Avian Pathology

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Chicken anaemia virus infection: Molecular basis

of pathogenicity

Mathieu H. M. Noteborn & Guus Koch

To cite this article: Mathieu H. M. Noteborn & Guus Koch (1995) Chicken anaemia virus infection:
Molecular basis of pathogenicity, Avian Pathology, 24:1, 11-31, DOI: 10.1080/03079459508419046

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Avian Pathology (1995) 24, 11-31

REVIEW ARTICLE

Chicken anaemia virus infection: molecular basis

of pathogenicity

MATHIEU H. M. NOTEBORN 1 & GUUS KOCH 2

1
Laboratory for Molecular Carcinogenesis, Sylvius Laboratory, Leiden University,
PO Box 9503, 2300 RA Leiden, and 2 Department for Porcine and Avian Virology,
DLO-Institute of Animal Science and Health, PO Box 365, 8200 AJ Lelystad, The
Netherlands

SUMMARY

Chicken anaemia virus (CAV) is a small virus of a unique type with a particle
diameter of 23 to 25 nm and a genome consisting of a circular single-stranded
(minus-strand) DNA. This DNA multiplies in infected cells via a circular double-
stranded replicative intermediate, which was recently cloned. DNA analysis of CAV
strains isolated in different continents revealed only minor differences among the
various isolates. Apparently, all CAV isolates belong to a single serotype. CAV is not
related to other known animal single-stranded circular-DNA viruses, such as porcine
circovirus and psittacine beak-and-feather-disease virus.

The major transcript from the CAV genome is an unspliced polycistronic mRNA of
about 2100 nucleotides encoding three proteins of 51.6 kDa (VP1), 24.0 kDa (VP2)
and 13.6 kDa (VP3 or apoptin). All three predicted CAV proteins are synthesized in
CAV-infected cells. Immunization with (recombinant) VP1 and VP2 synchronously
synthesized in the same cells elicits a protective response and can be used as subunit
vaccine against chicken infectious anaemia.

CAV causes clinical and subclinical disease in chickens, and is recognized as an

important avian pathogen worldwide. In young chickens, CAV causes a transient
severe anaemia due to destruction of erythroblastoid cells in the bone marrow
and immunodeficiency due to depletion of cortical thymocytes. The depletion
of the cortical thymocytes is considered to cause a (transient) immunodeficiency
resulting in enhanced concurrent infections and to vaccination failures. The
depletion of thymocytes and most likely also of erythroblastoid cells occurs
via CAV-induced apoptosis. The CAV-encoded protein apoptin is the main inducer
of this phenomenon.

INTRODUCTION
Since McNulty (1991) published his review article about chicken anaemia agent
(renamed to chicken anaemia virus (CAV); Gelderblom et al, 1989; Noteborn et

Received 8 September 1994; Accepted 8 September 1994.

0307-9457/95/010011-21 © 1995 Avian Pathology

12 M. H. M. NOTEBORN & G. KOCH

al, 1991) our knowledge of the molecular aspects of CAV infection has increased
enormously. In this review, we shall describe the molecular biology of CAV and
the causal mechanism of the (cyto)pathogenic effects of CAV infections, i.e.
virus-induced apoptosis. The development of diagnostic assays and vaccines
based on the molecular biology of CAV will be discussed.

CAPSID AND GENOME

CAV particles are non-enveloped and have an average diameter of 23 to 25 nm.
CAV has a buoyant density of 1.33-1.34 g/ml (Gelderblom et al, 1989; Todd et
al> 1990). The virus capsid is composed of 32 structural subunits arranged in a
class P = 3 icosahedron with a triangulation number of 3 (McNulty et al, 1990c).
CAV particles are resistant to acetone, chloroform, ether and pH 3.0. CAV is
partially inactivated after heating at 80°C for 30 min and completely inactivated
after 10 min at 100°C (Goryo et al, 1985; Taylor, 1992; Urlings et al, 1993).
Todd et al, 1990) reported that the CAV capsid contains only one protein
species of 50 kDa. Electron-microscopic analysis of the capsid DNA showed that
it is a circular single-stranded (ss) DNA of about 2300 nucleotides (nt)
(Gelderblom et al, 1989; Todd et al, 1990). When DNA from purified CAV was
analysed in Southern-blot assays with synthetic oligomer probes derived from the
CAV DNA plus or minus strand (Noteborn et al, 1991) or with probes represent-
ing the plus or minus strand of the complete CAV genome (Phenix et al, 1994),
only the 'plus' oligomer hybridized with the viral DNA. In addition, the viral
DNA proved to be sensitive to SI nuclease. Apparently, the virus particle
contains circular minus-strand DNA, which is complementary to the transcribed
RNAs.
The thymi and spleens of chickens from flocks with signs of chicken infectious
anaemia contain large amounts, and their livers and bursas relatively
small amounts of circular ss CAV DNA (Noteborn et al, 1994c). Cultured,
CAV-infected cells contain, in addition to ss DNA, equal amounts of double-
stranded (ds) replicative DNA forms. The latter are involved in the generation
of viral RNA and new circular ss CAV genomes (Figure 1). Experiments
performed by ourselves (Noteborn et al, 1994c) and Meehan et al. (1992)
demonstrated the existence of three major replicative forms in infected cells:
ds open circular, linear and closed-circular and ss circular CAV DNA. In some
preparations, a slower migrating minor DNA was observed. The above-
mentioned data suggest that CAV DNA replicates via the rolling-circle model,
which has been described for the circular ss DNA bacteriophage phiX174
(Kornberg, 1980).
In our laboratory, low-molecular-weight DNA obtained from chicken cells
infected with CAV-CUX-1 (Biilow et al, 1983) was digested separately with
several restriction enzymes with a single cleavage site in CAV ds DNA, e.g. EcoRl
or Pstl. The DNA samples were size-fractionated on an ethidium-bromide
agarose gel. DNA fragments of about 2.3 kb were isolated and cloned into a
 CHICKEN ANAEMIA VIRUS INFECTION 13

