Avian bornavirus (ABV) is a newly discovered mem-
ber of the family Bornaviridae that has been associated with
the development of a lethal neurologic syndrome in birds,
termed proventricular dilatation disease (PDD). We suc-
cessfully isolated and characterized ABV from the brains
of 8 birds with confi rmed PDD. One isolate was passed 6
times in duck embryo fi broblasts, and the infected cells were
then injected intramuscularly into 2 healthy Patagonian
conures (Cyanoliseus patagonis). Clinical PDD developed
in both birds by 66 days postinfection. PDD was confi rmed
by necropsy and histopathologic examination. Reverse
transcription–PCR showed that the inoculated ABV was in
the brains of the 2 infected birds. A control bird that received
uninfected tissue culture cells remained healthy until it was
euthanized at 77 days. Necropsy and histopathologic ex-
aminations showed no abnormalities; PCR did not indicate
ABV in its brain tissues.
ed for >50 species of psittacine birds as well as many other
bird species (1). It is considered a serious disease because
many of these birds are highly endangered, and several af-
fected species depend on captive breeding for their survival.
The clinical signs of PDD vary and may be predominately
neurologic (weakness, ataxia, proprioceptive defi cits, sei-
zures, blindness), gastrointestinal (weight loss, passage of
undigested food, regurgitation, delayed crop emptying), or
roventricular dilatation disease (PDD) is a progressive,
invariably fatal neurologic disease that has been report-
a combination thereof (2). The gastrointestinal signs, espe-
cially proventricular dilatation, are secondary to pseudo-
obstruction brought about by damage to the enteric nervous
system. PDD is characterized by severe lymphoplasmacyt-
ic infl ammation in peripheral, central, and autonomic ner-
vous tissues (3–5). Defi nitive diagnosis of PDD requires
demonstration of lymphoplasmacytic ganglioneuritis in the
Recently, 2 independent groups of investigators identi-
fi ed a new member of the family Bornaviridae, named avian
bornavirus (ABV), in parrots with histopathologically con-
fi rmed PDD. Honkavuori et al. used unbiased high-through-
put sequencing to identify the virus in several parrots with
histopathologically confi rmed PDD (6). Quantitative PCR
confi rmed the presence of the virus in brain, proventriculus,
and adrenal gland in 3 birds with PDD but not in 4 unaffect-
ed birds. Kistler (7) used a panviral microarray to identify a
bornavirus hybridization signature in 5 of 8 birds with PDD
and 0 of 8 controls. These investigators used ultra high-
throughput sequencing combined with conventional PCR-
based cloning to recover a complete viral genome sequence.
Before this discovery, the family Bornaviridae contained
only 1 species, Borna disease virus (BDV). BDV causes
a neurologic syndrome, Borna disease, which is restricted
to central Europe, where it is found primarily in horses and
sheep. The virus infects neurons and astrocytes, and the re-
sulting disease appears to be mediated by an immunopatho-
logic response of the host to the virus.
BDV can be grown in mammalian cell culture, where
it causes a noncytolytic persistent infection. Borna disease
appears as a sporadic infection affecting small numbers of
animals each year. Its epidemiology is unclear, but it may
be carried by certain species of shrews (8). BDV has also
been detected in the feces of wild birds and in captive os-
triches, but the epidemiologic signifi cance of this observa-
Use of Avian Bornavirus Isolates
to Induc e Proventric ular Dilatation
Disease in Conures
Patricia Gray, Sharman Hoppes, Paulette Suchodolski, Negin Mirhosseini, Susan Payne,
Itamar Villanueva, H.L Shivaprasad, Kirsi S. Honkavuori, Thomas Briese, Sanjay M. Reddy,
and Ian Tizard
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 3, March 2010 473
Author affi liations: Texas A&M University, College Station, Texas,
USA (P. Gray, S. Hoppes, P. Suchodolski, N. Mirhosseini, S. Payne,
I. Villanueva, S.M. Reddy, I. Tizard); California Animal Health and
Food Safety Laboratory System, Fresno, California, USA (H.L.
