaker flask culture and to determine freezing conditions. The results demonstrate that expres SF+®cells quickly adapt to EX-CELL TM420, with no observed decrease in cell growth or viability. expres SF+®cell viabilities and densities in EX-CELL TM420 were comparable to those seen in cultures grown in Sf-900 I I SFM, (I nvitrogen Corporation) and expres SF+®cells frozen in fresh EX-CELL TM420 medium with 10% dimethyl sulfoxide (DMSO) exhibited viabilities of about 90% upon thawing from storage in liquid nitrogen.MaterialsCells• expres SF+®Serum-Free I nsect Cells, Protein Sciences Corporation, Catalog No. 1000Media and Supplements• Sf-900 II SFM, Invitrogen Corporation, Catalog No. 10902 • EX-CELL TM420, SAFC Biosciences, Inc., Catalog No. 14420• L-Glutamine Solution 200 mM, SAFC Biosciences, I nc., Catalog No. 59202• DMSO, Sigma-Aldrich Co., Catalog No. D-2650 Methodsexpres SF+®Culture Initiationexpres SF+®cells were handled and subcultured per the manual (Version 1.4) provided by the manufacturer. As such, a new vial of frozen expres SF+®cells was thawed rapidly with agitation in a 28 C water bath. Once a small ice pellet remained, the contents of the vial were added to 100 mL pre-warmed Sf-900 II SFM in a 250 mL spinner flask. The flask was incubated at 28 C in a non-CO2atmosphere, with constant stirring at 100 rpm. The cells were subsequently subcultured into triplicate shaker flasks (50 mL volume per 125 mL flask) at a seeding density of 1.5 x 106cells/mL. Flasks were incubated at 28 C, on an orbital shaker at 135 rpm. Cell counts and viability were monitored using trypan blue exclusion methods. Flasks were subcultured every 2 - 3 days using the same seeding density as above.Direct Adaptation and Continuous Growth of expres SF+®Cells in EX-CELL TM420Cultures initiated in Sf-900 II SFM were passed five times to ensure the cultures were well established. On the sixth pass, the cells were directly subcultured into 100% EX-CELL TM420 medium at a seeding density of 1.5 x 106cells/mL. These cultures were monitored for an additional five passes to assess cell density and viability in EX-CELL TM420. On the sixth pass, a kinetic growth curve was generated by obtaining daily cell counts, until culture viability dropped below 50%.Growth Characteristics of the expres SF+®Serum-Free Insect Cell Line in EX-CELL TM420 Serum-Free MediaTechnical BulletinUnited States SAFC Biosciences, Inc. 13804 W. 107th Street EuropeSAFC Biosciences Ltd.Smeaton Road, West PortwayAsia PacificSAFC Biosciences Pty. Ltd.18-20 Export DriveFreeze/T haw Evaluation of expres SF+®Cells in EX-CELL TM 420To evaluate freezing conditions in EX-CELL TM 420, a cell bank of expres SF+®cells was prepared. Mid-log phase cells ( > 95%viable) were harvested and centrifuged to remove spent medium. The cells were resuspended at 3 x 107cells/mL in a cryopreservation medium consisting of 90% fresh EX-CELL TM 420 medium and 10% DMSO. Cells were frozen at a controlled rate and stored under liquid nitrogen. Cell viability post-thaw was determined by thawing 3 vials (1 mL each) of cells and combining