Insertion of reticuloendotheliosis virus long terminal repeat into the genome of CVI988 strain of Marek's disease virus results in enhanced growth and protection.

Marek's disease (MD) is a lymphoproliferative disease of chickens caused by serotype 1 MD virus (MDV). Vaccination of commercial poultry has drastically reduced losses from MD, and the poultry industry cannot be sustained without the use of vaccines. Retrovirus insertion into herpesvirus genomes is an efficient process that alters the biological properties of herpesviruses. RM1, a virus derived from the virulent JM strain of MDV, by insertion of the reticuloendotheliosis (REV) long terminal repeat (LTR), was attenuated for oncogenicity but retains properties of the parental virus, such as lymphoid organ atrophy. Here we show that insertion of the REV LTR into the genome of vaccine strain CVI988 resulted in a virus (CVRM) that replicated to higher levels than parental CVI988 in cell culture and that remained apathogenic for chickens. In addition, CVRM showed protection indices similar or superior to those afforded by CVI988 virus in laboratory and field protection trials, indicating that it could be developed as a safe and efficacious vaccine to protect against very virulent plus MDV.

Cloning of a very virulent plus, 686 strain of Marek's disease virus as a bacterial artificial chromosome.

Bacterial artificial chromosome (BAC) vectors were first developed to facilitate propagation and manipulation of large DNA fragments. This technology was later used to clone full-length genomes of large DNA viruses to study viral gene function. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that causes rapid induction of T-cell lymphomas in chickens. Based on the virus's ability to cause disease in vaccinated chickens, MDV strains are classified into pathotypes, with the most virulent strains belonging to the very virulent plus (vv+) pathotype. Here we report the construction of BAC clones of 686 (686-BAC), a vv+ strain of MDV. Transfection of DNA isolated from two independent clones into duck embryo fibroblasts resulted in recovery of infectious virus. Pathogenesis studies showed that the BAC-derived 686 viruses were more virulent than Md5, a vv strain of MDV. With the use of a two-step red-mediated mutagenesis process, both copies of viral interleukin 8 (vIL-8) were deleted from the MDV genome, showing that 686-BACs were amenable to mutagenesis techniques. The generation of BAC clones from a vv+ strain of MDV is a significant step toward understanding molecular basis of MDV pathogenesis.

Properties of a meq-deleted rmd5 Marek's disease vaccine: protection against virulent MDV challenge and induction of lymphoid organ atrophy are simultaneously attenuated by serial passage in vitro.

We have previously shown that deletion of the meq gene from the genome of Cosmid-cloned rMd5 strain of Marek's disease virus (MDV-1) resulted in loss of transformation and oncogenic capacity of the virus. The rMd5deltaMeq (Meq null) virus has been shown to be an excellent vaccine in maternal antibody positive (MAb+) chickens challenged with a very virulent plus (vv+) strain of MDV, 648A. The only drawback was that it retained its ability to induce bursa and thymus atrophy (BTA) like that of the parental rMd5 in maternal antibody negative (MAb-) chickens. We recently reported that the attenuated Meq null virus did not induce BTA at the 40th cell culture passage onward. Its protective ability against challenge with vv+ MDV, strain 686 was similar to the original virus at the 19th passage in MAb- chickens. In this study, we compared the same series of attenuated meq null viruses in commercial chickens. In commercial chickens with MAb, the attenuated viruses quickly lost protection with increasing cell culture attenuation. These data suggest that although attenuation of these meq null viruses eliminated BTA, it had no influence on their protective efficacy in MAb- chickens. However, in commercial chickens (MAb+), the best protection was provided by the original 19th passage; the attenuated 40th passage was as good as one of the currently commercial CVI988/Rispens vaccine, and it did not induce BTA. Therefore, protection against virulent MDV challenge and induction of lymphoid organ atrophy are simultaneously attenuated by serial passage in vitro.

Identification and in vitro characterization of a Marek's disease virus-encoded Ribonucleotide reductase.

