Avian infectious bronchitis virus (IBV) still causes serious economic losses in the poultry industry. Currently, there are multiple prevalent genotypes and serotypes of IBVs. It is imperative to develop a new diagnosis method that is fast, sensitive, specific, simple, and broad-spectrum. A monoclonal hybridoma cell, N2D5, against the IBV N protein was obtained after fusion of myeloma SP2/0 cells with spleen cells isolated from the immunized Balb/c mice. The N2D5 monoclonal antibody (mAb) and the previously prepared mouse polyclonal antibody against the IBV N protein were used to target IBV as a colloidal gold–mAb conjugate and a captured antibody, respectively, in order to develop an immunochromatographic strip. The optimal pH and minimum antibody concentration in the reaction system for colloidal gold–mAb N2D5 conjugation were pH 6.5 and 30 μg/mL, respectively. Common avian pathogens were tested to evaluate the specificity of the strip and no cross-reaction was observed. The sensitivity of the strip for detecting IBV was 10−1.4522 EID50/mL. The strip showed a broad-spectrum cross-reactive capacity for detecting IBV antigens, including multiple IBV genotypes in China and all of the seven serotypes of IBV that are currently prevalent in southern China. Additionally, the result can be observed within 2 min without any equipment. The throat and cloacal swab samples of chickens that were artificially infected with three IBV strains were tested using the developed strip and the qPCR method; the strip test demonstrated a high consistency in detecting IBV via qPCR gene detection. In conclusion, the immunochromatographic strip that was established is rapid, sensitive, specific, simple, practical, and broad-spectrum; additionally, it has the potential to serve as an on-site rapid detection method of IBV and can facilitate the surveillance and control of the disease, especially in resource-limited areas.

[目的]构建鼠源Ⅲ型中间丝蛋白Desmin真核/原核表达载体,明确鼠源Desmin蛋白生物学功能,为在体外及细胞内表达系统中鉴定Desmin互作蛋白及探索其作用网络提供理论依据.[方法]通过RT-PCR克隆Desmin基因,分别构建原核表达载体pGEX-4T-1-Desmin.F1ag和真核表达载体pcDNA3.0-Desmin-Flag,以IPTG对原核表达载体进行诱导表达,并通过ProtParam、ProtScale、TMHMM-2.0、SignalP-5.0、SOPMA 和 SWISS-MODEL等在线 软件对Desmin蛋白进行生物信息学分析.[结果]鼠源Desmin基因长1410 bp,选用pGEX-4T-1载体和pcDNA3.0载体分别成功构建了原核表达载体pGEX-4T-1-Desmin-Flag和真核表达载体pcDNA3.0-Desinimin-Flagn.原核表达载体pGEX-4T-1-Desmin-Flag转化大肠杆菌后经IPTG诱导,成功表达出70 kD的融合蛋白DesminiFlag;真核表达载体pcDNA3.0-DesminF1ag转染BHK-21细胞,通过Western blotting在56 kD处检测到Flag标签,即Desmin蛋白能在真核细胞成功表达,且主要定位在细胞质.Desmin蛋白由470个氨基酸残基组成,分子量为54 kD,分子式为C2299H3722N688O755S13,理论等电点(pI)为5.21,属于不稳定蛋白;蛋白脂溶系数为79.94,平均亲水系数(GRAVY)为-0.721,推测为亲水性蛋白;无跨膜结构域和信号肽;其二级结构由α-螺旋(占67.59％)、无规则卷曲(占22.39％)、β-转角(占1.92％)和延伸链(占8.10％)构成.[结论]鼠源Desmin蛋白在原核表达体系中主要以包涵体形式进行表达,在真核细胞中表达主要定位于细胞质,呈骨架结构分布,其理化性质不稳定,属亲水性蛋白,无跨膜结构域和信号肽.Desmin作为一种重要的Ⅲ型中间丝蛋白,在神经肌肉组织信号转导及与相关蛋白发生相互作用方面发挥重要作用.