Shivaprasad); and Columbia University, New York, New York, USA
(K.S. Honkavuori, T. Briese)
tion is unclear (9,10). Studies undertaken in this laboratory
have demonstrated some histopathologic similarities, in
particular in the selective destruction of cerebellar Purkinje
cells, between ABV and BDV infections of the brains of
birds and mammals, respectively (11).
Seven ABV genotypes have been identifi ed based on
partial genome sequencing (12,13). In general, these ABV
strains show only ≈65% sequence identity with BDV. Nev-
ertheless, the overall structure of the bornaviral genome is
well conserved (6,7). Thus, the number and order of genes
is unchanged, as is the structure of transcription initiation
and termination sites. Recently, Rinder et al. (14) have
shown that the region between the N and X gene in ABV
is shorter than that in BDV. ABV apparently lacks a 22-
nt fragment that serves a regulatory function for the genes
coding for viral proteins X and P.
Although these discoveries suggest that ABV is a
plausible cause of PDD, as described in Koch’s postulates,
proof of a causal relationship requires isolation of the agent
from infected birds; its propagation in culture; and, after
reintroduction of the isolate into a susceptible host, mani-
festation of the disease (15). We describe the isolation and
culture of ABV from the brains of 8 psittacine birds with
histopathologically confi rmed PDD. After 6 passages, 1
of the cultured isolates was intramuscularly injected into
2 healthy Patagonian conures (Cyanoliseus patagonis).
Typical PDD subsequently developed in each bird, and the
inoculated virus was found in the brain.
Materials and Methods
From independent sources we obtained 8 parrots that
had clinical signs of PDD, were clinically judged to be in
the late stages of the disease, and were euthanized for hu-
mane reasons. The 8 birds were 1 green-winged macaw
(Ara chloroptera), 1 scarlet macaw (A. macao), 2 blue and
yellow macaws (A. ararauna), 2 yellow-collared macaws
(Primolius auricollis), 1 African gray parrot (Psittacus er-
ithracus), and 1 umbrella cockatoo (Cacatua alba). Four
parrots with conditions not related to PDD and euthanized
for humane reasons were also included in the study as neg-
Immediately after euthanasia, complete necropsies
were performed on all birds. Tissue samples from brain,
spinal cord, peripheral nerves, lungs, heart, liver, spleen,
pancreas, adrenal glands, kidneys, crop, proventriculus,
ventriculus, intestine, and cloaca were placed in 10% buff-
ered formalin for histopathologic examination. Tissue sec-
tions were stained with hematoxylin and eosin to confi rm
the clinical diagnosis. Half of each brain was retained for
virus isolation, Western blot, and reverse transcription–
Specifi c pathogen–free duck eggs were obtained from
the US Department of Agriculture Avian Disease Labora-
tory (East Lansing, MI, USA). Embryos 9–10 days old were
harvested, macerated, and cultured. Primary duck embryonic
fi broblasts (DEFs) were used for virus isolation and propa-
gation. DEFs were maintained in Leibowitz L15–McCoy
5A medium (LM; Sigma-Aldrich, St. Louis, MO, USA)
supplemented with 5% bovine calf serum (Sigma-Aldrich)
and 1% penicillin-streptomycin at 37°C in an atmosphere of
5% CO2. DEFs were seeded to confl uency 24 h before in-
oculation. DEFs not inoculated with tissue homogenate were
maintained in parallel throughout the experiment.
Virus Isolation and Culture
Brain tissue was harvested immediately after eutha-
nasia. Sections of the cerebrum and cerebellum were ho-
mogenized, minced, and then passed through an 18-gauge
needle in LM complete medium. In instances where im-
mediate culture inoculation was not possible, the brain tis-
sue was frozen at –80°C within minutes of being harvested
and was thawed in a 37°C water bath immediately before
inoculation. One milliliter of the brain suspension was used
to inject previously plated DEF monolayers that were then
incubated for 24 h. The injected DEF cultures were then
washed once with phosphate-buffered saline (PBS), re-
placed with fresh LM medium supplemented with 2% fetal
calf serum, and incubated for 5–7 days. Infected DEFs were
trypsinized and cocultivated with freshly plated DEFs. This
procedure was repeated for a minimum of 3 passages.
