MHC-restricted cytotoxic response of chicken T cells: expression, augmentation, and clonal characterization

Major histocompatibility complex (MHC)-restricted cytotoxicity of chicken lymphocytes was studied by using three reticuloendotheliosis virus (REV)-transformed cell lines as targets in 51Cr-release assays. The cell lines, designated RECC-UG5, RECC-UG6, and RECC-UG8, were developed from bone marrow cells of REV-infected line G-B1, line G-B2, and (G-B1 X G-B2)F1 chickens respectively. Effector cells were obtained from spleens of G-B1, G-B2, F1, and F2 chickens 7 days after inoculation of REV. The inbred G-B1 (MHC genotype B13/B13) and G-B2 (MHC genotype B6/B6) lines originate from a common partially inbred line. Initial studies with effector cells from G-B1 and G-B2 chickens showed that significant cytotoxicity occurred only with syngeneic target cells. The degree of cytotoxicity was markedly enhanced by neonatally treating effector cell donors with cyclophosphamide (CY) and delaying virus challenge until the birds were 4 wk old. Augmentation of cytotoxicity was presumed to be due to elimination of bursal-dependent suppressor T cells by CY. The results with spleen cells from REV-inoculated F2 birds clearly showed that cytotoxicity was MHC restricted; i.e., significant lysis only occurred if effector cells and target cells had a common B system antigen. Lysis of RECC-UG5 targets was three to four times higher than lysis of RECC-UG6 targets when effector cells were from heterozygous (B6/B13)F1 and F2 birds. Because these two target cell lines generally showed a similar degree of lysis by effector cells from syngeneic B homozygous birds, the differences obtained with effector cells from B heterozygous birds was most likely due to differences in the number of effector cells with specificity for each target line. Evidence for an additive cytotoxic effect, considered to be due to the lytic activity of two separate T cell clones, was obtained when F1 effector cells were tested with the F1-derived RECC-UG8 targets. The results of other experiments indicated that the effector cells were of T cell lineage and that their activity was probably directed against virus-induced antigens on the transformed target cells.     

Horizontal transmission of Marek's disease virus requires US2, the UL13 protein kinase, and gC

Marek's disease virus (MDV) causes a general malaise in chickens that is mostly characterized by the development of lymphoblastoid tumors in multiple organs. The use of bacterial artificial chromosomes (BACs) for cloning and manipulation of the MDV genome has facilitated characterization of specific genes and genomic regions. The development of most MDV BACs, including pRB-1B-5, derived from a very virulent MDV strain, involved replacement of the US2 gene with mini-F vector sequences. However, when reconstituted viruses based on pRB-1B were used in pathogenicity studies, it was discovered that contact chickens housed together with experimentally infected chickens did not contract Marek's disease (MD), indicating a lack of horizontal transmission. Staining of feather follicle epithelial cells in the skins of infected chickens showed that virus was present but was unable to be released and/or infect susceptible chickens. Restoration of US2 and removal of mini-F sequences within viral RB-1B did not alter this characteristic, although in vivo viremia levels were increased significantly. Sequence analyses of pRB-1B revealed that the UL13, UL44, and US6 genes encoding the UL13 serine/threonine protein kinase, glycoprotein C (gC), and gD, respectively, harbored frameshift mutations. These mutations were repaired individually, or in combination, using two-step Red mutagenesis. Reconstituted viruses were tested for replication, MD incidence, and their abilities to horizontally spread to contact chickens. The experiments clearly showed that US2, UL13, and gC in combination are essential for horizontal transmission of MDV and that none of the genes alone is able to restore this phenotype.     

Attenuation of Marek's disease virus by deletion of open reading frame RLORF4 but not RLORF5a

