表达H5亚型禽流感病毒HA基因的重组马立克氏病病毒的构建

采用聚合酶链反应或反转录聚合酶链反应扩增出H5亚型禽流感病毒(AIV)的HA基因、网状内皮增生症病毒的长末端重复序列(LTR)、马立克氏病病毒(MDV)Rispens CVI988毒株基因组的sorf 1和sorf 2序列、两端带loxp位点的lac/smGFP标志基因,构建含这些基因的转移载体质粒pMHA;以MDV Rispens CVI988毒株的基因组DNA和PMHA质粒DNA共转染鸡胚成纤维细胞(CEF),采用同源重组方法将LTR、lac/smGFP和HA基因插入到MDV基因组,获得重组病毒rMDV-HA/GFP;以cre介导的同源重组去除lac/smGFP标志基因,再转染CEF,获得仅带LTR启动子和HA基因的重组MDV疫苗毒株rMDV-HA.rMDV-HA仍保留了MDV RispensCVI988疫苗毒株的复制特点,并能稳定表达AIV的HA.     

Evaluation of factors affecting vaccine efficacy of recombinant Marek's disease virus lacking the Meq oncogene in chickens

We previously reported that deletion of the Meq gene from the oncogenic rMd5 virus rendered it apathogenic for chickens. Here we examined multiple factors affecting Marek's disease vaccine efficacy of this nonpathogenic recombinant Meq null rMd5 virus (rMd5deltaMeq). These factors included host genetics (MHC haplotype), strain or dose of challenge virus, vaccine challenge intervals, and maternal antibody status of the vaccinated chicks. Studies on host genetics were carried out in five chicken lines comprising four different MHC B-haplotypes. Results showed that chicken lines tested were highly protected, with protective indexes of 100% (B*2/*15), 94% (B*2/*2), 87% (B*19/*19), and 83% (B*21/*21). At a challenge dose above 8000 plaque-forming units, differences in protection were observed between the two highly virulent strains examined (648A and 686). The interval between vaccination and challenge indicated a protective efficacy from 0 to 2 days varied greatly (12%-82%) after challenge with vv+686, the most virulent virus. Less variation and significant protection began at 3 days post vaccination and reached a maximum at 5 days post vaccination with about 80%-100% protection. Taken together, our results indicate that the factors examined in this study are important for vaccine efficacy and need to be considered in comparative evaluations of vaccines.     

A comparative evaluation of the protective efficacy of rMd5deltaMeq and CVI988/ Rispens against a vv+ strain of Marek's disease virus infection in a series of recombinant congenic strains of White Leghorn chickens

Marek's disease (MD) is a lymphoproliferative disease of domestic chickens caused by a highly infectious, oncogenic alpha-herpesvirus known as Marek's disease virus (MDV). MD is presently controlled by vaccination. Current MD vaccines include attenuated serotype 1 strains (e.g., CVI988/Rispens), avirulent serotype 2 (SB-1), and serotype 3 (HVT) MDV strains. In addition, recombinant MDV strains have been developed as potential new and more efficient vaccines to sustain the success of MD control in poultry. One of the candidate recombinant MDV strains, named rMd5deltaMeq, was derived from Md5, a very virulent strain of MDV lacking the MDV oncogene Meq. Our earlier reports suggest that rMd5deltaMeq provided protection equally well or better than commonly used MD vaccines in experimental and commercial lines of chickens challenged with very virulent plus (vv+) strains of MDV. In this study, maternal antibody-positive (trial 1) and negative (trial 2) chickens from a series of relatively MD resistant lines were either vaccinated with the rMd5deltaMeq or CVI988/Rispens followed by infection of a vv+ strain of MDV, 648A, passage 10. This report presents experimental evidence that the rMd5deltaMeq protected significantly better than the CVI988/Rispens (P < 0.01) in the relatively resistant experimental lines of chickens challenged with the vv+ strain of MDV. Together with early reports, the rMd5deltaMeq appeared to provide better protection, comparing with the most efficacious commercially available vaccine, CVI988/Rispens, for control of MD in lines of chickens regardless of their genetic background.     

