鸭肠炎病毒UL41、UL42基因的克隆与分析

依据GenBank登录的鸭肠炎病毒(DEV)核苷酸序列设计引物,利用长片段PCR技术扩增了DEV基因组UL36与UL43基因之间的未知序列,扩增所得片段长度约为15 kb.经EcoRV单酶切,将其中的3.9 kb片段克隆到pUC18中.序列分析表明该3.9 kb EcoR V片段含有2个完整的转录方向相反的与单纯疱疹病毒(HSV)UL41和UL42基因同源的ORF,命名为DEV UL41和ULA2基因.通过氨基酸序列比对发现:DEV UL41基因含有5个高度保守位点,而UL42含有2个,进化树分析表明DEV与疱疹病毒科a疱疹病毒亚科的马立克病毒、火鸡疱疹病毒的进化关系非常相近,为DEV的分类提供了参考依据.     

Protection and synergism by recombinant fowl pox vaccines expressing multiple genes from Marek's disease virus

Recombinant fowl poxviruses (rFPVs) were constructed to express genes from serotype 1 Marek's disease virus (MDV) coding for glycoproteins B, E, I, H, and UL32 (gB1, gE, gI, gH, and UL32). An additional rFPV was constructed to contain four MDV genes (gB1, gE, gI, and UL32). These rFPVs were evaluated for their ability to protect maternal antibody-positive chickens against challenge with highly virulent MDV isolates. The protection induced by a single rFPV/gB1 (42%) confirmed our previous finding. The protection induced by rFPV/gI (43%), rFPV/gB1UL32 (46%), rFPV/gB1gEgI (72%), and rFPV/gB1gEgIUL32 (70%) contributed to additional knowledge on MDV genes involved in protective immunity. In contrast, the rFPV containing gE, gH, or UL32 did not induce significant protection compared with turkey herpesvirus (HVT). Levels of protection by rFPV/gB1 and rFPV/gl were comparable with that of HVT. Only gB1 and gI conferred synergism in rFPV containing these two genes. Protection by both rFPV/gB1gEgI (72%) and rFPV/gB1gEgIUL32(70%) against Marek's disease was significantly enhanced compared with a single gB1 or gI gene (40%). This protective synergism between gB1 and gI in rFPVs may be the basis for better protection when bivalent vaccines between serotypes 2 and 3 were used. When rFPV/gB1gIgEUL32 + HVT were used as vaccine against Md5 challenge, the protection was significantly enhanced (94%). This synergism between rFPV/gB1gIgEUL32 and HVT indicates additional genes yet to be discovered in HVT may be responsible for the enhancement.     

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.     

Coinfection of specific-pathogen-free chickens with Marek's disease virus (MDV) and chicken infectious anemia virus: effect of MDV pathotype

Both Marek's disease virus (MDV) and chicken infectious anemia virus (CIAV) infections are prevalent in chickens throughout the world. In the past decade, MDV strains with increased virulence (very virulent plus MDV pathotype [vv+MDV]) have been isolated. The purpose of this experiment was to determine the effects of coinfection of chickens with CIAV and a vv+MDV isolate. Specific-pathogen-free chickens were inoculated at 1 day posthatch with RB1B (very virulent MDV pathotype [vvMDV]) only, 584A (vv+MDV) only, CIAV only, RB1B + CIAV, 584A + CIAV, or nothing. Samples of spleen, thymus, and bursa of Fabricius were collected at 4, 7, 10, and 13 days postinoculation (DPI). Thymic and bursal atrophy at 13 DPI and final mortality at 30 DPI were significantly greater in chickens inoculated with 584A with or without added CIAV, or with RB1B plus CIAV, compared with birds inoculated with RB1B alone. Both amounts of virus reisolated and levels of virus detected by quantitative-competitive polymerase chain reaction were greater at 4 DPI in 584A inoculates compared with RB1B inoculates. To monitor the early cytolytic infection, northern analysis was done with a probe for the MDV immediate early gene ICP4 (infected cell protein 4). In the absence of CIAV, ICP4 expression was more apparent in chickens inoculated with 584A than in those inoculated with RB1B. CIAV coinfection increased ICP4 expression in the spleens of chickens infected with RB1B. These results indicated that inoculation of chickens with the 584A isolate caused a more robust early cytolytic infection compared with inoculation with RB1B alone and support the classification of 584A as a vv+MDV strain. Coinfection with CIAV exacerbated vvMDV strain RB1B infection. The extent of this exacerbation was less evident when birds were coinfected with 584A and CIAV.     

