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Chicken Infectious Anaemia Virus | 259

et al., 1991; Noteborn et al., 1992) suggesting that this region is sequenced a number of isolates including the pathogenic BD-3
important for binding of the capsid protein to the viral genome isolate which has an alanine at AA 287, while some other iso-
(Noteborn and Koch, 1995; Todd et al., 2001). lates had serine or threonine at this position.
Koch et al. (1994, 1995) had shown that VP1 and VP2 Because clone 10 was unstable, passage 320 (P320) of Cux-1
need to be synthesized in Sf9 cells simultaneously infected was examined as a potential vaccine candidate (Todd et al., 1998).
with recombinant baculoviruses expressing VP1 and VP2 to P320 produced very low levels of CAV-specific staining in MSB1
produce neutralizing antibodies in chickens. Progeny of these cells using immunofluorescence assays. Inoculation of 1-day-old
chickens vaccinated with the baculovirus constructs were chicks confirmed that P320 was attenuated and that chickens
protected against challenge with CAV confirming that the developed antibodies. However, challenge experiments to deter-
antibodies neutralized the virus. Studies using neutralizing mine if P320 protects have not been reported. Subsequently, Scott
monoclonal antibody (MAb) 2A9 (McNulty et al., 1990b) or et al. (1999) used 10 molecular clones derived from passage 310
CVI-CAV-132.1 (Koch unpublished data, cited in Noteborn et for further studies. All clones produced virus neutralizing anti-
al., 1998a) showed that these MAbs react with conformational bodies. Interestingly, clone 33 was even more pathogenic than the
epitope(s) (Todd et al., 1994; Noteborn et al., 1998a). The control p13 virus pool. Clone 33, but not the apathogenic clones,
absence of VP2 in viral particles combined with the need to be reacted strongly with MAb 2A9, which has high levels of virus
present in order to form conformational epitopes suggested that neutralizing activity. Sequence analysis showed only one differ-
VP2 is a scaffold protein. Using immunoprecipitation assays ence at AA 87 between clone 33 and the apathogenic clones. The
with VP2-specific MAb 111.1 confirmed that VP2 and VP1 can latter had alanine, while clone 33 and p13 had threonine at AA 87.
bind to each other but only when the proteins were synthesized It is not clear if this change was the marker for decreased patho-
in the same cells (Noteborn et al., 1998a). Trinh et al. (2015), genicity or that changes in VP2 and/or VP3 were responsible for
using neutralizing MAbs, identified two epitopes. One epitope the change, because sequence information was not available for
involved AA 144 because an escape mutant had a change from VP2 and VP3. One of the clones resistant to neutralization with
glutamic acid to glycine. The second epitope appeared to be MAb 2A9 was inducing virus neutralizing antibodies in chickens
a conformational epitope because an escape mutant lacking (Scott et al., 2001) indicating that in addition to the epitope rec-
T89+A90 was not neutralized by MoCAV/F2 and MoCAV/F8 ognized by MAb 2A9, other epitopes recognized by polyclonal
and an escape mutant I261T was not neutralized by MoCAV/ virus-neutralizing chicken sera are present on the surface of the
F8 or MoCAV/F2. virus particles. Clearly, VP1 contributes to the pathogenicity
The importance of VP1 for in vivo and in vitro pathogenicity of CAV, but no single AA can be identified that determines the
has been suggested by several groups, but there is a lack of pathogenicity.
consensus on the determinants of the pathogenicity. Renshaw
et al. (1996) identified a hypervariable region important for Viral protein 2
infectivity of cell lines (see section on cell culture) but was, Douglas et al. (1995) reported that VP2 is produced early
unfortunately, not allowed to test the recombinant viruses in during the infection and that large quantities are present in the
chickens. Yamaguchi et al. (2001) demonstrated that glutamine nucleus, where it is associated with electron-dense aggregates
at AA position 394 was linked to pathogenicity. Changing first described by McNulty et al. (1990a). A functional nuclear
glutamine to histidine resulted in a decrease in pathogenicity. localization signal (NLS) motif has been identified spanning AA
Eltahir et al. (2011b) sequenced 25 Chinese isolates obtained 133–138 with the following AA sequence: KRAKKR (Cheng et
from diseased chickens which all had glutamine at AA 394. al., 2012). A putative nuclear export signal (AA120 to 128 with
The authors claim that this finding supports the importance the sequence LEEAILRPL) was also predicted by Cheng et al.
of glutamine at AA 394 as a marker for pathogenicity. How- (2012) although the prediction score was lower than the thresh-
ever, experimental studies were not conducted to verify their old expected value of the NetNES 1.1 program. Subsequently,
statement. Moreover, several substitutions in the deduced AA Cheng et al. (2012) showed that VP2 does not bind chromosome
sequence for VP1, VP2 and VP3 were noticed. Using maximum- region maintenance 1 protein (CRM1), the major receptor for
likelihood models, Wang et al. (2009) suggested that aspartic the export of proteins from the nucleus. However, VP2 binds
acid at AA 287 is important for pathogenicity. This suggestion is to chromatin and associates with the minichromosome mainte-
not supported by a series of studies by Todd and his associates nance complex component 3 (MCM3). This protein is part of a
(Todd et al., 1995, 2003; Meehan et al., 1997). The attenuated hexameric protein complex involved in the initiation of eukary-
clone 10 was derived after 173 passages of Cux-1 in MSB1 cells otic genome replication. The binding of VP2 to MCM3 does not
followed by molecular cloning of RF molecules. Unfortunately, require the dual specificity protein phosphatase (DSP) activity
the attenuation was unstable and the 10th chick passage yielded described below.
a revertant with some pathogenicity. Sequencing of clone 10 In addition to the scaffold function (see ‘Viral protein 1’), VP2
identified differences at 17 nt positions throughout the genome is also a DSP (Peters et al., 2002). CAV VP2 and ORF2 of TLMV
including one that changed AA 287 to alanine from aspartic possess protein-tyrosine phosphatase (PTPase) and serine/
acid. However, some revertant clones obtained after seven threonine phosphatase (S/T PPase) activity. The catalytic motif
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94
additional passages in MSB1 cells did have aspartic acid at AA in CAV is I CNCGQFRKH with the bold letters indicating
287 and were fully attenuated. Moreover, Islam et al. (2002) the conserved signature AA. The presence of a second cysteine at