Marek's disease virus (MDV) encodes a ribonucleotide reductase (RR), a key regulatory enzyme in the DNA synthesis pathway. The gene coding for the RR of MDV is located in the unique long (UL) region of the genome. The large subunit is encoded by UL39 (RR1) and is predicted to comprise 860 amino acids whereas the small subunit encoded by UL40 (RR2) is predicted to be 343 amino acids long. Immunoprecipitation analysis of MDV-1 (GA strain)-infected cells with T81, a monoclonal antibody specific for RR of MDV, identified two major proteins of 90,000 and 40,000 daltons, corresponding to RR1 and RR2, respectively. In addition, RR was abundantly expressed in the cytoplasm of cells infected with 51 strains of MDV belonging to MDV serotypes 1, 2, and 3 as demonstrated by immunofluorescence staining. Northern blot analysis of RNA extracted from MDV-infected cells showed a major band of around 4.4 kb in size corresponding to the RR1 and RR2 transcripts. In vivo, RR was abundantly expressed in lymphoid organs and feather follicle epithelium of MDV-infected chickens during early cytolytic infection, as determined by immunohistochemistry. There was, however, no expression of RR in MDV-induced tumors in lymphoid organs. The abundant expression of RR in MDV-infected chicken may suggest an important role of RR in the conversion of ribonucleotides to deoxyribonucleotides for MDV DNA synthesis.

Deletion of Marek's disease virus large subunit of ribonucleotide reductase impairs virus growth in vitro and in vivo.

Marek's disease virus (MDV), a highly cell-associated lymphotropic alphaherpesvirus, is the causative agent of a neoplastic disease in domestic chickens called Marek's disease (MD). In the unique long (UL) region of the MDV genome, open reading frames UL39 and UL40 encode the large and small subunits of the ribonucleotide reductase (RR) enzyme, named RR1 and RR2, respectively. MDV RR is distinguishable from that present in chicken and duck cells by monoclonal antibody T81. Using recombinant DNA technology we have generated a mutant MDV (Md5deltaRR1) in which RR1 was deleted. PCR amplification of the RR gene in Md5deltaRR1-infected duck embryo fibroblasts (DEF) confirmed the deletion of the 2.4 kb RR1 gene with a resultant amplicon of a 640-bp fragment. Restriction enzyme digests with SalI confirmed a UL39 deletion and the absence of gross rearrangement. The biologic characteristics of Md5deltaRR1 virus were studied in vitro and in vivo. The Md5deltaRR1 replicated in DEF, but significantly slower than parental Md5-BAC, suggesting that RR is important but not essential for replication in fibroblasts. In vivo studies, however, showed that the RR1 deletion virus was impaired for its ability to replicate in chickens. Inoculation of specific-pathogen-free (SPF) chickens with Md5deltaRR1 showed the mutant virus is nonpathogenic and does not induce MD in birds. A revertant virus, Md5deltaRR1/R, was generated with the restored phenotype of the parental Md5-BAC in vivo, indicating that RR is essential for replication of the virus in chickens. Protection studies in SPF chickens indicated that the Md5deltaRR1 virus is not a candidate vaccine against MD.

Competition between two virulent Marek's disease virus strains in vivo.

Previous studies have demonstrated the presence of multiple strains of Marek's disease virus simultaneously circulating within poultry flocks, leading to the assumption that individual birds are repeatedly exposed to a variety of virus strains in their lifetime. Virus competition within individual birds may be an important factor that influences the outcome of co-infection under field conditions, including the potential outcome of emergence or evolution of more virulent strains. A series of experiments was designed to evaluate virus competition within chickens following simultaneous challenge with two virulent serotype 1 Marek's disease virus strains, using either pathogenically similar (rMd5 and rMd5/pp38CVI) or dissimilar (JM/102W and rMd5/pp38CVI) virus pairs. Bursa of Fabricius, feather follicle epithelium, spleen, and tumour samples were collected at multiple time points to determine the frequency and distribution of each virus present using pyrosequencing, immunohistochemistry and virus isolation. In the similar pair, rMd5 appeared to have a competitive advantage over rMd5/pp38CVI, which in turn had a competitive advantage over the less virulent JM/102W in the dissimilar virus pair. Dominance of one strain over the other was not absolute for either virus pair, as the subordinate virus was rarely eliminated. Interestingly, competition between two viruses with either pair rarely ended in a draw. Further work is needed to identify factors that influence virus-specific dominance to better understand what characteristics favour emergence of one strain in chicken populations at the expense of other strains.