表达鸡马立克氏病病毒gB基因重组鸡痘病毒的遗传稳定性及生物安全性评价

为了评价表达鸡马立克氏病病毒gB基因重组鸡痘病毒(rFPV-gB/R)的遗传稳定性,我们将纯化后的重组病毒在CEF单层上连续传30代,引起细胞病变的速度和形态均未发生明显变化;覆盖含X-Gal的琼脂引起的空斑均为蓝色;间接免疫荧光实验证明rFPV-gB/R中的gB基因始终能稳定表达;序列测定结果表明,重组病毒在细胞上连续传30代、在SPF鸡上连续传5代后,gB基因序列没有发生任何变化;以0、10、20、30代重组病毒制成冻干疫苗进行的实验室免疫效力实验表明,细胞连续传代后rFPV-gB/R仍然保持了原有的免疫原性。可见重组鸡痘病毒gB基因的结构和免疫原性都是高度稳定的。为了评价rFPV-gB/R的生物安全性,我们将rFPV-gB/R通过SPF鸡连续传5代,检测病毒在鸡体的存在部位及其消长、生长繁殖性能和毒力变化;将rFPV-gB/R免疫鸡与未免疫鸡同笼饲养,攻击FPV-102E6强毒,以检测rFPV-gB/R感染鸡的接触传染性。结果显示,rFPV-gB/R在鸡体的存在时间大约为7d,在体内仅存在于接种部位;鸡体传代后痘病毒毒力有一定程度下降,gB基因核苷酸序列未发生任何变化;同居未免疫SPF鸡在痘病毒强毒攻击后全部发痘,可见重组病毒免疫鸡没有接触传染性,能在鸡体内稳定地传代,rFPV-gB/R具有高度的生物安全性。     

Marek's disease in turkeys. II. Characterization of the viral glycoprotein B gene and antigen of a turkey strain of Marek's disease virus

Marek's disease virus (MDV) causes immunosuppression and tumors in chickens. As sporadic cases of Marek's disease (MD) were recorded in turkeys, the antigenic and genomic characteristics of the MDV glycoprotein B (gB) gene and antigen of turkeys were compared to the chicken MDV gB. The whole chicken and turkey gB genes were sequenced and found identical. By immunoblotting of infected-cell culture lysates using chicken convalescent and gB monoclonal antibodies, the antigenic epitopes of the chicken and turkey viruses were found to differ. The turkey MDV had a unique epitope, compared to the chicken MDV and compared with our previous findings. While the chicken MDV had two epitope types, heat-labile but dithiothreitol (DTT)-stable and heat-stable but DTT-labile, the turkey MDV gB epitope is both heat and DTT-labile.     

不同代次缺失meq基因的重组马立克病毒SC9-1株主要基因序列同源性分析

分析缺失meq基因的重组马立克病病毒SC9-1原代、10代及40代主要基因序列的同源性。提取缺失meq基因的重组马立克病病毒SC9-1原代、10代及40代DNA,设计引物PCR扩增其与致瘤相关pp38基因、缺失meq基因后残余的FRT位点序列及囊膜蛋白基因gB、gC、gD、gE、gH、gI、gK,连接pMD18-T载体,转化DH5α感受态细胞,挑取阳性克隆进行测序,DNAStar软件对3代次各基因片段进行同源性比对。不同代次SC9-1病毒株的致瘤相关pp38基因、残留FRT位点序列及囊膜蛋白基因gB、gC、gD、gE、gH、gI、gK的核苷酸序列同源性均为100%,核苷酸序列差异性分析结果显示序列完全一致,无位点上的变化。缺失meq基因的重组马立克病病毒SC9-1在鸡胚成纤维细胞(CEF)上的传代过程中其致瘤相关pp38基因、残留FRT位点序列及囊膜蛋白基因gB、gC、gD、gE、gH、gI、gK没有发生变异,侧面反映了SC9-1在CEF细胞传代过程中具有很好的遗传稳定性。     

