Molecular Cloning and Sequencing of a Specific cDNA Mapping to the Bam HI-I2 and -LFragments within the Inverted Repeats of Unique Long Region(IRL) from the Genom e of the Marek′s Disease Herpesvirus (MDV) Oncogenic Strain Beijing-1

Isolation and identification of a specific cDNA mapping to the BamHI I2 and L fragments from the inverted repeats of unique long region(IRL) in the genome of Marek′s disease herpesvirus (MDV) oncogenic strain Beijing 1 had been previously performed by us. In this study, the specific cDNA was cloned into phagemid vectors PUC118 and 119 on the basis of prefabricated two recognized sites in synthesized primers. Recombinants were further identified with restriction pattern, molecular hybridization, and DNA sequencing analysis. It was demonstrated that this cDNA with 720 base pair (bp) contained sequences including a potentially incomplete open reading frame (ORF) encoded a 238 amino acids (aa) predicted polypeptide which was significantly homologous not only to part of N terminus of meq, but also to that of XbaI ClaI subfragment of BamHI L. In accordance with these data, following results could be deduced:①the 720 bp cDNA represented a spliced transcript;②meq transcription could be extended from the right hand end of BamHI I2 to the adjacent BamHI L fragment;③the L region was transcribed in varying degrees\ in MDV induced lymphoblastoid tumors.

Isolation and Identification of a Specific cDNA Mapping to the Bam HI-I2 and -LFragments within the Inverted Repeats ofUnique Long Re-gion (IRL) in the Genom e ofMarek′s Disease Herpesvirus (MDV) Oncogenic Strain Beijing-1

Objective\ To understand the transcription of BamHI L DNA fragment from genome of strong virulent GA strain of Marek′s disease herpesvirus (MDV) in lymphoblastoid tumor tissue induced by oncogenic strain Beijing 1 (a specific local strain in China) of MDV. Methods\ Two oligonucleotide primers were synthesized according to the reported sequence of \%meq\% gene an ideal oncogenic candidate and our previously determined sequence of BamHI L fragment of Marek′s disease herpesvirus (MDV), respectively. Reverse transcriptase PCR(RT PCR) assay was performed by using these primers and the mRNA as a template which was isolated from visceral lymphoblastoid tumors obtained from chickens artificially infected with strain Beijing 1 of oncogenic MDV. Southern blot molecular hybridization was further carried out to detect the product of RT PCR with digoxigenin labeled nucleotide probe from BamHI I2 and L fragment in the gene library of MDV strain GA, respectively. Results\ Two probes could simultaneously hybridize this cDNA amplified by RT PCR with a length of about 730 bp. Conclusion\ It is suggested that \%meq\% transcription could extend from the right hand end of BamHI I2 to the adjacent BamHI L, and the BamHI L region was likely to be transcribed in MDV induced lymphoblastoid tumors.

Are the two polymorphic sites of anti-Marek’s disease in White Leghorn chickens also suitable for Partridge Shank chickens?

Background:The selection of Marek’s disease(MD)-resistant breeds in Partridge Shank chicken,a popular local chicken breed in Henan Province of China,has practical value.We hypothesized that the two polymorphic sites(rs14527240 located in SMOC1 and GGaluGA156129 located in PTPN3)related to MD resistance in White Leghorn chickens are also applicable to Partridge Shank chickens.Methods:In this experiment,we screened 10 live hens and 2 live roosters with the double GG genotype by genotyping the two sites from 6500 Partridge Shank chickens.Nineteen one-day-old chicks with the double GG genotype were obtained by artificial insemination.Seventy-two one-day-old chickens(19 from the population expansion test and 53 randomly selected from chicken farms)were injected with 2000 plaque-forming units of the Md5 virus strain.After 100 days of infection,all chickens were examined by pathological anatomical examination,histological sectioning,genotyping,and a quantitative polymerase chain reaction of SMOC1 and PTPN3.Results:There was only one site(rs14527240 located in SMOC1)associated with MD in Partridge Shank chickens(p<0.05),but the GG genotype of SMOC1 in Partridge Shank chickens indicated susceptibility to MD.SMOC1 expression in MD-susceptible chickens was also significantly higher than that in MDresistant chickens(p<0.05).Conclusion:Therefore,the MD resistance sites selected from White Leghorn chickens were not completely suitable for Partridge Shank chickens,but they can be used as a reference.This study indicated that SMOC1 plays an important role in screening for MD resistance in poultry.

