Marek's disease in turkeys. II. Characterization of the viral glycoprotein B gene and antigen of a turkey strain of Marek's disease virus

Marek's disease virus (MDV) causes immunosuppression and tumors in chickens. As sporadic cases of Marek's disease (MD) were recorded in turkeys, the antigenic and genomic characteristics of the MDV glycoprotein B (gB) gene and antigen of turkeys were compared to the chicken MDV gB. The whole chicken and turkey gB genes were sequenced and found identical. By immunoblotting of infected-cell culture lysates using chicken convalescent and gB monoclonal antibodies, the antigenic epitopes of the chicken and turkey viruses were found to differ. The turkey MDV had a unique epitope, compared to the chicken MDV and compared with our previous findings. While the chicken MDV had two epitope types, heat-labile but dithiothreitol (DTT)-stable and heat-stable but DTT-labile, the turkey MDV gB epitope is both heat and DTT-labile.     

Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 by using polymerase chain reaction DNA amplification.

Differentiation of oncogenic and nononcogenic strains of Marek's disease virus type 1 (MDV1) was attempted by polymerase chain reaction (PCR) using the primers chosen from the sequence within the long inverted repeats of MDV1 DNA. PCR of the DNAs extracted from oncogenic-strain-infected cells and Marek's disease tumor cell lines produced a major product containing two or three copies of 132-base-pair (bp) repeat units, whereas PCRs of the DNAs extracted from nononcogenic-strain-infected cells yielded amplified products with various sizes corresponding to the number of 132-bp repeat units. The primers chosen from the glycoprotein A genes of MDV1 and herpesvirus of turkeys also were used for determination of their serotype specificity. The PCR procedure was found to be a simple and sensitive procedure for identification of MDV1 and herpesvirus of turkeys and for estimation of oncogenicity of MDV1.     

Antibody directed against Marek's disease-associated tumor surface antigen can be eluted from Marek's disease tumor cells.

Antibody directed against Marek's disease-associated tumor surface antigen (MATSA) was eluted from tumor cells of lymphomas and peripheral blood lymphocytes that were isolated from Marek's disease virus-infected chickens. Feather follicular Marek's disease virus (MDV) antigen could not be demonstrated with this antibody by indirect immunofluorescent (IF) staining. Monoclonal antibody directed against MATSA could completely block the activity of eluted antibody and vice versa. By indirect IF staining using eluted antibody and fluorescein isothiocyanate (FITC) labelled antichicken globulin conjugate. MATSA-bearing cells were detected in MDV infected and herpes virus of turkey (HVT) vaccinated birds. Blocking of immunoglobulin molecules present on B-cells by anti-chicken globulin is critical in this test.     

Isolation and Identification of a Field Strain of Marek's Disease Virus and Analysis of Major Pathogenic Genes

In a certain area of Shandong province, Marek's disease (MD) occurred in diseased chickens that had been vaccinated by turkey herpesvirus.In order to isolate the virus strain and detect the virus pathogenicity, agar diffusion test, cell culture and indirect immunofluorescence assay (IFA) were used to isolate the Marek's virus from chicken's blood and feather marrow.The isolated strain was adapted to grow in chick embryo fibroblasts (CEF).Genes involved in pathogenesis of MDV, such as meq, pp38 and 132 bp repeat sequence were amplified by PCR.The obtained sequences were compared with that of standard strains published in GenBank by DNAStar software.The results showed that pp38 gene of the SDAU-1 shared homology from 100% with standard virulent sequence.Analysis of 132 bp repeat sequence and meq gene sequences of the viral genome showed that the isolated virus belongs to the highly virulent MDV strains.     

一株鸡马立克氏病病毒的分离鉴定及主要致病基因分析

山东省某地区鸡马立克氏病疫苗免疫鸡群暴发马立克氏病(MD),为分离得到致病毒株,检测其致病性,采用琼脂扩散试验、细胞培养和间接免疫荧光试验(IFA)等方法从发病鸡的血液及羽髓中分离到一株适应鸡胚成纤维细胞(CEF)生长的马立克氏病病毒。采用PCR方法扩增分离毒株的meq、pp38、132bp重复序列等病毒致病相关基因,所得序列用DNAStar软件与GenBank上登录的参考毒株进行比对分析。结果显示,该分离株SDAU-1的pp38基因与标准强毒序列同源性为100%,132bp重复序列的拷贝数及meq基因的变异均符合MDV强毒株的序列特征。     

Marek's disease in turkeys: lack of protection by vaccination

Herpesvirus of turkeys, a highly effective vaccine against Marek's disease (MD) in chickens, was ineffective in protecting turkeys against MD. Another tissue-culture attenuated vaccine virus also protected chickens, but not turkeys, from MD. Intact and immunosuppressed turkey poults inoculated with herpesvirus of turkey developed a persistent viremia, but did not have detectable gross or microscopic lesions.     