Western Blot Analysis
Infected DEFs passaged a minimum of 3 times were
used for Western blot analyses. Samples from infected
DEFs were collected by trypsinization and pelleted by cen-
trifugation, and pellets resuspended in PBS were sonicated
on ice at 50% intensity for 5 min (Sonifi er 250; Branson
Ultrasonics Corp, Danbury, CT, USA); 50% intensity).
Western blotting was performed as described by Towbin
et al. (16) by using 10% polyacrylamide gels and a Mini-
Protean II gel electrophoresis apparatus (Bio-Rad, Hercu-
les, CA, USA). The tissue culture preparations were diluted
in sample loading buffer containing β-mercaptoethanol at
a ratio of 1:1 and heated to 95°C for 5 min before being
loaded (30 μg/slot). A prestained sodium dodecyl sulfate–
polyacrylamide gel electrophoresis standard covering the
6.5- to 200-kDa range was used for molecular weight esti-
The size-fractionated antigen preparations were trans-
ferred to Immobilon polyvinylidene fl uoride transfer mem-
474 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 3, March 2010
Propagation of Avian Bornavirus
branes (Millipore, Bedford, MA, USA) as described by
Towbin et al. (16). Transfer effi ciency was indicated by
the presence of prestained bands on the membranes. After
transfer, the membranes were incubated for 2 h in PBS,
0.05% Tween-20, 3% skimmed milk (PBST blocking buf-
fer), then with histopathologically confi rmed PDD-positive
parrot serum diluted 1:5,000 in PBST blocking buffer for
2 h and with horseradish peroxidase–conjugated goat anti-
macaw immunoglobulin Y (Bethyl Inc., Montgomery, TX,
USA) diluted 1:10,000 in PBST blocking buffer for 1 h.
Membranes were washed with PBST after each step, and
all steps were performed at room temperature under con-
stant shaking. Finally, the membranes were incubated for
30 min in Sigma-Fast 3,3′-diaminobenzidine developing
substrate (Sigma-Aldrich) and then rinsed in distilled wa-
ter. The serum from a confi rmed PDD-positive parrot used
in this experiment has been shown to contain antibodies
specifi c for the 38-kDa ABV N-protein by its reaction with
2 preparations of recombinant protein prepared in Escheri-
chia coli and in mammalian cell systems (11).
Indirect Immunofl uorescent Assay
Infected DEF were washed 2 times for 5 min each in
0.02M PBS, fi xed for 10 min in 2% paraformaldehyde in
0.02 M PBS, and washed 2 times for 5 min each in 0.02 M
PBS. Cells were permeabilized in 1% Triton X-100/0.02
M PBS for 10 min and washed 3 times for 5 min each in
0.3% Tween/0.02 M PBS. Blocking was performed for 2 h
in 5% dried milk/0.3% Tween/0.02 M PBS. The cells were
incubated in a humidifi ed chamber for 30 min at 37°C with
the primary antibody (serum from a parrot with histopatho-
logically confi rmed PDD) at a 1:500 dilution in 1% dried
milk/0.3% Tween/0.02 M PBS. Cells were washed 3 times
for 5 min each in 0.03% Tween/0.02 M PBS. The cultures
were then incubated in a humidifi ed chamber for 30 min at
37°C with the secondary antibody (horseradish peroxidase–
or fl uorescein isothiocyanate–conjugated goat anti-macaw
immunoglobulin G; Bethyl Inc.) at a 1:500 dilution in 1%
dried milk/0.3% Tween/0.02M PBS. Cells were washed 3
times for 5 min each in 0.03% Tween/0.02M PBS and then
rinsed in distilled water and mounted with ProLong anti-
fade reagent with DAPI (Invitrogen, Carlsbad, CA, USA).
Total RNA was isolated from collected brain tissue
and passaged DEF by using the RNeasy Mini Kit (QIA-
GEN, Valencia, CA, USA). First-strand cDNA was gener-
ated by using the High Capacity cDNA Reverse Transcrip-
tion Kit (Applied Biosystems, Foster City, CA, USA), with
1μg RNA and random primers. PCR for ABV N-protein
was performed by using 1–2 μL cDNA and forward (5F:
5′-GCGGTAACAACCAACCAGCAA3-′) and reverse
primers, which were developed using GenBank submissions
NC_001607.1, FJ169441.1, and FJ169440.1 for reference.