them directly into a 125 mLTo demonstrate the kinetic growth curve of expres SF+®cellsin continuous culture, expres SF+®cells were maintained in EX-CELL TM 420 for seven days (see Figure 2). Cell densities increased exponentially over the first two days then appeared to plateau between days two to four, reaching maximum densities of approximately 7 x 106cells/mL. Culture viabilities remained high (above 95%) until day four, after which viabilities dropped to below 50% by day seven. Based on these results, it was confirmed that the optimal time for subculture is during the exponential growth phase of the cells, which is between two to three days in culture.Conclusions• expres SF+®cells can be transferred directly into EX-CELL TM420 from Sf-900 I I SFM without an adaptation period.• expres SF+®cells grown in EX-CELL TM420 achieve densities of 6-7 x 106cells/mL with viabilities greater than 95%.• expres SF+®cells can be frozen and recovered in EX-CELL TM420 with only the addition of 10%DMSO. References1. Protein Sciences Corporation, Meriden, CT2. expres SF+®Serum-Free I nsect Cell Line, Manual Version1.4, Protein Sciences Corporation3. Dianne E. Potts, Justine A. Malinski, Laura T. Kakach andSarah L. Gilliland, Commercially Available Serum-Free Insect Media:A Comparison of Sf9 Growth Dynamics and Protein Production, SAFC Biosciences Literature Reference R011, 2000.4. Susan E. Lenk, Thomas W. Irish and Karen J. Etchberger,EX-CELL TM420 Serum-Free Medium for the Growth of Spodopteran (Sf9 and Sf21) Insect Cells, SAFC Biosciences Literature Reference R015, 2000.Warranty, Limitation of RemediesSAFC Biosciences warrants to the purchaser for a period of one year from date of delivery that this product conforms to its specifications. Other terms and conditions of this warranty are contained in SAFC Biosciences’ written warranty, a copy of which is available upon request. ALL OTHER WARRANTIES, EXPRESSED OR IMPLIED, INCLUDING THE IMPLIED WARRANTY OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE, ARE EXCLUDED. In no case will SAFC Biosciences be liable for any special, incidental, or consequential damages arising out of this product or the use of this product by the customer or any third party based upon breach of warranty, breach of contract, negligence, strict tort, or any other legal theory. SAFC Biosciences expressly disclaims any warranty against claims by any third party by way of infringement or the like. THIS PRODUCT IS INTENDED FOR PURPOSES DESCRIBED ONLY AND IS NOT INTENDED FOR ANY HUMAN OR THERAPEUTIC USE.Additional Terms and Conditions are contained in the product Catalog, a copy of which is available upon request.EX-CELL™ is a trademark of SAFC Biosciences, Inc.expres SF+®is a registered trademark of Protein Sciences Corporation.© 2006 SAFC Biosciences, Inc.Issued February 2006 T0660204 0805United States SAFC Biosciences, Inc. 13804 W. 107th Street Lenexa, Kansas 66215 EuropeSAFC Biosciences Ltd.Smeaton Road, West PortwayAndover, Hampshire SP10 3LFAsia PacificSAFC Biosciences Pty. Ltd.18-20 Export DriveBrooklyn, Victoria 3025。