伪狂犬病病毒Ea株UL6基因的克隆、序列分析及表达

以伪狂犬病病毒Ea株基因组DNA为模板,通过PCR扩增UL6全长基因,将PCR产物克隆于pMD18-T载体,并采用双脱氧终止法进行序列测定.序列分析显示UL6全长1 938 bp,可编码646个氨基酸.将该基因克隆到插入原核表达载体pET28a的6×His下游,获得原核表达质粒pET28a-UL6,转化大肠埃希菌BL21,经IPTG诱导在大肠埃希菌中成功表达获得分子质量约70 ku的融合表达蛋白6×His-UL6,Western blot证实,表达的融合蛋白能与抗6×His的单克隆抗体发生特异性反应.根据测定的序列,设计一对能扩增UL6基因完整编码区的引物,PCR扩增UL6基因并将其插入真核表达载体pEGFP-C2中EGFP基因的3'端,获得与EGFP融合表达的真核表达质粒pEGFP-UL6,转染Hela细胞,通过激光共聚焦显微镜观察发现,转染48 h,融合蛋白EGFP-UL6主要定位在胞浆,为进一步研究伪狂犬病病毒Ea株UL6基因的结构和功能奠定了基础.     

Comparative analysis of Marek's disease virus (MDV) glycoprotein-, lytic antigen pp38- and transformation antigen Meq-encoding genes: association of meq mutations with MDVs of high virulence

Marek's disease (MD) is a highly contagious lymphoproliferative and demyelinating disorder of chickens. MD is caused by Marek's disease virus (MDV), a cell-associated, acute-transforming alphaherpesvirus. For three decades, losses to the poultry industry due to MD have been greatly limited through the use of live vaccines. MDV vaccine strains are comprised of antigenically related, apathogenic MDVs originally isolated from chickens (MDV-2), turkeys (herpesvirus of turkeys, HVT) or attenuated-oncogenic strains of MDV-1 (CVI-988). Since the inception of high-density poultry production and MD vaccination, there have been two discernible increases in the virulence of MDV field strains. Our objectives were to determine if common mutations in the major glycoprotein genes, a major lytic antigen phosphoprotein 38 (pp38) or a major latency/transformation antigen Meq (Marek's EcoRI-Q-encoded protein) were associated with enhanced MDV virulence. To address this, we cloned and sequenced the major surface glycoprotein genes (gB, gC, gD, gE, gH, gI, and gL) of five MDV strains that were representative of the virulent (v), very virulent (vv) and very virulent plus (vv+) pathotypes of MDV. We found no consistent mutations in these genes that correlated strictly with virulence level. The glycoprotein genes most similar among MDV-1, MDV-2 and HVT (gB and gC, approximately 81 and 75%, respectively) were among the most conserved across pathotype. We found mutations mapping to the putative signal cleavage site in the gL genes in four out of eleven vv+MDVs, but this mutation was also identified in one vvMDV (643P) indicating that it did not correlate with enhanced virulence. In further analysis of an additional 12 MDV strains, we found no gross polymorphism in any of the glycoprotein genes. Likewise, by PCR and RFLP analysis, we found no polymorphism at the locus encoding the pp38 gene, an early lytic-phase gene associated with MDV replication. In contrast, we found distinct mutations in the latency and transformation-associated Marek's EcoRI-Q-encoded protein, Meq. In examination of the DNA and deduced amino acid sequence of meq genes from 26 MDV strains (9 m/vMDV, 5 vvMDV and 12 vv+MDVs), we found distinct polymorphism and point mutations that appeared to correlate with virulence. Although a complex trait like MDV virulence is likely to be multigenic, these data describe the first sets of mutations that appear to correlate with MDV virulence. Our conclusion is that since Meq is expressed primarily in the latent/transforming phase of MDV infection, and is not encoded by MDV-2 or HVT vaccine viruses, the evolution of MDV virulence may be due to selection on MDV-host cell interactions during latency and may not be mediated by the immune selection against virus lytic antigens such as the surface glycoproteins.     

