试验对安徽某蛋鸡场采集的病料进行病毒分离,获得2株鸡传染性支气管炎病毒(IBV),命名为AH01/2020和AH02/2020.对分离的病毒进行鸡胚接种、S1基因测序以及遗传进化分析.结果 显示,AH01/2020为LSC/99 Ⅰ型,AH02/2020为QX型,2株病毒均可导致鸡胚出现不同程度IBV典型病变,表现为发育不良、蜷缩等.AH01/2020、AH02/2020与参考株S1基因的核苷酸相似性分别为76.7％～97.8％和78.1％～90.9％,与常用疫苗株的S1同源性均偏低,仅76.7％～79.4％,试验结果揭示了我国IBV的基因多样性,为IBV进行遗传监测和研发新型疫苗提供了参考.

为建立快速测定猪肺炎支原体抗原含量的方法,试验应用P46单抗建立不同CCU含量的猪肺炎支原体抗原蛋白免疫印迹图谱,测定不同批次的猪肺炎支原体抗原CCU含量,并与Western blot(WB)测定的CCU进行比较.结果 显示:曝光5s,107 CCU/mL含量以上的猪肺炎支原体抗原在46 kDa处有条带,且条带粗细、明暗程度随着CCU含量的增高而增强;中试生产的7批抗原中,有3批抗原CCU和WB2种方法测定的含量相同,两批抗原测定的含量基本相同;两批抗原测定的含量有差异,但差值在1.5～5.0倍之间.结果 表明,WB可用于猪肺炎支原体抗原含量的快速测定.

用相同量猪肺炎支原体(Mhyo)和不同量猪圆环病毒2型(PCV2)混合后的4组疫苗分别免疫BALB/c小鼠、新西兰大耳白兔和健康仔猪,测定小鼠、兔和仔猪血清中PCV2和Mhyo抗体水平,并在仔猪二免后进行Mhyo济南系强毒攻击.结果:小鼠和兔免疫疫苗后产生的Mhyo抗体水平随着疫苗中PCV2抗原含量的增加而提高.仔猪免疫疫苗后,ELISA与IHA检测结果都显示Myho抗体水平随着疫苗中PCV2抗原含量的增加而提高;Mhyo攻毒后肺部病变程度也与疫苗中PCV2抗原浓度呈负相关.以上结果说明PCV2、Mhyo二联疫苗中PCV2全病毒抗原对Mhyo免疫有增强作用,且随着PCV2全病毒抗原含量的增加,Mhyo的免疫保护效果也随之增强,这个结果提示了 PCV2抗原对Mhyo抗原的免疫效果有促进作用.

本研究通过对比不同中空纤维超滤膜孔径、洗滤液及剪切速率,建立猪肺炎支原体中空纤维浓缩纯化工艺;并采用该工艺对猪肺炎支原体活菌液和灭活菌液进行浓缩纯化,测定回收率、杂蛋白去除率及纯化前后抗原免疫原性变化.结果表明:750kD和0.1μm的中空纤维膜柱纯化效果较好,杂蛋白去除率达80％以上;不含猪血清的Friis基础液洗滤,抗原回收率高于PBS洗滤;浓缩纯化过程中剪切速率可控制在2000～6000s-1.猪肺炎支原体活菌液经0.1μm中空纤维膜浓缩纯化抗原回收率达100％;灭活抗原纯化前后按相同含量配制疫苗免疫兔子,猪肺炎支原体抗体效价没有明显差别,说明纯化后免疫原性未损失.

为分离鉴定湖南地区2个猪场的猪圆环病毒2型(PCV2),从临床表现为断奶仔猪多系统衰竭综合征(PMWS)猪体采集组织病料,验证为PCV2感染的病料,用无污染猪肾细胞系(PK15)细胞分离培养.采用间接免疫荧光试验(IFA)、分子克隆及生物信息学等方法对分离病毒进行分析.结果 显示,分别获得了2株PCV2:1株为PCV2d(命名为ChenZ-2-1,GenBank号MH718995),另1株为PCV2b(命名为YiY-3-27,GenBank号KU317482),且两分离株的病毒效价TCID50分别为1×10566/mL和1×105.5/mL.本研究有利于提高PCV2的诊断水平,对深入研究PCV2的发病机理及开发PCV2新型疫苗奠定基础.