Marek's disease (MD) in chickens is caused by the alphaherpesvirus MD virus (MDV) and is characterized by the development of lymphoblastoid tumors in multiple organs. The recent identification and cloning of RLORF4 and the finding that four of six attenuated strains of MDV contained deletions within RLORF4 suggested that it is involved in the attenuation process of MDV. To assess the role of RLORF4 in MD pathogenesis, its coding sequence was deleted in the pRB-1B bacterial artificial chromosome clone. Additionally, RLORF5a was deleted separately to examine its importance for oncogenesis. The sizes of plaques produced by MDV reconstituted from pRB-1BdeltaRLORF5a (rRB-1BdeltaRLORF5a) were similar to those produced by the parental pRB-1B virus (rRB-1B). In contrast, virus reconstituted from pRB-1BDeltaRLORF4 (rRB-1BdeltaRLORF4) produced significantly larger plaques. Replication of the latter virus in cultured cells was higher than that of rRB-1B or rRB-1BdeltaRLORF5a using quantitative PCR (qPCR) assays. In vivo, both deletion mutants and rRB-1B replicated at comparable levels at 4, 7, and 10 days postinoculation (p.i.), as determined by virus isolation and qPCR assays. At 14 days p.i., the number of PFU of virus isolated from chickens infected with rRB-1BdeltaRLORF4 was comparable to that from chickens infected with highly attenuated RB-1B and significantly lower than that from rRB-1B-infected birds. The number of tumors and kinetics of tumor production in chickens infected with rRB-1BdeltaRLORF5a were similar to those of P2a chickens infected with rRB-1B. In stark contrast, none of the chickens inoculated with rRB-1BdeltaRLORF4 died up to 13 weeks p.i.; however, two chickens had tumors at the termination of the experiment. The data indicate that RLORF4 is involved in attenuation of MDV, although the function of RLORF4 is still unknown.     

Protective Effect of Avian Myelomonocytic Growth Factor in Infection with Marek’s Disease Virus

Marek's disease virus (MDV) is a herpesvirus that induces T lymphomas in chickens. The aim of this study was to assess the role of the macrophage activator chicken myelomonocytic growth factor (cMGF) in controlling MDV infection. B13/B13 chickens, which are highly susceptible to MD, were either treated with cMGF delivered via a live fowlpox virus (fp/cMGF) or treated with the parent vector (fp/M3) or were left as untreated controls. Seven days later, when challenged with the very virulent RB-1B strain of MDV, the spleens of chickens treated with fp/cMGF showed increased expression of the inducible nitric oxide synthase (iNOS) gene compared to those of control chickens and fp/M3-treated chickens. Increased iNOS gene expression was also accompanied by greater induction of gamma interferon and macrophage inflammatory protein (K203) gene expression, both possible activators of iNOS. fp/cMGF treatment also increased the number of monocytes and systemic NO production in contrast to fp/M3 treatment. Even though cMGF treatment was unable to prevent death for the chickens, it did prolong their survival time, and viremia and tumor incidence were greatly reduced. In addition, cMGF treatment improved the partial protection induced by vaccination with HVT (herpesvirus isolated from turkeys) against RB-1B, preventing 100% mortality (versus 66% with vaccination alone) and greatly reducing tumor development. Treatment with fp/M3 did not have such effects. These results suggest that cMGF may play multiple roles in protection against MD. First, it may enhance the innate immune response by increasing the number and activity of monocytes and macrophages, resulting in increased NO production. Second, it may enhance the acquired immune response, indicated by its ability to enhance vaccine efficacy.     

马立克氏病病毒超强毒感染鸡羽髓蛋白质组分析

【目的】羽毛是细胞游离马立克氏病病毒(Marek’s disease virus,MDV)释放的部位,为了解感染MDV后鸡羽中宿主基因表达的变化及对病毒感染的应答,进行了MDV感染鸡的羽髓蛋白质组学分析。【方法】1日龄无特定病原体(specific pathogen free,SPF)鸡人工感染MDV超强毒RB1B株(1000PFU),感染后21d采集鸡羽毛,提取羽髓蛋白,以17cm,pH5-8的IPG胶条进行二维电泳,以未感染病毒的SPF鸡羽髓蛋白为对照,使用PDQuest软件对二维电泳图谱进行差异蛋白分析,并选取部分差异斑点进行质谱鉴定。【结果】PDQuest软件分析发现攻毒组和对照组表达差异大于两倍的蛋白点有41个,其中攻毒组表达上调的蛋白点25个,下调的蛋白点7个,新出现的蛋白点有9个。质谱分析共成功鉴定了21个斑点,对应于20个蛋白。如载脂蛋白AI(apolipoprotein AI)、14-3-3 sigma(两个斑点均为该蛋白)、癌蛋白18(stathmin)等。【结论】功能预测表明这些蛋白涉及到宿主的抗病毒应答、物质代谢、细胞骨架成分、细胞增殖相关等方面。     

Inhibitory effects of nitric oxide and gamma interferon on in vitro and in vivo replication of Marek's disease virus

