Dynamic Pathology and Antigen Location Study on Broiler Breeders with Coinfection of Marek's Disease Virus and Reticuloendotheliosis Virus

To further understand the generation and development of coinfection of Marek＇s disease virus （MDV） and reticuloendotheliosis virus （REV） in broiler breeders, and then find the method and optimal time of differential diagnosis for complex clinic multiple infection, the authors studied the pathohistological changes, apoptosis, immunohistochemistry （immunofluorescence）, and ultrastructure of tumor tissues of broiler breeders inoculated with MDV and REV. The study showed that proliferation of small lymphocytes was seen in the main organs at the age of 1 week, then immature lymphocytes, all kinds of lymphocytes, primitive reticulum cells, and Marek＇s disease cells （MDCs） were observed at 2-9 weeks. Apoptosis of lymphocytes could not be seen until the age of 10 weeks in the immune system. Immunohistochemistry detection showed that the positive signs of MDV and REV antigen were observed in the main organs at 2 weeks of age. Multi-morphology lymphocytes, MDV, and REV, mitotic figures and apoptosis of lymphocytes were observed with the help of transmission electron microscopy. MDV cooperating with REV promotes the course of disease of coinfection. Differential diagnosis can be done by immunohistochemistry in the early stage （before 2 weeks）, and histopathology in the late stage （post 4 weeks）. MDCs, primitive reticulum cells, immature lymphocytes, and two kinds of virions can serve as a basis for bistopathology differential diagnosis.

Comparison of Immune Effect of Four Commercial Marek's Disease Vaccines in Wenchang Chicken

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）.The aim of this study was to compare the efficacy of four commercial MDV vaccines in Wenchang chicken.The 1-day old Wenchang chickens tested were injected with one of four different vaccines or not unvaccinated as control;five days later,they were then challenged by virulent MDV strain MD5.The results showed that,in comparison with HVT vaccines,the CVI988 vaccine gave the immunized chickens more potent immunities against challenges of MDV strain MD5.

MDV对雏鸡血浆MDA含量与SOD活性的影响

为探讨丙二醛(MDA)和超氧化物歧化酶(SOD)在雏鸡马立克病(MD)发病过程中的作用,研究了雏鸡在人工攻毒后血浆MDA含量与SOD活性的动态变化。结果表明,攻毒组接种马立克病病毒(MDV)后第14天,血浆中MDA的含量显著高于对照组(P<0.05),第7天和第28天高于对照组,第56天时基本接近;血浆中SOD活性在第7天时对照组显著高于攻毒组(P<0.05),第14天和第28天高于攻毒组,但差异不显著。表明雏鸡在感染MDV后血浆中MDA含量显著升高,SOD活性明显降低,对雏鸡MD的发病诊断有重要的指导作用。

MDV Rispens 株B抗原基因的酶切及序列分析

将马立克氏病病毒（ＭＤＶ）血清Ｉ型Ｒｉｓｐｅｎｓ株接种原代鸡胚成纤维细胞（ＣＥＦ），待出现８０％以上细胞病变后收获，提取细胞和病毒的总ＤＮＡ．以此为模板，根据Ｂ抗原基因核苷酸序列设计一对引物，通过聚合酶链反应（ＰＣＲ）扩增出一条约２．８５ｋｂ的特异性条带，双酶消化后克隆至质粒ｐＵＣ１９，酶切分析表明该病毒的Ｂ抗原基因上ＨｉｎｄⅢ、ＥｃｏＲⅤ、ＢａｍＨⅠ、ＥｃｏＲⅠ的酶切位点分布与ＲＢ１Ｂ株、ＧＡ株的完全相同，进一步序列分析表明。

Expression and intercellular trafficking of the VP22 protein of CVI988/Rispens vaccine strain of Marek’s disease virus

The viral protein 22 (VP22) in the tegument of Marek’s disease virus serotype 1 (MDV-1) plays an im-portant role in cell-to-cell spread and viral propagation. Antiserum against the carboxyl terminus of VP22 was prepared by immunizing mice with recombinant VP22 expressed in E. coli, and used to in-vestigate its expression in chicken embryo fibroblast (CEF) cells infected with different MDV-1 strains. At an infection dose of PFU=50, intercellular trafficking of the VP22 into the nuclei of the surrounding receipt cells was detected as early as 3 hours post infection. By 6 hours after infection (before viral plague formation), the protein was detected in the whole nuclei of the recipient cells with no difference among MDV-1 strains CVI988/Rispens, GA and RB1B. Intra-nuclear accumulation of the VP22 protein was further increased when the viral plagues started to form. These results indicate that, albeit the ex-istence of the 201TKSERT206 deletion, the VP22 of the CVI988/Rispens vaccine strain has also intercel-lular-trafficking function, which might serve as a potential alternative delivering protein instead of virulent strains VP22.