Microfilaments and microtubules, two crucial structures of cytoskeletal networks, are usurped by various viruses for their entry, egress, and/or intracellular trafficking, including the Rabies virus (RABV). Intermediate filaments (IFs) are the third major component of cytoskeletal filaments; however, little is known about the role of IFs during the RABV infection. Here, we identified the IF protein desmin as a novel host interactor with the RABV matrix protein, and we show that this physical interaction has a functional impact on the virus lifecycle. We found that the overexpression of desmin facilitates the RABV infection by increasing the progeny virus yield, and the suppression of endogenous desmin inhibits virus replication. Furthermore, we used confocal microscopy to observe that the RABV-M co-localizes with desmin in IF bundles in the BHK-21 cells. Lastly, we found that mice challenged with RABV displayed an enhanced expression of desmin in the brains of infected animals. These findings reveal a desmin/RABV-M interaction that positively regulates the virus infection and suggests that the RABV may utilize cellular IFs as tracks for the intracellular transport of viral components and efficient budding.

试验通过CCK8法检测并筛选螺旋藻多糖提取物对南美白对虾血淋巴细胞安全浓度,探究螺旋藻多糖提取物对南美白对虾血淋巴细胞免疫功能的影响.试验设置对照组、黄芪多糖对照组、3个螺旋藻多糖提取物组(50、100、200mg/L).螺旋藻多糖提取物处理对虾血淋巴细胞12h,测定其对南美白对虾血淋巴细胞中酸性磷酸酶(ACP)、碱性磷酸酶(AKP)和过氧化物酶(POD)的活性.结果 显示,螺旋藻多糖提取物浓度范围在50～400mg/L对南美白对虾血淋巴细胞的存活率无显著影响(P＞0.05).与对照组相比,50、100、200mg/L螺旋藻多糖提取物均极显著提高ACP的活性(P＜0.01);50 mg/L螺旋藻多糖提取物极显著提高AKP、POD的活性(P＜0.01),200mg/L螺旋藻多糖提取物极显著提高POD的活性(P＜0.01),400mg/L螺旋藻多糖提取物极显著提高POD (P＜0.01),显著提高AKP活性(P＜0.05).研究表明,螺旋藻多糖提取物浓度范围在50～400mg/L时,对南美白对虾血淋巴细胞的存活率无显著影响,能够提高南美白对虾血淋巴细胞的免疫功能.

[目的]明确猪源肿瘤坏死因子α(TNF-α)多克隆抗体的特异性和反应性,并探索猪瘟病毒(CSFV)感染对PK-15细胞分泌TNF-α的影响,为揭示CSFV的致病机理打下基础.[方法]提取CSFV病料基因组RNA,通过RT-PCR扩增TNF-α基因,构建原核表达载体pGEX-4T-1-TNF-α并转化BL21感受态细胞诱导表达融合蛋白,经纯化和浓缩后免疫SPF级昆明小鼠制备TNF-α多克隆抗体;同时构建真核表达载体pcDNA3.0-TNF-α,分别转染HEK-293T细胞和PK-15细胞表达融合蛋白,通过Western blotting、ELISA和间接免疫荧光分析等方法检测TNF-α多克隆抗体效价、反应性及特异性.[结果]以原核表达载体pGEX-4T-1-TNF-α转化BL21感受态细胞,经IPTG诱导后能表达出约43 kD的融合蛋白,且主要以包涵体形式进行表达.以真核表达载体pcDNA3.0-TNF-α转染HEK-293T细胞可表达出25 kD的融合蛋白,主要在细胞质中表达,且均匀分布.制备获得的TNF-α多克隆抗体能与转染HEK-293T细胞表达的融合蛋白TNF-α及PK-15细胞的内源蛋白TNF-α发生良好反应,即具有较好的反应特异性,其抗体效价高达1:8000.CSFV能刺激PK-15细胞分泌蛋白TNF-α上调表达,且TNF-α与其下游因子(TRAF1)的表达变化趋势基本一致,即二者间存在一定关联性.[结论]制备获得的TNF-α蛋白抗体具有效价高、反应性好及特异性强的特点,可用于检测CSFV感染后真核细胞中过表达的TNF-α水平.TNF-α可刺激TRAF1产生,参与TRAF1相关信号通路而发挥其生物学功能,CSFV感染PK-15细胞后TNF-α和TRAF1的表达变化趋势基本一致,说明CSFV能刺激TNF-α和TRAF1信号通路,使机体产生炎症反应.

试验选用高剂量(26.40 g/kg)、中剂量(13.20 g/kg)和低剂量(6.60 g/kg)的三黄连散饲喂人工感染无乳链球菌的罗非鱼,观察并记录其临床症状和死亡率,旨在明确三黄连散对罗非鱼人工感染无乳链球菌的预防作用.结果 显示,高剂量和中剂量的三黄连散保护率分别为77.77％和55.55％,且与市售三黄散相比,效果较稳定.研究表明,三黄连散对罗非鱼无乳链球菌的预防作用较好.

