Marek's disease virus (MDV) causes an acute lymphoproliferative disease in chickens, resulting in T cell lymphomas in visceral organs and peripheral nerves. Earlier studies have determined that the repeat regions of oncogenic serotype 1 MDV encode a basic leucine zipper protein, Meq, which structurally resembles the Jun͞Fos family of transcriptional activators. Meq is consistently expressed in MDV-induced tumor cells and has been suggested as the MDVassociated oncogene. To study the function of Meq, we have generated an rMd5⌬Meq virus by deleting both copies of the meq gene from the genome of a very virulent strain of MDV. Growth curves in cultured fibroblasts indicated that Meq is dispensable for in vitro virus replication. In vivo replication in lymphoid organs and feather follicular epithelium was also not impaired, suggesting that Meq is dispensable for lytic infection in chickens. Reactivation of the rMd5⌬Meq virus from peripheral blood lymphocytes was reduced, suggesting that Meq is involved but not essential for latency. Pathogenesis experiments showed that the rMd5⌬Meq virus was fully attenuated in chickens because none of the infected chickens developed Marek's disease-associated lymphomas, suggesting that Meq is involved in lymphocyte transformation. A revertant virus that restored the expression of the meq gene, showed properties similar to those of the parental virus, confirming that Meq is involved in transformation but not in lytic replication in chickens.

Marek's disease virus (MDV) genetics has lagged behind that of other herpesviruses because of the lack of tools for the introduction of site-specific mutations into the genome of highly cellassociated oncogenic strains. Overlapping cosmid clones have been successfully used for the introduction of mutations in other highly cell-associated herpesviruses. Here we describe the development of overlapping cosmid DNA clones from a very virulent oncogenic strain of MDV. Transfection of these cosmid clones into MDV-susceptible cells resulted in the generation of a recombinant MDV (rMd5) with biological properties similar to the parental strain. To demonstrate the applicability of this technology for elucidation of gene function of MDV, we have generated a mutant virus lacking an MDV unique phosphoprotein, pp38, which has previously been associated with the maintenance of transformation in MDV-induced tumor cell lines. Inoculation of Marek's disease-susceptible birds with the pp38 deletion mutant virus (rMd5⌬pp38) revealed that pp38 is involved in early cytolytic infection in lymphocytes but not in the induction of tumors. This powerful technology will speed the characterization of MDV gene function, leading to a better understanding of the molecular mechanisms of MDV pathogenesis. In addition, because Marek's disease is a major oncogenic system, the knowledge obtained from these studies may shed light on the oncogenic mechanisms of other herpesviruses.

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The biological diversity within viruses is one of the largest found in all other forms of nature. Many mechanisms contribute to virus diversity and include incorporating genetic material from the host, recombination between viruses belonging to the same or to a different family, and even recombination between viruses normally infecting different hosts. In particular, avian viruses can utilize all three of these mechanisms to generate new viruses. It is well documented that recombinations can occur between Marek's disease virus (MDV), an oncogenic herpesvirus, fowlpox virus (FPV), and various avian retroviruses. In addition, chicken infectious anemia virus (CIAV), a circovirus, was created by several inter-family recombination events, which occurred between plant and animal viruses. The circovirus represents the ancestral creation of a recombination between a plant DNA virus (nanovirus) and a mammalian RNA virus (calicivirus), through a transition of RNA to DNA made by an endogenous mammalian retrovirus. The present review will discuss the current knowledge on recombination events that have occurred between avian herpesviruses and retroviruses following dual infections in vitro and in vivo. In addition, we will discuss recombinations between fowlpox viruses and the avian retrovirus reticuloendotheliosis (REV). Finally, the review will address the creation of CIAV and how it evolved from recombinations between a plant virus and an animal virus.