Amplifi cation conditions were as follows: 1 cycle at 94°C
for 2 min; 35 cycles at 94°C for 30 sec, 50°C for 30 sec,
and 72°C for 80 sec; fi nal extension at 72°C for 7 min. PCR
products were cloned in TOPO-TA vector (Invitrogen), and
individual clones were sequenced after transformation into
E. coli. DNA sequencing reactions were performed by us-
ing the ABI BigDye Terminator Cycle Sequencing Kit, and
sequences were generated with an ABI PRISM 3100 Ge-
netic Analyzer (Applied Biosystems). Sequences were as-
sembled and aligned by using Geneious Pro 4.6.2 software
(www.geneious.com). Isolates were assigned to previously
defi ned ABV groups by comparing a 397-nt region to se-
quences representing avian bornaviruses 1–5 (GenBank
accession nos. FJ002329, FJ603688, FJ002328, FJ603687,
FJ002335). Evolutionary distances were computed by us-
ing a Kimura 2-parameter model with MEGA4 software
Experimental infections were performed under Animal
Use Protocol no. 2009–033B approved by the Texas A&M
University Institutional Animal Care and Use Committee.
Three adult Patagonian conures were shown to be serone-
gative by Western blotting and to be ABV negative by fe-
cal PCR. All 3 birds were known to be chronic carriers of
psittacine herpes virus; 1 had a cloacal papilloma, but all
were otherwise in good health. Psittacine herpes virus has
never been implicated in PDD. Two birds were placed in
isolation and inoculated by intramuscular injection with in-
fected DEFs containing 8 × 104 focus-forming units (17) of
an ABV4 (M24) originally isolated from a yellow-collared
macaw. A large batch of the M24 strain was grown for 5
days on passage 6, and 500-μL aliquots of this batch were
frozen at –80°C in freezing medium. Two of the 500-μL
aliquots were grown for 5 days, and immunohistostaining
(by using the immunofl uorescent antibody [IFA] assay de-
scribed, substituting the fl uorescein isothiocyanate–labeled
antibodies with horseradish peroxidase–labeled antibodies)
was used to visualize and quantify the focus-forming units.
Isolation and Culture of Avian Bornavirus Isolates
Cytopathic effects were not observed in any of the 12
DEF cultures inoculated with brain tissue harvested from
birds with or without clinical signs of PDD. Western blot
analyses showed a pronounced ABV N-protein band in ex-
tracts of 8 of the 12 cultures. Only DEF cultures inoculated
with samples from parrots displaying histopathologically
confi rmed PDD were positive by Western blotting (Figure 1).
ABV N-protein was not detectable in the 4 cultures injected
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 3, March 2010 475
with brain tissue from birds with no histologic evidence of
PDD. IFA of infected DEFs also demonstrated ABV N-pro-
tein within cells. Foci of antigen-positive cells were appar-
ent 3 days after culture inoculation. Many cells showed both
nuclear and diffuse cytoplasmic staining. Other cells showed
the characteristic punctate nuclear staining of infected cul-
tures (Figure 2). No positive immunofl uorescence was ob-
served in uninfected DEFs or in DEFs inoculated with brain
tissue from negative control birds.
Characterization of Avian Bornavirus Isolates
RNA was isolated from the brain tissues and infected
DEF cultures of 8 parrots with PDD and 4 parrots that were
PDD negative. A 397-bp region of the ABV N-gene was
amplifi ed from all 8 PDD brain and tissue culture samples
but not those from the 4 negative parrots. The amplicons
were cloned and their sequences compared with previously
described ABV groups (7). One isolate, M25, was most
closely related to ABV group 1, whereas the other 7 ABV
isolates were most closely related to ABV group 4 (Table).
Pairwise comparisons among the group 4 isolates ranged
from 94.2% to 99.7% nucleotide identity. When 2–3 com-
plete N-protein gene sequences (1,143 nt) originating from
any bird were compared, nucleotide sequence identity
ranged from 99.2% to 100% (data not shown).