Contributions of neurotropic human herpesviruses herpes simplex virus 1 and human herpesvirus 6 to neurodegenerative disease pathology

Human herpesviruses （HVs） have developed ingenious mechanisms that enable them to traverse the defenses of the central nervous system （CNS）. The ability of HVs to enter a state of latency, a defining char- acteristic of this viral family, allows them to persist in the human host indefinitely. As such, HVs represent the most frequently detected pathogens in the brain. Under constant immune pressure, these infections are largely asymptomatic in healthy hosts. However, many neurotropic HVs have been directly connected with CNS pathology in the context of other stressors and genetic risk factors. In this review, we discuss the potential mechanisms by which neurotropic HVs contribute to neurodegenerative disease （NDD） patholo- gy by highlighting two prominent members of the HV family, herpes simplex virus 1 （HSV-1） and human herpesvirus 6 （HHV-6）. We （i） introduce the infectious pathways and replicative cycles of HSV-1 and HHV-6 and then （ii） review the clinical evidence supporting associations between these viruses and the NDDs Alzheimer＇s disease （AD） and multiple sclerosis （MS）, respectively. We then （iii） highlight and dis- cuss potential mechanisms by which these viruses exert negative effects on neurons and glia. Finally, we （iv） discuss how these viruses could interact with other disease-modifying factors to contribute to the initiation and/or progression of NDDs.

St udy on Hsp90 Expression in Different Tissues and Its Antibody in Serum of Chickens Infected with Marek's Diseases

To investigate the dynamic change of heat shock protein 90 （Hsp90） in the genesis and development of tumor, we successfully established tumor animal model using Marek’s disease and then determined the location of Hsp90 in the tumor tissue using immunohistochemistry method, the antibody titer level of Hsp90 in the serum and the expression level in the tissue using enzyme-linked immunosorbent assay （ELISA） method. Our result showed that Hsp90 location in the tumor tissue was signiifcantly associated with the tumor cell and most in the cytoplasm of the tumor cell, and Hsp90 expression level in the tissue and the antibody titer level in the serum was most signiifcantly increased with the development of tumor. This is the ifrst report to show the presence of Hsp90 in tumor tissues induced by the Marek’s disease, with its expression correlated to the tumoral grading. These data may also be valuable for developing new molecular anti-cancer therapies.

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.

Vaccine by Chicken Line Interaction Alters the Protective Efficacy against Challenge with a Very Virulent plus Strain of Marek’s Disease Virus in White Leghorn Chickens

Marek’s disease (MD) is a lymphoproliferative disease of domestic chickens caused by Marek’s disease virus (MDV), an oncogenic and highly contagious α-herpesvirus. MD has been controlled by vaccination but sporadic outbreaks of MD still occur in some parts of the world. Efforts to improve vaccine efficacy have continued in both research communities and vaccine industries. We reported the host genetic variation affecting Marek’s disease vaccine-induced immunity in chickens earlier. In this study, we evaluated chicken lines, vaccines, and line by vaccine interaction on the protective efficacy of vaccination against MD. Specific pathogen free chickens from the relatively resistant line 63 and the highly susceptible line 72 were primarily used to evaluate the protection by three kinds of vaccines (rMd5ΔMeq, CVI988/Rispens, and HVT) upon challenge with a very virulent plus strain of MDV, vv+648A. Our data confirmed that both the chicken line and the vaccine significantly affected the protective efficacy of vaccination and showed that a chicken line by vaccine interaction, in most of the trials, also altered vaccine protective efficacy. More interestingly, although the protective index of all vaccine strains was higher in resistant than in susceptible line of chickens, the difference for HVT protection was striking and warrants further study. The findings may have important implications for vaccine development as well as for selective use of particular vaccines in specific lines of chickens to achieve maximum protection at minimized costs.

Knockdown of the Meq gene in Marek’s disease tumor cell line MSB1 might induce cell apoptosis and inhibit cell proliferation and invasion

Marek’s disease(MD),a highly cell-associated and contagious disease of chickens caused by Marek’s disease virus(MDV)can result in neural lesions,immunosuppression and neoplasia in chicken.The Meq gene is an important oncogene in the MDV genome,and it is expressed highly in MD tumor tissues and MD T-lymphoblastoid cell lines.An experiment was conducted to elucidate the role of Meq in MD tumor transformation.RNA interference technology was used to block its expression,and then analyzed the biological effects of Meq knockdown on the MD tumor cell line MSB1.A small interfering RNA with an interference efficiency of 70%(P<0.01)was transfected into MSB1 cells to knock down the expression of Meq gene.The cell proliferation,cycle and apoptosis were detected post-Meq knockdown.The results showed that MSB1 cell proliferation was downregulated remarkably at 48 h(P<0.01),60 h(P<0.05)and 72 h(P<0.01)post-Meq knockdown.The cell cycle was unaffected(P>0.05).B-cell lymphoma 2 gene(BCL2)was anti-apoptotic and caspase-6 was the effector in the apoptosis pathway.The activity of caspase-6 was upregulated(P<0.05)significantly and BCL2 gene expression was downregulated(P<0.05)significantly post-Meq knockdown,suggesting cell apoptosis might be induced.MSB1 cell migration did not exhibit any obvious change(P>0.05)post-Meq knockdown,but the expression of two genes(matrix metalloproteinase 2(MMP2)and MMP9)that are correlated closely to cell invasion was downregulated(P<0.05)remarkably post-Meq knockdown.The Meq knockdown might affect the main features of tumorous cells,including proliferation,apoptosis,and invasion,suggesting that the Meq gene might play a crucial role in interfering with lymphomatous cell transformation.