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.

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.

Homodimerization of Marek's disease virus-encoded Meq protein is not sufficient for transformation of lymphocytes in chickens.

Marek's disease virus (MDV), the etiologic agent of Marek's disease, is a potent oncogenic herpesvirus. MDV is highly contagious and elicits a rapid onset of malignant T-cell lymphomas in chickens within several weeks after infection. MDV genome codes an oncoprotein, Meq, which shares resemblance with the Jun/Fos family of bZIP transcription factors. Similar to Jun, the leucine zipper region of Meq allows the formation of homo- and heterodimers. Meq homo- and heterodimers have different DNA binding affinities and transcriptional activity; therefore, they may differentially regulate transcription of viral and cellular genes. In this study we investigated the role of Meq homodimers in the pathogenicity of MDV by generating a chimeric meq gene, which contains the leucine zipper region of the yeast transcription factor GCN4 (meqGCN). A recombinant virus (rMd5-MeqGCN) containing the chimeric meqGCN gene in place of parental meq was generated with overlapping cosmid clones of Md5, a very virulent MDV strain. The rMd5-MeqGCN virus replicated in vitro and in vivo but was unable to transform T cells in infected chickens. These data provide the first in vivo evidence that Meq homodimers are not sufficient for MDV-induced transformation.

Characterization of reticuloendotheliosis virus isolates obtained from broiler breeders, turkeys, and prairie chickens located in various geographical regions in the United States.

Nine reticuloendotheliosis virus (REV) isolates obtained from broiler breeders, turkeys, and prairie chickens located in three different geographical regions in the USA, and three isolates obtained from known contaminated live-virus vaccines were characterized using polymerase chain reaction (PCR) and indirect immunofluorescence (IFA) assays. All isolates were propagated in chicken embryo fibroblasts obtained from a specific pathogen free breeder flock. PCR analysis of all 12 isolates resulted in the amplification of the 291-bp REV long-terminal repeat region (LTR); none of the isolates exhibited a different pattern or shift from the expected PCR product of REV LTR. The subtype of the REV isolates was determined by IFA using REV-specific monoclonal antibodies, 11B118.22, 11C237.8, and 11D182. Results from sub-typing indicated that all nine isolates from broiler breeders, turkeys, and prairie chickens belonged to subtype 3, and are antigenically related to the chick syncytial virus (CSV) strain of REV, the prototype of subtype 3 REV. In contrast, the three isolates from contaminated vaccines were classified as subtype 2, and were antigenically related to spleen necrosis virus (SNV) strain of REV, the prototype of subtype 2 REV. Three isolates representing REV isolated from broiler breeders, turkeys, and prairie chickens were cloned and further evaluated by DNA sequence analysis of the envelope gene. Results from DNA sequence analysis confirmed those from sub-typing and indicated that the three REV isolates representing those from broiler breeders, turkeys, and prairie chickens are closely related to CSV of REV, with an amino acid homology of 98% or greater as compared with SNV with an amino acid homology of 95% or less. Data from this study clearly indicate that subtype 3 is the most common subtype of REV circulating in three different avian species, namely broiler breeders, turkeys and prairie chickens, located in three different geographical regions in the United States.

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.

In vitro characterization of the Meq proteins of Marek's disease virus vaccine strain CVI988.

