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site approximately 160 bases upstream from the EcoR1 restric- haemorrhages. Thrombocytes are not only important for blood
tion site. This site is located within a putative cruciform-loop clotting but also play an important role in the innate immune
structure, which may function as the origin of replication. Todd responses. Thrombocytes are also highly phagocytic (Carlson et
et al. (2001) suggested that the complimentary strand is initiated al., 1968) and constitutively express transcripts for pro- and anti-
by cellular DNA primase together with RNA primers followed inflammatory cytokines. Stimulation of toll-like receptors (TLR)
by elongation by cellular DNA polymerase to generate the RF. on thrombocytes increases pro-inflammatory responses (James-
Meehan et al. (1992) and Todd et al. (1996) described two forms Berry et al., 2010), and these cells respond to infection with
of RF: the closed circular duplex (RFI) and relaxed or open cir- H5N1 avian influenza virus by up-regulation of TLR3 (Schat et
cular duplex (RFII). The latter is probably involved in the process al., 2012).
of DNA replication. The process of DNA replication from the RF Infection of the hemocytoblasts can first be detected between
also has not been elucidated. According to Cheng et al. (2012) 4 and 6 days pi and continues to 12 to 20 days pi, causing
VP2 associates with MCM3 (see also section ‘Viral protein 2’), hypoplasia and replacement of haemocytoblasts by adipocytes.
suggesting that VP2 is important in the activation of viral genome Repopulation with immature hemocytoblasts typically starts
replication. In addition, VP1 may also be important for the repli- after 20 days pi. Infected hemocytoblasts are enlarged and are
cation of viral DNA from the RF. Based on GenBank sequences, present in the intra- and extravascular spaces. The nuclei are also
Ilyina and Koonin (1992) identified three conserved sequence enlarged with coarsely granular chromatin often containing single
motifs in VP1 which are associated with the initiator proteins or multiple eosinophilic intranuclear inclusions. Macrophages
needed in the RCR mechanism. Cheng et al. (2012) suggested containing degenerated hemocytoblasts are frequently scattered
that VP2 binds with VP1 to form a complex that might regulate in the intravascular spaces (Taniguchi et al., 1983; Goryo et al.,
the RCR reaction during the early phase of infection. However, 1989a; Smyth et al., 1993). The ultrastructural changes of CAV-
there is a large difference in the timing of the appearance of VP2 infected haemocytoblasts have been described by Goryo et al.
at 12 hours PI versus VP1 at 30 hours in the nucleus (Todd et al., (1989b). Electron-opaque inclusion bodies were frequently seen
1994; Douglas et al., 1995). It is therefore quite possible that VP2 in the nuclei. The inclusions consisted of non-membrane-bound
is important for the change of RFI to RFII which is an essential homogeneous, finely granular material. Other changes included
part for the replication of the viral DNA. the increased presence of pseudopodia and the presence of
electron-opaque and tubular structures in the cytoplasm. Occa-
Encapsidation, maturation and release sionally aggregations of virus-like particles were observed. The
The process of encapsidation and maturation of CAV has not actual cause of cell death has not been established but apoptosis
been studied in detail. The following relevant facts have been is likely involved although necrosis has not been ruled out.
discussed in previous sections: (1) production of infectious virus Thymocytes in the thymic cortex are the other major cell
in susceptible cell lines, and by extension in chickens, requires population affected by CAV infection causing a major decrease
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the production of phosphorylated VP3 (Prasetyo et al., 2009; of CT1 (a pan T-cell marker), CD4 and CD8 cells (Jeurissen
Kucharski et al., 2016); (2) VP2 is needed as a scaffold protein for et al., 1989; Hu et al., 1993b) resulting in severe thymus atrophy
the correct folding of VP1 (Koch et al., 1995); (3) the NH2 termi- (Fig. 9.9). Smyth et al. (1993) detailed the changes in thymocytes
nal region of VP1 has a significant degree of similarity to histone after infection of 1-day-old SPF chickens. Starting at 4 days pi
proteins (Claessens et al., 1991; Noteborn et al., 1991), which is enlarged lymphoblasts with karyomegaly and occasionally small
likely to be important for binding to the viral genome. Electron circular eosinophilic nuclear inclusions were detected. These cells
microscopic examination of CAV-infected MSB1 or 1104-X5 were often positive for CAV antigens in the nuclei. Degenerated
cells showed the presence of electron-dense rings and aggregates cells and cell debris were accumulating starting at 6 days pi. Goryo
of virus-like particles at 45 to 48 hours pi in a few cells (McNulty et al. (1989a) noticed scattered lymphocytes with karyorrhexis
et al., 1990a; Jeurissen et al., 1992b). Unfortunately, earlier time starting at 6 days pi. During this process the medulla remained
points were not examined, and it is therefore not known if virus basically normal (Jeurissen et al., 1989). Thymus repopulation
particles are assembled before 45 hours. The release of virus from was starting around 20 days pi (Taniguchi et al., 1983; Goryo et
the infected cell is most likely the consequence of apoptosis. In al., 1989a; Jeurissen et al., 1989; Hu et al., 1993b; Smyth et al.,
support of this hypothesis, Jeurissen et al. (1992b) found virus- 1993). Electron microscopy studies indicated the presence of
like particles in apoptotic vacuoles in epithelial cells in thymus chromatin aggregation, nuclear fragmentation and the presence
tissues 13 days pi of 1-day-old chickens. of apoptotic bodies (Jeurissen et al., 1992b) which were also seen
after infection of 3- and 6-week-old chickens (Smyth et al., 2006).
Effects on the host cell However, some of the features described by Smyth et al. (1993)
In vitro infection of susceptible cell lines clearly leads to apoptosis such as cytomegaly, karyomegaly and dispersed chromatin are
as discussed above. Infection of susceptible chickens (See ‘Patho- normally not associated with apoptosis as discussed by Smyth
genesis’) causes severe destruction of the hemocytoblasts in the and Schat (2013). Originally, Jeurissen et al. (1992a) suggested
bone marrow and thymocytes in the thymus. Hemocytoblasts are that thymocytes in the thymus are only susceptible to infection
precursors for erythrocytes, heterophils and thrombocytes and between ED 13 and 21 days of age, when the second wave of thy-
the reduction or elimination of hemocytoblasts by CAV explains mocyte stem cells are present (Le Douarin et al., 1984). However,
the development of anaemia, increased bacterial infections and embryonally bursectomized chickens infected at 38 days were