MDV-1 VP22 conjugated VP2 enhancing immune response against infectious bursal disease virus by DNA vaccination in mice

VP22 of Marek’s disease virus serotype 1 (MDV-1) could function in protein transduction. In this study, an infectious bursal disease virus VP2 gene was fused to the carboxyl termini of VP22. It showed that the fusion protein did not spread into the bystander cells from the cells transfected with pVP22-VP2, as the VP22 alone could. The VP22 proteins were found to be translocated into all the nuclei in the neighboring COS-1 cells, as analyzed by a fluorescence assay. Although mice were immunized with the recombinant DNAs mixed with polyethylenimine (PEI) at a dose of 1:2, it failed to enhance the antibody response against IBDV VP2, as measured by the indirect ELISA assay, yet the cell mediated immune response was significantly increased. The ratio of CD8+/CD4+ T cells was significantly increased in the immunized group with the fusion genes, compared with the group immunized with VP2 (P<0.05). Our results demonstrated that VP22 indeed enhances the cell-mediated response in the fused VP2 in a mice model system, possibly due to the fact that the IBDV VP2 could be carried into the surrounding cells at a limited level under pressure from MDV VP22.

Study on the structure of heteropolymer pp38/pp24 and its enhancement on the bi-directional promoter upstream of pp38 gene in Marek’s disease virus

In the latest report, Chloramphenicol acetyltransferase (CAT) gene was used as a reporter to investi-gate the influence of pp38 on its upstream bi-directional promoter, and it was found that the co-expression of pp38 and pp24 can significantly enhance the transactivity of the bi-directional pro-moter between pp38 gene and 1.8-kb mRNA transcript in genome of Marek’s disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) gene was used as another reporter to further investigate the promoter activity. The transfection shows the promoter has the complete activity under the condition of co-expression of pp38 and pp24 in the same cells. Immunoprecipitation test was used to verify the structure of pp38/pp24 heteropolymer. The pp38-specific monoclonal antibody H19 was used in this test, and pp38, pp24 or both were prepared from the pcDNA-pp38, pcDNA-pp24 or pBud-pp38-pp24 transfected chicken embryonic fibroblast (CEF), respectively. Immunoprecipitation indicates that pp24 could be co-precipitated with pp38 by MabH19, implying that pp24 and pp38 were able to form a heteropolymer in the natural condition. The two separated tests clarify that pp38 and pp24 form a heteropolymer, which enhances the activity of the promoter.

Analysis of DNA methylation of CD79B in MDV-infected chicken spleen

Marek’s disease(MD),an immunosuppressive disease induced by Marek’s disease virus(MDV),provides an ideal model for studying diseases caused by a carcinogenic virus.CD79 B is a B-cell antigen receptor complex-associated protein β-chain precursor which is involved in the activation,proliferation,differentiation of B-cell and the transmission of downstream signals.This study analyzed CD79 B gene mRNA expression and methylation by two schemes#20(5′flanking to intron 1)and#27(intron 2 to intron 3),between MDV-infected tumorous spleens(TS)and non-infected spleens(NS).Results showed that average methylation levels of CpGs in #20 and #27 were higher in TS than in NS(P<0.05),while,CD79 B mRNA expression was lower in TS than in NS(P<0.01).Six of 40 CpG sites showed significantly(P<0.05)different methylation levels between TS and NS.Correlation analysis showed that the average methylation level rather than a single site methylation level in #20 affected(P<0.05)mRNA expression.Collectively,it was found that the change of CD79 B gene expression after MDV infection might be partly explained by modification of DNA methylation.

