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interfere with TGF-β signalling in the transformed cells. This is Marker-rescue method
a strategy often used by tumour viruses to protect infected cells In early MDV research, the generation of mutant viruses was
from growth arrest and apoptosis. Meq engages with c-Jun to achieved by marker-rescue introducing a selectable marker in
form heterodimers and activates the v-Jun oncogenic pathway the viral genome followed by several rounds of plaque purifica-
(Levy et al., 2005). The c-Jun connection and the transactivation tion (Parcells et al., 1995, 2001; Bublot et al., 1999). As MDV is a
function are critical to Meq’s ability to transform chicken embryo highly cell associated virus, this process was laborious and moreo-
fibroblasts. The involvement of the Meq–Jun partnership in ver, unwanted secondary mutations were introduced during
T-cell transformation has not been rigorously tested, largely due repeated rounds of purification (Reddy et al., 2002). Owing to
to the lack of proper research tools. With the availability of next these disadvantages, marker-rescue method was replaced by a
generation sequencing tools, it is now possible to study how Meq faster and more reliable manipulation method that did not require
intercept to induce uncontrolled cell growth. purification of recombinant viruses.

Transcriptional repression: the CtBP oncogenic Overlapping cosmid technology
pathway Overlapping cosmid is one of the first methods that replaced
While activation of cellular genes involved in cell cycle, survival, marker-rescue method to study MDV gene function. As the
and migration is important in oncogenesis, increasing evidence cosmid clones accommodate up to 45,000 bp of foreign DNA,
suggests that transrepression of specific cohorts of genes is also a series of vectors containing overlapping segments of the her-
vital to the oncogenesis pathway. Genes involved in terminal dif- pesvirus genome were required to facilitate the generation of
ferentiation, growth arrest, and cell cycle restriction are targets for recombinant viruses. This approach was first tested in 1988 by
transrepressor oncogenes. A prime example is the v-ErbA onco- co-transfection of up to five overlapping cloned subgenomic frag-
gene, which represses differentiation-associated genes induced ments, which together covered the entire genome of pseudorabies
by retinoic acid receptor (RAR) and thyroid hormone recep- virus, resulted in the efficient reconstitution of the virus (van Zijl
tor (THR) leading to promyelocytic leukaemia development. et al., 1988). After that, the technique was utilized to generate a
CtBP was originally discovered as a protein interacting with the large number of herpesvirus mutants for HSV-1, EBV, and VZV
C-terminus of E1A, the oncoprotein of adenovirus (Boyd et al., (Cohen and Seidel, 1993; Cunningham and Davison, 1993; Tom-
1993). Subsequently, CtBP was found to associate with EBV kinson et al., 1993). The MDV genome from a vv strain (Md5)
oncoprotein EBNA3C and Evi-1, a translocated oncogene found was cloned as five overlapping fragments in 2002 (Reddy et al.,
in myeloid leukaemia. In the latter two cases, CtBP was found 2002). Using this technique, it was shown that pp38 plays an
to be crucial for the transformation potential of the respective important role in early cytolytic infection in the lymphoid organs
oncogenes (Touitou et al., 2001; Skalska et al., 2010). CtBP inter- but not transformation of lymphocytes. Similarly, using these
acts with a consensus motif, PXDLS, found in its target proteins, clones it was proved that Meq is essential for lymphocyte trans-
including Meq. CtBP does not bind DNA by itself, thus it relies formation but not for early cytolytic infection in lymphocytes
on the target proteins to localize it to promoters where it exerts (Lupiani et al., 2004). Although useful, the overlapping cosmid
repression function by recruiting histone deacetylase and methyl- approach had some limitations. First, it was difficult to find suit-
transferase G9a to compact the surrounding chromatin (Shi et al., able restriction sites for the introduction of mutations. Second,
2003). For MDV oncogenicity, Meq protein was found to associ- several recombination events were necessary to re-assemble the
ate with CtBP in vitro and in vivo, and the association was found to full-length genome.
be essential to induce tumours in chickens (Brown et al., 2006).
Bacterial artificial chromosomes (BACs)
The limitations or the disadvantages of the early techniques used to
Genome manipulation manipulate herpesvirus genomes were solved by cloning, mutat-
The large herpesvirus genome contains genes with a role in dif- ing and maintaining viral genomes in bacteria where the accuracy
ferent steps of the life cycle of the virus including attachment, of the bacterial polymerase allows for clonal maintenance of viral
penetration, replication, assembly, egress, latency and patho- sequences in Escherichia coli. In 2000, Schumacher et al. (2000)
genesis of the associated diseases (Pellet and Roizman, 2013). first cloned MDV-1 strain 584Ap80C (cell culture attenuated
Studies on MDV gene function lagged behind those of other strain) into E. coli as a BAC and characterized an MDV-1 mutant
herpesviruses because of the lack of efficient tools to manipulate with glycoprotein B deletion. Since then, various MDV strains
MDV genome. Generation of MDV mutants using marker-rescue have been cloned as a BAC, including CVI998, RB-1B, Md5 and
method was difficult due to the highly cell-associated nature of 686 (Petherbridge et al., 2003, 2004; Reddy et al., 2013). In 2006,
the virus. This was overcome after the application of overlapping Tischer et al. (2006) described the two-step red-mediated recom-
cosmid clones and bacterial artificial chromosome (BAC) tech- bination method that combines red recombination and cleavage
nologies to MDV, because these techniques do not rely on plaque with the endonuclease I-SceI to modify BAC constructs, includ-
purification of recombinant viruses. More recently, clustered ing insertion, deletion or site mutation. The establishment of
regularly interspaced short palindromic repeats (CRISPR)-Cas9 infectious MDV BAC clones allowed researchers to elucidate the
system has been used to manipulate the genome of large DNA function of individual MDV gene and proteins in virus replication
viruses, including MDV (Yao, 2016; Tang et al., 2018). and pathogenesis and it has also become a widely used method to