The replication of Marek's disease herpesvirus (MDV) and herpesvirus of turkeys (HVT) in chicken embryo fibroblast (CEF) cultures was inhibited by the addition of S-nitroso-N-acetylpenicillamine, a nitric oxide (NO)-generating compound, in a dose-dependent manner. Treatment of CEF culture, prepared from 11-day-old embryos, with recombinant chicken gamma interferon (rChIFN-gamma) and lipopolysaccharide (LPS) resulted in production of NO which was suppressed by the addition of N(G)-monomethyl L-arginine (NMMA), an inhibitor of inducible NO synthase (iNOS). Incubation of CEF cultures for 72 h prior to treatment with rChIFN-gamma plus LPS was required for optimal NO production. Significant differences in NO production were observed in CEF derived from MDV-resistant N2a (major histocompatibility complex [MHC], B(21)B(21)) and MDV-susceptible S(13) (MHC, B(13)B(13)) and P2a (MHC, B(19)B(19)) chickens. N2a-derived CEF produced NO earlier and at higher levels than CEF from the other two lines. The lowest production of NO was detected in P2a-derived CEF. NO production in chicken splenocyte cultures followed a similar pattern, with the highest levels of NO produced in cultures from N2a chickens and the lowest levels produced in cultures from P2a chickens. Replication of MDV and HVT was significantly inhibited in CEF cultures treated with rChIFN-gamma plus LPS and producing NO. The addition of NMMA to CEF treated with rChIFN-gamma plus LPS reduced the inhibition. MDV infection of chickens treated with S-methylisothiourea, an inhibitor of iNOS, resulted in increased virus load compared to nontreated chickens. These results suggest that NO may play an important role in control of MDV replication in vivo.     

Protection against Marek''s disease by a fowlpox virus recombinant expressing the glycoprotein B of Marek''s disease virus.

Fowlpox virus (FPV) recombinants expressing the glycoprotein B and the phosphorylated protein (pp38) of the GA strain of Marek's disease virus (MDV) were assayed for their ability to protect chickens against challenge with virulent MDV. The recombinant FPV expressing the glycoprotein B gene elicited neutralizing antibodies against MDV, significantly reduced the level of cell-associated viremia, and, similar to the conventional herpesvirus of turkeys, protected chickens against challenge with the GA strain and the highly virulent RB1B and Md5 strains of MDV. The recombinant FPV expressing the pp38 gene failed to either elicit neutralizing antibodies against MDV or protect the vaccinated chickens against challenge with MDV.     

Retention of oncogenicity by a Marek's disease virus mutant lacking six unique short region genes.

We previously reported the construction of Marek's disease virus (MDV) strains having mutations in various genes that map to the unique short (US) region of the viral genome (J.L. Cantello, A.S. Anderson, A. Francesconi, and R.W. Morgan, J. Virol. 65:1584-1588, 1991; M.S. Parcells, A.S. Anderson, and R.W. Morgan, Virus Genes 9:5-13, 1994; M.S. Parcells, A.S. Anderson, and R.W. Morgan, J. Virol. 68:8239-8253, 1994). These strains were constructed by using a high-passage-level serotype 1 MDV strain which grew well in chicken embryo fibroblasts. Despite the growth of the parent and mutant viruses in cell culture, in vivo studies were limited by poor growth of these strains in chickens. One of the mutants studied lacked 4.5 kbp of US region DNA and contained the lacZ gene of Escherichia coli inserted at the site of the deletion. The deletion removed MDV homologs to the US1, US2, and US10 genes of herpes simplex virus type 1 as well as three MDV-specific open reading frames. We now report the construction of a mutant MDV containing a similar deletion in the US region of the highly oncogenic RB1B strain. This mutant, RB1B delta 4.5lac, had a growth impairment in established chicken embryo fibroblasts similar to that described previously for MDVs lacking a functional US1 gene. In chickens, RB1B delta 4.5lac showed decreased early cytolytic infection, mortality, tumor incidence, and horizontal transmission. Several lymphoblastoid cell lines were established from RB1B delta 4.5lac-induced tumors, and virus reactivated from these cell lines was LacZ+. These results indicate that the deleted genes are nonessential for the transformation of chicken T cells or for the establishment and maintenance of latency. On the basis of the growth impairment observed for RB1B delta 4.5lac in cell culture and in vivo, we conclude that deletion of these genes affects the lytic replication of MDV. This is the first MDV mutant constructed in the RB1B oncogenic strain, and the methodology described herein provides for the direct examination of MDV-encoded determinants of oncogenicity.     