Two Patagonian conures were challenged with ABV4,
strain M24. They were tested by fecal PCR before challenge
and at 33, 43, 60, and 62 days postchallenge. Both conures
were seronegative by Western blotting before challenge but
seropositive for antibodies to the 38-kDa N-protein on day
33 and thereafter. Fecal PCR testing showed that both birds
were negative on days 33 and 43. One bird was weakly
positive on day 60, but both were strongly positive on day
62. One inoculated bird died on day 66. Necropsy showed
a dilated proventriculus and gross lesions characteristic of
PDD. Subsequent histopathologic examination confi rmed
that the bird had a lymphoplasmacytic ganglioneuritis typi-
cal of PDD in the crop, proventriculus, gizzard, and intes-
tine (Figure 3). This gangloneuritis included mild to severe
infi ltration of lymphocytes and a few plasma cells in the
serosa, subserosal nerves, and ganglia. The bird also had
adrenalitis, encephalitis, and neuritis, as well as a myo-
carditis. The heart showed a lymphocytic infi ltration of the
476 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 3, March 2010
1 2 3 4 5 6 7 8 9
Figure 1. Western blot of infected duck embryonic fi broblasts
(DEFs) showing avian bornavirus N-protein during culture. Lanes
1–4 are supernatant fl uids. Lane I is from an African gray parrot
(AG5). Lanes 2 and 3 are from a yellow-collared macaw (M24).
Lane 4 is from uninfected DEFs. Lanes 5–8 are sonicated cell
extracts. Lane 5 from AG5; 6 and 7 from M24; and Lane 8 from
uninfected DEFs. Lane 9 is an infected brain control. The virus is
strongly cell associated.
Figure 2. A) Avian bornavirus (ABV)–infected duck embryonic
fi broblast (DEF) cell culture 6 days after injection with
hindbrain tissues from an African gray parrot with confi rmed
proventricular dilatation disease (AG5) and staining by an
indirect immunofl uorescence assay for ABV N-protein. Speckled
immunofl uorescence is typical of bornavirus infection. Original
magnifi cation ×40. B) DEFs 3 days after injection with forebrain
from a yellow-collared macaw with confi rmed proventricular
dilatation disease (M24). Nuclear and cytoplasmic fl uorescence in
DEFs stained by immunofl uorescence assay for ABV N-protein.
Original magnifi cation ×40.
Propagation of Avian Bornavirus
epicardial ganglia as well as in and around Purkinje fi bers.
Thus, the brain and spinal cord showed multifocal perivas-
cular cuffi ng and gliosis (Figure 4). The adrenal medulla
was infi ltrated with lymphocytes and plasma cells.
The second inoculated bird was examined on day 66
and was found to be emaciated and had clinical signs con-
sistent with PDD. It was euthanized for humane reasons.
This bird also had gross and histopathologic lesions char-
acteristic of PDD, essentially identical to those described
above. The brains of both conures were subjected to PCR
for ABV N-protein as described above. Results for both
were positive (Figure 5). Sequence analysis of the PCR
products confi rmed that bird brains contained ABV4 iden-
tical to the M24 challenge strain. Brain homogenates from
these 2 birds were also cultured on DEFs, and a strong posi-
tive PCR signal was obtained at day 16 of culture.
The third conure in this study received uninfected
DEFs by both intramuscular and oral routes as described
for experimentally infected birds. This bird was housed in
an aviary separate from the isolation facilities of the infect-
ed birds. It was in apparent good health when euthanized
on day 77. Necropsy of the bird conducted, including his-
topathologic examination of its tissues, and PCR was per-
formed on 4 regions of its brain. No evidence of PDD was
seen during necropsy or histopathologic examination, and
all 4 brain samples were negative for ABV nucleic acid.