Diversity (polymorphism) of the meq gene in the attenuated Marek's disease virus (MDV) serotype 1 and MDV-transformed cell lines

The meq gene encoding a 339-amino-acid bZIP transactivator protein has been identified as a candidate oncogene of Marek's disease virus serotype 1 (MDV1), which induces malignant lymphomas in chickens. We have previously reported that, in addition to meq, L-meq, in which a 180-bp sequence is inserted into the region encoding the transactivation domain of meq, is also detected in chickens experimentally infected with MDV. To further analyze the diversity in meq, PCR was performed using a primer set which specifically amplify the proline-rich repeat (PRR) region in the transactivation domain of meq. In CVI988/R6, a vaccine strain of MDV1, and JM, an MDV1 strain attenuated by prolonged passage in vitro, a major band of a 0.8 kb corresponding to L-meq as well as a minor band of 0.6 kb corresponding to meq was detected by PCR. Furthermore, extra 0.5- and 0.3-kb bands, corresponding to genes termed as short meq (S-meq), and very short meq (VS-meq), respectively, were also detected. These genes were also detected in MDV-transformed cell lines, MSB1 and MTB1. In Md5, an oncogenic MDV1, attenuated by prolonged passage in vitro, the 0.6-kb meq was consistently detected, and 0.5-kb S-meq was occasionally detected. This diversity in meq was due to the difference in the copy number of the PRR region: L-meq and meq contained 9 and 6 copies of PRR while 4 and 2 copies of PRR were present in S-meq and VS-meq, respectively. Thus, the meq gene is polymorphic in the attenuated MDV1 and the MDV-transformed cell lines, and gene products from different meq genes may have different functions from each other.     

Differentiation of infectious laryngotracheitis virus isolates by restriction fragment length polymorphic analysis of polymerase chain reaction products amplified from multiple genes

Infectious laryngotracheitis (ILT) has been identified in most countries around the world and remains a threat to the intensive poultry industry. Outbreaks of mild to moderate forms of ILT are common in commercial layer flocks, while sporadic outbreaks of ILT in broiler flocks have also been recognized as an emerging problem in several countries. Examination of viral isolates using restriction fragment length polymorphism of polymerase chain reaction (PCR-RFLP) from individual ILTV genes has suggested that some of these outbreaks were caused by vaccine strains. In this study, PCR-RFLP of a number of ILTV genes/genomic regions including gE, gG, TK, ICP4, ICP18.5, and open reading frame (ORF) B-TK was used to examine a number of historical and contemporary Australian ILTV isolates and vaccine strains. PCR-RFLP of gE using restriction endonuclease EaeI failed to distinguish between any of the isolates including the vaccine strains. PCR-RFLP of gG, TK, and ORFB-TK using restriction endonucleases MspI and FokI, respectively, divided all the isolates into two groups. PCR-RFLP of ICP18.5 and ICP4 using restriction endonuclease HaeIII separated the isolates into three different groups with some field isolates only able to be distinguished from vaccine strains by PCR-RFLP of ICP18.5. A combination of groupings including gG, TK, ICP4, ICP18.5, and ORFB-TK PCR-RFLP classified the ILTV isolates under investigation into five different groups with most isolates distinguishable from vaccine strains. Results from this study reveal that to achieve reliable identification of strains of ILTV, the examination of multiple gene regions will be required, and that most of the recent ILT outbreaks in Australia are not being caused by vaccine strains.     

伪狂犬病病毒Bartha-K61株UL49基因的克隆和序列分析

根据已经发表的伪狂犬病病毒(PRV)国内Ea株UL49基因序列,设计并合成了1对引物,通过PCR方法扩增到了PRV Bartha-K61株UL49基因的编码区,并克隆到pMD18-T载体中.重组质粒pMD-UL49经XhoI酶切和PCR鉴定后,进行了序列测定和分析.结果表明,重组质粒pMD-UL49含有PRV Bartha-K61株UL49基因的编码区.同源性分析显示,PRV Bartha-K61株UL49基因序列与国外Kaplan株、国内Ea株相应基因的核苷酸同源性分别为98.9 %和94.1 %,推导的氨基酸序列同源性分别为96.7 %和87.2 %.     

