A Marek's disease virus vIL-8 deletion mutant has attenuated virulence and confers protection against challenge with a very virulent plus strain

Marek's disease virus (MDV) is an alpha-herpesvirus that causes rapid development of T-cell lymphomas in chickens. MDV-encoded vIL-8 is homologous to the cellular IL-8 gene, and its function in MDV pathogenesis has yet to be determined. Using overlapping cosmid clone-based technology, we have generated an MDV vIL-8 deletion mutant virus, rMd5/delta vIL-8. In vivo experiments with this mutant virus demonstrated that deletion of vIL-8 results in attenuation of the virus and induction of significantly less gross tumor, both in viscera and nerves, when compared to the parental virus. Reintroduction of the vIL-8 gene in the genome of the mutant virus restored the virulence of the virus to the wild-type levels, indicating that vIL-8 plays a role in MDV-induced pathogenesis. In this study, we show that there is a significant difference in the reduction of B cells and activation of T cells in the spleen cells of chickens inoculated with parental rMd5 and vIL-8 deletion mutant virus. These results indicate that vIL-8 is involved in the early phase of pathogenesis, presumably by attracting target cells to the initial site of infection. In addition, protection studies with the vIL-8 mutant virus showed that this mildly virulent virus protects susceptible maternal antibody-positive viruses at a higher level than the commonly used serotype 1 CVI988 vaccine. These results confirm the potential of partially attenuated viruses as vaccines against very virulent plus strains and the usefulness of recombinant DNA technology to generate the next generation of MDV vaccines.     

Derivation, safety and efficacy of a Marek's disease vaccine developed from an Australian isolate of very virulent Marek's disease virus

Objective To develop a serotype 1 Marek's disease (MD) vaccine from a very virulent MDV (vvMDV) pathotype and demonstrate safety and efficacy against early challenge with very virulent field strains in the presence of maternal antibody.
Study design Strain BH 16 was isolated and attenuated by serial cell culture passage. One of two cloned passages was selected for vaccine development following early laboratory-scale protection trials in commercial birds. Comparative protection trials were carried out on the BH 16 vaccine and on a CVI 988 Rispens vaccine using commercial and SPF chickens. Challenge viruses used were either a low passage strain BH 16 virus, the Woodlands No. 1 strain or MPF 57 strain of MDV. The BH 16 vaccine was back-passaged in SPF chickens six times and virus recovered from the final passage and the original vaccine virus were tested for safety. The immunosuppressive potential of the BH 16 and Rispens vaccines was also assessed in parallel.
Results The BH 16 and Rispens vaccines induced comparable levels of protection when used as monovalent or multi-valent vaccines, although protection achieved with the mono-valent vaccines was lower. No gross tumour formation was evident in any birds receiving the BH 16 vaccine or bird-passaged virus, although microscopic lesions were present in 2/12 birds that received the bird-passaged virus. In tests for immunosuppression, there was no histological evidence of damage to either the bursa of Fabricius or the thymus.
Conclusion The BH 16 vaccine was shown to be safe and at least as protective as the Rispens vaccine against three highly virulent MD challenge viruses.     

Derivation,safety and efficacy of a Marek's disease vaccine developed from an Australian isolate of very virulent Marek's disease virus