表达日本乙型脑炎病毒E蛋白重组伪狂犬病病毒SA215（E）的构建

将日本乙型脑炎病毒(Japanese encephalitis virus,JEV)CQRC-1株的E基因通过RT-PCR产生,克隆到pMD18-T simple载体中,再亚克隆到转移载体pPI-2,EGFP中,命名为PPE.PPE质粒和伪狂犬病毒(PRV)SA215株DNA通过磷酸钙共转染方法转染到Vero细胞上.通过有限稀释法、噬斑纯化、PCR检测、Southern杂交、West杂交,获得了表达JEV E蛋白的重组伪狂犬病病毒,命名为SA215(E).结果表明,SA215(E)在生长曲线、噬斑形态及大小、在Vero细胞上的病变与PRV SA215一致.安全性试验表明,SA215(E)对小鼠和兔具有较高的安全性,SA215(E)具有很强的免疫原性.接种兔能预防致死剂量的PRV Fa肌肉内攻毒;接种小鼠能预防致死剂量的JEV cQRC-1株腹膜内攻毒.表明该重组病毒是养猪业控制伪狂犬病和乙脑最适合的候选疫苗株之一.     

Protection of chickens from Newcastle and Marek's diseases with a recombinant herpesvirus of turkeys vaccine expressing the Newcastle disease virus fusion protein.

Recombinant strains of herpesvirus of turkeys (HVT) were constructed that contain either the fusion protein gene or the hemagglutinin-neuraminidase gene of Newcastle disease virus (NDV) inserted into a nonessential gene of HVT. Expression of the NDV antigens was regulated from a strong promoter element derived from the Rous sarcoma virus long terminal repeat. Recombinant HVT strains were stable and fully infectious in cell culture and in chickens. Chickens receiving a single intra-abdominal inoculation at 1 day of age with recombinant HVT expressing the NDV fusion protein had an immunological response and were protected (> 90%) against lethal intramuscular challenge at 28 days of age with the neurotropic velogenic NDV strain Texas GB. Recombinant HVT expressing the NDV hemagglutinin-neuraminidase provided partial protection (47%) against the same challenge. Chickens vaccinated with recombinant HVT vaccines had low levels of protection against NDV replication in the trachea when challenged ocularly. Recombinant HVT vaccines and the parent HVT strain provided similar levels of protection to chickens challenged with the very virulent RB1B strain of Marek's disease virus, indicating that insertion of foreign sequences into the HVT genome did not compromise the ability of HVT to protect against Marek's disease.     

Bovine herpesvirus-1 (BHV-1) recombinant expressing pseudorabies virus (PrV) glycoproteins B and C induces type 1 immune response in BALB/c mice

Bovine herpesvirus 1 (BHV-1) attached poorly and penetrated into a mouse cell line, BALB 3T3/A31, but a recombinant BHV-1/TF7-6, which expresses pseudorabies virus (PrV) gB and gC genes, did attach and penetrated into cells more efficiently. In this study the gene green fluorescent protein (GFP) has been integrated into genome of BHV-1/TF7-6 and its parental line of BHV-1. When the mouse mesenteries were incubated in vitro and infected with BHV-1/TF7-6/GFP, strong fluorescence was observed while BHV-1/GFP infection hardly demonstrated fluorescence, suggesting that BHV-1 recombinant expressing PrV gB and gC can infect mouse tissue cells more efficiently than the parental BHV-1 does. When BALB/c mice were inoculated with purified BHV-1/TF7-6 or its parental BHV-1, the former induced lower level of anti-BHV-1 immunoglobulin G (IgG) than the latter did. When sub-classes of anti-BHV-1 IgG were analyzed, it was found that mice immunized with BHV-1/TF7-6 or the parental BHV-1 demonstrated the same level of IgG2a. Since anti-BHV-1 IgG1 level was lower in mice inoculated with BHV-1/TF7-6, the IgG2a:IgG1 ratio was higher in BHV-1/TF7-6 inoculated mice than in the parental BHV-1 inoculated ones. These results indicate that BHV-1/TF7-6 induces type 1 predominant immune to BALB/c mice.     