The enhancement effect of pp38 gene product on the activity of its upstream bi-directional promoter in Marek's disease virus

There was a bi-directional promoter between gene 38 kd phosphorylated protein (pp38) gene; 1.8-kb mRNA transcript gene family in the genome of Marek's disease virus (MDV). In this study, enhanced green fluorescence protein (EGFP) reporter plamids, pP(pp38)-EGFP; pP(1.8-kb)-EGFP, were constructed under this bi-directional promoter in two directions. The two plasmids were transfected into uninfected chicken embryo fibroblast (CEF), MDV clone rMd5 infected CEF (rMd5-CEF); pp38-deleted derivative rMd5Δpp38 infected CEF (rMd5Δpp38-CEF) respectively. Transfection analysis showed that EGFP was only expressed in rMd5-CEF,; no EGFP could be detected in uninfected CEF or rMd5Δpp38-CEF, implying that pp38 was a factor influencing the activity of the promoter. The pp38-expressing recombinant plasmid pcDNA-pp38 was constructed to co-transfect CEF or rMd5Δpp38-CEF with pP(pp38)-EGFP or pP(1.8-kb)-EGFP. In this case, EGFP could be detected only in rMd5Δpp38-CEF but still not in uninfected CEF, implying that pp38 needs other protein(s) to work together for the complete trans-acting activity. Another MDV gene, 24 kd phosphorylated protein pp24 gene was cloned into pcDNA3.1 as a pp24-expressing recombinant plasmid pcDNA-pp24. When uninfected CEF was co-transfected with pcDNA-pp38, pcDNA-pp24; EGFP expressing plasmids pP(pp38)-EGFP or pP(1.8-kb)-EGFP, the EGFP could be detected. These results indicated that pp38; pp24 could enhance the activity of the promoter when they worked together. DNA mobility shift assay showed that pp38 would bind to the bi-directional promoter with the co-existing of pp24, although neither of them alone influenced mobility of the promoter DNA. All the above suggested that MDV pp38 could transactivate the bi-directional promoter when combined with pp24. The results also indicated that the activity of the promoter in the direction of 1.8-kb mRNA was significantly stronger than that of pp38 direction.

传染性法氏囊病病料中MDV、CAV、REV的共感染检测

从江苏、山东、浙江、河南、上海、广西、云南等地收集根据临床表现及病理变化诊断为传染性法氏囊病( IBD)的病鸡法氏囊样品 66份 ,用相应的核酸探针及斑点杂交法分别检测样品中鸡传染性贫血病病毒( CAV)、马立克氏病病毒 ( MDV)、网状内皮细胞增生病病毒 ( REV)的感染 ;用抗 IBD血清做琼扩检测 IB-DV。结果显示 ,部分地区病料中这些病毒有不同程度的二重感染、三重感染甚至四重感染。提示在研究特定毒株 IBDV的毒力时 ,应考虑多重感染对致病性的影响。

抗鸡早幼粒细胞白血病蛋白单克隆抗体的制备及其在荧光检测中的应用

【目的】探究鸡早幼粒细胞白血病蛋白核小体(PML NBs)在马立克病病毒(Marek’s disease virus,MDV)感染过程中的生物学功能,制备抗鸡早幼粒细胞白血病蛋白(Ch-PML)单克隆抗体,建立可视化检测Ch-PML的方法。【方法】通过原核表达系统制备Ch-PML重组蛋白,并将其作为免疫原免疫BALB/c小鼠,采用细胞融合技术将骨髓瘤细胞和免疫小鼠的脾细胞进行融合,用ELISA方法筛选出分泌特异性抗体的杂交瘤细胞株,通过体内诱生法制备腹水并用Protein A/G纯化抗Ch-PML单克隆抗体,利用Western blotting和ELISA方法检测单克隆抗体的特异性、亲和力和亚类,最后利用该单克隆抗体建立用于鸡PML NBs的间接免疫荧光试验(IFA)检测方法。【结果】Ch-PML蛋白在大肠杆菌中可溶性表达,分子质量大小为19 ku;以此蛋白成功制备了1株抗Ch-PML单克隆抗体,该抗体具有良好的特异性和高亲和力,经亚类和型检测,其为IgG2b亚类,轻链为Kappa型。利用制备的抗Ch-PML单克隆抗体建立的IFA检测方法发现,内源性PML NBs在宿主细胞核中呈点状分布,过表达的Ch-PML在宿主细胞核中形成团块状聚集体,且MDV感染宿主细胞后核内PML NBs数量比未感染细胞显著减少(P<0.05),说明MDV感染导致核内的PML NBs组装受到抑制。【结论】本研究利用制备的抗Ch-PML单克隆抗体建立了鸡PML NBs的IFA检测方法,并发现MDV抑制宿主细胞PML NBs组装这一现象,为MDV致病机制的解析提供了新的研究思路和工具。