Gallid herpesvirus 2 (GaHV-2), commonly known as Marek's disease virus serotype-1 (MDV-1), causes T cell lymphomas in chickens. Vaccines prepared from the attenuated CVI988/Rispens MDV-1 strain currently offer the best protection. Although attenuated CVI988/Rispens is non-oncogenic, it codes for at least two forms of the MDV oncoprotein Meq, and these proteins (CVI-Meq and CVI-LMeq) have not been fully characterized. Here, we report that both CVI-Meq proteins, like the Meq protein of Md5 (a very virulent oncogenic strain), were capable of transforming Rat-2 and NIH3T3 cells. Both CVI-Meq and CVI-LMeq proteins activated the meq promoter only in the presence of chicken c-Jun (CK-Jun) whereas Md5-Meq activated the same promoter irrespective of CK-Jun co-expression. However, Meq proteins of both Md5 and CVI988 bound the meq promoter in a ChIP assay regardless of whether CK-Jun was co-expressed. To understand the role of Meq DNA binding and transactivation/repression domains in transcription, we constructed three chimeric Meq proteins, namely, Md5-CVI-Meq, CVI-Md5-Meq, and Md5-CVI-L by exchanging domains between Md5 meq and CVI meq genes. Although these chimeric Meq proteins, unlike CVI-Meq proteins, transactivated the meq promoter, the activation was significantly less than Md5-Meq. To determine the role of individual amino acids, point mutations were introduced corresponding to the amino acid changes of CVI-Meq into Md5-Meq. Amino acid residues at positions 71 and 320 of the Md5-Meq protein were found to be important for transactivation of the meq promoter. All three Meq proteins activated the MDV gB, MMP-3 and Bcl-2 promoters and suppressed transcription from the MDV pp38/pp14 bidirectional promoter. Although no significant differences were observed, decreased transactivation activity was observed with CVI-Meq proteins when compared to Md5-Meq. Collectively, the data presented here indicate that CVI-Meq proteins are generally weak transactivators, which might contribute to the non-oncogenic phenotype of CVI988 virus in chickens.

Marek's disease virus infection in the eye: chronological study of the lesions, virus replication, and vaccine-induced protection.

Marek's disease virus (MDV) infection in the eye was studied chronologically after inoculating 1-day-old chickens with a very virulent MDV strain, Md5. The ocular lesions could be classified as early lesions (6-11 days postinoculation [dpi]) and late lesions (26 and 56 dpi), based upon the location and severity of the lesions. The early lesions involved iris, ciliary body, and choroid layer, and were characterized by endothelial cell hypertrophy, vasculitis, and infiltration of lymphocytes (mainly CD8+), plasma cells, macrophages, and heterophils. Expression of early MDV-antigen pp38 in the cells infiltrating choroid layer was detected as early as 11 dpi. Late lesions consisted of severe lymphohistiocytic uveitis, keratitis, pectenitis, vitreitis, retinitis, and segmental to diffuse retinal necrosis. Cell infiltration included macrophages, granulocytes, plasma cells, and both CD4+ and CD8+ cells of various sizes. Expression of early MDV-antigen pp38 was readily found within the retina, uveal tract, and corneal epithelium. No expression of late-antigen gB or oncoprotein meq was detected in any of the eyes examined. A second experiment was conducted to study the effect of vaccination on the development of ocular lesions. Both HVT and CVI988 were able to protect against the development of early ocular lesions in chickens infected with very virulent plus strain MDV 648A. However, only CVI988 conferred complete protection against the development of late ocular lesions. HVT conferred partial protection, as it reduced the frequency and severity of the late ocular lesions. These results enhance our understanding of the nature and pattern of MDV infection in the eye.

Characterization of LORF11, a unique gene common to the three Marek's disease virus serotypes.

The unique open reading frame 11 (LORF11) of Marek's disease virus (MDV) is present in all three serotypes of MDV and is located in the unique long region of the MDV genome. In the serotype 1 Md5 genome, LORF11 comprises 2711 nucleotides and encodes a predicted protein of 903 amino acids. In order to study the biological function of LORF11 we deleted it from the MDV cosmid A6 by using the RecA-assisted restriction endonuclease cleavage method. The recombinant cosmid, A6DeltaLORF11, was transfected into duck embryo fibroblasts (DEF) in conjunction with parental SN5, P89, SN16, and B40 cosmid clones. Recombinant rMd5DeltaLORF11 plaques were evident at 12-13 days after transfection. Polymerase chain reaction amplification of DEF cells infected with rMd5DeltaLORF11 viruses confirmed the deletion of a 2.57-kb fragment resulting in a 296-bp fragment. Three rMd5DeltaLORF11 mutants were generated and their biological functions were studied in vitro and in vivo. In vitro growth characteristics of rMd5DeltaLORF11 viruses were similar to those of parental rMd5, indicating that LORF11 is not essential for replication in vitro. In vivo studies of rMd5DeltaLORF11 mutants showed that they were impaired in viral replication in the lymphoid organs and had 100x lower viremia than chickens infected with the parental rMd5 virus. Furthermore, rMd5-infected chickens horizontally transmitted the virus to contact controls whereas no horizontal transmission occurred in rMd5DeltaLORF11-infected chickens. Three independent deletion mutants were tested and showed the same phenotypes, so it is unlikely that the observed phenotype is because of any random mutation in the genome. Therefore the LORF11 gene of MDV is essential for normal virus replication in chickens and deletion of LORF11 renders an attenuated virus.