Propagation of virulent and avirulent turkey hemorrhagic enteritis virus in cell culture

Virulent and apathogenic isolates of turkey hemorrhagic enteritis virus (HEV) were successfully propagated in lymphoblastoid cell lines of turkey origin, whereas spleen and kidney cell cultures from HEV-infected turkeys failed to replicate the virus. The lymphoblastoid cell lines used were MDTC-RP16 and MDTC-RP19, which were previously established from tumors induced by Marek's disease virus in turkeys. Virus replication followed co-cultivation of lymphoblastoid cells with spleen cells from HEV-infected turkeys. Virus replication was demonstrated by immunofluorescence, by agar-gel-precipitin tests, and by electron microscopy. Supernatant fluid of cultures infected with virulent HEV caused death and specific lesions in turkey poults. Poults vaccinated with apathogenic HEV were protected against death and lesions after challenge with pathogenic HEV, which was recovered from infected cultures. The MDTC-RP19 cell line appeared far more susceptible than the MDTC-RP16 cell line to infection with HEV.     

Partial protection against Marek's disease in chickens immunized with glycoproteins gB purified from turkey-herpesvirus-infected cells by affinity chromatography coupled with monoclonal antibodies

Glycoproteins gB of Marek's disease virus (MDV) and herpesvirus of turkeys (HVT) related to virus neutralization were purified from HVT-infected cells by affinity chromatography. Immunization of chickens with purified glycoproteins gB resulted in partial protection against MD. Neutralizing antibodies were detected in chickens immunized with HVT-gB.     

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.     

The effect of the time interval between exposures on the susceptibility of chickens to superinfection with Marek's disease virus

Marek's disease virus (MDV) is ubiquitous within commercial poultry flocks because current vaccines do not prevent MDV infection or transmission. In order for newly-evolved MDV strains to become established within a flock, it seems inevitable that any new strain would need to infect and replicate in chickens previously infected with resident MDV strains. This phenomenon is difficult to detect and there is no clear evidence that it is even possible. Four experiments were performed to demonstrate superinfection and evaluate the effect of time between challenges on the effect of superinfection with the use of two pairs of fully virulent MDV strains that could be discriminated by novel technology: 1) JM/102W and rMd5//38CVI, and 2) rMd5 and rMd5//38CVI. Feather follicle epithelium (FFE), spleen, and tumor samples were collected at single or multiple time points from the same bird to determine the frequency and distribution of each virus present following superinfection, with the use of pyrosequencing and immunohistochemistry. Superinfection was observed in 82 of 149 (55%) FFE samples following short-interval challenge (24 hr) compared to only 6 of 121 (5%) samples following long-interval challenge (13 days), indicating a strong influence of challenge interval. In cases where the first inoculated virus was weak or delayed, the second inoculated virus was detected in 42 of 95 (44%) birds. In tumors from dually challenged birds, the second virus was again present much more often following short-interval challenge (68%) compared to long-interval challenge (11%). Virus mixtures in tumors were less common compared to those in FFE samples. Vaccination with turkey herpesvirus had no significant effect on the virus frequency for either virus pair or challenge time interval, suggesting these conclusions may be applicable to vaccinated chickens in the field. These studies demonstrated superinfection for the first time with two fully virulent MDV strains and suggest that short-interval challenge exposure and/or weak initial exposures may be important factors leading to superinfection--a prerequisite for the establishment of a second virus strain in the population. This model system should be useful to elucidate this important phenomenon further.     

Response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses.

OBJECTIVE: To examine effects of virus exposure on embryonic lymphoid organ structure, apoptosis, and lymphoid cell subpopulations. ANIMALS: Eggs of specific pathogen free (SPF) White Leghorn chickens at embryonation day (ED) 17. PROCEDURES: Eggs were inoculated with 2,000 plaque-forming units (PFU) of serotype 1 herpesvirus (Marek's disease virus [MDV 1]), 2,000 PFU of herpesvirus of turkeys (MDV 3), or 1,000 embryo infectious doses (EID50) of infectious bursal disease virus (IBDV). On post-inoculation days (PID) 3 and 5, lymphoid organ to body weight ratios were determined, and bursa of Fabricius, thymus, and spleen were evaluated for lesions and apoptosis. Proportions of lymphoid cell subpopulations of PID-3 chicken embryos and 7- to 10-day-old chicks were quantitated by flow cytometry. RESULTS: Lymphoid organ weights were similar in virus-free, MDV1, and IBDV groups. Embryos inoculated with 2,000 PFU MDV 3/egg had lower bursal weights than virus-free controls. In a repeated trial, MDV 3 (1,000 PFU to 4,000 PFU) did not reduce bursal weights among groups. Histologic changes were seen in bursae after MDV 1 and IBDV inoculation. Apoptosis was greater in bursae of MDV 1-infected embryos than controls. Lymphoid cell subpopulations were similar among all groups with the exception of CD8+ and IgM+ cells in spleens of IBDV-infected 10-day-old chicks. CONCLUSIONS AND CLINICAL RELEVANCE: Infection with pathogenic strains of MDV 1 and IBDV did not alter lymphocyte subpopulations in embryos or cause complete destruction of lymphoid organs. Changes in lymphoid cell subpopulations exposed as embryos to IBDV were seen only after hatching.     