猪圆环病毒2型(Porcine circovirus Type 2,PCV2)被认为是猪圆环病毒相关疾病(PCVAD)的主要病原体,分为多个基因型和亚型,具有很高的突变率,给猪场的疫病防控带来了巨大的挑战.虽然PCV2不同基因型间存在一定的交叉免疫保护作用,但是,在国内外多个接种了PCV2疫苗的猪群中,报道分离得到了突变型PCV2d毒株(又称PCV2b-1C,PCV2 mutant或mPCV2b).为更好地防控PCV2d毒株的流行,文中就PCV2d的分子生物学特征和流行病学情况分别进行了综述.

本研究通过整理分析2012～2018年国内获得临床试验批件的兽用生物制品的数量、分类、治疗靶动物、申报单位、成功转化新兽药情况等内容,挖掘兽用生物制品领域的研究开发趋势和方向.

Although porcine circovirus-like particles can function as a vector to carry foreign peptides into host cells, displaying foreign peptides on the surface of virus-like particles (VLPs) remains challenging. In this study, a plateau, consisting of the middle portion of Loop CD (MP-Lcd) from two neighboring subunits of PCV2 capsid protein (Cap), was identified as an ideal site to insert various foreign peptides or epitopes and display them on the surface of PCV2 VLPs. One of the goals of this work is to determine if the surface pattern of this plateau can be altered without compromising the neutralizing activity against PCV2 infections. Therefore, biological roles of MP-Lcd regarding VLPs assembly, cell entry, and antigenicity were investigated to determine whether this was a universal site for insertion of foreign functional peptides. Three-dimensional (3D) structure simulations and mutation assays revealed MP-Lcd was dispensable for PCV2 Cap assembly into VLPs and their entry into host cells. Notably, substitution of MP-Lcd with a foreign peptide, caused surface pattern changes around two-fold axes of PCV2 VLPs based on 3D structure simulation, but was not detrimental to VLPs assembly and cell entry. Moreover, this substitution had no adverse effect on eliciting neutralizing antibodies (NAbs) against PCV2 infection in pigs. In conclusion, MP-Lcd of the PCV2 Cap was a promising site to accommodate and display foreign epitopes or functional peptides on the surface of PCV2 VLPs. Furthermore, chimeric VLPs (cVLPs) would have potential as bivalent or multivalent vaccines and carriers to deliver functional peptides to target cells.

Porcine circovirus type 2 (PCV2) is one of the smallest, nonenveloped, single-stranded DNA viruses. The PCV2 capsid protein (Cap) is the sole viral structural protein and main antigenic determinant. Previous sequence analysis has revealed that the N terminus of the PCV2 Cap contains a nuclear localization signal (NLS) enriched in positively charged residues. Here, we report that PCV2's NLS can function as a cell-penetrating peptide (CPP). We observed that this NLS can carry macromolecules, e.g. enhanced GFP (EGFP), into cells when they are fused to the NLS, indicating that it can function as a CPP, similar to the classical CPP derived from HIV type 1 transactivator of transcription protein (HIV TAT). We also found that the first 17 residues of the NLS (NLS-A) have a key role in cellular uptake. In addition to entering cells via multiple endocytic processes, NLS-A was also rapidly internalized via direct translocation enabled by increased membrane permeability and was evenly distributed throughout cells when its concentration in cell cultures was ≥10 μm. Of note, cellular NLS-A uptake was ∼10 times more efficient than that of HIV TAT. We inferred that the externalized NLS of the PCV2 Cap may accumulate to a high concentration (≥10 μm) at a local membrane area, increasing membrane permeability to facilitate viral entry into the cell to release its genome into a viral DNA reproduction center. We conclude that NLS-A has potential as a versatile vehicle for shuttling foreign molecules into cells, including pharmaceuticals for therapeutic interventions.