Characterization of a very virulent Marek's disease virus mutant expressing the pp38 protein from the serotype 1 vaccine strain CVI988/Rispens.

Marek's disease virus (MDV), a highly cell-associated oncogenic chicken herpesvirus, causes Marek's disease in domestic chickens. A unique phosphoprotein of MDV, pp38, has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. However, recently, the biological properties of a deletion mutant virus (rMd5Deltapp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. Thus, pp38 is important for early cytolytic infection and not for transformation. The pp38 protein of the MDV serotype 1 vaccine strain CVI988/Rispens differs by one amino acid when compared to the pathogenic strains of MDV. Monoclonal antibody, H19, recognizes all serotype 1 MDV strains except CVI988/Rispens. Previous studies have also shown that the unique pp38 epitope in CVI988/Rispens induced high antibody response. In order to study the role of this epitope in the protective properties of CVI988/Rispens, we generated a mutant rMd5 virus in which the wild type pp38 gene has been substituted with that of CVI988/Rispens (rMd5/pp38CVI). The replication properties of rMd5/pp38CVI, both in vitro and in vivo, and tumor induction were examined. We found that the biological properties of rMd5/pp38CVI were similar to the wild type rMd5 virus with regards to in vivo replication, antibody response and tumor induction. This shows that the pp38 derived from CVI988/Rispens is not involved in protective properties as was previously suggested.

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.

The efficacy of recombinant fowlpox vaccine protection against Marek's disease: its dependence on chicken line and B haplotype.

Earlier studies have shown that the B haplotype has a significant influence on the protective efficacy of vaccines against Marek's disease (MD) and that the level of protection varies dependent on the serotype of MD virus (MDV) used in the vaccine. To determine if the protective glycoprotein gene gB is a basis for this association, we compared recombinant fowlpox virus (rFPV) containing a single gB gene from three serotypes of MDV. The rFPV were used to vaccinate 15.B congenic lines. Nonvaccinated chickens from all three haplotypes had 84%-97% MD after challenge. The rFPV containing gB1 provides better protection than rFPV containing gB2 or gB3 in all three B genotypes. Moreover, the gB proteins were critical, since the B*21/*21 chickens had better protection than chickens with B*13/*13 or B*5/*5 using rFPV with gB1, gB2, or gB3. A newly described combined rFPV/gB1gEgIUL32 + HVT vaccine was analyzed in chickens of lines 15 x 7 (B*2/*15) and N (B*21/*21) challenged with two vv+ strains of MDV. There were line differences in protection by the vaccines and line N had better protection with the rFPV/gB1gEgIUL32 + HVT vaccines (92%-100%) following either MDV challenge, but protection was significantly lower in 15 X 7 chickens (35%) when compared with the vaccine CVI988/Rispens (94%) and 301B1 + HVT (65%). Another experiment used four lines of chickens receiving the new rFPV + HVT vaccine or CVI988/Rispens and challenge with 648A MDV. The CVI 988/Rispens generally provided better protection in lines P and 15 X 7 and in one replicate with line TK. The combined rFPV/gB1gEgIUL32 + HVT vaccines protected line N chickens (90%) better than did CVI988/Rispens (73%). These data indicate that rFPV + HVT vaccines may provide protection against MD that is equivalent to or superior to CVI988/ Rispens in some chicken strains. It is not clear whether the rFPV/gB1gEgIUL32 + HVT vaccine will offer high levels of protection to commercial strains, but this vaccine, when used in line N chickens, may be a useful model to study interactions between vaccines and chicken genotypes and may thereby improve future MD vaccines.

Tissue tropism and bursal transformation ability of subgroup J avian leukosis virus in White Leghorn chickens.