我国部分地区蛋鸡群ALV-J及与REV、MDV、CAV混合感染检测

为了解J亚群禽白血病病毒(ALV-J)及其与禽网状内皮细胞增生症病毒(REV)、马立克氏病病毒(MDV)和鸡传染性贫血病病毒(CAV)的混合感染现象,本研究从宁夏、湖北、广东、山东、辽宁、吉林、黑龙江7个省的39个蛋鸡群收集临床表现和剖检病理变化疑似禽白血病的病料样品184份,采用PCR、病毒分离和IFA检测样品中ALV-J、REV、MDV和CAV。结果表明,7个省蛋鸡场均存在ALV-J感染,病料样品阳性率为60.9%,检测鸡群阳性率为82.1%,与REV、MDV、CAV的混合感染率分别为13.6%、24.5%、22.8%,其中存在较为严重的双重感染(29.0%)和3重感染(18.8%),甚至4重感染(1.7%)。研究结果表明,我国蛋鸡群中普遍存在ALV-J感染,而且与REV、MDV、CAV混合感染严重;提示ALV-J已经可以引起蛋鸡群发病,在临床诊断和致病性研究中,应考虑到多重感染的影响。

用斑点杂交法同时检测鸡群中的CAV MDV和REV

为研究鸡传染性贫血病毒(CAV)在鸡群中的感染状况以及马立克氏病病毒(MDV)和网状内皮细胞增生病病毒(REV)在鸡群中的发病率,用斑点杂交法对山东省4个肉鸡场和2个肉种鸡场进行CAV、MDV和REV的检测,结果表明除一个肉种鸡场没有检测出REV以外,其他鸡场均同时检测出CAV、MDV和REV,并且发现直接从病料中检测CAV的阳性率(20％)远远低于将病料接种SPF鸡胚后的检出率(80％)。

我国自然发病鸡群中MDV、REV和CAV共感染的检测

从山东、河南、河北、北京、江苏、广东、广西、四川、吉林、辽宁、台湾11 省42 个不同鸡群收集临床有发病表现的828只病、死鸡的病理组织样品,用点杂交方法检测各个样品中马立克氏病病(Marek′s disease virus,MDV)、网状内皮细胞增生病病毒(Reticuloendotheliosis virus,REV) 、鸡传染性贫血病病毒(Chicken anemia virus,CAV)的感染情况。结果表明:828 只病鸡中这三种病毒的检出率均相当高,分别为83. 94% (MDV)、61. 0% (CAV)和57.25%(REV),并且存在非常严重的双重感染(31. 16%)和三重感染(44. 69%),单重感染和阴性个体仅占15.34%和8.82%;在42个鸡群中的29个存在三重感染,占鸡群总数的69.05%,5个鸡群存在MDV和CAV的共感染,3个鸡群存在MDV和REV的共感染,二重感染占鸡群总数的19.05%,仅有2个鸡群未检测到这三种病毒;在地域分布上,11个省份均检测到了MDV、CAV和REV,表明了这三种病毒在我国的广泛分布及其在生产鸡群中的混合感染是导致当前我国养禽业生产性能下降、条件致病性疾病发生严重、临床腺胃肿大症状发生的重要原因。

抗MDV-1 VP22羧基端单克隆抗体的制备与免疫学特性鉴定

目的:制备抗血清I型马立克氏病病毒(MDV-1)VP22的单克隆抗体(mAb),并鉴定其免疫学特性。方法:在原核系统中表达VP22羧基端区域(94-243aa),获得融合表达产物GST-VP22C。将该表达产物切胶免疫小鼠,利用杂交瘤技术制备抗MDV-1 VP22C的mAb,并通过ELISA、间接免疫荧光(IFA)、Western blot鉴定其特性。结果:获得了2株可稳定分泌抗MDV-1 VP22C的mAb的杂交瘤细胞,命名为3F7、4E4。IFA鉴定表明,两株mAb均能与感染MDV-1的成纤维细胞反应,其中,3F7 mAb染色呈现MDV空斑,而4E4mAb呈现整个单层的细胞核荧光。ELISA和Western blot分析表明,3F7能与杆状病毒表达的VP22反应,4E4不具备该特性。对3F7 mAb进一步鉴定,确定了该mAb针对的具体位置在94-193aa处,是蛋白转导域的预测位置。结论:成功地制备了抗MDV-1 VP22C的mAb,为深入研究VP22蛋白的转导功能提供了有用的试剂。