DNA

RNA

PROTEIN

VP2 VP3 VP1

Figure 1. Schematic representation of the molecular processes of CAV. A single-stranded circular

DNA is present in the virus capsids jingle circle). In infected cells, this ss-DNA genome becomes
a ds circular replicative intermediate (double circle) from which 1 polyadenylated RNA
species is transcribed (line). This polycistronic RNA contains three partially overlapping genes
each with its own initiation (x) and termination (•) codons. Three CAV-encodedproteins VP1,
VP2 and VP3 (apoptin) are synthesized in CAV-infected cells. VP1 is probably the capsid
protein.

bacterial vector. The cloned material was found to represent the complete CAV
ds DNA and to contain all the elements required for the CAV replication cycle
and for its pathogenicity. First, transfection of the recircularized, cloned insert
into chicken cell lines caused a cytopathogenic effect, which could be prevented
by addition of a chicken serum containing neutralizing antibodies directed against
CAV. Secondly, when 1-day-old chickens were inoculated with CAV collected
from cell lines that were transfected with bacterially cloned CAV DNA, they
developed clinical signs of CAV (Noteborn et al., 1991).
Both strands of the cloned CAV DNA were sequenced. The CAV genome
is 2319 bp long and contains three partially or completely overlapping major
open reading frames (ORF), which all lie on one strand, the plus-DNA strand.
A computer search revealed no significant homology between the CAV-encoding
sequences and known sequences in the databanks. A search for known tran-
scription-factor-binding sites showed that only one region had potential pro-
moter/enhancer properties. The CAV genome contains only one polyadenylation
signal. Both transcription elements are, just as the above-mentioned ORFs,
located on the plus-DNA strand (Noteborn et al, 1991; accession number
M55918).
Recently, Meehan et al (1992; accession number M81223) have also published
14 M. H. M. NOTEBORN & G. KOCH

the DNA sequence data of the CUX-1 isolate. Claessens et al (1991; D10068)
and Soine et al. (1994; L14767) determined the complete DNA sequence of two
different USA isolates, Kato et al. (1994; D31965) that of a Japanese isolate and
Pallister et al. (1994) the main part of an Australian isolate. All these sequence
data proved to be very similar to our CAV sequence (Noteborn et al., 1991).
When DNA isolated from a large number of CAV isolates was examined via
Southern-blot analysis with 32P-labelled DNA probes from cloned CAV DNA
(Noteborn et al., 1991), similar ds replicative forms and ss genomic DNA were
found. Restriction-enzyme mapping revealed only minor differences among the
various CAV isolates (Noteborn et al, 1992b; Todd et al, 1992). Todd et al.
(1991a) developed a dot-blot hybridization assay capable of detecting CAV-
specific DNA in tissues of infected birds. It uses a 32P-labelled DNA probe from
cloned CAV-specific fragments representing the entire virus genome. In our
laboratory, we have shown that a non-radioactive hybridization test based on
digoxigenin-labelled DNAs representing the complete CAV DNA genome detects
all CAV isolates analysed so far (Noteborn et al, 1992b). To increase the
sensitivity of detection, CAV-specific PCR has been developed by several research
groups (Noteborn et al, 1992b; Tham & Stanislawek, 1992; Todd et al, 1992;
Soine et al, 1993). CAV DNA equivalent to a single cell could be detected by
dot-blot (Tham & Stanislawek, 1992; Todd et al, 1992) or Southern-blot
hybridization (Noteborn et al, 1992b; Soine et al, 1993). For routine application
for laboratory diagnosis or for testing vaccines for the presence of extraneous
CAV, PCR should be as sensitive as virus isolation; ideally, it should detect a
single copy of CAV in a large amount of background DNA. Until now, such a
sensitivity was only attained in a nested PCR (Soine et al, 1993). However, this
technique may be sensitive to contaminations (Dren et al, 1994). Recently, Dren
et al. (1994) developed a sensitive PCR assay for CAV consisting of the addition
of primers to the Taq polymerase during the hot phase of the amplification cycle,
the so-called hot-start PCR (Chou et al, 1992). By direct analysis of the amplified
DNA on ethidium bromide-stained agarose gels, we were able to detect CAV in
the equivalent of one infected cell or 10 T d D 5 0 . These results constitute an
improvement over our original PCR procedure (Noteborn et al, 1992b) by a
factor of 100. When the fragments were hybridized with a non-radioactively-la-
belled CAV DNA probe, the detection limit was increased at least 10-fold.
Furthermore, Dren et al (1994) reports a spiked PCR assay to validate negative
PCR results and for estimating the number of CAV genomes present in the
analysed samples. The determination of the amount of viruses might be import-
ant because McNulty et al. (1990a) have reported that the virus dose influences
the level of pathogenicity.
In conclusion, the above DNA analyses based on cloned CAV DNA from
various strains isolated across the world revealed only minor differences among
the CAV isolates. This confirms the observation of McNulty et al. (1989, 1990b)
that all known CAV isolates constitute a single serotype. Therefore, diagnostic
tests based on the described cloned CAV DNAs are suitable for detection of CAV
worldwide.
 CHICKEN ANAEMIA VIRUS INFECTION 15