猪圆环病毒2型(PCV2)和副猪嗜血杆菌(HPs)混合感染十分普遍,造成我国养猪业严重经济损失.本研究将杆状病毒表达的重组PCV2 Cap蛋白(BCap)和PCV2灭活病毒液(iPCV2),分别与HPs血清5型(HPs5)灭活菌液混合,加入水性佐剂混合制备成BCap/HPs5与iPCV2/HPs5二联灭活疫苗,进行猪体免疫保护试验.仔猪免疫保护试验结果为:两种疫苗免疫后均可诱导猪体产生PCV2和HPs抗体.免疫后35天用PCV2攻击,两免疫组猪均无明显临床症状,相对日增重(RDWG)与空白对照组相似,但高于攻毒对照组(P＜0.05);攻毒后14天,病毒血症明显低于攻毒对照组(P＜0.05);攻毒后28天剖解,腹股沟淋巴结PCV2载量明显低于攻毒对照组(P＜0.05),病理学变化明显轻于攻毒对照组.免疫后35天用HPs攻击,对照组猪均出现明显临床症状,但两免疫组猪无明显临床症状;攻毒后14天剖检,两免疫组病理学变化方面相似,但明显轻于攻毒对照组.结果表明,BCap/HPs5与iPCV2/HPs5两种二联苗免疫仔猪后均能诱导机体产生免疫应答,产生免疫保护作用,具有较好应用前景.

猪瘟是严重危害我国和世界养猪业发展的重要传染病.我国研制的猪瘟兔化弱毒疫苗(即C株)在世界范围内的猪瘟免疫防控和最终根除过程中起到了极其重要的作用.尽管如此,目前我国养猪业仍在承受该病的困扰,因种种原因造成的疫苗免疫力下降或免疫失败现象时有发生,每年都让养殖业蒙受巨大的经济损失.文章就我国猪瘟流行特点、现有猪瘟疫苗特点及合理使用猪瘟ST传代细胞苗、科学免疫猪瘟等问题作一概述和探讨分析.

猪圆环病毒2型(Porcine circovirus 2,PCV2)是引起猪圆环病毒病(Porcine circovirus disease,PCVD)的主要病原.该病毒可破坏动物机体免疫系统,引起严重的免疫抑制,最终导致猪的生产性能下降、进而造成巨大的经济损失.目前,国内共有6种不同的PCV2疫苗获得了新兽药注册证书.文章就目前国内外PCVD的流行状况、我国当前PCV2疫苗特点及PCVD的科学免疫等方面作一简述,以期为PCVD的疫苗选择和科学防控等提供参考和借鉴.

Since the end of 2010,a disease characterized by diarrhea has been prevailing in sucking piglets in many provinces in China,resulting in heavy economic losses in swine industry.In order to identify the major pathogens of the disease,a total of 153 samples of diarrhea suckling piglets were collected from 47 pig farms and porcine epidemic diarrhea virus(PEDV) was detected by RT-PCR.The results showed that 88 samples from 26 pig farms were positive.The farm positive rate was 55.32% and the sample positive rate was 57.52%.ORF3 genes of 11 isolates were cloned into pMD18-T and sequenced.The result of sequencing showed that all the ORF3 genes were 675 bp which encoded 224 amino acids.The nucleotide homologies of the 11 isolates were 95.9% to 99.9% and amino acid homologies were 96.4% to 100%,respectively.In addition,comparing with those of European strain CV777,vaccine strain CV777 truncated and Korean strains,the nucleotide homologies were 96.3% to 97.0%,96.3% to 97.0% and 95.0% to 98.5% and the amino acid homologies were 95.1% to 96.4%,96.8% to 97.6% and 95.5% to 99.6%,respectively.Furthermore,based on the phylogenetic relationship of their ORF3 genes,the PEDV isolates and PEDV reference strains were divided into four sub-groups.And most of the PEDV isolates belonged to sub-group 1 and had a close phylogenetic relationship with Korean strains.But there were genetically different from the PEDV vaccine strains used in China.It suggested that PEDV could be one of the main pathogens of the diarrhea in suckling piglets and these isolates had a significant genetic variation.