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356 | Lupiani et al.

generate novel recombinant vaccine candidates. Although BAC the pathogenesis of MDV and subsequent development of more
clone technology has a number of advantages, like the stabil- effective vaccines.
ity of insert propagation over multiple generations and mirror
endogenous gene expression far more accurately than previously
established cloning systems, it also has some limitations, like the Life cycle
time-consuming and labour-intensive generation and screen- MD infected chickens shed virus through dander, which remains
ing of recombinant BAC constructs, the oversized BAC DNA infectious for several months. Under commercial rearing condi-
constructs which are more easily sheared and degraded during tions, chickens are exposed soon after hatching by inhaling dander
manipulation before transfection, and some random recombina- shed by infected chickens (Fig. 12.5) (Calnek, 1986, 2001). The
tion events that may occur during the mutagenesis process. lung appears to be the first site of entry of MDV into the chick-
ens. The exact role of the lung in MD pathogenesis remains to be
CRISPR-Cas9 system explored but is generally accepted that lung phagocytes play a role
In recent years, the CRISPR-Cas9 system has emerged as a fast in transmitting the virus to lymphocytes (Gimeno, 2008). MDV
and reliable genome editing method. The CRISPR-Cas9 system then establishes primary cytolytic infection in lymphoid tissues
is a bacterial adaptive immune response mechanism used to pro- from 2–7 days post infection, peaking at day 4. Primary infection
tect bacteria from virus infection. Three types of CRISPR systems occurs in B-lymphocytes, which leads to activation of T-cells.
have been identified and the type II CRISPR-Cas9 system is the Unlike resting T-cells, which are fairly refractory to infection,
most widely used for genome modification. Type II CRISPR- activated T-cells are susceptible to infection (Schat et al., 1991).
Cas9 system includes RNA-guided Cas9 endonuclease, a single In the lymphocytes the virus remains strictly cell-associated.
guide RNA (sgRNA) and the trans-activating crRNA (tracr- After 6–7 days post infection, antigen expression in lymphoid
RNA) (Tang et al., 2018). The success of CRISPR-Cas9 system in organs is down regulated leading to a switch from cytolytic to
eukaryotic cells genome editing provide a novel method for large latent phase. Some of these latently infected T-cells eventually
DNA virus genome modification. In a recent study, CRISPR- become transformed, within 2 weeks post infection, leading to a
Cas9 technology was used to generate HVT recombinants by lymphoproliferative disease in chickens. The virus is then carried
expressing the VP2 gene of infectious bursal disease virus (IBDV) to the skin by latently infected or transformed lymphocytes by
providing a more feasible and efficient method to introduce genes day 10–14, where the feather follicular epithelial cells become
into the HVT genome for rapid development of recombinant infected. Fully infectious MDV is then shed trapped in the kerati-
vaccines (Tang et al., 2018). A recent report demonstrated that nized epithelial cells that are very stable (Calnek, 2001).
CRISPR-Cas9 could be used to study MDV gene function by It has recently been reported that B cells are dispensable for
deleting Meq and pp38 from the MDV vaccine strain CVI988 MDV replication, spread and tumour formation by utilizing newly
(Zhang et al., 2018). Henceforth, the CRISPR-Cas9 technology generated knockout chickens that lack mature and peripheral B
will speed up the process to study the function of all MDV genes cells (Bertzbach et al., 2018). This study suggests that MDV could
+
leading to better understanding of the molecular mechanism of rapidly infect and replicate in CD4 and CD8 T-cells and further
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Days post infection (dpi)

0 7 14 21

Inhalation of Early Latently Fully productive Shedding of
infectious cytolytic infected infection in infectious virus
dander infection of T cells feather follicle in dander
B cells & epithelium (FFE)
activated T
cells Transient
paralysis (TP) Paralysis of leg/wings,
cachexia, blindness
Persistent immuno-suppression
neurologic
Respiratory Apoptosis disease (PND)
tract & lung of B and T
phagocytic cells Transformation Lymphomas in
cells of T cells various organs

Figure 12.5 Schematic representation of MDV life cycle.
Figure 5