TRANSCRIPTION OF THE CAV GENOME

Noteborn et al (1992a) and Phenix et al. (1994) studied the transcription of the
CAV genome in cultured cells infected with CAV-CUX-1. One (major) CAV
transcript of about 2.1 kb was detected in the poly(A)+ RNA fraction (Figure 1).
This RNA was CAV-specific, being absent in mock-infected cells. Dot-blot
analysis showed that CAV RNA was already weakly detectable 8 h after infection,
but its level rose dramatically at 32 h after infection. By means of nuclease-Sl
mapping and primer extension analysis it was established that transcription
initiates from a single site. SI mapping or amplification, cloning and sequencing
of the 3' end of the major transcript showed that there is only one transcription
termination point. These experiments placed the transcription start point 30 nt
downstream of a TATA-box at nt 324 and 26 nt upstream from the initiation
codon of the first major ORF (nt 380). The numbering used in this review is
according that of Noteborn et al. (1991). The polyadenylation site is located 25
nt from the only perfect polyadenylation signal (AAUAAA; nt 2317) in CAV
RNA. Northern blot analysis using a series of genomic probes indicated that the
2.1-kb transcript had not undergone any splicing (Phenix et al, 1994). CAV RNA
is a polycistronic mRNA which encodes three proteins (see below). This implies
that, apart from the 5'-proximal initiation codon 2 additional internal AUG's are
used as start codons. The initiation codons of the 2 ORF's at nt 380 and 486
occur in an unfavourable context for translation (Kozak, 1986), whereas the
initiation codon of the third ORF is the only one in the CAV genome following
the Kozak consensus: an A residue at — 3, and a G at + 4. It is assumed that
ribosomes scan the CAV mRNA and will start translation alternately at the
initiation codons of either the first, second or third ORF.
Analysis of the sequences of the cloned DNA derived from the CAV-CUX-1
genome (Noteborn et al., 1991; Meehan et al., 1992) showed that a complete set
of promoter-enhancer elements is situated upstream from the transcription start
point beginning with the TATA box at nt 324. Upstream of the TATA box, an
SPl-binding site (5'-GGGCGG-3'; position 305-310) and the 'CCAAT-TF'
binding site (position 260-266) are located. Again further upstream, a remarkable
array of four or five (depending on the strain) near-perfect 21-bp direct repeats
interrupted by an 12-bp insert is located, that terminates 3 bp upstream of the
'CCAAT' box. This non-transcribed region was shown to possess promoter
activity, stimulating the expression of the human growth hormone (Phenix et al,
1994) or chloramphenicol-acetyl transferase (Noteborn et al., 1994b) reporter
genes in a transient gene-expression assay. The repeat region was proven to be the
main transcription-activation element, with enhancer-like characteristics. Either a
single direct-repeat unit or the 12-bp insert can akeady enhance transcriptional
activity, but do so with relatively low efficiency (Noteborn et al, 1994b). Analysis
of various CAV isolates in our laboratory (Noteborn et al, 1994b) and in that of
others (Claessens et al, 1991; Meehan et al, 1992; Kato et al, 1994; Soine et al,
1994) revealed that the direct-repeat elements and the 12-bp insert apparently are
representative for CAV isolates worldwide. In most virus isolates four direct
16 M. H. M. NOTEBORN & G. KOCH

repeats with a 12-bp insert in the middle were found instead of five as reported
for two European (Noteborn et al, 1991, 1994b) and a Japanese strain 82-2
(Kato et al, 1994). Electrophoretic-mobility-shift assays showed that the indi-
vidual repeat units as well as the 12-bp insert can bind to nuclear factors of
chicken T cells, indicating that all these elements are involved in the regulation of
the transcription of the CAV genome. Competition assays revealed that the repeat
units bound to other factor(s) than the 12-bp insert. The latter element showed
strong affinity for purified (human) SP1 transcription factor (Noteborn et al,
1994b).

CAV PROTEINS
Several research groups have provided evidence that all three CAV ORFs encod-
ing VP1 (51.6 kDa), VP2 (24.0 kDa) and VP3 (13.6 kDa) are, indeed, expressed
in CAV-infected cells (Figure 1).
Chandratilleke et al. (1991) have described three viral proteins in CAV-infected
cells which migrate on a polyacrylamide-SDS gel as products with estimated sizes
of 45, 30 and 16 kDa. Buchholz (1994), by Western blotting, analysed the
proteins synthesized in MDCC-MSB1 cells (Akiyama & Kato, 1974) that had
been infected with the CAV strains CUX-1 (Bulow et al, 1983, Gifu-1 (Yuasa et
al, 1979) or TK5803 (Goryo et al, 1985). In all three cases, three viral proteins
with molecular weights of 48, 30 and 16 kDa were stained with a CAV-specific
hyperimmune serum. Todd et al (1994) have carried out investigations using
monoclonal antibodies and antisera raised to N- and C-terminal ORF peptides
sequences, which reveal that all three ORFs are expressed in infected cells.
VP1 most likely is the capsid protein. Todd et al (1990) have detected one
major polypeptide of M r 50,000 in purified virus collected from CsCl gradients:
when purified CAV preparations that contained approximately 500 ng of the
50-kDa protein were size-fractionated and analysed by silver staining, no other
proteins were detected. In vitro expression of the ORF encoding VP1 revealed a
product of 52 kDa (Noteborn et al, 1992a). The N-terminal region of VP1
contains an amino-acid tract, which is highly reminiscent of histones (e.g. Swiss-
Prot data-base, accession Nos P14402 and P80001). Histones are known for their
arginine content and ability to bind and to protect DNA. Thus, the N-terminal
region of VP1 might bind to the ss DNA within the capsid.
In vitro expression of the ORFs encoding VP2 or VP3 yielded products of 28
to 30 and 16 kDa, respectively. The monoclonal antibody (MAb) CVI-CAV-85.1
was shown to stain specifically CAV-infected cells in the thymus cortex and bone
marrow. MAb 85.1 precipitated a 16-kDa protein from lysates of insect cells
infected with a recombinant baculovirus expressing only ORF 3 encoding VP3.
The 16-kDa protein had a mobility identical to VP3 of CAV-infected MDCC-
MSB1 cells as estimated from PAA-SDS gels (Noteborn et al, 1994a). These
data were the first to show a correlation between a CAV-specific MAb and the
product of one of the ORFs of the CAV genome. Recently, we have found
evidence that another CAV-specific MAb is directed against the 30-kDa VP2
 CHICKEN ANAEMIA VIRUS INFECTION 17

product, synthesized by a recombinant baculovirus. This 30-kDa protein is

present in lysates of CAV-infected MDCC-MSBl'.cells (S. Veldkamp, unpub-
lished results).

