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136 | Liu et al.

Morphology ‘sacs’ within the egg (Hawkes, 1979). The shell membrane lies
Under the electron microscope, CoV virions appear roughly directly under the shell, a tough fibrinous membrane forming the
spherical and 82 nm in diameter (Becker et al., 1967), and have air sac in the region of the broader end of the egg (Fig. 5.4). In
distinct ‘club-like’ projections radiating from the virion surface. contrast to the shell membrane, the chorioallantoic membrane
The exterior projections formed by trimers of S protein render the (CAM), amniotic membrane and yolk membrane comprised
virus particle to resemble like a crown and based on this morpho- largely of epithelium, providing potential sites for IBV replication.
logical feature these group of viruses are called ‘coronaviruses’ CAM, which lies directly underneath the shell membrane, is a
(the rootword ‘corona’ means crown in Latin) (Fig. 5.3). In addi- highly vascular membrane serving as the respiratory organ of the
tion to the S protein, structural M and E proteins are also found embryo. In addition, the CAM is the largest of all embryo mem-
on the membrane, while the helically symmetrical nucleocapsid branes, and consequently encloses the largest cavity within the
which contains the N protein and viral RNA genome (Fig. 5.3). egg known as the allantoic cavity. In an embryonated chicken egg,
The genome is enclosed within the N protein core (Fig. 5.3). this cavity can hold up to 10 ml of fluid depending on the stage of
embryonation. The amniotic membrane encloses the embryo and
forms the amniotic cavity and may contain approximately 1 ml of
Propagation fluid in an embryonated chicken egg. The yolk sac attached to the
embryo contains nutrients for use during embryonic develop-
Embryonated eggs ment and post-hatch period.
Embryonated chicken eggs are utilized as a laboratory host system The developing embryo and its membranes (CAM, amniotic
for isolation and propagation of a variety of avian CoVs, such as and yolk) provide diverse cell types necessary for successful repli-
turkey CoV (TCoV) (Adams and Hofstad, 1971) and pheasant cation of different viruses, including IBV. Virus may be inoculated
CoVs (Gough et al., 1996). Pioneering work on IBV propagation into embryonated eggs through depositing onto the CAM or
in embryonated chicken eggs in the 1970s established that IBV within the allantoic, amniotic and yolk sacs (Senne, 2008). For
grows well in embryonated chicken eggs, and the passage of IBV avian CoVs, inoculation of eggs by allantoic or amniotic routes
field isolate via the allantoic route of 9-day-old embryos became has been shown to provide access to specific cell types which sup-
the method of choice for virus isolation (Cunningham, 1970; port their replication (Gough et al., 1996; Cavanagh and Naqi,
Fabricant, 1998). Furthermore, embryonated chicken eggs have 2003; Guy, 2013). IBV is an epitheliotropic virus that replicates
been utilized for production of IBV vaccines on a commercial in a variety of epithelial tissues in post-hatch chickens such as the
scale (Britton et al., 2012). Collectively, embryonated chicken respiratory tract, gastrointestinal tract, kidney, bursa of Fabricius
eggs provide a potential host system for isolation and propaga- and oviduct (Cavanagh, 2003), and replicates well regardless of
tion of CoVs, and it may be used for studies aimed for identifying inoculation route in the embryonated egg. However, the allantoic
novel CoVs. route is favoured as the virus replicates extensively in the epithe-
The embryonated chicken egg comprises the developing lium of CAM and high titres of IBV are shed into the allantoic
embryo and supporting membrane which enclose cavities or fluid (Jordan and Nassar, 1973).

Figure 3. Schematic of a coronavirus virion. The structural proteins of coronaviruses, the spike, membrane, nucleocapsid and envelope proteins are shown
Figure 5.3 Schematic of a coronavirus virion. The structural proteins of coronaviruses, the spike, membrane, nucleocapsid and envelope
along with the nucleocapsid encapsidated (+)sense RNA genome. The molecular weight of each structural protein monomer is shown. The size of an infectious
proteins are shown along with the nucleocapsid encapsidated (+)sense RNA genome. The molecular weight of each structural protein
bronchitis virus (IBV) virion is approximately 82 nm.
monomer is shown. The size of an infectious bronchitis virus (IBV) virion is approximately 82 nm.