OBJECTIVE: To develop a serotype 1 Marek's disease (MD) vaccine from a very virulent MDV (vvMDV) pathotype and demonstrate safety and efficacy against early challenge with very virulent field strains in the presence of maternal antibody. STUDY DESIGN: Strain BH 16 was isolated and attenuated by serial cell culture passage. One of two cloned passages was selected for vaccine development following early laboratory-scale protection trials in commercial birds. Comparative protection trials were carded out on the BH 16 vaccine and on a CVI 988 Rispens vaccine using commercial and SPF chickens. Challenge viruses used were either a low passage strain BH 16 virus, the Woodlands No. 1 strain or MPF 57 strain of MDV. The BH 16 vaccine was back-passaged in SPF chickens six times and virus recovered from the final passage and the original vaccine virus were tested for safety. The immunosuppressive potential of the BH 16 and Rispens vaccines was also assessed in parallel. RESULTS: The BH 16 and Rispens vaccines induced comparable levels of protection when used as monovalent or multivalent vaccines, although protection achieved with the monovalent vaccines was lower. No gross tumour formation was evident in any birds receiving the BH 16 vaccine or bird-passaged virus, although microscopic lesions were present in 2/12 birds that received the bird-passaged virus. In tests for immunosuppression, there was no histological evidence of damage to either the bursa of Fabricius or the thymus. CONCLUSION: The BH 16 vaccine was shown to be safe and at least as protective as the Rispens vaccine against three highly virulent MD challenge viruses.     

The detection of the meq gene in chicken infected with Marek's disease virus serotype 1

In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.     

感染MDV鸡羽髓组织中CyP、LASP1和PCBP1蛋白的表达分析

本实验室前期的蛋白质组学研究显示,鸡羽髓组织中亲环素蛋白(CyP)、LIM和SH3蛋白1(LASP1)和多聚胞嘧啶结合蛋白1(PCBP1)3种蛋白为马立克氏病病毒(MDV)感染过程中差异表达蛋白。为进一步检测这3种蛋白在病毒感染过程中的表达水平,本研究以MDV GA强毒株感染SPF鸡,应用荧光定量PCR和westernblot检测病毒感染14 d和21 d后这3种蛋白在羽髓组织中的表达水平。检测结果表明,病毒感染14 d后,羽髓组织中这3种蛋白的mRNA和蛋白表达水平均有不同程度的下调,其中LASP1 mRNA和PCBP1蛋白分别下调62%和48%(p<0.05);在21 d,除CyP蛋白表达基本不变外,其他mRNA和蛋白均不同程度下调,其中LASP1蛋白下调达到46%(p<0.05)。结果表明在两个不同的MDV感染时期,MDV均可以不同程度的抑制羽髓组织中这3种蛋白的表达,本研究为进一步阐释MDV与羽髓组织的相互作用机理提供有价值的参考。     

Major histocompatibility complex class I is downregulated in Marek's disease virus infected chicken embryo fibroblasts and corrected by chicken interferon

The major histocompatibility complex (MHC) is a part of the immune system which presents epitopes of intracellular antigens on the cell surface. MHC molecules have receptor-ligand binding affinities with T lymphocytes, permitting the latter to detect foreign intracellular infectious agents. Some pathogens, such as herpesviruses, have developed strategies of evading the host response by MHC. This pressure on the immune system brought, in turn, improvements in the antigen-presenting pathway, for example through the effect of interferon (IFN), which can upregulate MHC expression. The main objective of this work was on the one hand, to determine the abilities of three strains of Marek's disease virus (MDV), a chicken herpesvirus, in interfering with the expression of MHC class I molecules in chicken embryo fibroblasts. On the other hand, we analyzed the ability of IFN to reinstate this important immune capability to the infected cells. Our results show that only an oncogenic serotype 1 strain of MDV (RB1B) was able to markedly decrease MHC class I expression, and that addition of IFN reversed this MDV effect.     

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.     

Early cell-mediated immune responses to Marek's disease virus in two chicken lines with defined major histocompatibility complex antigens

N2a and P2a chickens, resistant and susceptible to Marek's disease (MD), respectively, were used to examine relationships between major histocompatibility complex (MHC)-restricted cytotoxic T lymphocytes (CTL) and natural killer (NK)-like cell activity with resistance to infection with Marek's disease virus (MDV). Ten-day-old chickens were infected with MDV and euthanatized at selected times to evaluate for NK cell and MHC-restricted cytotoxicity. The N2a MDV-infected chickens had an early cell-mediated immune response characterized by a sustained NK-like cytotoxicity that coincided with a measurable MHC-cytotoxicity that was lower than controls. Although MHC-restricted and NK cell cytotoxicity was demonstrated in P2a MDV-infected chickens at 8 dpi, both abruptly decreased and remained low for the remainder of the 20-day experiment. The critical time point that may determine the resistance to MD appears to be within the first 2 weeks post-infection. Improvement of the chicken NK cell activity may be a good candidate for both selection and immunomodulation MD control programs.     