CAV BELONGS TO A NOVEL VIRUS FAMILY

The previous sections concerned the molecular characterization of CAV. Apart
from CAV, two other non-enveloped, icosahedral animal viruses with a circular
ss-DNA genome have been described: porcine circovirus (PCV; Tischer et al.,
1982) and psittacine beak-and-feather-disease virus (PBFDV; Ritchie et al,
1989). These viruses, too, could not be assigned to any known family of animal
viruses on the basis of their biochemical and structural characteristics. Studdert
(1993) have proposed that CAV, together with PCV and PBFDV, constitutes a
new virus family, Circoviridae. However, CAV, PBFDV and PCV exhibit no
DNA-sequence similarities or common antigenic determinants. Negative-contrast
electron microscopy showed that PCV and PBFDV particles are 30% smaller
than CAV particles and lack the surface structure of CAV (Todd et al, 1991b).
Furthermore, the fact that the CAV genome expresses only one polycistronic
transcript of CAV (Noteborn et al, 1992a; Phenix et al, 1994) distinguishes it
from PCV, from which at least three RNAs are transcribed (Mankertz & Buhk,
1990). Together, these data justify the conclusion that CAV belongs to a novel
virus family independent from PCV and PBFDV and that the classification of
CAV remains to be determined by taxonomists.

DIAGNOSTIC TEST AND VACCINES BASED ON RECOMBINANT

ANTIGEN
Pallister et al. (1994) developed an ELISA for the detection of serum antibody to
CAV based on recombinant antigen. The VP1 gene (ORF 3) of an Australian
isolate was cloned in a bacterial expression vector. There are only a few differ-
ences between the Australian and the other published VP1 sequences (see above).
The bacterially produced VPl-fusion protein was used successfully to develop an
ELISA for detection of chicken antibodies to CAV.
Koch et al. (in press) studied the immunogenic and protective properties of the
CAV protein(s). The genes encoding VPl, VP2 or VP3 were each inserted into
a baculovirus vector and expressed in insect cells. The baculovirus expression
system is known for its high production levels of specific proteins with immuno-
genic properties (Vlak & Keus, 1990). The recombinant CAV proteins produced
separately in insect cells did not induce neutralizing antibodies when inoculated
into chickens. Only when insect cells were infected simultaneously with at least
VPl- and VP2-recombinant baculoviruses, the produced proteins elicited neutral-
izing CAV-antibodies in the sera of inoculated chickens. Inoculation of a mixture
of lysates of cells that had been infected separately with VP1-, VP2- and VP3-re-
combinant baculovirus did not induce significant levels of neutralizing antibodies
directed against CAV. Furthermore, neutralizing MAb 2A9 (McNulty et al,
18 M. H. M. NOTEBORN & G. KOCH

1990d) does not bind to insect cells synthesizing either VP1 or VP2, but does
when the cells produced both VP1 and VP2 (M. H. M. Noteborn, unpublished
results). The fact that the simultaneous synthesis and not a simple mixture of
recombinant CAV proteins VP1 and VP2 is required for the formation of a
neutralizing epitope suggests that VP2 is a non-structural protein that at some
stage of infection is required for virus assembly. Possibly, VP2 might act as an
scaffold protein that is necessary during the assembly of the virion, but absent
from the final product, as reported, inter alia, for the P/a2 and 39-kDa proteins
of adenovirus (Leibowitz & Horwitz, 1975; D'Halluin et ah, 1978] Persson et ah,
1979). In general, viral proteins are more immunogenic as polymers, than in a
monomeric form (Morein et at, 1990). However, it cannot be entirely excluded
that small amounts of VP2, that remained undetected in electroblots of purified
CAV preparations (Todd et al, 1990) associated with VP1 in virions to form
neutralizing epitopes.
Vielitz et al. (1991) reported that hens exposed to CAV form large amounts of
antibodies, which are passively transferred to the offspring per ovum. These
antibodies protect against chicken infectious-anaemia-associated disease. CAV-
neutralizing antibodies were detected in yolk of eggs produced by hens that had
been inoculated with lysates of cells that had been co-infected with CAV-recom-
binant baculovirus and produced either all three CAV proteins, or mainly VP1
and VP2. Specific clinical signs did not develop in CAV-challenged progeny that
hatched from these eggs.
Vielitz & Landgraf (1988) have developed a vaccine against infectious anaemia,
which is based on CAV propagated in chicken embryos. Currently, this is the only
commercially available vaccine. The offspring of vaccinated breeders was pro-
tected against infectious anaemia. Vaccination is possible because when maternal
immunity vanished, the birds are susceptible to virus infection without developing
the disease. Obviously, the use of live vaccines based on non-attenuated virus
harbours risks. Experimental infection with CAV of 3-week-old chickens resulted
in a comprehensive decrease of the functions of the immune system (McConnell
et al, 1993a,b) in the absence of disease. In line with this finding, Mcllroy et al.
(1992) have provided evidence that CAV also causes considerable economic
losses in the absence of disease: the subclinical disease negatively affected feed
conversion and average body weight and increased medication, both of which
result in a considerable financial burden. So far, non-pathogenic CAV has not
been isolated. Also, the production of an inactivated CAV vaccine has not been
feasible due to the difficulty of obtaining high CAV litres in embryos or cell
cultures. Furthermore, the only chicken cell lines known to be susceptible to CAV
have been immortalized by Marek's disease virus or avian leukosis virus and,
therefore, CAV produced in these cells are contaminated with these viruses
(Billow, 1991).
The recombinant CAV proteins synthesized by means of the baculovirus
expression system can be used as a subunit vaccine. The recombinant CAV
proteins VP1 and VP2 have been proven to protect chicks by maternal immunity.
Since the baculovirus vector is an insect-specific virus, known to be non-patho-
 CHICKEN ANAEMIA VIRUS INFECTION 19

genie for vertebrates, it can be cultured and used in chickens without undue risks
(Vlak and Keus, 1990).
In general, live-virus vaccines induce a better immune response and are less
expensive than sub-unit vaccines. Therefore, knowledge about the immunogenic-
ity of the various CAV proteins can be used to construct other recombinant-virus
vectors, such as avian herpes viruses (Nakamura et al, 1992; Morgan et al, 1993)
or fowlpox virus (Nazerian et al, 1992; Boyle and Heine, 1993). These recombi-
nant viruses should express the CAV proteins VP1 and VP2, in addition to their
own proteins, and may be applicable as vaccines for both layer breeders and
broiler breeders to protect their offspring against infectious anaemia. In addition,
such vector vaccines may be used to protect maternally immune broilers against
subclinical disease.