Although it has long been proposed that a viral patho-
gen was responsible for PDD, past attempts to identify a
causal agent through inoculation of chick embryos and a
variety of tissue cultures were unsuccessful. Because no
cytopathic effects were detected in the DEF cultures af-
ter several passages, prior attempts to grow the agent may
have been successful but had not been recognized because
of lack of immunologic or PCR detection tools. We were
able to isolate and propagate ABV from all studied birds
with clinical PDD. IFA of infected DEF using this same an-
tiserum showed the punctate nuclear staining that is typical
of cells infected with bornaviruses and appears to be the re-
sult of the formation of N-and P-protein complexes known
as Joest-Degen inclusion bodies (18–20). It is noteworthy
that we were unable to grow ABV in primary chicken em-
bryo fi broblasts handled the same as the DEFs. Rinder et
al. reported successful propagation of ABV in the chicken
LMH hepatoma cell line (14). However, they noted slow
growth and only a few positive cells compared with propa-
gation in the quail fi broblast cell line CEC32 and the quail
skeletal muscle cell line QM7. Thus ABV appears to have
constraints in host cell range. PDD in chickens has not been
reported. Rinder et al., like ourselves (P. Gray et al., unpub.
data), were unable to grow ABV in cell lines of mammalian
origin, such as Vero cells or MDCK cells in which BDV
grows routinely. This research fi nding suggests that ABV
may be unable to infect mammals.
Bornavirus has a nonsegmented negative strand ge-
nome. It encodes at least 6 proteins: N, X, P, M, G ,and L.
The N or nucleoprotein interacts with the viral RNA and
accumulates in the nucleus during the life cycle of the vi-
rus (21,22). The nucleoprotein of BDV exists in 2 isoforms
of 40 and 38 kDa (23,24). P40 is primarily nuclear, and
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 3, March 2010 477
Table. Percent nucleotide identity between partial N genes in avian bornavirus isolates
Gene ABV Type 1a* M25
ABV Type 1a 100.0
M20 80.9 82.0
AG5 80.9 82.0
M10 80.9 82.0
06 81.4 81.8
ABV Type 4b 80.681.4
*ABV genotype 1 accession no. FJ002329.
†ABV genotype 4 accession no. FJ603687.
M20AG5 M15 M10M14 M24 06ABV Type 4b†
Figure 3. Proventriculus wall from conure PG8 showing
characteristic lymphoplasmacytic infi ltration of the subserosal
enteric ganglia as well as infi ltration of submucosa. This bird had
been inoculated 55 days earlier with avian bornavirus, genotype 4.
Original magnifi cation ×325.
P38 is primarily cytoplasmic. Both isoforms can bind to
the viral phosphoprotein. The immunofl uorescent staining
pattern observed with ABV-infected DEFs, which showed
a punctate nuclear staining combined with a more diffuse
cytoplasmic staining, is thus compatible with the known
properties of the BDV nucleoprotein. Of 8 isolates reported
here, 7 were of genotype 4 and 1 was of genotype 1. This
fi nding may suggest that genotype 4 is a more pathogenic
type associated with disease, or it may simply be the pre-
dominant strain circulating in Texas.
Rinder et al. (14) reported on 6 isolates from Germa-
ny, 4 of which were genotype 4 and the others were geno-
type 2. This fi nding supports the suggestion that geno-
type 4 may be predominant worldwide and possibly more
virulent than other genotypes. The experimental infection
of 2 Patagonian conures with cultured virus that result-
ed in clinical PDD 66 days postinfection fulfi lls Koch’s
postulates. PCR and sequencing of the amplifi ed product
demonstrated large amounts of ABV4 in the brains of the
challenged birds. The birds did seroconvert for anti-N
antibodies at 33 days, whereas fecal shedding was not
detected until days 60–62. This fi nding is in contrast to
observations on naturally infected birds in which fecal
shedding may precede seroconversion by many months
(25). ABV RNA was detected by RT-PCR after a mini-
mum of 3 passages in DEF primary cell culture subse-
quent to inoculation with brain tissue from all 8 necropsy-
confi rmed PDD-positive birds. PCR detection in the brain
tissue and ready isolation of the virus from freshly har-
vested brain tissue are compatible with the concept that
PDD originates as a viral encephalitis (11).