洛阳市郊区四个鸡场鸡马立克氏病(MAREK′S DISEASE)诊断报告

一、引言 鸡马立克氏病(MD)在我省的检出是1978年3月我们对三只仙居鸡进行了大体剖检和组织学检查之后确诊的。之后79年元月在处理牧场病残鸡时又检出了数只。1981年许昌地区畜牧兽医工作站报道了许昌某鸡场MD暴发的情况。1981年11月我们又在洛阳市郊区四个鸡场检出MD病鸡。仅将洛阳市郊MD诊断结果报告于后。 二、诊断方法 1、群检:在四个鸡场中任选1——2圈鸡群进行视检,从中挑出弱病鸡备检。 2、在上述挑出的备检鸡中随机取若干鸡,从翅、腋下、胸部拔取新鲜羽毛6根。

Cloning and Identification of L-meq Gene of Marek's Disease Virus

[Objective]To clone and identify Marek＇s disease virus （ MDV） serum gene. [Method]MDV genomic DNA was extracted from lymphoid tissues of the diseased chickens with latent MDV infection. The MDV L-meq gene was amplified by gradient PCR,inserted into pMD18-T vector and sequenced. The sequence was analyzed using DNAman software. [Result] The obtained sequence had 100% similarity with the published se- quence of L-meq gene,showing successful amplification of target gene. [Conclusion]The paper provides new ideas and new methods for preven- tion and treatment of Marek＇s disease in chickens.

Marek’s disease virus challenge induced immune-related gene expression and chicken repeat 1 (CR1) methylation alterations in chickens

Marek’s disease virus (MDV) challenge induces lymphoma in susceptible chickens. Host genes, especially immune related genes, are activated by the virus. DNA methylation is an epigenetic mechanism that governs gene transcription. In the present study, we found that expression of signal transducer and activator of transcription 1 (STAT1) was upregulated at 10 days post infection (dpi) in MD susceptible chickens, whereas interleukin 12A (IL12A) was elevated in both resistant and susceptible chickens. However, we did not observe MDV-induced DNA methylation variations at the promoter CpG islands (CGIs) in STAT1 and IL12A. Interestingly, the methylation levels at Chicken Repeat 1 (CR1), the transposable elements (TEs) located upstream of two genes, were different between resistant and susceptible chickens. Furthermore, a mutation was identified in the CR1 element near IL12A. The impact of the point mutation in transcriptional factor binding is to be examined in the near future.

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.

A case of rapid growing colonic NK/T cell lymphoma complicated by Crohn's disease

A 37-year-old man developed abdominal pain and bloody diarrhea 11 months before admission. The colonoscopy revealed multifocal ulcers in the colon. Histology showed active chronic inflammation. Although anti-tuberculosis medication was effective, his symptoms repeated 2 months later. The subsequent colonoscopy revealed more extensive irregular ulcers than before, and he was clinically suspected with intestinal malignant lymphoma. He underwent subtotal colectomy and was histologically suggested Crohn＇s disease, then 5-aminosalicylic and a combination of prednisone and azathioprine were administered in succession postoperatively, but they achieved minimal relief of symptoms for a period of 7 months. The third colonoscopy showed a large irregular ulcer in the ileocolon stomas, and primary colonic NK/T cell lymphoma was diagnosed through histological and immunophenotypic studies. Malignant lymphoma should be taken into consideration when clinically diagnosed Crohn＇s disease was refractory to medication or when its clinical course became aggressive.

Prevention and Treatment of KSHV-associated Diseases with Antiviral Drugs

Kaposi’s sarcoma-associated herpesvirus (KSHV) was first identified as the etiologic agent of Kaposi’s sarcoma (KS) in 1994. KSHV infection is necessary,but not sufficient for the development of Kaposi sarcoma (KS),primary effusion lymphoma (PEL),and multicentric Castleman disease (MCD). Advances in the prevention and treatment of KSHV-associated Diseases have been achieved,even though current treatment options are ineffective,or toxic to many affected persons. The identification of new targets for potential future therapies and the randomized trial to evaluate the efficacy of new antivirals are required.