IMMUNOSUPPRESSION
CAV is prevalent in most countries with a poultry industry of importance
(McNulty, 1991; McNulty et al., 1991). The first observations on the pathogen-
esis of CAV infections were made by Yuasa et al. (1979). In newly hatched
chickens, CAV transiently causes severe anaemia due to destruction of erythrob-
lastoid cells and immunodeficiency due to depletion of cortical thymocytes
(Billow & Witt, 1986; Otaki et al., 1987; Engstrom et al., 1988; Goryo et al,
1989; Jeurissen et al., 1989; Lucio et al., 1990; McNulty et al., 1990b; Adair et al,
1991, 1993; Hu et al, 1993a,b). In experimentally infected 1-day-old chicks, the
depletion of their thymus is at its maximum 2 weeks after infection. When the
thymus sections of these chicks are stained with a MAb specific for CAV-VP3,
positive cells are detected only in the cortex. The number of infected cells is at its
maximum at 6 to 7 days after infection; it takes several days before infected cells
appear and, again, several days before the entire cortex is depleted.
Chickens older than 3 weeks of age are still susceptible to infection but do not
develop disease. Jeurissen et al. (1992a) could not detect any infected cell or
thymus depletion at this age, which may have been caused by the fact that only
a few thymus lobules are affected and not the cortex of all thymus lobules. In
chickens infected at 6 weeks of age, we detected infected cells in the cortex of one
thymus lobule in two out of 10 infected animals (G. Koch et al, unpublished
results). The differences in susceptibility for the disease of very young and
chickens older than 3 weeks is an intriguing one. Jeurissen et al. (1992a)
suggested that it may be caused by lack of susceptibility of thymic precursor cells
of the third wave that populate the thymus after hatching. However, this does not
explain why cells of the erythroid lineage are not susceptible to CAV, either,
unless the susceptibility of the erythroid cells change in a similar way to that of
thymocytes. Another explanation of the destruction of erythroid cells is the lack
of regulating signals from the thymus. However, this seems to be unlikely since
large numbers of CAV-infected cells are also detected in the bone marrow of very
young chicks suggesting that the destruction of erythroid precursor cells is not
caused by secondary effects. Another explanation in the difference of susceptib-
20 M. H. M. NOTEBORN & G. KOCH

ility to disease would be that the immune system matures in the first 2 weeks after
hatching. Indeed, chickens infected at older ages with CAV show seroconversion
already after 4 to 7 days, whereas in chicks infected at day 1, antibody can be
detected only after 2 to 3 weeks (Hoop & Reece, 1991). The earlier immune
response will neutralize CAV and thus will reduce the effective virus dose. It has
been shown in 1-day-old chicks that the severity of the disease is dependent on
the virus dose (McNulty et al., 1990a). Furthermore, embryonally bursectomized
chickens are susceptible to infectious anaemia for a longer period (Hu et al,
1993a). Nevertheless, macrophage and lymphocyte functions are decreased in
chickens infected at 3 weeks of age (McConnell et al, 1993a,b).
The depletion of the cortical thymocytes is considered to cause an im-
munodeficiency resulting in enhanced concurrent infections and to vaccination
failures. Chickens with dual infections of CAV and Marek's disease virus, infec-
tious bursal disease virus, lentogenic Newcastle disease virus, reticulo-endothelio-
sis virus, adenovirus or reovirus may develop aggravated signs associated with the
concurrent infection (Yuasa et al, 1980; Von Bulow et al, 1983; Von Biilow &
Witt, 1986; Engstrom et al, 1988; Rosenberger & Cloud, 1989; De Boer et al,
1992, 1994).
For a more extensive description of the clinical consequences of CAV infections
as well as their economic impact the reader is referred to the review by McNulty
(1991).

CAV CAUSES DESTRUCTION OF THYMOCYTES VIA APOPTOSIS

Jeurissen et al. (1992b) observed a number of phenomena in the thymi of
CAV-inoculated SPF chickens that were absent in thymi of control chickens. Ten
days after infection, the entire cortex contained cells whose chromatin had
condensed in areas adjacent to the nuclear membrane. More or less spherical
electron-dense bodies were seen sporadically in the cytoplasm of epithelial cells.
At day 13 after infection, the cortex was severely depleted of thymocytes, whereas
epithelial cells, many of them containing electron-dense bodies and other non-
lymphoid cells were still present. DNA isolated from thymuses of chickens
infected with various field isolates of CAV displayed the typical laddering of
oligonucleosomal breakdown in an electropherogram. These observed morpho-
logical cellular and biochemical changes were also observed in CAV-infected
cultured avian lymphoblastoid cells (Jeurissen et al, 1992b) and myeloid cells (G.
Koch, unpublished results).
The above phenomena are characteristic of the physiological process of pro-
grammed cell death or apoptosis (Kerr et al, 1972; Wyllie et al, 1980). Apoptosis
is involved in specific developmental stages, i.e. it contributes to the morphogen-
esis of organs. Many different cell species can undergo apoptosis, but immature
T and B cells are particularly susceptible to apoptotic cell death (Hartley et al,
1991). Negative selection of T lymphocytes in the thymus is known to involve
elimination by apoptosis of the self-reactive thymocytes (Raff, 1992). A variety of
stimuli, such as glucocorticoids and triggering of cell-surface receptors induce
 CHICKEN ANAEMIA VIRUS INFECTION . 21

