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resulting in the production of fully infectious virus particles. de Cux-1 which includes the hypervariable region. In addition, chi-
Villiers et al. (2011) reported the replication of viral DNA of TTV meric viruses Cux/CIA N-B and Cux/CIA S-B were generated
after transfection of the human embryonic kidney cell line 293TT, using the Cux-1 backbone in which nt 1191–1508 and 844–1508
but this did not result in production of infectious virions. were replaced with the CIA-1 fragments (Fig. 9.4). CIA/Cux
CAV, like other members of the Anelloviridae, lacks the genes N-B and CIA/Cux S-B replicated to the same degree as Cux-1
coding for proteins needed for DNA replication. As a conse- in MSB1(S), while Cux/CIA N-B and Cux/CIA S-B replicated
quence, CAV requires dividing cells for virus replication. Yuasa at the same level as CIA-1 (Fig. 9.5) suggesting that the hyper-
(1983) infected four MDCC cell lines (MSB1, JP2, RP1 and BP1) variable region is important for the rate of virus spread in MSB1
and three leukosis/sarcoma-derived chicken cell lines (LSCC- (S). Infection of MSB1(L) on the other hand was only successful
1104B1, -1104X5, and TLT). Only MSB1, JP2, and 1104B1 were with CIA/Cux S-B. Apparently, additional upstream differences
able to replicate CAV for nine passages, when the experiments in VP1 and/or differences in VP2 and possible VP3 may also be
were terminated. Eight different monolayer cell cultures derived important for the infection and replication of CIA-1 and similar
from chickens or chicken embryos were refractory to infection. isolates (Renshaw et al., 1996; Islam et al., 2002) in MSB1. On
Limited information suggests that mammalian lymphoblastoid the other hand, Nogueira et al. (2007) did not detect amino acid
cell lines are not susceptible to infection (Haridy et al., 2010). substitutions in VP2 and VP3 among the 12 isolates or consist-
Subsequently, Yuasa et al. (1983a) compared isolation of CAV ent differences in VP1 between isolates that could or could not
in 1-day-old chicks and MSB1 cells using 99 samples represent- replicate in the 3 MSB1 sublines. Both MSB1(L) and MSB1(S)
+
–
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ing 12 flocks. CAV was isolated in 69% of the samples after chick are characterized as CD4 CD8 TCR2 (Calnek et al., 2000) but
inoculation and in 58% of the samples in MSB1 cells. In 11% of it is possible that other cell surface markers differ between the two
the samples the chick inoculations were positive, while MSB1 sublines. It is also possible that differences in cell metabolism or
cells were negative. This difference may depend on the passage cytokine expression play a role in the ability to replicate CAV.
level of the cell line or the virus strain. MSB1 cells have been Yuasa (1983) reported that, in contrast to MSB1 and JP2,
widely distributed and differences in susceptibility to infection some other MD-derived cell lines were unable to become
between sublines using the same virus strain have been reported infected with CAV. In an attempt to determine if specific pheno-
(von Bülow et al., 1986a; Renshaw et al., 1996; Calnek et al., types of T-cells are more susceptible to CAV infection, Calnek
2000). Von Bülow et al. (1985, 1986) noticed that continuous et al. (2000) examined 24 MD cell lines in addition to the two
culture of MSB1 cells may result in a decreased susceptibility to MSB1 sublines. The cell lines represented the following phe-
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–
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infection. Some CAV isolates cannot be isolated or propagated notypes: CD4 CD8 TCR2 (n = 8 including the two MSB1
–
+
+
–
in certain MSB-1 sublines (Soiné et al., 1993; Renshaw et al., sublines), CD4 CD8 TCR3 (n = 2), CD4 CD8 TCR2 (n = 5),
+
+
+
+
–
+
–
–
1996; Islam et al., 2002; Nogueira et al., 2007; van Santen et al., CD4 CD8 TCR3 (n = 4), CD4 CD8 TCR2 (n = 5) and CD4 –
+
–
2007). For example, the CIA-1 strain (Lucio et al., 1990) could CD8 TCR3 (n = 2). No clear-cut differences in susceptibility
not be isolated or propagated in the MSB1(L) subline but could were detected in relation to the phenotype (Table 9.2), thus it
be propagated in the MSB1(S) subline (Renshaw et al., 1996) does not seem that CD4, CD8 or TCR are uniquely important as
and replicated to higher titres in MDCC-CU147 than MSB1(S) a virus receptor.
+
(Calnek et al., 2000). The CU147 cell line also yielded higher Interestingly, CU147 (CD4 CD8 TCR3 ) was replicating
–
+
titres with the Cux-1 isolate than in MSB1(S). Nogueira et al. the Cux-1 and CIA-1 isolates equally well and Cux-1 to a 10- to
(2007) tried to isolate 12 Brazilian CAV strains in three different 100-fold higher titre than in MSB1. Van Santen et al. (2007) used
MSB1 sublines but eight of these strains could not be propagated CU147 to regenerate a CAV virus from PCR fragments obtained
in any of the three MSB1 sublines. from a field case but were unsuccessful using MSB1. Unfortu-
The reasons for the inability of some CAV strains to replicate nately, CU147 is more difficult to grow in culture than MSB1 (van
in MSB1 cells or to replicate at reduced rates are not clear. Pos- Santen et al., 2007; Schat, unpublished observations) and the cell
sible explanations include minor sequence differences among line is no longer available for distribution.
strains and differences in the expression of surface antigens All susceptible cell lines consist of lymphoblastoid cells
between the different sublines of MSB1. Renshaw et al. (1996) growing in suspension and need to be passaged every 2–3 days.
analysed sequences of ORF 1, coding for VP1, from isolates that It is therefore difficult to determine the presence of cytopathic
are able to replicate in MSB1(L) and compared them with the effects (CPE). Infected cells become swollen followed by lysis,
sequences of two isolates (CIA-1 and L-028) which do not rep- which can occur as early as 30 hours post inoculation (pi) (von
licate in MSB1(L). A hypervariable region in VP1 was identified Bülow et al., 1985, 1986a), but this may be difficult to detect
spanning amino acid positions 139–151 with Q at position 139 if the virus titre of the inoculum is low. If CAV is present most
and 144 in CIA-1 and L-028 instead of K and D in Cux-1. This cells will die, and the cultures become alkaline, but this may
hypervariable region is part of a hydrophilic domain of VP1 and take 10–30 passages depending on the titre in the sample and
modelling suggests that it is expressed on the surface of the virus specific laboratory conditions. Succesful propagation needs to be
particles. To examine the relevance of the hypervariable region verified by showing the presence of viral proteins in the nucleus
for infection of MSB1, chimeric viruses CIA/Cux N-B and CIA/ using indirect immunofluorescence with CAV-specific chicken
Cux S-B were generated consisting of the CIA-1 VP1 back bone IgY (Fig. 9.6) or monoclonal antibodies to CAV VP3 and
with 317 or 744 nucleotides (nt 1191–1508 and 844–1508) from appropriate secondary reagents. Careful titration of the primary