Evidence for widespread epistatic interactions influencing Marek's disease virus viremia levels in chicken

Marek's disease (MD), a T cell lymphoma induced by the Marek's disease virus (MDV), is the main chronic infectious disease concern threatening the poultry industry. Enhancing genetic resistance to MD in commercial poultry is an attractive method to augment MD vaccines, which is currently the control method of choice. In order to implement this control strategy through marker-assisted selection (MAS), it is necessary to identify quantitative trait loci (QTL) or genes that influence MD incidence. Previous studies have demonstrated that it is possible to identify QTL that confer MD resistance in both experimental and commercial chickens. With the advent of the chicken genome sequence and new genomic tools, and evidence that interactions are important in understanding complex traits, the line 6 x 7 F(2) experimental resource population was re-evaluated with finer resolution for epistatic interactions. The F(2) population, consisting of 272 individuals and previously genotyped with 133 genetic markers, was combined along with 576 additional single nucleotide polymorphisms (SNPs) genotyped on 80 individuals in each of the distribution tails for MD and other associated traits, and tested for the presence of main effects and two-way epistatic interactions accounting for MD incidence, viremia titers, and length of survival. Main effects were generally not significant but a large number of highly significant interactions, involving loci located throughout the genome, were identified that account for MDV viremia titers in infected birds. These results suggest that resistance to MD is highly complex and will require the incorporation of epistatic interaction analyses and functional genomic approaches to reveal the underlying genetic basis.     

敲除meq的鸡马立克氏病毒强毒株对超强毒的免疫保护作用

【目的】比较和评价一株敲除了meq基因的马立克氏病毒(MDV)的致病性及其诱发的保护性免疫作用。【方法】将1日龄SPF鸡150只随机分为5组,每组30只,分别饲养于正压过滤空气的SPF动物饲养隔离罩内。1日龄时,第1和第5组鸡以2000PFU/只的剂量腹腔接种GX0101Δmeq,第2组鸡以2000PFU/只的剂量腹腔接种CVI988/Rispens疫苗株,第3和第4组鸡不接种任何病毒作为对照组。免疫接种5d后,第1、2、3组分别以500PFU/只的剂量攻击MDV超强毒株vvrMd5。饲养90d,观察死亡情况,对各组死亡鸡只剖检,并取疑似马立克特有病变脏器做石蜡切片,于攻毒后90d处死全部存活鸡并随机取心脏、肝脏、脾脏做病理切片。【结果】单独接种GX0101Δmeq的第5组没有任何马立克氏病临床症状和特有的组织学病变,接种GX0101Δmeq再感染超强毒株vvrMd5的第1组也没有马立克病特有的组织学病变,但CVI988/Rispens免疫后感染超强毒株vvrMd5的第2组显示马立克病特有病变的病理切片比例为9/42,单独接种超强毒株vvrMd5的第3组死亡率为87%,死亡鸡出现可眼观典型肿瘤率为25%,免疫接种GX0101Δmeq和CVI988/Rispens的第1组和第2组对超强毒株vvrMd5攻击的保护指数分别为100%和89%。【结论】本实验构建的MDVmeq基因缺失株-GX0101Δmeq可在体外稳定复制,不仅对SPF鸡没有致病性和致瘤性,而且能诱导比CVI988/Rispens疫苗株更好的对超强毒MDV的免疫保护效果。     

Persistence of Marek''s disease virus in a subpopulation of B cells that is transformed by avian leukosis virus, but not in normal bursal B cells.

Previous studies have described an augmentation of avian leukosis virus (ALV)-induced lymphoid leukosis in chickens that were coinfected with a serotype 2 Marek's disease virus (MDV) strain, SB-1. As a first step toward understanding the mechanism of this augmentation, we have analyzed the tropism of the MDV for the ALV-transformed B cell. After hatching, chickens were coinfected with ALV and a nonpathogenic strain of MDV, SB-1. Seventy primary and metastatic ALV-induced lymphomas that developed in chickens between 14 and 20 weeks of age were found, with only one exception, to carry SB-1 DNA. The MDV genome was maintained in cell lines derived from the tumors. However, MDV DNA could not be detected in nontransformed bursal B cells from chickens carrying ALV lymphomas. Moreover, during and after the lytic phase of MDV infection, SB-1 DNA was near or below the level of detection in bursal cells, suggesting that MDV most likely infects only a small subpopulation of bursal cells. By contrast, ALV-transformed B cells from MDV-free chickens could be persistently infected with MDV in vitro. These findings indicate that ALV lymphoma cells, unlike nontransformed bursal B cells, are susceptible to persistent MDV infection and can serve as a reservoir of MDV that can potentially influence the physiology of the transformed cell.     