apoptosis in thymocytes (Cohen, 1992). Apoptosis is also a mechanism by which

the immune system can kill infected cells (Golstein etal, 1991). Apoptosis occurs
in a controlled and programmed manner (Arends & Wyllie, 1991) that requires
de novo gene transcription and translation (Bursch et al, 1992). During apoptosis,
cells shrink, degrade their chromatin to oligonucleosomal fragments and as a
consequence condense it. Rapidly, these events are followed by separation of the
nucleus to discrete masses of condensed chromatin, and finally fragmentation of
the cell into several membrane-bound electron-dense vesicles (apoptotic bodies)
do occur (Wyllie et al, 1980; Martin et al, 1994). The remnants of the dead cells
are recognized and rapidly phagocytosed by neighbouring cells in the tissue (Savill
et al., 1993). This efficient elimination of cell debris probably does not elicit any
inflammatory reaction (Wyllie, 1980; Duvall & Wyllie, 1986), in contrast to
necrosis which constitutes a non-physiological process of cell death. Another
important characteristic of apoptosis is that it can be prevented by hormones and
other growth stimuli, whereas necrosis cannot (Bursch et al, 1992).
Apoptosis is usually initiated by a variety of physiological stimuli, although
pathological stimuli, such as viral infections, can also be the triggering factor. On
the other hand, transforming viruses have developed strategies to prevent apopto-
sis of their target cells (Razvi & Welsh, in press). In view of these phenomena it
is not surprising that CAV, too, induces apoptosis in chicken thymocytes and
probably also in erythroblastoid cells (Jeurissen et al, 1992). Recently, Vascon-
celos et al. (1994) reported that immunosuppression induced by infectious bursal
disease virus (IBDV) is caused, at least in part, by the activation of apoptosis in
chicken lymphocytes. Acquired immune-deficiency syndrome (AIDS) is associ-
ated with a persistent infection of human immunodeficiency virus (HIV) that
results in a gradual reduction in the number of CD4 + T cells leading to severe
immune suppression. Several studies have suggested that apoptosis is the under-
lying mechanism of HIV-induced cell death (Ameisen & Capron, 1991; Meyaard
et al., 1992). Parvoviruses are small ss-DNA viruses that grow only in rapidly
dividing cells. Morey et al. (1993) reported that human parvovirus B19 can
induce apoptosis in erythroblasts. Several strains of influenza A virus of avian,
equine, swine and human origin, and human influenza B were shown to induce
DNA fragmentation in human cells in vitro (Hinshaw et al, 1994). Finally, the
infectious prion protein which is associated with the neurodegenerative disorder
spongiform encephalopathy, causes apoptotic morphology and generation of
DNA ladders in cultured neuronal cells (Forloni et al, 1993). Thus far, the
benefit to these viruses of inducing apoptosis is not understood. It might be that
apoptosis is a prerequisite for their replication.
Viruses cannot only induce apoptosis, but they can inhibit this process. Aden-
oviruses encode a number of proteins that either enhance or inhibit apoptosis in
infected cells (Debbas et al, 1993) and baculoviruses which grow in insect cells,
produce an anti-apoptosis protein, p35 (Hershberger et al, 1992). Cells infected
with a p35-mutant baculovirus strain, were found to undergo apoptosis and
because of that this mutant virus multiplies to low titres. This suggests that the
prevention of apoptosis is essential for viral replication. The Epstein-Barr virus
22 M. H. M. NOTEBORN & G. KOCH

(EBV) is known to transform B cells by inhibiting the apoptotic process. The

EBV gene LMP-1 induces expression of the cellular gene bcl-2 in infected cells.
The bcl-2 protein protects the cells from apoptosis (Henderson et al., 1991).
Although many (viral) agents are known to induce or repress apoptosis, the
underlying mechanism by which these components function is poorly understood.
A whole cascade of mostly unknown processes is involved, which result in DNA
fragmentation. This DNA fragmentation is believed to involve activation of an
endogenous endonuclease (Martin et al., 1994). Changes in accessibility of DNA
to endonucleases could be mediated by topoisomerases, which elicit conforma-
tional changes in DNA by making ss cuts (D'Arpa & Liu, 1989). Recent studies
have suggested an intricate relationship between the cell cycle and apoptosis;
p34cdc2 kinase, which stimulates cell-division seem to render cells susceptible to
apoptosis (Shi et al., 1994). The tumour-suppressor gene p53 induces or mediates
apoptosis (Yonish-Rouach et al., 1991). However, there also exist a p53-indepen-
dent pathway of apoptosis, since radiation cannot induce apoptosis in p53-minus
thymocytes, whereas the antigen-induced negative selection of the same thymo-
cytes via apoptosis is unaffected (Clarke et al., 1993; Lowe et al, 1993). Without
knowing precisely the required apoptotic processes, one can conclude that there
exists several distinct pathways in one cell which result in programmed cell death.
The question arises how CAV induces apoptosis. Two views exist. First,
apoptosis may represent only a side-effect of CAV infection, indirectly caused by,
for instance, increased glucocorticoid levels. Glucocorticoid and other non-viral
stimuli are well-known inducers of apoptosis (see above). Secondly, apoptosis
forms an obligatory phase of the virus replication. This view is supported by the
fact that CAV is highly specific for bone marrow and thymus, and can be cultured
only in actively proliferating lymphoid and haematopoietic cell lines. Among
other things, all these cells have in common that they are able to undergo
apoptosis. Thus, it appears that the ability of CAV to replicate in a certain cell
type coincides with the ability of this cell type to undergo apoptosis. On the other
hand, apoptosis may be the cellular defense mechanism to prevent virus spread by
pre-mature host-cell death. The infected cells are removed without inducing
life-threatening processes, i.e. inflammatory reactions.