In Experiment 1, a monoclonal antibody against the envelope glycoprotein (gp85) of subgroup J avian leukosis virus (ALV-J) was used to study the distribution of ALV-J in various tissues of White Leghorn chickens inoculated as embryos with the strain ADOL-Hcl of ALV-J. At 2 and 6 wk of age, various tissues from infected and control uninfected chickens were tested for the presence of ALV-J gp85 by immunohistochemistry. In Experiment 2, using the methyl green-pyronine (MGP) stain, sections of bursa of Fabricius (BF) from chickens of line 15I5 x 7(1), inoculated with ALV-J or Rous-associated virus-1 (RAV-1), a subgroup A ALV, at hatch were examined for transformation of bursal follicles at 4 and 10 wk of age. In Experiment 1, specific staining indicative of the presence of ALV-J gp85 was noted at both 2 and 6 wk of age in the adrenal gland, bursa, gonads, heart, kidney, liver, bone marrow, nerve, pancreas, proventriculus, spleen, and thymus. In Experiment 2, by 10 wk of age, transformed bursal follicles were detected in MGP-stained sections of BF in only one of five (20%) chickens inoculated with ALV-J at hatch, compared with five of five (100%) chickens inoculated with RAV-1. The data demonstrate distribution of ALV-J gp85 in various tissues of White Leghorn chickens experimentally inoculated as embryos with the virus. The data also confirm our previous observation that ALV-J is capable of inducing transformation of bursal follicles, albeit the incidence is less frequent than that induced by subgroup A ALV.

Cell culture attenuation eliminates rMd5ΔMeq-induced bursal and thymic atrophy and renders the mutant virus as an effective and safe vaccine against Marek's disease.

Marek's disease virus (MDV) encodes a basic leucine zipper oncoprotein, Meq, which structurally resembles jun/fos family of transcriptional activators. It has been clearly demonstrated that deletion of Meq results in loss of transformation and oncogenic capacity of MDV. The rMd5ΔMeq virus provided superior protection than CVI988/Rispens vaccine in 15×7 chickens when challenged with a very virulent plus (vv+) strain of MDV, 648A. The rMd5ΔMeq construct was also shown to be an effective vaccine in commercial chickens that were challenged under field conditions by exposure to seeder chicken inoculated with MDV strain 686, a vv+ and arguably the most pathogenic strain of MDV. Although deletion of Meq gene renders the virus non-oncogenic, it still induces lymphoid organ atrophy like that of the parental rMd5, in highly susceptible MDV maternal antibody negative (MAb-) chickens. We have generated 50 cell culture passages of attenuated rMd5ΔMeq viruses and found no significant lymphoid organ atrophy beginning at 40(th) passage onward when compared with the normal control chickens. The protective ability of these attenuated Meq null viruses against challenge with vv+ MDV strain 686 is similar to the original virus at 19(th) passage in maternal antibody negative chickens. The data indicate that attenuation of these Meq null viruses has no influence on their protective efficacy, but eliminated lymphoid organ atrophy and rendered them safe to use even in MAb- chickens, a characteristic that should facilitate commercialization and licensing by vaccine manufacturers.

Both homo and heterodimers of Marek's disease virus encoded Meq protein contribute to transformation of lymphocytes in chickens.

Marek' disease virus serotype-1, also know as Gallid herpesvirus 2 (GaHV-2), elicits T-cell lymphomas in chickens. The GaHV-2 genome encodes an oncoprotein, Meq, with similarity to the Jun/Fos family of proteins. We have previously shown that Meq homodimers are not sufficient to induce lymphomas in chickens. In this study, we investigated the role of Meq heterodimers in the pathogenicity of GaHV-2 by generating a chimeric meq gene, which contains the leucine zipper region of Fos (meqFos). A recombinant virus containing the meqFos gene in place of parental meq, rMd5-MeqFos, was not capable of transforming chicken lymphocytes, indicating that heterodimerization of Meq alone is not sufficient for transformation. In addition, the recovery of the oncogenic phenotype by a recombinant virus encoding one copy each of MeqGCN (homodimer) and MeqFos (heterodimer) conclusively demonstrates that both homo and heterodimerization of Meq are required for oncogenesis.