Spodoptera frugiperda（草地夜蛾）卵巢细胞形态：上皮细胞生长特性：贴壁注释：Sf-9细胞是由G.E. Smith 和C.L. Cherry 在1983年从细胞株IPLB-SF 21 AE得来的一个克隆。

IPLB-SF 21 AE是1977年由Vaughn等从草地夜蛾蛹的卵巢组织得到的。

培养液：TNM-FH培养液：照厂家的说明配好Grace's Antheraea 培养液，每升培养液加3.3g 酵母粉和3.3g乳白蛋白水解物，用1M KOH调pH到6.2-6.4过滤除菌，4℃保存。

完整培养液在使用前加入10%热灭活的胎牛血清。

传代：用吸液管吹洗细胞使其悬浮，或用手从侧面拍打细胞培养瓶(当用大细胞培养瓶时)重悬细胞。

杆状病毒表达系统(baculovirus expression system)是近年来应用较多的真核表达系统，它可以在昆虫细胞中表达多种外源基因，包括真菌，植物，细菌，病毒的基因。

杆状病毒是一类以昆虫细胞为天然宿主的双链DNA病毒，具有高度的种属特异性，不感染脊椎动物，对人、畜无害。

目前研究较多的是苜蓿银纹夜蛾核多角体病毒(AcNPV) ，其宿主是草地贪夜蛾。

而sf9细胞，正是杆状病毒表达系统的宿主细胞之一。

sf9细胞属于半贴壁型细胞系，可以进行悬浮培养，也可以进行贴壁培养，在应用于蛋白的表达与制备纯化中，有着许多的优点。

杆状病毒表达系统的主要优越性有：①表达的重组蛋白能正确折叠，并形成二硫键；②翻译后可进行修饰加工如糖基化、磷酸化、酰胺化及信号肽切割等，使重组蛋白在结构和功能上更接近天然蛋白；③与其他真核表达系统相比，杆状病毒表达系统可以高效表达外源基因，表达量最高可达被感染昆虫细胞总蛋白量的5 0 ％；④可以容纳大片段外源基因。

杆状病毒表达系统——有效的VLP构建工具刘拂晓1,2 柳增善2 王志亮1【摘要】杆状病毒表达系统是以杆状病毒为外源基因载体，昆虫细胞或活体昆虫为受体的真核表达系统。

相对于其他表达系统，杆状病毒表达系统具有特殊的优势：杆状病毒基因组作为表达载体可以容纳更多外源基因；杆状病毒极晚期启动子能有效调控外源蛋白的表达；昆虫细胞作为受体能够对外源蛋白进行加工修饰；杆状病毒通常只感染节肢动物，不会对人畜构成危害。

因此，该系统越来越受到人们的重视，并已应用于亚单位疫苗的研发与生产，特别其对于构建病毒样颗粒，即由一种或多种病毒结构蛋白自行装配而成且不含病毒基因组的蛋白颗粒，具有不可比拟的优势。

对此做详细评述并展望病毒样颗粒疫苗的发展趋势。

【期刊名称】生物技术通报【年(卷),期】2012(000)006【总页数】7【关键词】杆状病毒昆虫细胞病毒样颗粒杆状病毒表达系统疫苗1983年，Smith等［2］成功实践了美国学者Miller的提出的杆状病毒作为载体在昆虫细胞中表达外源基因的可行性理论：他们将干扰素基因插入至苜蓿银纹夜蛾核型多角体病毒（Autographa californi-ca nuclear polyhedrosis virus，AcNPV）表达载体，然后将其转染草地贪夜蛾（Spodoptera frugiperda，Sf）细胞，成功表达了具有生物活性的人β干扰素。

此后，杆状病毒蛋白表达技术逐步建立并完善起来。

相对于传统大肠杆菌、酵母及哺乳动物细胞蛋白表达技术，杆状病毒技术在病毒样颗粒（virus-like particle，VLP）的构建及应用方面具有不可比拟的优势。

VLP是含有一个或多个病毒结构蛋白的空心颗粒，高度模仿真实病毒的衣壳空间构象而不含其基因组。

大多数VLP 是良好的免疫原，既可诱导体液免疫又可诱导细胞免疫［3-5］。

随着杆状病毒蛋白表达技术的成熟，利用其构建VLP的报道也屡见不鲜。

1 杆状病毒分子生物学根据国际病毒分类委员会最新病毒分类报告，杆状病毒科（Baculoviridae）分为4个属，即α杆状病毒属（Alphabaculovirus）、β杆状病毒属（Betabaculovirus）、δ杆状病毒属（Deltabaculovirus）、γ杆状病毒属（Gammabaculovirus）。

·研究论文·Chinese Journal of Animal Infectious Diseases中国动物传染病学报摘 要：为解决杆状病毒表达系统存在的表达量低和毒种种子批代次窄问题，加快杆状病毒表达系统产业化进程。

本研究首次通过Red 和Cas9两种重组技术，先后对影响蛋白表达的V-cath 、ChiA 、P10和重组杆状病毒基因组稳定性的FP25K 和DA26基因进行缺失。