A SINGLE CAV PROTEIN INDUCES APOPTOSIS

Early after infection of cultured chicken mononuclear cells, the CAV-encoded
protein apoptin (VP3) colocalizes with cellular chromatin. Later after infection,
apoptin forms aggregates, the cells become apoptotic, i.e. the cellular DNA is
fragmented and, as a result, becomes condensed (Figure 2). It was shown by
means of immunogold electron microscopy that apoptin is present in apoptotic
structures. In vitro, expression of apoptin in chicken lymphoblastoid T cells and
myeloid cells induced apoptosis in these cells. These data indicate that apoptin by
itself can trigger the apoptotic pathway in CAV-infected cells (Noteborn et al.,
1994a).
How does apoptin cause apoptosis? Apoptin is a small protein of 121 amino
 CHICKEN ANAEMIA VIRUS INFECTION 23

Figure 2. MDCC-MSB1 cells were infected tvith CA V-Cux-1. Identical cells were stained with
either monoclonal antibody CVI-CAV-85.1 directed against apoptin (VP3;panelA) or propidium
iodide (PI; panel B). PI stains intact DNA strongly whereas apoptotic DNA is stained only weakly
or irregularly. The CA V-infected cell in the upper half of the photographs is a cell containing
fine-granular apoptin in the nucleus with intact DNA, for it is strongly stained by PI. The two
CA V-infected cells at the bottom of each panel are apoptotic. They contain aggregates of apoptin in
the nucleus and their DNA is stained weakly/irregularly by PI.

acids which contains two proline-rich stretches and two positively-charged re-
gions. The amino-acid sequence of apoptin does not show any obvious similarity
to the sequences of the receptors (Watanabe et al, 1992) or cellular products that
mediate or induce apoptosis, such as certain endonucleases or p53 (see above).
So, it is unlikely that apoptin mimics the activity of one of these cellular proteins.
Due to its basic and proline-rich character, apoptin might act as a transcriptional
regulator. Further experiments have to be carried out to examine whether apoptin
increases the synthesis of proteins that induce apoptosis or decreases the pro-
duction of products that inhibit apoptosis. Apoptin is located strictly within the
cellular chromatin structures. Truncation of the C-terminal basic stretch of
apoptin results in a reduced nuclear location and a significantly reduced apoptotic
activity (Noteborn et al., 1994a). The small size and rather basic character of
apoptin may allow it to interact with histone and/or non-histone proteins within
the chromatin structure. The presence of apoptin in the chromatin structure
could possibly result in a breakdown of the supercoiled organization leading to
subsequent DNA fragmentation and condensation, as has been reported for
topoisomerase-induced apoptosis (see abovej Kyprianou et ah, 1991).
Apart from the obvious relevance of apoptin for the cytopathogenic effect of
CAV infections, this CAV protein may also have an interesting curative appli-
cation: apoptin also induces apoptosis in several cell lines derived from human
24 M. H. M. NOTEBORN & G. KOCH

leukemias or lymphomas (Zhuang et al, 1994a). Some of these cell lines contain
bcl-2 (Tsujimoto et al, 1985; Kluin-Nelemans et al, 1991) or BCR-AB1 (McGa-
hon et al, 1994), known to block apoptosis induced by chemotherapeutic com-
pounds. Apoptin induces apoptosis in human osteosarcoma cells independently
of p53 (Zhuang et al., 1994b). Mutation of p53 in tumours has been reported to
correlate with poor response to treatment with ionizing radiation and anti-cancer
drugs (Hollstein et al, 1991). These data indicate that apoptin may become a
candidate for the destruction of such resistant cells.

CONCLUDING REMARKS
Four years ago, McNulty (1991) stated in his review on CAA: "The replication
strategy of such a small virus is obviously of interest, as is the molecular basis of
its pathogenicity. The relative simplicity of CAA and its ability to cause a severe
disease experimentally may make it a useful model system to expand our knowl-
edge of the interaction of animal viruses with their hosts".
The past few years have seen considerable progress in our understanding of the
molecular biology of CAV. CAV was shown to be a unique virus, which does not
share properties with known animal viruses. DNA-sequence analysis revealed that
the CAV genome contains three genes, which are all expressed in CAV-infected
cells. The regulation of transcription has been partially unraveled, whereas the
replication mechanism is yet poorly understood.
The underlying pathogenic mechanism of CAV infection was unraveled. CAV
causes depletion of lymphoblastoid and erythroblastoid cells via induction of
apoptosis. A single CAV protein, apoptin (VP3), can trigger CAV-induced
apoptosis. The fact, that we are able to induce apoptosis by expression of only a
single CAV protein makes it very useful as model for studying the apoptotic
process. Apoptin might have also medical applications for it can induce apoptosis
in human malignant cells in vitro.
Worldwide, the CAV genome is highly uniform, particularly in the coding
regions and the regulatory elements. Therefore, diagnostic assays and vaccines
based on the described cloned CAV DNAs are universally applicable. Several
sensitive and rapid diagnostic methods have been developed. The economic
impact of CAV infections, both clinical and subclinical, warrant the search for
and the use of efficient vaccines. A recombinant subunit vaccine based on the
recombinant CAV proteins VP1 and VP2, has been shown to protect the progeny
from vaccinated mothers.

ACKNOWLEDGEMENTS
We thank Dr H. van Ormondt for critical reading of the manuscript and C. A. J.
Verschueren for excellent art work. This research was made possible with re-
search grants from the Dutch Ministry of Economic Affairs and Aesculaap BV,
Boxtel, The Netherlands.
 CHICKEN ANAEMIA VIRUS INFECTION 25

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RESUME
Infection du poulet par le virus de 1'anémie: base moléculaire de la
pathogenicite
Le virus de l'anémie du poulet (CAV) est un petit virus de type unique d'un diamètre de
particule de 23 à 25 nm possédant un génome d'ADN circulaire à simple brin. Cet ADN se
multiplie dans les cellules infectées par un intermédiate réplicatif circulaire à double brin qui
a ete recemment clone. L'analyse de l'ADN des souches de CAV isolées dans différents
continents n'ont révélé que des differences mineures parmi les divers isolats. Apparemment,
toutes les souches de CAV appartiennent a un seul sérotype. Le CAV ne peut etre rattache a
d'autres virus connus chez l'animal à ADN circulaire à simple brin tel que les sircovirus du
pore et les virus de la maladie du bee et des plumes des psittacides.