Differential expression of immune-related genes in the bursa of Fabricius of two inbred chicken lines following infection with very virulent infectious bursal disease virus

Among different inbred chickens’ lines, we previously showed that lines P and N of Institute for Animal Health, Compton, UK are the most susceptible and the least affected lines, respectively, following infection with very virulent infectious bursal disease virus (vvIBDV). In this study, the differential expressions of 29 different immune-related genes were characterized. Although, birds from both lines succumbed to infection, line P showed greater bursal lesion scores and higher viral copy numbers compared to line N. Interestingly, line N showed greater down-regulation of B cell related genes (BLNK, TNFSF13B and CD72) compared to line P. While up-regulation of T-cell related genes (CD86 and CTLA4) and Th1 associated cytokines (IFNG, IL2, IL12A and IL15) were documented in both lines, the expression levels of these genes were different in the two lines. Meanwhile, the expression of IFN-related genes IFNB, STAT1, and IRF10, but not IRF5, were up-regulated in both lines. The expression of pro-inflammatory cytokines (IL1B, IL6, IL18, and IL17) and chemokines (CXCLi2, CCL4, CCL5 and CCR5) were up-regulated in both lines with greater increase documented in line P compared to line N. Strikingly, the expression of IL12B was detected only in line P whilst the expression of IL15RA was detected only in line N. In conclusion, the bursal immunopathology of IBDV correlates more with expression of proinflammatory response related genes and does not related to expression of B-cell related genes.     

马立克氏病病毒对鸡肿瘤相关基因TNFSF13B和ST18表达水平的影响

将150只1日龄雏鸡随机分成2组,100只作为攻毒组,50只作为对照组。攻毒组腹腔注射2 000PFU的马立克氏病病毒（MDV）-GA细胞毒0.2mL;对照组注射同等剂量的病毒稀释液。2组鸡严格隔离,在相同条件的负压隔离器中饲养。攻毒后60d,屠宰所有存活鸡。在对照组中取4个正常鸡的脾脏（HS）;在攻毒组中取4个感染MDV后产生肿瘤鸡的脾脏（TS）及4个感染MDV后未产生肿瘤并表现为健康鸡的脾脏（ANS）,并对TNFSF13B和ST18基因在这些脾脏组织中的表达水平进行荧光定量分析。结果表明,肿瘤相关基因TNFSF13B和ST18的表达在产生肿瘤鸡的脾脏中都是显著性下调的。MDV通过抑制宿主TNFSF13B基因的表达来抑制B细胞分化、成熟进而对宿主免疫系统进行抑制;同时MDV通过下调肿瘤抑制基因ST18的表达,促使机体肿瘤发生。在对MDV有明显抵抗能力的鸡（ANS组）中,TNFSF13B和ST18的表达水平与对照组健康鸡基本一致,表明这2个基因在MDV抗病过程中起着重要作用。     

Differential expression of inducible nitric oxide synthase and cytokine mRNA in chicken lines divergent for cutaneous hypersensitivity response