MDV-1 VP22羧基端在大肠杆菌中高效可溶性表达

VP22是血清I型马立克氏病病毒(MDV-1)的复制和传播必不可少的组分。本研究从MDV-1无致病性的CVI988/Rispens疫苗感染的鸡胚成纤维细胞DNA中扩增得到VP22基因,并在大肠杆菌中表达其C端功能区。SDS-PAGE电泳发现,VP22C端得到高效可溶性表达,大小为42kDa左右。将获得的阳性条带经切胶免疫和细菌超声波裂解的上清作为抗原免疫6周龄Balb/C小鼠,均能诱导产生特异性抗VP22C端抗体。通过免疫荧光试验,检测到VP22在感染MDVCEF所有细胞核中呈特异性表达。这一抗体对深入研究VP22蛋白转导功能起到重要的作用。

重组马立克病病毒CVI988／Rispens的构建

根据I型马立克病病毒(MDV)强毒GA株基因序列,设计两对引物,用PCR方法扩增出CVI988／Rispens株的US10及其侧翼序列,分别克隆入pUC18载体中。经测序检测正确后,进一步插入含CMV启动子的绿色荧光蛋白 (EGFP)基因表达盒,获得了含EGFP报告基因的转移载体质粒pPUC18-US10-EGFP。通过同源重组,成功地筛选出表达EGFP的重组病毒rCVI988-EGFP,经传代证明重组rCVI988-EGFP在感染的CEF细胞中能稳定表达EGFP。结果表明:构建的重组转移载体质粒正确,US10是MDV复制非必需片段,为进一步利用US10区构建重组MDV多价基因工程疫苗奠定了基础。

鸡传染性贫血病毒与马立克氏病病毒共感染SPF鸡群免疫抑制协同作用的研究

将鸡传染性贫血病毒(Chicken infectious anemia virus,CIAV or CAV)和马立克氏病病毒(Marek s dis-ease virus,MDV)人工单一和共同感染1日龄的SPF鸡,感染后分别于14、21、28、35日龄检测鸡体红细胞压积的变化,并检测鸡群疫苗免疫3周后的抗体反应,以探讨CAV与MDV共感染对鸡体的免疫抑制是否有协同作用。结果表明,在血液分析方面,CAV与MDV共感染组较病毒单一感染组与对照组差异极显著,共感染不仅加重了鸡群贫血现象,而且延长了贫血的病理症状;而在禽流感病毒(Avian influenza virus,AIV)H5/H9疫苗、新城疫病毒(Newcastle disease virus,NDV)疫苗和传染性法氏囊病毒(Infectious bursal disease virus,IBDV)疫苗免疫后3周的抗体检测中,CAV与MDV共感染组较其它各实验组差异极显著,抗体滴度大大低于其它实验组;此外,CAV与MDV共感染组,鸡体生长状况明显差于实验各组,有6只鸡只死亡(6/25),比病毒单一感染时的死亡率大大增加。综上研究证明,CAV与MDV共感染在免疫抑制作用上有协同作用。

致弱Ⅰ型MDV pp38基因同源物的克隆和序列分析

将致弱Ⅰ型马立克氏病病毒（ＭＤＶ）感染的细胞基因组ＤＮＡ的ＥｃｏＲⅠ酶切产物建于ｐＵＣ１８质粒载体文库中，以ｄｉｇｏｘｉｎｇｅｎｉｎ标记的含有强毒ＧＡ株ＭＤＶｐｐ３８基因克隆片段作为探针，进行原位杂交反应，初步筛选出阳性重组质粒，进一步用ＥｃｏＲⅠ酶切分析筛选到含ｐｐ３８基因同源物的重组ｐＵＣ１８质粒．序列分析表明该ｐｐ３８基因同源物与ｐｐ３８基因有极高的同源性。

MDV和AIV主要保护性抗原基因在鸡痘病毒中的联合表达

将强启动子Ps插入载体7S中构建成插入载体7SP,将鸡马立克氏病病毒(MDV)糖蛋白B(gB)基因和禽流感病毒(AIV)血凝素H9A基因共同克隆到插入载体7SP中,获得重组转移质粒7SPGA,通过酶切鉴定获得Ps-gB与P7.5-HA同向的转移质粒,再将报告基因盒PL11-LacZ插入转移质粒7SPGA得7SPLGA,将质粒7SPLGA与禽痘病毒疫苗株共转染鸡成纤维细胞,通过蓝白斑筛选纯化得到重组病毒rFPV-Ps-gB-P7.5-HA,以间接免疫荧光法证实gB基因和HA基因同时得到表达。该研究为探索新型基因工程苗打下了良好的基础。