Le produit de transcription majeur du génome du CAV est un mARN polyscistronique non

épissé d'environ 2100 nucléotides codant pour 3 proteines de 51,6 kDa (VP2), 24,0 kDa
(VP2) et 13,6 kDa (VP3 ou apoptine). Toutes ces protéines déduites du CAV sont synthétisées
dans les cellules infectées par le virus. L'immunisation avec (recombinant) VP1 et VP2
synthétisée en même temps dans les mêmes cellules, produit une réponse protégeant les
animaux et peut etre utilisée comme vaccin sub-unité contre l'anémie infectieuse du poulet.

Le CAV entraîne une maladie clinique et sub-clinique chez le poulet et est reconnu comme un
agent pathogéne important dans le monde entier. Chez les jeunes poulets, le CAV entraine une
anémie sévère transitoire due à la destruction des cellules érythroblastoïdes dans le moëlle
osseuse et une immunodéficience due à la déplétion des thymocytes de la corticale, respons-
30 M. H. M. NOTEBORN & G. KOCH

ables d'une augmentation des infections concomitantes et des échecs de vaccination. La

déplétion des thymocytes et plus vraisemblablement des cellules érythroblastoïdes intervient
par l'apoptose induite par le CAV. La protéine apoptine codant pour le CAV est la principale
cause de ce phénomène.

ZUSAMMENFASSUNG
Hühneranämievirus-Infektion: Molekulare Grundlagen der Pathogenität
Hühneranämievirus (CAV) ist ein einzigartiges kleines Virus mit einem Partikeldurchmesser
von 23 bis 25 nm und mit einem Genom, das aus einer zirkulären einzelsträngigen (Minus-
strang) DNA besteht. Diese DNA vermehrt sich in infizierten Zellen über eine zirkuläre
replikative Zwischenform, die kürzlich kloniert wurde. Die DNA-Analyse von CAV-Stämmen,
die auf verschiedenen Kontinenten isoliert wurden, ergab nur geringfügige Unterschiede
zwischen den verschiedenen Isolaten. Alle CAV-Isolate gehören anscheinend zu einem einzigen
Serotyp. CAV ist nicht mit anderen bekannten animalen Virusarten mit zirkulärer Einzel-
strang-DNA, wie dem porcinen Circovirus und dem Virus der Schnabel- und Federkrankheit
der Psittaziden, verwandt.

Das Haupttranskript vom CAV-Genom ist eine ungespleißte polycystronische mRNA mit
etwa 2.100 Nukleotiden, die drei Proteine von 51,6 kDa (VP1), 24,0 kDa (VP2) und 13,6 kDa
(VP3 oder Apoptin) kodieren. Alle drei vorausgesagten CAV-Proteine werden in CAV-
infizierten Zellen synthetisiert. Die Immunisierung mit (rekombinantem) VP1 und synchron
in denselben Zellen synthetisiertem VP2 fuhrt zu einem Impfschutz. Die beiden Proteine
können deshalb als Subunit-Vakzine gegen die infektiöse Anämie der Hühner verwendet
werden.

CAV verursacht bei Hühnerküken klinische und subklinische Erkrankungen und wird weltweit
als wichtiger Krankheitserreger beim Geflügel anerkannt. Bei jungen Küken verursacht CAV
eine vorübergehende schwere Anämie infolge der Zerstörung von erythroblastoiden Zellen im
Knochenmark und eine Immunschwäche infolge eines Zellschwunds in der Thymusrinde. Der
Zellschwund in der Thymusrinde wird als Ursache für eine (vorübergehende) Immunschwäche
angesehen, die zur Verschlimmerung gleichzeitiger anderer Infektionen und zum Versagen von
Schutzimpfungen führt. Die Verminderung der Thymozyten und höchstwahrscheinlich auch
die der erythroblastoiden Zellen erfolgt über CAV-induzierte Apoptose. Das CAV-kodierte
Protein Apoptin ist der Hauptverursacher dieses Phänomens.

RESUMEN
Infección por el virus de la anemia del pollo: bases moleculares de su
patogenicidad
El virus de la anemia del pollo (CAV) es un virus pequeño con un diámetro del virión de 23
a 25 nm y un genoma a base de una hebra (—) de ADN circular. El ADN se multiplica en
las células infectadas mediante un ADN de doble hebra intermediario replicativo que se ha
clonado recientemente. Al Análisis del ADN de cepas de CAV aisladas en diferentes continen-
tes reveló que sólo existían diferencias menores entre los distintos aislamientos. Aparente-
mente, todos los aislamientos de CAV pertenecen al mismo serotipo. CAV no está relacionado
con otros virus animales con ADN circular de una sóla hebra como el circovirus porcino y el
virus de la enfermedad de las plumas y el pico de las psitácidas.
 CHICKEN ANAEMIA VIRUS INFECTION 31

El transcripto principal del genoma del CAV es un ARNm policistrónico no montado de

alrededor 2.100 nucleótidos codificando para tres proteínas de 51.6 kDa (VP1), 24.0 kDa
(VP2) y 13.6 kDa (VP3 o apoptina). Estas tres proteínas son sintetizadas en células infectadas
por el CAV. La inmunización con VP1 (recombinante) y VP2 sintetizados sincrónicamente en
las mismas células desencadena una respuesta protectora y puede ser empleada como una
vacuna de subunidades para la anemia infecciosa del pollo.

Este virus produce cuadros clínicos y subclínicos en pollos y es reconocido como un

importante agente patógeno aviar en todo el mundo. En pollos jóvenes, CAV produce una
anemia severa transitoria debido a la destrucción de células eritroblastoides en la médula ósea
e inmunodeficiencia debido a depleción de timocitos corticales. Se considera la depleción de
timocitos corticales como la causa de la inmunodeficiencia transitoria que produce un
incremnento de infecciones concurrentes así como fallos vacunales. La depleción de timocitos
y, probablemente, también de células eritroblastoides ocurre vía apoptosis inducida por CAV.
La proteína apoptina, codificada por CAV, es la principal inductora de este fenónemo.