Novel applications of feather tip extracts from MDV-infected chickens; diagnosis of commercial broilers, whole genome separation by PFGE and synchronic mucosal infection

Marek's disease virus (MDV) productive replication occurs in the feather follicle epithelium and the feather tips are valuable both for research and disease diagnosis. Three novel applications of feather tip extracts are described now: (A). As a source of DNA for amplifying either MDV and/or ALV-J. In two clinical situations a marked advantage was obtained compared to blood and organs; in broiler breeder flocks with a mixed MDV and ALV-J infection, and in young broilers with neurological Marek's disease (MD). (B). Separation of the large ( approximately 200 kbp) MDV genome directly from the infected chickens. Using pulsed field gel electrophoresis, the DNA extracted from tumors or feather tips was separated and hybridized to a 132 bp tandem repeat MDV probe. Compared to 2/55 polymerase chain reaction (PCR) positive tumor samples, 15/61 feather tip extracts contained whole MDV genomes. (C). Experimental MDV infection was induced by the mucosal route by dripping feather tip extract to the eye and mouth of the bird. That attempted to reproduce the native infection process, however the use of extracts, instead of dry feather dust was a compromise, aimed to synchronize the infection. In one trial, tumors were induced 6 weeks after dripping day-old broilers, while in another, feather tips were PCR positive 16 days after dripping of 2-month-old layers.     

Genetic control of Rous sarcoma regression in chickens: Linkage with the major histocompatibility complex

Studies with two closely related inbred chicken strains show that regression of Rous sarcoma virus-induced tumors is a dominantly inherited trait, controlled by a gene within, or closely linked to, the major histocompatibility complex (B region). Strain G-B2 birds are capable of regressing Rous tumors, while strain G-B1 birds are uniformly susceptible to progressive Rous tumor development. Evidence for crossing over between the genes controlling serologically determined MHC antigens on erythrocytes and genes controlling Rous sarcoma growth was obtained. The MHC-linked gene which confers the ability to regress Rous sarcomas is designatedR-Rs-1. The allelic gene which allows for progressive tumor growth in homozygous birds is designatedr-Rs-1.     

表达禽流感病毒M2基因的重组马立克氏病病毒的构建

将禽流感病毒M2基因克隆于真核表达质粒pIRES-EGFP中,使其位于pCMV启动子的调控下,并与绿色荧光蛋白基因（EGFP）串联后,将上述串联基因插入到含MDV CVI988的非必需区US基因的重组质粒pUS2中,构建带标记的重组质粒,然后将此重组质粒转染感染了MDV CVI988的鸡胚成纤维细胞,利用同源重组的方法,筛选了表达禽流感病毒M2基因的重组病毒MDV1。经PCR、Dot-blotting,Western-blotting等实验的结果表明,禽流感病毒M2基因的确插入到MDV1（CVI988）基因组中并获得表达。重组MDV1免疫1日龄SPF鸡21天后,用ELISA可检测到M2蛋白的特异性抗体。接种了重组病毒rMDV的鸡体内针对H9N2疫苗血凝素的抗体滴度(p<0.05)明显提高,以禽流感病毒AIV A/Chicken/Guangdong/00（H9N2）攻毒后进行病毒重分离试验的结果发现,重组病毒能有效地降低病毒的排出量(p<0.01),说明该重组病毒可以用于防制禽流感的免疫。     

Effects of increased major histocompatibility complex dosage on chicken monocyte-macrophage function

The influence of major histocompatibility complex (B complex) dosage on monocyte-macrophage function was examined using 4- to 6-week-old trisomic strain chickens. Di- (B15B15), tri- (B15B15B15), and tetrasomic (B15B15B15B15) progeny were produced from trisomic x trisomic crosses. Although mononuclear leukocytes from tetrasomics exhibited enhanced chemotactic activity in response to both f-met-leu-phe and Enterobacter cloacae culture supernatant as compared with that of cells from other groups, the ability to generate peritoneal exudate cells in response to intraperitoneal Sephadex stimulation was similar in all groups. Among peritoneal exudate cells, tetrasomic birds produced a significantly lower percentage of adherent macrophages with a higher proportion of Fc receptor-positive and CMTD-2-reactive macrophages than either disomic or trisomic chickens. Both tetrasomic and trisomic peritoneal macrophages exhibited a reduced phagocytic activity for unopsonized but not opsonized SRBC than was found with disomic macrophages. Thus, the number of major histocompatibility complex copies present in cells appears to influence monocyte-macrophage function.