Gancz et al. (26) have induced PDD in cockatiels
(Nymphicus hollandicus) after inoculation of brain homo-
genates from PDD-affected, ABV-positive birds. Although
the fi ndings of Gancz et al. support our results and are in
line with previous fi ndings (27), interpretation of their re-
sults is diffi cult because of evidence for an autoimmune
component in PDD similar to that which occurs in Guillain
Barré syndrome (28). We also have detected autoantibodies
to myelin basic protein and other nervous system autoanti-
gens in PDD cases, suggesting that in this study, the brain
homogenate may have contributed to the abnormalities ob-
served. (25). The known pathogenesis of mammalian bor-
navirus infections fi ts well with the causative role of ABV
in PDD. Both PDD and mammalian Borna disease share
many attributes, including a viral encephalitis and polyneu-
ritis with selective destruction of Purkinje cells, lympho-
cyte infi ltration, and dysfunction of the central, peripheral,
and autonomic nervous systems (29–31).
In conclusion, the results reported here together with
previous fi ndings confi rm unequivocally that the long-
sought cause of proventricular dilatation disease is indeed
478 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 3, March 2010
Figure 4. Lymphoplasmacytic encephalitis with multifocal peri-
vascular cuffi ng in the cerebrum of conure PG8 inoculated 55 days
earlier with avian bornavirus genotype 4. Original magnifi cation
Figure 5. PCR of avian bornavirus N-protein in different areas of
the brains of A) 2 Patagonian conures (PG7 and PG8) inoculated
55 days earlier with avian bornavirus–infected duck embryonic
fi broblasts and B) control, uninfected bird, PG5. HB, hindbrain; FB,
forebrain; MB, midbrain; Cerebr., cerebrum.
Propagation of Avian Bornavirus Download full-text
avian bornavirus. Investigations into this virus and the com-
plex disease that it causes may provide useful insights into
the pathogenesis of mammalian Borna disease. The origin
and epidemiology, as well and prevention and treatment, of
this infection remain to be elucidated.
This research was supported by the Richard M. Schubot En-
dowment at Texas A&M University.
Dr Gray is a resident in avian studies and works at the
Schubot Exotic Bird Health Center, where she has been conduct-
ing research on proventricular dilatation disease.
1. Gregory CR, Latimer KS, Niagro FD, Ritchie BW, Campagnoli RP,
Norton TM, et al. A review of proventricular dilatation syndrome. J
Assoc Avian Vet. 1994;8:69–75.
2. Steinmetz A, Pees M, Schmidt V, Weber M, Krautwald-Junghanns
M-E, Oechtering G. Blindness as a sign of proventricular dilata-
tion disease in a grey parrot (Psittacus erithracus erithracus). J
Small Anim Pract. 2008;49:660–2. DOI: 10.1111/j.1748-5827
3. Shivaprasad HL, Barr BC, Woods LW, Daft BM, Moore JD, Kinde
H, et al. Spectrum of lesions (pathology) of proventricular dilation
syndrome. Proceedings of the Association of Avian Veterinarians;
1995 Aug 28–Sep 2; Philadelphia, Pennsylvania, USA. p. 507–8.
4. Schmidt RE, Reavill DR, Phalen DN. Pathology of pet and aviary
birds. Ames (IA): Iowa State Press, 2003. p. 47–55.
5. Berhane Y, Smith DA, Newman S, Taylor M, Nagy E, Binning-
ton B, et al. Peripheral neuritis in psittacine birds with proven-
tricular dilatation disease. Avian Pathol. 2001;30:563–70. DOI:
6. Honkavuori KS, Shivaprasad HL, Williams BL, Quan P-L, Hornig
M, Street C, et al. Novel bornavirus in psittacine birds with proven-
tricular dilatation disease. Emerg Infect Dis. 2008;14:1883–6. DOI:
7. Kistler AL, Gancz A, Clubb S, Skewes-Cox P, Fischer K, Sorber
K, et al. Recovery of divergent avian bornaviruses from cases of
proventricular dilatation disease: identifi cation of a candidate etio-
logic agent. Virol J. 2008;5:88. DOI: 10.1186/1743-422X-5-88
8. Hilbe M, Herrsche R, Kolodziejek J, Nowotny N, Zlinszky K,
Ehrensperger F. Shrews as reservoir hosts of Borna disease virus.
Emerg Infect Dis. 2006;12:675–7.