The nucleotide sequences of the terminal repeat long (TR(L)) and internal repeat long regions (IR(L)) in the genomes of 13 strains of Marek's disease virus type 1 (MDV-1) were determined and represent the largest collection of sequencing data from a contiguous region (12.8 kb) in the serotype 1 genomes. The collection of strains used in this study has been well characterized with respect to their virulence and contains members of each pathotype (4 attenuated, 1 mildly virulent, 3 virulent, 2 very virulent and 3 very virulent plus). It has previously been reported that two loci (meq and RLORF4) in the RL regions are likely to encode virulence factors based on comparative genomic studies involving vaccine and virulent strains. Additional studies using knockout mutants have provided stronger evidence that indeed RLORF4 and meq or the overlapping genes 23 kD and RLORF6 are involved in virulence. In this report, we provide evidence that additional open reading frames (ORFs) in the RL regions differ significantly between the extremes of the pathotypes (attenuated vs. nonattenuated). A deletion of 10 base pairs has been identified in RLORF12 from two attenuated strains CVI988 BP-5, p48 and RM-1, p40; and the lower virulence strain JM/102W. A deletion of 40 bp was also identified in RLORF4 of the attenuated strain R2/23, passage 106. A 177 bp insertion within the meq loci has been identified in most of the attenuated strains examined. Interestingly, R2/23 did not contain this insertion but instead truncated proteins are predicted for the three overlapping ORFs (meq, 23 kD and RLORF6) due to a frameshift mutation. Single nucleotide polymorphisms (SNPs), which loosely partition between attenuated and nonattenuated strains, have been identified in the ORFs encoding RLORF12, RLORF8, meq, 23 kD, RLORF6, RLORF4, RLORF3 and ICP0 and three previously unidentified short ORFs: MHLS, MLHG and MPSG. Although no single nucleotide polymorphism in the RL regions could predict virulence, their overall contribution to virulence can now be examined in defined mutants containing additional insertions or deletions in ORFs, suspected of encoding virulence factors, identified by this research.

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There are no simple, direct methods to reliably distinguish oncogenic serotype 1 Marek's disease viruses (MDVs) from their attenuated variants. The present study was an attempt to apply polymerase chain reaction (PCR) to develop a rapid and sensitive assay for the presence of the MDV genome. PCR oligos were chosen to flank the 132-base-pair tandem direct repeats in the serotype 1 MDV genome. The PCR reaction was specific for serotype 1 MDVs, amplifying fragments corresponding to one to three copies of the tandem repeats present in Md11/8, JM/102W, and GA viruses. A high-molecular-weight DNA smear was observed when the DNA from an attenuated Md11/100 was PCR-amplified. Use of the PCR technique allowed the detection of two copies of the 132-base-pair repeat in the DNA extracted from MDV-induced lymphomas removed from two chickens. No DNA was amplified from the DNA extracted from lymphomas induced by either an avian leukosis virus (RAV-1) or reticuloendotheliosis virus (chick syncytial virus).

Avian leukosis viruses (ALVs) are common in many poultry flocks and can be detected using an enzyme-linked immunosorbent assay or any other test designed to identify p27, the group-specific antigen located in gag. However, endogenous retroviruses expressing p27 are often present and can be confused with exogenous ALVs. A more specific and informative assay involves targeting the variable envelope glycoprotein gene (gp85) that is the basis for dividing ALVs into their different subgroups. We designed polymerase chain reaction (PCR) primers that would specifically detect and amplify viruses from each of the six ALV subgroups: A, B, C, D, E, and J. Subgroup B and D envelopes are related, and our B-specific primers also amplified subgroup D viruses. We also designed a set of common primers to amplify any ALV subgroup virus. To demonstrate the usefulness of these primers, we obtained from the Center for Veterinary Biologics in Iowa culture supernatant from chicken embryo fibroblasts infected with an ALV that was found to be a contaminant in two commercial Marek's disease vaccines. Using our PCR primers, we demonstrate that the contaminant was a subgroup A ALV. We cloned and sequenced a portion of the envelope gene and confirmed that the ALV was a subgroup A virus. Unlike typical subgroup A viruses, the contaminant ALV grew very slowly in cell culture. We also cloned and sequenced a portion of the long terminal repeat (LTR) from the contaminant virus. The LTR was found to be similar to those LTRs found in endogenous ALVs (subgroup E) and very dissimilar to LTRs normally found in subgroup A viruses. The E-like LTR probably explains why the contaminant grew so poorly in cell culture.