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182 | Kibenge et al.

icosahedral double-layered capsid of ≈ 75 nm diameter (Fig. 6.2). inoculation (dpi) oedematous swellings were observed in the
The precise diameter of the particles from electron cryomicros- CAM of 9–10-day Specific Pathogen Free (SPF) ECE (Schwartz
copy image analysis is 85.7 nm (Zhang et al., 2005). The two et al., 1976). Mackenzie and Bains (1977) reported gross thick-
capsid shells are identified as the ‘core’ made of λA and σA pro- ening and pock lesions of 0.5–1.0 mm in diameter on the CAM
teins and ‘outer capsid’ made of µB and σB proteins (Benavente and embryo death with stunting and liver necrosis at 4–6 dpi. In
and Martínez-Costas, 2007). Protein λA forms the inner core addition to formation of plaques on the CAM, Wu et al. (2004)
shell that encloses the 10 viral genome segments, the viral RNA noted curling and stunting of embryos. Nersessian et al. (1986)
polymerase λB and its cofactor µA. This shell is stabilized by pro- reported turkey reovirus produced oedema, greyish yellow pocks
tein σA, which is seated on top of λA and acts as a bridge between on the CAM and embryo stunting when inoculated on CAM of
the inner core and the outer capsid. Pentamers of the λC protein ECE.
form the turrets that extend from the inner core through the outer When avian orthoreovirus grew in the yolk sac, depending
capsid, and trimers of the cell attachment protein σC protrude on the amount of inoculum used, mortality usually occurred in
from the turret tops. The reovirion structure is completed by the 4–6 dpi, and frequently gave rise to a markedly haemorrhagic and
addition of the outer capsid shell, which is formed by proteins µB purplish embryo (Olson and Kerr, 1966). Embryos that survived
and σB (Benavente and Martínez-Costas, 2007). Thus, the inner- beyond the 15th day of incubation revealed an enlarged liver and
capsid layers and proteins are primarily involved in virus assembly spleen with necrotic foci (Olson and Kerr, 1966). According to
and replication whereas the outer-capsid proteins are involved in Guneratne et al. (1982) yolk sac inoculation of 5- to 6-day-old
virus transmission, cell attachment and penetration and show ECE was a superior method compared with other routes of egg
much greater variation reflecting differences in the targeted host inoculation, and therefore recommended for primary isolation
species (Mertens, 2004). Avian reovirus particles are very stable of avian orthoreoviruses (Glass et al., 1973). Avian reoviruses
and resistant to lipid solvents and a wide pH range (Jones, 2013). from wild birds may be propagated on chicken embryos by yolk
sac inoculation (Styś-Fijoł et al., 2017). In general, the death
Propagation of embryos after yolk sac inoculation occurs more rapidly than
Avian reoviruses replicate in a diverse range of culture systems allantoic cavity inoculations (Rekik et al., 1991).
including embryonated chicken eggs and primary cell cultures
of chicken embryo as well as certain established mammalian and
avian cell lines. Primary cell cultures
Viruses are obligatory dependents on host systems for their
Embryonated eggs propagation and animal cells are excellent hosts for viruses (Hos-
Avian reoviruses are pathogenic for chicken embryos and have a sain et al., 2006). Avian orthoreoviruses are readily propagated in
predilection for the chorioallantoic membrane (CAM) and the many avian origin primary cell cultures (Guneratne et al., 1982;
yolk sac of 5- to 7-day-old embryonated chicken eggs (ECE) Barta et al., 1984), including chicken embryo lung (CELu) (Petek
(Deshmukh and Pomeroy, 1969; Wood et al., 1980), killing the et al., 1967; Guneratne et al., 1982), fibroblast (CEF) (Lee et al.,
embryos and producing pock-like lesions on the CAM (Fahey 1973; Guneratne et al., 1982), Kidney (CEK) (Glass et al., 1973;
and Crawley, 1954). Inoculation of CAM has been reported to be Guneratne et al., 1982), liver (CELi) (McFerran et al., 1976;
more successful than via the allantoic cavity for avian orthoreovi- Guneratne et al., 1982) and tendon (CET) (Huang, 1995);
rus propagation (Hollmén and Docherty, 2007). At 5 days post chicken kidney (CK) (Green et al., 1976), lung (CL), testicular
cell cultures (CTCC) (Sahu and Olson, 1975), bone marrow
derived macrophages (von Bülow and Klasen, 1983) and leuco-
cytes (Mills and Wilcox, 1993); turkey kidney cells (Fujisaki et
al., 1969); duck embryo fibroblast cells (Lee et al., 1973); and
Muscovy duck embryo fibroblast cells (Hollmén and Docherty,
2007). Of the primary cell cultures, CELi has been reported to
be the most sensitive (Guneratne et al., 1982; Barta et al., 1984);
however, CEF is the most commonly used for avian orthoreovi-
rus replication (Deshmukh and Pomeroy, 1969; Mustaffa-Babjee
et al., 1973; van der Heide, 1977; Wood et al., 1980; Guneratne et
al., 1982).
The cytopathic effects (CPE) produced in cell cultures infected
with all characterized avian reovirus isolates is the formation of
multinucleated giant cells (Fig. 6.3) called syncytia (Duncan
and Sullivan, 1998), that detach from the cell monolayer surface
leaving small holes (Deshmukh and Pomeroy, 1969; Robertson
Figure 6.2. Electron micrographs of purified American crow and Wilcox, 1986). This CPE is more commonly associated
orthoreovirus particles negatively stained with 2% phosphotungstic with enveloped virus replication (Duncan et al., 1996) and can
acid. Note the double capsid structure. The scale bar represents
100 nm. be utilized to identify the replication of avian orthoreoviruses