[目的]明确产蛋鸡源传染性支气管炎病毒(IBV)分离株(GX-YL170808)的结构基因及抗原变异情况,为广西种鸡场的传染性支气管炎(IB)防控提供科学依据.[方法]通过鸡胚尿囊液血凝试验、鸡胚侏儒化试验及3'端非编码区(3'-UTR)测序对分离株进行鉴定;采用RT-PCR扩增S1、E、M和N基因,以MegAlign和MEGA 6.0分别进行核苷酸序列相似性及系统发育进化树分析,运用RDP4和SimPlot对S1、E、M和N基因进行重组分析,利用NetNGlyc 1.0和NetOGlyc 4.0进行糖基化位点预测分析,并通过中和试验进行血清型鉴定.[结果]分离株的鸡胚尿囊液血凝试验呈阴性,鸡胚盲传5代后出现侏儒胚典型病变,其3'-UTR序列与IBV的3'-UTR序列相似性为99.13％;综合病鸡临床症状及其病理变化可确定该病毒为IBV,命名为GX-YL170808.GX-YL170808分离株S1、E、M和N基因的开放阅读框(ORF)长度分别为1620、327、675和1230 bp,对应编码540、109、225和410个氨基酸残基,与参考株的核苷酸序列相似性分别为60.4％～96.5％、81.8％～97.2％、85.6％～93.5％和85.7％～92.0％;GX-YL170808分离株的S1、M和N基因属于LX4型,而E基因属于LDT3型;4个结构基因内部均无重组区域;除S1基因同时具有N-糖基化和O-糖基化位点外,E、M和N基因均只有N-糖基化位点;S1蛋白裂解位点为HRRRR,与参考株LX4的裂解位点相同.GX-YL170808分离株属于血清4型,不同于常用的疫苗株H120(血清3型)和4/91(血清5型),也不同于广西主要侵害雏鸡的IBV优势血清型.[结论]产蛋鸡源IBV分离株并非疫苗株,其基因型和血清型均已发生变异,且该毒株的血清型不同于广西地区侵害雏鸡的优势血清型,提示了广西地区IB防控的严峻性及新型多价疫苗研发的紧迫性.

为了解鸡传染性支气管炎病毒(IBV)变异情况,对2018-2019年多次发生疑似鸡传染性支气管炎(IB)的广西某公司发病鸡群进行IBV的分离鉴定,并对分离株进行S1基因序列测定、同源性、系统进化树和重组分析.结果 显示:共分离到5株IBV,其S1基因核苷酸相似性为85.2％～99.9％;5株IBV中3株S蛋白裂解位点为HRRRR,2株为RRFRR;2018年分离的2株IBV属于LX4型中的QXⅡ亚型,与疫苗株QXL87处于同一个分支;2019年分离的3株IBV为LDT3-A型,且均为重组毒株,来源于分离株GX-YL161022(QXⅡ亚型,主亲本)与参考株CK/CH/LSC/99I(次亲本)的重组.研究表明这5株IBV主要为LX4型毒株及其参与的重组株,未及时免疫与流行株基因型相同的疫苗以及重组导致的变异可能是造成该养殖场免疫失败的主要原因.

2009-2017年从广西地区采集了556份发病鸡群气管、肺脏以及肾脏样本,利用RT-PCR和基因测序等方法分离鉴定出64份鸡传染性支气管炎病毒(infectious bronchitis virus,IBV)阳性样品,从检出率、发病日龄、季节、地域、症状和病变、与H120疫苗株的同源性等方面进行详细统计分析.结果 显示:(1)2009-2017年间IBV检出率为11.5％,IBV单一感染检出率为35.9％,IBV混合感染检出率为64.1％,其中IBV与细菌混合感染率高达39.1％;(2)2～4周龄发病鸡群IBV检出率最高;(3)IB的高发阶段是当年的10月份到次年的4月份,具有明显的季节性;(4)广西14个地区中IBV检出率较高的地区分别为南宁市、钦州市、玉林市,检出率分别为14.7％、13.0％和9.9％;(5)IB症状和病变分别以呼吸道症状和肾脏肿大为主;(6)大部分IBV毒株(89.1％,57/64,)S1基因序列与H120疫苗株的相似性很低.本试验通过对广西地区近10年IBV流行病学调查,为今后广西防控IB提供科学依据,对国内其他地区制定有效的IB防控措施也具有重要的参考价值.