Immunogenic properties of a bovine herpesvirus-1 (BHV-1) recombinant expressing major pseudorabies virus (PrV) glycoproteins in combination

A recombinant bovine herpesvirus type 1 (BHV-1), designated BHV-1/TF17-1, which expresses pseudorabies virus (PrV) glycoproteins gB, gC, gD, gE and gI in combination was constructed. To test the protective immunity, 10 mice were inoculated with BHV-1/TF17-1 and three weeks later 10 mice were intraperitoneally (i.p.) challenged with 20 LD50 virulent PrV (YS-81). BHV-1/TF17-1 protected all the mice from the PrV lethal challenge while all the control mice died in around 3 days. Mice vaccinated with BHV-1/TF17-1 acquired high PrV-neutralizing antibody titers and demonstrated strong delayed type hypersensitivity responses and moderate in vitro lymphocyte proliferative responses to PrV antigen. Since the major PrV glycoproteins were integrated into virions (probably into viral envelope), BHV-1/17-1 was neutralized with anti-PrV antiserum. However, the susceptibility of BHV-1/TF17-1 to anti-PrV antiserum is 2- to 4-fold lower than that of PrV vaccine lines. Our results demonstrated the possibility of BHV-1/17-1 as a vaccine to protect piglets from Audjesky's disease where maternal antibodies against PrV interfere attenuated live PrV vaccines.     

表达MERS-CoVS1蛋白重组狂犬病病毒rSRV9-MERS_(S1)纯化方法的建立

建立一种可线性放大、应用于表达中东呼吸综合征冠状病毒(Middle East respiratory syndrome coronavirus,MERS-CoV)S1蛋白重组狂犬病病毒rSRV9-MERS_(S1)的规模化纯化方法。将收获的病毒去除细胞碎片、灭活后,分别通过3种方法(醋酸锌沉淀+Sepharose 4FF凝胶过滤层析、500 000中空纤维超滤柱+Sepharose 4FF凝胶过滤层析、500 000中空纤维超滤柱+Cellufine~(TM )Sulfate亲和层析)进行纯化,经比较筛选出一种温和、目的蛋白回收率高、纯度高的纯化方法。结果显示,500 000中空纤维超滤柱+Sepharose 4FF凝胶过滤层析回收的抗原含量最高,50 mL重组病毒原液经纯化后可得到1.82 mg蛋白,杂蛋白去除率达到97.02%,病毒纯度较高,电镜下可见到病毒囊膜刺突。结合成本等方面综合考虑,此方法优于其他2种纯化方法。本试验筛选获得了一种适用于rSRV9-MERS_(S1)的纯化方法,为rSRV9-MERS_(S1)规模化纯化工艺的建立与MERS新型疫苗的研制奠定了基础。     

表达禽腺病毒4型中国流行株Fiber2蛋白重组新城疫病毒的构建与鉴定

利用NDV LaSota弱毒疫苗株反向遗传操作系统,将禽腺病毒4型(FAdV-4)中国流行株的全长Fiber2基因插入到NDV基因组的P和M基因之间,构建并拯救出重组病毒rLaSota-Fiber2。利用RT-PCR和序列测定,以及Western blot对重组病毒进行了鉴定。结果显示,Fiber2基因插入位置和方向正确,Fiber2蛋白得到正确表达,表达形式为可溶的游离形式而非嵌入到NDV颗粒中。第10代重组病毒的鸡胚致病性试验和在鸡胚中的生长特性研究结果显示,重组病毒的半数鸡胚感染量(EID_(50))最高可达10~(8.5)/100μL,鸡胚平均致死时间(MDT)为120 h, 1日龄雏鸡脑内接种指数(ICPI)和6周龄鸡静脉接种致病指数(IVPI)均为0,重组病毒保持了LaSota弱毒疫苗亲本毒株的低致病特性和对鸡胚良好的高滴度生长适应性。重组病毒与亲本LaSota株生长滴度在相近时间达到峰值,生长动力学特性与亲本株无明显差异。本试验构建的表达FAdV-4中国流行株Fiber2基因的重组病毒rLaSota-Fiber2有望为同时防控NDV和FAdV-4的感染提供安全、廉价和高效的辅助工具。     

A comparison of the onset of protection induced by Newcastle disease virus strain B1 and a fowl poxvirus recombinant Newcastle disease vaccine to a viscerotropic velogenic Newcastle disease virus challenge

