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.     

Selection of a recombinant Marek's disease virus in vivo through expression of the Marek's EcoRI-Q (Meq)-encoded oncoprotein: characterization of an rMd5-based mutant expressing the Meq of strain RB-1B

Marek's disease (MD) is a highly contagious viral disease of chickens (Gallus gallus domesticus) caused by MD virus (MDV), characterized by paralysis, neurologic signs, and the rapid onset of T-cell lymphomas. MDV-induced T-cell transformation requires a basic leucine zipper protein called Marek's EcoRI-Q-encoded protein (Meq). We have identified mutations in the coding sequence of Meq that correlated with virus pathotype (virulent, very virulent, and very virulent plus). The aim of this study was to determine whether recombinant viruses could be isolated based on Meq expression through in vivo selection. Chicken embryo fibroblasts (CEFs) were cotransfected with an rMd5 strain-based Meq deletion virus (rMd5deltaMeq) and meq loci from strains representing different pathotypes of MDV. Transfected CEFs were inoculated into chickens in two independent studies. We were able to isolate a single recombinant virus, rMDV-1137, in a contact-exposed chicken. rMDV-1137 had recombined two copies of the meq gene of RB-1B and was found to have pathogenicity similar to both RB-1B and rMd5 parental strains. We found the RB-1B- and rMd5-induced lymphomas showed differences in composition and that rMDV-1137-induced lymphomas were intermediate in their composition. We were able to establish cell lines from both RB-1B- (MDCC-UD35, -UD37) and rMDV-1137 (MDCC-UD36, -UD38)-induced, but not rMd5-induced, lymphomas. To date, no rMd5- or parent Md5-transformed T-cell lines have been reported. Our results suggest that 1) a recombinant MDV can be selected on the basis of oncogenicity; 2) changes in Meq sequence seem to affect tumor composition and the ability to establish cell lines; and 3) in addition to meq, other genomic loci affect MDV pathogenicity and oncogenicity.     

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.     

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.     

Development of a nested polymerase chain reaction method to detect oncogenic Marek's disease virus from feather tips.

For the easy survey of Marek's disease virus (MDV), feather tip-derived DNA from MDV-infected chickens can be used because feather tips are easy to collect and feather follicle epithelium is known to be the only site of productive replication of cell-free MDV. To develop a diagnostic method to differentiate highly virulent strains of MDV from the attenuated MDV vaccine strain, CVI988, which is widely used, nested polymerase chain reaction (PCR) was performed to detect a segment of the meq gene in feather tip samples of chickens experimentally infected with MDV. In chickens infected with Md5, a strain of oncogenic MDV, the meq gene was consistently detected, whereas the L-meq gene, in which a 180-base pair (180-bp) sequence is inserted into the meq gene, was detected in CVI988-infected chickens. Moreover, the meq gene was mainly detected even in chickens co-infected with both Md5 and CVI988. These results suggest that this method is appropriate for the surveillance of the highly virulent MDV infection in the field.     

Fluorescent tagging of VP22 in N-terminus reveals that VP22 favors Marek’s disease virus (MDV) virulence in chickens and allows morphogenesis study in MD tumor cells

Marek’s disease virus (MDV) is an alpha-herpesvirus causing Marek’s disease in chickens, mostly associated with T-cell lymphoma. VP22 is a tegument protein abundantly expressed in cells during the lytic cycle, which is essential for MDV spread in culture. Our aim was to generate a pathogenic MDV expressing a green fluorescent protein (EGFP) fused to the N-terminus of VP22 to better decipher the role of VP22 in vivo and monitor MDV morphogenesis in tumors cells. In culture, rRB-1B EGFP22 led to 1.6-fold smaller plaques than the parental virus. In chickens, the rRB-1B EGFP22 virus was impaired in its ability to induce lymphoma and to spread in contact birds. The MDV genome copy number in blood and feathers during the time course of infection indicated that rRB-1B EGFP22 reached its two major target cells, but had a growth defect in these two tissues. Therefore, the integrity of VP22 is critical for an efficient replication in vivo, for tumor formation and horizontal transmission. An examination of EGFP fluorescence in rRB-1B EGFP22-induced tumors showed that about 0.1% of the cells were in lytic phase. EGFP-positive tumor cells were selected by cytometry and analyzed for MDV morphogenesis by transmission electron microscopy. Only few particles were present per cell, and all types of virions (except mature enveloped virions) were detected unequivocally inside tumor lymphoid cells. These results indicate that MDV morphogenesis in tumor cells is more similar to the morphorgenesis in fibroblastic cells in culture, albeit poorly efficient, than in feather follicle epithelial cells.     