[目的]对从广西某鸭场发生呼吸道感染的11天龄樱桃谷肉鸭分离到的病毒株进行鉴定,并探索此鸭源病毒分离株的遗传变异情况.[方法]通过血凝试验、鸡胚接种实验、3'端非编码区（3'UTR)基因扩增与序列测定对分离株进行鉴定,并对该分离株的结构基因S1、E、M和N分别进行序列测定以及相似性、系统进化树分析和血清型鉴定.[结果]血凝试验为阴性,接种鸡胚盲传5代后出现侏儒胚,3'UTR基因测序结果表明为传染性支气管炎病毒(IBV)序列.该分离株S蛋白的裂解位点为RRSRR,S1、E、M和N基因与IBV毒株H120、4/91、LTD3核苷酸相似性分别为:78.6％-99.7％、85.4％-100.0％、91.6％-93.2％、86.7％-91.7％.除N基因存在点突变外,S1、E和M基因均存在氨基酸的突变、插入和（或）缺失.系统进化树分析显示,其S1基因属于4/91型,E、M和N基因均为LDT3型.血清型分析表明,该分离株的血清型不同于疫苗株H120和4/91.[结论]此鸭源病毒分离株为IBV,且该分离株的基因型与血清型均发生了变异.本研究结果暗示禽类传染性支气管炎的防控面临着更严峻的挑战.

The high mutation rates of infectious bronchitis virus (IBV) pose economic threats to the poultry industry. In order to track the genetic evolutionary of IBV isolates circulating in yellow chickens, we continued to conduct the genetic analyses of the structural genes S1, E, M, and N from 64 IBV isolates in southern China during 2009-2017. The results showed that the dominant genotypes based on the four genes had changed when compared with those during 1985-2008. Based on the S1 gene phylogenetic tree, LX4-type (GI-19) was the most dominant genotype, which was different from that during 1985-2008. The second most dominant genotype was LDT3-A-type, but this genotype disappeared after 2012. New-type 1 (GVI-1) isolates showed increasing tendency and there were four aa (QKEP) located in the hypervariable region (HVR) III and one aa (S) insertion in all the New-type 1 isolates. Both the analyses of amino acid entropy and molecular evolutionary rate revealed that the variations from large to small were S1, E, M, and N. Purifying selection was detected in the S1, E, M, and N gene proteins, which was different from the positive selection during 1985-2008. Six isolates were confirmed to be recombinants, possibly generated from a vaccine virus of the 4/91-type or LDT3-A-type and a circulating virus. The estimated times for the most recent common ancestors based on the S1, E, M, and N genes were the years of 1744, 1893, 1940, and 1945, respectively. Bayesian skyline analysis revealed a sharp decrease in genetic diversity of all the four structural genes after 2010 and since late 2015, the viral population rapidly rose. In conclusion, the IBVs circulating in southern China over the past decade have experienced a remarkable change in genetic diversity, dominant genotypes, and selection pressure, indicating the importance of permanent monitoring of circulating strains and the urgency for developing new vaccines to counteract the emerging LX4-type and New-type IBVs.

根据GenBank发表的禽偏肺病毒(aMPV)F基因序列,设计一对特异性引物,建立C亚型aMPV的SYBR Green I实时荧光定量PCR方法.对该反应体系进行条件优化,建立了标准曲线,并进行特异性、敏感性及重复性试验,然后将建立的方法应用于临床样品和攻毒样品的检测.结果显示:标准曲线循环阈值与模板浓度呈现良好的线性关系;建立的方法只能检测出C亚型aMPV,最低可以检测到0.8x101拷贝/μL的核酸模板,重复性试验的变异系数小于4％.应用建立的方法对43份临床样品检测,结果显示均为阴性;对42份35日龄SPF鸡人工感染后1～21 d的气管和肺脏样品进行检测,结果显示攻毒样品均为阳性.因此,研究建立的特异、灵敏、重复性好的C型aMPV实时荧光定量PCR方法,既可适合于该病的早期诊断和流行病学调查,又为该病毒的致病机制研究提供技术支撑.