Phytohemagglutinin (PHA)-induced delayed-type hypersensitivity is an immunocompetent trait considered an indicator of cell-mediated immune or T-cell responses. Divergent selection was performed to generate high and low lines for response to PHA-P. Extreme-responder birds of the F2 generation in each line were used to study possible differences in macrophage activity and the associated functional genes. To evaluate macrophage activity, nitric oxide (NO) was estimated both systemically in serum and in in vitro monocyte culture. Semi-quantitative RT-PCR was used to detect the differential mRNA expression patterns of iNOS and MIP-1beta in monocyte culture, whereas T(H)1 cytokines (IL-2 and IFN-gamma) were studied in peripheral blood mononuclear cells (PBMC) at different time intervals after lipopolysaccharide (LPS) induction. The high line showed strong systemic, as well as in vitro NO production, compared to the low line, upon stimulation with NDV and LPS, similar to early and high iNOS mRNA expression. Following the pattern of iNOS gene expression, an early strong expression of cytokines with powerful iNOS-inducing action, such as IFN-gamma and the chemokine MIP-1beta, was observed in the high line. In contrast, for response to PHA-P, low expression of IL-2 was observed in the high compared to the low line. In conclusion, the study revealed that divergent selection for response to PHA-P resulted in a divergent effect on T(H)1 cell activity, resulting in altered macrophage function in chickens. Selection, based on response to PHA-P, could lead to more resistant birds or birds with an enhanced immune response.     

IBDV超强毒GX8/99株的细胞适应毒和鸡体传代毒的致病性和VP5基因的比较

传染性法氏囊病病毒(Infectious Bursal Diseas Virus,IBDV)的超强毒株GX8/99经在鸡胚成纤维细胞(CEF)上连续盲传23代后,细胞适应毒株GXC23时鸡的致病性显著减弱,对4～6周龄SPF鸡的致死率从85%～90%减为0～5%.GXC23在96孔细胞培养板上经2次无限稀释法后得到3个克隆病毒.经SPF鸡回传20代后,相应的毒株GX-2820、GX-4820、GX-5820对SPF鸡的致死率仍维持在0～5%.序列比较表明,GX8/99在传代过程中,其细胞适应毒GXC23有8个碱基突变,其中5个碱基突变导致了氨基酸改变.bp#2 T→C突变,导致传代毒ORF中的起始编码子后移了4个氨基酸.而3个克隆化病毒回鸡20代后致病性不变,这几个位点也保持不变,且与3个疫苗株完全一致.进一步分析表明,有4个超强毒株和4个强毒株的VP5基因的5个位点与GX8/99原始毒相同,而另3个超强毒及6个强毒株与细胞适应毒GXC23一致.这些结果表明,超强毒GX8/99在细胞传代过程中VP5基因上5个氨基酸的变异主要与适应细胞培养相关而与致病性无关.     

Indications of immunodepression in chickens infected with various strains of Marek's disease virus

The effect of infection by various strains of Marek's disease virus (MDV) on the immune function of 3-week-old chickens was examined. MDV strains of low (CU-2, RB-7) and high (RB-3, MD-5, and MD-11) pathogenicity were compared with prototype JM-10 strain of moderate pathogenicity. Mortality, whole body weight, relative weights of lymphoid organs, histopathology, humoral antibody responses to thymus-dependent and -independent antigens, and in vitro lymphocyte responses to mitogen stimulation were investigated at 1, 2, and 3 weeks postinfection. MDV strains of high pathogenicity significantly depressed responses at 3 weeks postinfection, seeming to indicate the ability of these viruses to induce severe immunodepression. However, the fact that the moderately pathogenic and even some of the low-pathogenicity strains induced immunodepression suggests that other viral mechanisms are also important in its determination.     

Differential expression of inducible nitric oxide synthase is associated with differential Toll-like receptor-4 expression in chicken macrophages from different genetic backgrounds.