同时基于对CSFV-E2蛋白的结构和糖基化预测分析，突变影响同源二聚体二硫键形成的糖基化位点。

最后通过悬浮转染的方式提高拯救病毒的滴度。

结果表明对E2基因突变后，表达的E2蛋白95%以同源二聚体形式存在。

通过供体质粒优化和病毒载体基因缺失，E2蛋白的表达水平提高约4倍，可稳定表达蛋白的重组杆状病毒毒株代次从P7代延长至P20代。

悬浮转染获得的重组杆状病毒滴度提高约70倍，同时获得足量低代次的毒种，有效缩短从毒种构建到蛋白表达时间，有效解决杆状病毒表达系统表达量低和毒种种子批代次窄的问题，为猪瘟亚单位疫苗研发奠定基础。

关键词：杆状病毒；猪瘟病毒；基因缺失；E2蛋白中图分类号： S852.65文献标志码：A文章编号：1674-6422（2024）01-0048-08Construction and Application of Gene Deletion Baculovirus Vector收稿日期：2021-11-03基金项目：郑洛新自创区创新引领型产业集群专项（201200211200）作者简介：王同燕，女，硕士，兽医师，主要从事兽用基因工程疫苗研究与开发；仝晓丹，女，硕士，主要从事兽用基因工程疫苗研究与开发通信作者：谭菲菲，E-mail：**************；田克恭，E-mail:**************基因缺失杆状病毒载体的构建及应用王同燕1,2，仝晓丹1,2，苏晓蕊1,2，宋欢欢1,2，李伟国1,2，刘武杰1,2，谭菲菲1,2，田克恭1,2,3（1.国家兽用药品工程技术研究中心，洛阳471003；2.普莱柯生物工程股份有限公司，洛阳471000；3.洛阳普泰生物技术有限公司，洛阳471003）2024,32(1):48-55Abstract: In order to solve the low protein production and virus passages in baculovirus expression system to accelerate the industrialization process, the v-cath , ChiA , P10 that aff ected protein production and FP25K , DA26 genes that related to stability of the genome were deleted by Red and Cas9 recombinant techniques in this study. At the same time, the glycosylation site that aff ected the formation of disulfi de bonds were changed based on the structure and glycosylation prediction analysis of CSFV-E2. Finally, suspension transfection was performed to increase the titer of rescued viruses. The results showed that 95% of the mutant E2 protein was in the form of homodimer. The expression level of E2 protein was increased about 4 times by optimizing donor plasmid and deletion of viral vector gene and the passages of the recombinant baculovirus strain with stable expression of protein was extended from P7 to P20. The titer of recombinant baculovirus obtained by suspension transfection increased by about 70 times, and suffi cient low viral passages were obtained, which eff ectively shortened the time from strain construction to protein expression and solved the problems of low protein production of baculovirus expression system and virus passages of strain seeds. Therefore, the successful construction of the gene deletion baculoviruses laid a foundation for the research and production of a subunit swine fever vaccine.Key words: Baculovirus; Classical swine fever virus; gene deletion; E2 proteinWANG Tongyan 1,2, TONG Xiaodan 1,2, SU Xiaorui 1,2, SONG Huanhuan 1,2, LI Weiguo 1,2, LIU Wujie 1,2,TAN Feifei 1,2, TIAN Kegong 1,2,3(1. National Research Center of Veterinary Medicine, Luoyang 471003, China; 2. PULIKE Biological Engineering, INC., Luoyang 471000,China; 3. Luoyang Putai Biotechnology Co., Ltd, Luoyang 471000, China)· 49 ·王同燕等：基因缺失杆状病毒载体的构建及应用第32卷第1期猪瘟（Classical swine fever, CSF）是由猪瘟病毒（Classical swine fever Virus, CSFV）引起的一种急性、热性、高度接触性传染病，给中国和其他国家养猪业造成了严重危害，被世界动物卫生组织列为必须报告的动物疫病之一[1]。