Four-week-old specific-pathogen-free white rock chickens were immunized with either a commercial recombinant fowl poxvirus-vectored Newcastle disease vaccine (FPN) expressing the hemagglutinin-neuraminidase and fusion protein genes of Newcastle disease virus (NDV) strain B1 or live NDV B1. Vaccinates and controls were challenged by eyedrop and intranasal (E/I) route with a viscerotropic velogenic NDV at 14 days postvaccination to determine the time of clearance of challenge virus. In a subsequent experiment, chickens were challenged at 3, 6, or 10 days postvaccination to determine the onset of immunity. Chickens that received a recommended field dose (1x) or a 0.01x dose of FP-N subcutaneously (s.c.) and were seropositive by hemagglutination-inhibition test at 14 days postvaccination cleared the challenge virus by 14 days postchallenge. Clinical Newcastle disease and high challenge virus titers in tissues were seen only in seronegative FP-N 0.01x dose vaccinates and controls. In a comparison of vaccination with FP-N (1x, 10(4,9) median tissue culture infective dose) s.c., B1 (10(6) median egg infective dose [EID50]) s.c., or B1 (10(6) EID50) E/I, chickens vaccinated at 6 or 10 days before challenge with all vaccines were protected against clinical disease, but only those vaccinated with B1 E/I 10 days before challenge were protected against infection with the challenge virus. Vaccination at 3 days before challenge with B1 E/I provided early protection, but severe nervous signs developed later and reduced overall protection to 60%, whereas disease in chickens vaccinated with B1 s.c. and FP-N s.c. 3 days before challenge was similar to the challenge controls.     

Brain and spinal cord lesions in pigs inoculated with swine vesicular disease (UKG strain) virus and coxsackievirus B5.

Pigs inoculated intravenously with swine vesicular disease virus (UKG strain), those inoculated with coxsackievirus B5, and other pigs exposed by pen contact to the same viruses developed diffuse encephalomyelitis. Perivascular cuffing, with lymphocytes and formation of neuroglia cell foci, were most prominent in telencephalon, diencephalon, and mesencephalon. Encephalitis was of mild to severe intensity. Severity of lesions was more extensive and severe in the pigs exposed to swine vesicular disease virus. Pen contact exposure to either of the 2 viruses caused a more severe central nervous system reaction than did intravenous inoculation. The type and the distribution of lesions produced by the 2 viruses indicate that they may be related.     

Identification and characterization of Marek's disease virus serotype 1 (MDV1) ICP22 gene product: MDV1 ICP22 transactivates the MDV1 ICP27 promoter synergistically with MDV1 ICP4

A previous report [Virus Genes 6 (1992) 365-378] has shown that the US1 gene of Marek's disease virus serotype 1 (MDV1) encodes a homologue of herpes simplex virus type 1 infected cell protein No. 22 (ICP22). In the present study, we expressed and identified a product of the MDV1 US1 gene in chicken embryo fibroblasts (CEFs) with the aid of a recombinant baculovirus expressing a Flag epitope-tagged MDV1 US1 gene, under control of the SRalpha promoter (composed of the enhancer region of the simian virus 40 early promoter and the R region of the human T-cell leukaemia virus type 1 long terminal repeat). In CEF infected with the recombinant baculovirus, MDV1 ICP22 was specifically and efficiently expressed in the presence of n-butyric acid. The apparent M(r) of the expressed protein was 30,000. Reporter gene assays revealed that MDV1 ICP22 by itself transactivated an MDV1 ICP27 promoter/reporter construct weakly but specifically, and furthermore, worked synergistically with MDV1 ICP4 to efficiently up-regulate the MDV1 ICP27 promoter. MDV1 ICP22 may be a regulatory protein that stimulates viral promoters in co-operation with other viral regulatory proteins such as MDV1 ICP4.     

鸡传染性法氏囊病组织病毒液（B87株）生产工艺探讨

通过计算ELD50,将已知效价的鸡传染性法氏囊病（B87株）生产种毒分几个滴度进行稀释,分别接种SPF鸡胚,结果各组鸡胚随病毒接种量的提高,高峰期死亡时间逐渐靠前,高峰期死亡率也逐渐增加,抗原效价也逐渐提高,抗原产量也有明显变化,病毒滴度以102.0ELD50/0.1mL接种鸡胚更合适。