A comparative evaluation of the protective efficacy of rMd5deltaMeq and CVI988/ Rispens against a vv+ strain of Marek's disease virus infection in a series of recombinant congenic strains of White Leghorn chickens

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). MD is presently controlled by vaccination. Current MD vaccines include attenuated serotype 1 strains (e.g., CVI988/Rispens), avirulent serotype 2 (SB-1), and serotype 3 (HVT) MDV strains. In addition, recombinant MDV strains have been developed as potential new and more efficient vaccines to sustain the success of MD control in poultry. One of the candidate recombinant MDV strains, named rMd5deltaMeq, was derived from Md5, a very virulent strain of MDV lacking the MDV oncogene Meq. Our earlier reports suggest that rMd5deltaMeq provided protection equally well or better than commonly used MD vaccines in experimental and commercial lines of chickens challenged with very virulent plus (vv+) strains of MDV. In this study, maternal antibody-positive (trial 1) and negative (trial 2) chickens from a series of relatively MD resistant lines were either vaccinated with the rMd5deltaMeq or CVI988/Rispens followed by infection of a vv+ strain of MDV, 648A, passage 10. This report presents experimental evidence that the rMd5deltaMeq protected significantly better than the CVI988/Rispens (P < 0.01) in the relatively resistant experimental lines of chickens challenged with the vv+ strain of MDV. Together with early reports, the rMd5deltaMeq appeared to provide better protection, comparing with the most efficacious commercially available vaccine, CVI988/Rispens, for control of MD in lines of chickens regardless of their genetic background.     

马立克氏病胚胎免疫雏鸡体重和免疫器官增重变化

１８日龄鸡胚免疫接种火鸡疱疹病毒（ＨＶＴ）疫苗后,对出壳后雏鸡及其用马立克氏病（ＭＤ）病毒攻毒鸡体重与免疫器官增长效果的对比研究表明：相同饲养条件下胚胎免疫雏鸡的增长速度高于非免疫对照鸡,而且胚胎免疫组雏鸡免疫器官的生长发育普遍较相应的对照组雏鸡快。     

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.     

Early posthatch protection against Marek's disease in chickens vaccinated in ovo with a CVI988 serotype 1 vaccine

CVI988, a serotype 1 Marek's disease virus (MDV), was used as an in ovo vaccine in specific-pathogen-free chickens to determine if this virus induces early posthatch protection against Marek's disease as has been shown previously for turkey herpesvirus. MDV CVI988 was injected at embryonation day (ED) 17 (group 1) or at hatch (group 2). A third group (group 3) was left unvaccinated. At 1, 2, 3, 4, 5, and 7 days of age, chickens from each group were sampled and examined as follows: a) single-cell suspensions of spleen were inoculated onto chicken embryo fibroblast monolayers to isolate the virus; b) sections of bursal tissues were stained by indirect immunofluorescence assays with anti-pp38 monoclonal antibody to identify viral antigen expression; and c) chickens were exposed intra-abdominally to MDV RB1B, a virulent serotype 1 MDV. Results revealed that in chickens given MDV CVI988 at ED 17, virus and virus-encoded protein were not detected until chickens were 3 and 2 days old after hatching, respectively. Results also indicated that during the first 4 days after hatch, the chickens given MDV CVI988 at ED 17 were better protected against virulent MDV than those given MDV CVI988 at hatch (P < or = 0.001). These results suggested that MDV CVI988 proteins were adequately expressed in the embryo to initiate prehatch immunologic response. Additional efforts with more sensitive techniques than used in this study are needed to identify the nature of viral expression in embryos.     

Effects of IgY antibody on the development of Marek's disease.