The purpose of this study was to examine iNOS gene expression and activity in macrophages from different chicken genetic lines against various bacterial LPS. Furthermore, the possible involvement of surface LPS receptors as candidates for differential iNOS gene induction in these genetic lines of chicken was also examined. Sephadex-elicited abdominal macrophages (1 x 10(6)) as well as iNOS hyper-responder macrophages from a transformed chicken macrophage cell line, MQ-NCSU, were exposed to 5 microg/ml LPS from E. coli, Shigella flexneri, Serratia marcensces, and Salmonella typhimurium. Nitrite levels were quantitated in the culture supernatant fractions of macrophages after 24h by the Griess method. The results showed that macrophages from K-strain (B(15)B(15)) (range from two separate trials: 31-89 microM) and MQ-NCSU (22-81 microM) were high responders whereas macrophages from both GB1 (B(13)B(13)) (15-38 microM) and GB2 (B(6)B(6)) (7-15 microM) chickens were low responders against all LPSs used. Northern blot analysis revealed that K-strain macrophages expressed higher intensity of 4.5Kb iNOS mRNA (iNOS/beta-actin ratio) than macrophages from GB2 regardless of the LPS source. To elucidate possible molecular mechanism(s) involved in iNOS gene expression in these two strains of chickens, the constitutive expression of LPS-related macrophage cell surface receptors, CD14, Toll-like receptor-2 (TLR2), and Toll-like receptor-4 (TLR4), was examined via flow cytometry using anti-human CD14, TLR2 and TLR4 antibodies. CD14 surface expression and intensity was not different between macrophages from K-strain or GB2 chickens. In contrast, while the overall percentage of TLR4-positive macrophages was the same (K-strain, trial 1=92%, trial 2=62%; GB2, trial 1=91%, trial 2=64%), the mean fluorescence intensity (MFI), an indicator of receptor number, was significantly higher (P=0.05) in K-strain macrophages (MFI: trial 1=145; trial 2=131) than GB2 macrophages (MFI: trial 1=101; trial 2=98). Furthermore, TLR2 (a previously thought candidate as LPS signaling molecule) positive cell numbers were higher in K-strain than the GB2 macrophages in one of the two trials with no difference in the intensity of TLR2 expression in either trial. These findings suggest that the observed differences in iNOS expression and activity among the K-strain (hyper-responder) and GB2 (hypo-responder) chickens are, at least in part, due to differential expression of TLR4 (an LPS signaling molecule), leading to more intense LPS-mediated activation of K-macrophages.     

Interaction of Marek's disease virus and Cryptosporidium baileyi in experimentally infected chickens

Histocompatible B13/B13 white specific-pathogen-free leghorn chickens were used to investigate the effect of coinfection with Cryptosporidium baileyi and the HPRS 16 strain of Marek's disease virus (MDV) in chickens and to assess the pathogenicity of C. baileyi when MDV is given before or after the parasite. Groups of chickens concurrently infected with C. baileyi orally inoculated at day (D)4 and MDV inoculated at hatching (C4M0 group) or at D8 (C4M8 group) were compared with relevant control groups inoculated with only C. baileyi at D4 (C4 group), only MDV at hatching (M0 group) or at D8 (M8 group), and an uninoculated control group (UC group). The chickens were kept in isolator units until the end of the experiment at D62. Our results showed a considerable synergistic effect in concurrently infected chickens and more severe consequences when chickens received MDV before C. baileyi infection. In fact, except for a slight transitory weakness, the chickens in C4 group remained free of overt clinical signs and there was no mortality. However, coinfection with both pathogens induced more lasting or permanent oocyst shedding. Severe clinical cryptosporidiosis with weakness, anorexia, depression, growth retardation, and chronic and severe respiratory disease causing death occurred in all chickens in the C4M0 group between D12 and D43 and in 67% of the chickens in the C4M8 group between D17 and D57. Eighty-two percent and 33%, respectively, died before the development of specific Marek's disease lesions. Mortality rates were 27% and 33% in the M0 and M8 groups, respectively. The presence of MDV enhanced the establishment of more lasting cryptosporidial infection in the respiratory tract, esophagus, crop, proventriculus, and kidneys (only in C4M0 group) as well as in bursa of Fabricius, ceca, and cloaca. Serologic analysis showed that chickens with chronic cryptosporidiosis in the C4M8 group had an increased level of C. baileyi-specific immunoglobulin A. Our results may explain some cases of mortality in chickens naturally infected with MDV and Cryptosporidium.