鸡马立克氏病火鸡疱疹病毒间接免疫荧光检测方法的建立与比较

为建立鸡马立克氏病火鸡疱疹病毒(MDV HVT)的间接免疫荧光方法,将MDV HVT病毒接种96孔细胞板培养(CEF),72 h后用鸡抗马立克氏病病毒阳性血清作为一抗和兔抗鸡荧光抗体作为二抗,对反应条件进行优化,建立了间接免疫荧光方法(IFA)。并与经典的马立克蚀斑计数方法进行病毒滴度的测定比较,运用统计学方法分析,P小于0.05,相关系数为0.995,说明两种方法相关性很好,两种方法均可用于马立克氏病病毒的滴度测定。IFA方法较蚀斑计数方法操作简便快捷,可代替蚀斑计数方法用于马立克氏病活疫苗的病毒含量的初步测定,同时为2025年版《中国兽药典》马立克氏病活疫苗增加IFA鉴别检验方法积累实验数据。

基于高分辨率熔解曲线检测MD肿瘤微卫星不稳定性

本研究旨在通过高分辨率熔解曲线(high-resolution melting,HRM)方法检测马立克病(Marek′s disease,MD)肿瘤中存在的微卫星不稳定现象(microsatellite instability,MSI)。通过临床症状、病理剖检、组织病理学、PCR及测序确定为马立克病。采集5只发病鸡的肌肉、肝、脾、血液及肿瘤组织,提取DNA,采用15对扩增微卫星DNA序列的引物进行HRM检测,得到归一化处理的差异熔解曲线,比较分析各组织熔解曲线的差异,分析15个微卫星标记是否存在基因突变(即MSI)。结果表明,发病鸡的各种临床、病理表现符合为马立克病的特征,病毒meq基因序列与近年来国内流行的MDV强毒株有较高的同源性。HRM分析结果显示,MD发病鸡的肌肉、肝、脾、血液、肿瘤组织中微卫星标记PCR扩增后的熔解曲线存在明显的差异,即存在MSI现象,而且不同的病鸡个体和不同的微卫星标记发生MSI的频率不同。15个微卫星标记在5只鸡的病料中存在MSI的微卫星标记的个数分别为14、7、5、3和1。在15个微卫星标记中,MCWO200、MCW0220这两个微卫星标记在所有的病鸡中都存在MSI,而ADL0158微卫星标记在所有的病鸡中都不存在MSI。通过非变性聚丙烯酰胺凝胶电泳和测序分析进一步证明这些微卫星PCR扩增片段确实存在片段大小不同,碱基序列中存在微卫星重复单位的增多、减少或基因片段的插入或缺失。结果提示,通过HRM分析可以检测MD肿瘤中的MSI现象,是一种灵敏、准确、简便、高通量的方法。

人参皂苷Rh_2对MSB-1细胞的增殖抑制作用及其机理

以不同质量浓度的人参皂苷Rh2与马立克氏病肿瘤细胞系MSB-1细胞共同作用,利用改良MTT法测定了人参皂苷Rh2对MSB-l细胞的增殖抑制作用,并通过流式细胞术、荧光检测和电镜形态学观察等方法探讨其抑制机理。结果显示:人参皂苷Rh2对MSB-l细胞的增殖存在抑制作用,并呈剂量依赖性,人参皂苷Rh2与MSB-1细胞培养72 h后的半数抑制质量浓度为0.65 mg/L;流式细胞术测定表明,人参皂苷Rh2可阻滞MSB-1细胞于S期;荧光检测和透射电镜观察表明,人参皂苷Rh2可以诱导部分MSB-1细胞发生凋亡。

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。

用 Western Blot 法鉴定感染鸡外周血淋巴细胞中马立克氏病毒抗原

马立克氏病毒是鸡的一种高度致肿瘤性疱疹病毒，主要以淋巴瘤为特征，有关该病毒的致病机理还不清楚。本研究利用澳大利亚分离的ＭＤＶ致病毒株ＭＰＦ５７感染１日龄ＳＰＦ雏鸡，并在感染后４周对其外周血淋巴细胞中的ＭＤＶ特异性抗原进行了检测。结果表明，临床上呈急性感染并伴有明显症状的鸡的外周血淋巴细胞中的ＭＤＶ抗原分子量主要为２８、７３、６０和４９ｋＤａ；而无明显症状的鸡的外周血淋巴细胞中的ＭＤＶ抗原分子量主要为７３ｋＤａ。试验证明，Ｗｅｓｔｅｒｎｂｌｏｔ是一种较好的检测病毒抗原的方法，可以检测到荧光抗体方法检测不到的淋巴细胞中的ＭＤＶ特异抗原。

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。