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.     

Detection of the meq gene in the T cell subsets from chickens infected with Marek's disease virus serotype 1

The meq gene was thought to be only detected in Marek's disease virus serotype 1 (MDV 1) including a very virulent strain, Md5, while L-meq, in which a 180-bp sequence is inserted into the meq open reading frame, is found in other strains of MDV 1, such as CVI 988/R6. However, both meq and L-meq were previously detected by PCR in chickens infected with MDV 1, suggesting that MDV 1 may consists of at least two subpopulations, one with meq, the other with L-meq. To further analyze these subpopulations, we analyzed the time course changes in distribution of these subpopulations among T cell subsets from chickens infected with MDV 1. Both meq and L-meq were detected in CD4+ and CD8+ T cells infected with strain Md5 or CVI 988/R6. The shift in MDV subpopulations from one displaying meq to the other displaying L-meq and/or the conversion from meq to L-meq occurred mainly in the CD8+ T cell subset from Md5-infected chickens. PCR products corresponding to L-meq rather than meq were frequently amplified from the CD8+ T cell subset from CVI 988/R 6 -infected chickens. These results suggest that a dominant subpopulation of MDV 1 changes depending on the T cell subsets, and that L-meq is dominantly present in the CD8+ T cells which play a role in the clearance of pathogenic agents.     

Electron microscopic and immunohistochemical localization of Marek's disease (MD) herpesvirus particles in MD skin lymphomas.

Skin lymphomas induced in 11 specific-pathogen-free chickens by inoculation at 1 day of age with Marek's disease virus (MDV) were biopsied weekly and examined by electron microscopy and immunohistochemistry. In the sequentially biopsied lymphomas, immature MDV particles (abortive replication) were found only in the nuclei of necrotic lymphoblasts within necrotizing neoplasms. The necrotizing lymphomas were observed in two of the 11 experimental birds and were associated with prominent vascular endothelial cell injury, including fibrinoid necrosis of blood vessels. Nonnecrotizing lymphomas biopsied sequentially from the 11 experimental birds did not contain virus particles of any kind in the lymphoblasts and had no distinct vascular lesions. Immunohistochemically, MDV early antigen (pp38), but not late antigens (glycoproteins B and C), was detected only in the necrotizing lymphomas. These findings indicate that abortive MDV replication mainly occurred in necrotic lymphoblasts, which might have been induced by ischemia.     

Adaptation of Marek's disease virus to the Vero continuous cell line

Marek's disease virus (MDV) is a highly infectious, cell-associated oncogenic herpesvirus. Production of MD vaccines has been limited to primary chicken and duck embryo fibroblast (CEF and DEF) cultures. These have a limited life span and cannot be readily stored in liquid nitrogen. Moreover, the need to prepare CEF and DEF cells on a regular basis from 10 to 11 day-old embryos derived from a flock that must be tested continuously for the presence of avian pathogens adds to the cost of vaccine production. A continuous cell line that would support MDV replication could have significant advantages for the rapid large-scale preparation of MD vaccines. In this report, we describe the adaptation to growth of CEF-grown preparations of serotype 1 and serotype 3 (herpesvirus of turkeys; HVT) strains of MDV in cells of the Vero continuous cell line. Although both viruses produced typical CPE, higher levels of infectious progeny and more extensive virus-specific immunofluorescence were obtained for HVT than for the serotype 1 virus. PCR and pulsed field electrophoresis (PFE) analysis of the DNA from Vero cells infected with either virus confirmed the presence of virus-specific DNA.     