The effects of passive immunization with immunoglobulin Y (IgY) on the pathogenesis of Marek's disease (MD) were examined in an experimental line of White Leghorn chickens highly susceptible to MD. Purified IgY with anti-MDV antibody activity, when injected into chicks, delayed the development of MDV viremia and lesions until 9 days postinoculation (PI) with Marek's disease virus (MDV). The blastogenic response of spleen cells to concanavallin-A was depressed at 6 days PI in the birds without passive immunization, whereas it was not totally depressed until 17 days in birds passively immunized with IgY anti-MDV antibody.     

Properties of producer and non-producer clones of a Marek's disease turkey lymphoblastoid cell line

The MDTC-RP30 lymphoblastoid cell line established from Marek's disease (MD) tumors in turkeys consisted of a heterogeneous population of cells 10 to 25 micron in diameter. Large-cell fractions obtained from a bovine fetal serum gradient had a higher titer of cell-associated MD virus (MDV) than the small-cell fractions. Seven single-cell clones were established from MDTC-RP30 cell line: two consisted of large cells, and the other clones consisted of small cells. Infectious MDV was rescued from large-cell clones in chicken embryo fibroblast cultures but not from small-cell clones. All clones contained MDV DNA sequences when hybridized against cloned MDV DNA. All clones were positive for a Marek's-disease-tumor-associated surface antigen and surface immunoglobulins. All but two small-cell clones caused MD in susceptible chickens. The two large-cell-type clones were uniformly tetraploid, whereas one small-cell clone was diploid and the four others were a mixture of diploid and tetraploid, with an occasional triploid cell. Evidence of translocation involving the male (Z) chromosome and the chromosome #3 was seen in one clone. These results suggest that MDV transforms different subpopulations of lymphocytes.     

Natural killer cell activity in chickens exposed to Marek's disease virus: inhibition of activity in susceptible chickens and enhancement of activity in resistant and vaccinated chickens

Chickens of 2 genetic lines (lines P and N) were inoculated with a pathogenic strain of Marek's disease (MD) virus (MDV) and chronologically examined for disease response and natural killer (NK) cell expression. The NK cell reactivity was assayed in an in vitro cytotoxicity assay in which effector cells from the spleen of test chickens were reacted with 51Cr-labeled LSCC-RP9 target cells. Chickens of line P developed progressive debilitating disease and a high incidence of gross tumors and death. The NK cell reactivity of line-P chickens infected with MDV was significantly lower than that of uninfected control hatchmates. In contrast, NK cell levels were significantly elevated in MDV-inoculated line-N chickens that were resistant to MD and in chickens of lines P or N that had been inoculated with herpesvirus of turkeys (HVT). NK cell levels were also elevated in line P if chickens were vaccinated with HVT before infection with MDV. Inhibition of NK reactivity in susceptible chickens and elevation of reactivity in naturally resistant or vaccinated chickens may indicate a role for the NK cell system in regulating resistance to MD.     

Avian influenza adenovirus-vectored in ovo vaccination: target embryo tissues and combination with Marek's disease vaccine

We investigated embryo tissues targeted by replication competent adenovirus (Ad)-free recombinant Ad expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdH5) when injected into 18-day embryonated eggs. We also evaluated the effects of concurrent in ovo vaccination with the experimental AdH5 vaccine and commercially available Marek's disease virus (MDV) vaccine combinations Rispens/turkey herpesvirus (HVT) or HVT/SB-1. Computed tomography indicates that in ovo injection on day 18 of incubation places the solution in the amnion cavity, allantoic cavity, or both. Ad DNA was consistently detected in the chorioallantoic membranes as well as in the embryonic bursa of Fabricius, esophagus, and thymus 3 days postinoculation. H5 expression in these tissues also was detected by immunofluorescence assay. These results indicate possible swallowing of vaccine virus contained in the amnion. In contrast, vaccine localization in the allantoic fluid would have allowed bursal exposure through the cloaca. When the AdH5 vaccine was used in combination with MDV, chickens responding to the AdH5 vaccine had similar AI antibody levels compared with AdH5-only-vaccinated birds. However, combined vaccinated groups showed reduced vaccine coverage to AI, suggesting some level of interference. The combination of AdH5 with MDV Rispens/HVT affected the vaccine coverage to AI more severely. This result suggests that the replication rate of the more aggressive Rispens strain of serotype 1 may have interfered with the Ad-vectored vaccine. Increasing the Ad concentration produced similar AI antibody titers and AI vaccine coverage when applied alone or in combination with the HVT/SB-1 vaccine. Ad DNA was detected in hatched chickens 2 days after hatch but was undetectable on day 9 after hatch. MDV DNA was detected in feather follicles of all vaccinated birds at 12 days of age. Thus, Ad-vector vaccination does not interfere with the efficacy of MDV vaccination by using any of the commonly used vaccine strains.