马立克病毒UL36~（USP）蛋白的表达及其抗体制备

UL36USP是马立克病毒UL36基因编码的蛋白UL36上具有去泛素化酶活性的N-端部分片段（UL36USP）,本试验以提取MDCC-MSB1细胞的基因组为模板通过PCR获得目的基因,将UL36USP基因克隆入pMD18T载体,测序正确并酶切鉴定后,将目的基因亚克隆到pET28a原核表达载体,构建重组质粒pET28a-UL36USP。将重组质粒转化BL21（DE3）E.coli中,IPTG诱导表达并使用Ni-NTA Agarose亲和层析进行纯化得到目的蛋白。应用纯化后的UL36USP免疫獭兔制备多克隆抗体。使用昆虫细胞表达系统表达的UL36片段检测多克隆抗体的特异性。结果显示成功制备特异性的UL36USP抗体。     

Identification of virus-specific antigens in cultured cells infected with Marek's disease virus serotype 2 and CVI-988 strain

For the identification of serotype-specific antigens of Marek's disease virus (MDV) serotype 1 (MDV1) or serotype 2 (MDV2), a total of 24 hybridoma clones, secreting monoclonal antibodies (MAbs) against CVI-988 (MDV1) or HPRS-24 (MDV2) strain, were established and characterized by immunofluorescence assay, virus neutralization and immunoprecipitation analysis. Based upon the molecular weights (mol. wt.) of the immunoprecipitated polypeptides, the MAbs were subdivided into 7 groups. Among them, two groups of MAbs reacted with antigens that have not been reported, were identified. MAbs belonging to the first group reacted with CVI-988- and MDV2-specific antigens with mol. wt. ranging from 29 K to 34 K (29/34 K). This antigen was not found in cells infected with Md/5 and JM strains of MDV1, and the results of kinetic analysis of antigen expression showed this antigen appeared to be related to late membrane antigens. MAbs belonging to the second group immunoprecipitated MDV2-specific antigens with mol. wt. of 37 K, 33 K and 31 K from HPRS-24-infected cells or with those of 37 K, 34 K and 31 K from SB-1(MDV2)-infected cells, and these antigens appeared to be related to early antigens. MAbs belonging to the other 5 groups included those which recognized similar antigens reported previously or the antigens characterized insufficiently in this study.     

马立克氏病病毒“814”疫苗株基因组重复区序列测定及分析

为了解马立克氏病病毒(MDV)强、弱毒株主要致肿瘤相关基因变异情况,本研究根据MDV GA株基因组序列,设计合成扩增基因组重复区的引物,得到MDV814疫苗株病毒基因组中约26kb的序列片段。与GenBank登录的强、弱毒株进行比较分析表明,扩增的MDV1型814疫苗株的长重复区为12774bp,预测的开放阅读框(ORF)有48个;短重复区为11426bp,预测的ORF有38个。发现了4个MDV814株特有的ORF。814株在编码Meq、RLORF6和23ku的重叠基因内具有类似于疫苗株CVI988的177bp的插入;在RLORF12基因编码区内存在69bp的缺失,该缺失位于病毒复制起始位点内。同时,发现7个814疫苗株特有的氨基酸突变,分布在6个ORF内。单核苷酸多态性(SNP)的鉴定发现,多个基因具有单核苷酸的突变,主要分布于Meq基因,其中氨基酸A115V(丙氨酸-缬氨酸),N142D(天冬酰胺-天门冬氨酸)的变异是814疫苗株所特有的。MDV814疫苗株重复区的基因序列的比较分析将有助于MDV致肿瘤机制的研究。     

Identification of the cross-reactive antigens between Marek's disease virus and pseudorabies virus by monoclonal antibodies

Among the 33 monoclonal antibodies (MAbs) against pseudorabies virus (PRV) examined, three MAbs (24-17, 74-26, and 8) were found to react with cells infected with Marek's disease virus (MDV)-related viruses by immunofluorescence test. Two of the MAbs (24-17 and 74-26) reacted with the nuclei of cells infected with MDV serotype 1 (MDV1), MDV serotype 2 (MDV2), and herpesvirus of turkeys (HVT), whereas MAb 8 reacted with the cytoplasm of MDV2- and HVT-infected cells. However, none of the MAbs against MDV1, MDV2, and HVT that were examined reacted with PRV-infected cells. None of these three MAbs against PRV reactive with MDV-related viruses cross-reacted with the cells infected with other herpesviruses, such as herpes simplex virus type 1, herpes simplex virus type 2, varicella zoster virus, Epstein-Barr virus, or human herpesvirus 6. Southern-blot hybridization under stringent or less-stringent conditions showed that no significant DNA homology was detected between PRV DNA and MDV DNA.