Evaluation of factors affecting vaccine efficacy of recombinant Marek's disease virus lacking the Meq oncogene in chickens

We previously reported that deletion of the Meq gene from the oncogenic rMd5 virus rendered it apathogenic for chickens. Here we examined multiple factors affecting Marek's disease vaccine efficacy of this nonpathogenic recombinant Meq null rMd5 virus (rMd5deltaMeq). These factors included host genetics (MHC haplotype), strain or dose of challenge virus, vaccine challenge intervals, and maternal antibody status of the vaccinated chicks. Studies on host genetics were carried out in five chicken lines comprising four different MHC B-haplotypes. Results showed that chicken lines tested were highly protected, with protective indexes of 100% (B*2/*15), 94% (B*2/*2), 87% (B*19/*19), and 83% (B*21/*21). At a challenge dose above 8000 plaque-forming units, differences in protection were observed between the two highly virulent strains examined (648A and 686). The interval between vaccination and challenge indicated a protective efficacy from 0 to 2 days varied greatly (12%-82%) after challenge with vv+686, the most virulent virus. Less variation and significant protection began at 3 days post vaccination and reached a maximum at 5 days post vaccination with about 80%-100% protection. Taken together, our results indicate that the factors examined in this study are important for vaccine efficacy and need to be considered in comparative evaluations of vaccines.     

鸭肠炎病毒UL41基因缺失株的构建及其体外增殖能力分析

旨在构建鸭肠炎病毒（DEV）UL41基因缺失毒株,并对其生物学特性进行分析,本研究以克隆有DEV UL41基因的重组黏粒D1为骨架,利用Red/ET重组技术构建缺失UL41基因的重组黏粒D1 dUL41;将UL41基因缺失重组黏粒D1 dUL41与其他4个克隆有DEV基因组片段的亲本黏粒共转染鸭胚成纤维细胞（DEF）,拯救获得UL41基因缺失毒株rDEV-SD19/dUL41。将该基因缺失病毒感染DEF后,提取病毒基因组进行PCR鉴定及测序,并利用间接免疫荧光试验检测该病毒感染细胞中UL41基因表达情况;绘制拯救的基因缺失病毒的生长曲线,分析其体外复制特性。PCR及测序结果显示,本研究成功构建了缺失UL41基因的重组黏粒D1 dUL41。将该基因缺失黏粒与其他含有DEV基因组的亲本黏粒共转染DEF后能够产生典型的蚀斑病变。PCR及测序结果显示,UL41基因成功从DEV基因组中缺失;间接免疫荧光试验发现,基因缺失病毒rDEV-SD19/dUL41感染DEF后,未见UL41蛋白表达。综上表明,本研究成功构建了DEV UL41基因缺失病毒。体外生长曲线显示,rDEV-SD19/dUL41在DEF中的复制能力明显低于亲本病毒,提示UL41蛋白在DEV复制中发挥重要作用。UL41基因缺失DEV的构建为进一步研究UL41基因在DEV感染和致病中的作用机制奠定了基础。     

Effects of reticuloendotheliosis virus and Marek's disease virus infection and co-infection on IFN-gamma production in SPF chickens

Two experiments were used to examine the potential role of IFN-gamma in chickens infected with reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). First, chickens were infected with REV and/or MDV at 5 days of age and examined from 3 to 50 days post-infection (dpi). In REV+MDV co-infection chickens, IFN-gamma ELISA demonstrated a 3-fold increase at 7 dpi compared to the controls, while REV alone caused a 5-fold increase, the IFN-gamma levels peaked, and then gradually decreased. IFN-gamma levels significantly decreased in MDV infection at 3 dpi and 15 dpi. Second, experiments were designed to determine the effects of different viruses and ConA on IFN-gamma production. For REV- or MDV-infected chickens, the IFN-gamma levels decreased slightly after adding ConA. This is the first report of IFN-gamma production in SPF chickens infected with REV and MDV measured by directly quantitative method.