9. Berg M, Johansson M, Montell H, Berg A-L. Wild birds as a pos-
sible natural reservoir of Borna disease virus. Epidemiol Infect.
2001;127:173–8. DOI: 10.1017/S0950268801005702
10. Malkinson M, Weismann Y, Ashash E, Bode L, Ludwig H. Borna
disease in ostriches. Vet Rec. 1993;133:304.
11. Ouyang N, Storts R, Tian Y, Wigle W, Villanueva I, Mirhosseini N,
et al. Detection of avian bornavirus in the nervous system of birds
diagnosed with proventricular dilatation disease. Avian Pathol.
2009;38:393–401. DOI: 10.1080/03079450903191036
12. Weissenböck H, Bakonyi T, Sekulin K, Ehrensperger F, Doneley
RJT, Dürrwald R, et al. Avian bornaviruses in psittacine birds from
Europe and Australia with proventricular dilatation disease. Emerg
Infect Dis. 2009;15:1453–9. DOI: 10.3201/eid1509.090353
13. Weissenböck H, Sekulin K, Bakonyi T, Högler S, Nowotny N.
Novel avian bornavirus in a nonpsittacine species (canary; Seri-
nus canaria) with enteric ganglioneuritis and encephalitis. J Virol.
2009;83:11367–71. DOI: 10.1128/JVI.01343-09
14. Rinder M, Ackermann A, Kempf H, Kaspers B, Korbel R, Staeheli
P. Broad tissue tropism of avian bornavirus in parrots with proven-
tricular dilatation disease. J Virol. 2009;83:5401–7. DOI: 10.1128/
15. Koch R. Ueber bakteriologische Forschung, Verhandl. des X. In-
terntl. Med. Congr., Berlin 1890, August Hirschwald, Berlin; 1891.
16. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of pro-
teins from polyacrylamide gels to nitrocellulose sheets: procedure
and some applications. Proc Natl Acad Sci U S A. 1979;76:4350–4.
17. Pauli G, Grunmach J, Ludwig H. Focus-immunoassay for Bor-
na disease virus–specifi c antigens. Zentralbl Veterinarmed B.
18. Joest E, Degen K. Über eigentümliche Kerneinschlüsse der Gan-
glienzellen bei der enzootischen Gehirn-Rückenmarksentzündung
der Pferde. Z Infkrankh Haustiere. 1909;6:348–56.
19. Schwemmle M, Salvatore M, Shi L, Richt J, Lee CH, Lipkin WI.
Interactions of the borna disease virus P, N, and X proteins and their
functional implications. J Biol Chem. 1998;273:9007–12. DOI:
20. Sasaki S, Ludwig H. In borna disease virus infected rabbit neurons
100 nm particle structures accumulate at areas of Joest-Degen inclu-
sion bodies. Zentralbl Veterinarmed B. 1993;40:291–7.
21. Briese T, de la Torre JC, Lewis A, Ludwig H, Lipkin WI. Borna dis-
ease virus, a negative-strand RNA virus, transcribes in the nucleus
of infected cells. Proc Natl Acad Sci U S A. 1992;89:11486–9. DOI:
22. Schwemmle M, Lipkin WI. Models and mechanisms of Bornavirus
pathogenesis. Drug Discov Today. 2004;1:211–6. DOI: 10.1016/j.
23. Pyper JM, Gartner AE. Molecular basis for the different subcellular
localization of the 38-and 39-kilodalton structural proteins of Borna
disease virus. J Virol. 1997;71:5133–9.
24. Kobayashi T, Shoya Y, Koda T, Takashima I, Lai PK, Ikuta K, et al.
Nuclear targeting activity associated with the amino terminal region
of the Borna disease virus nucleoprotein. Virology. 1998;243:188–
97. DOI: 10.1006/viro.1998.9049
25. Tizard I, Villanueva I, Gray P, Hoppes S, Mirhosseini N, Payne S.
Update on avian bornavirus and proventricular dilatation disease.
In: Cross G, editor. Proceedings of the Association of Avian Vet-
erinarians, Australasian Chapter; 2009 Sep 14–17; Adelaide, South
Australia, Australia. p. 93–100.
26. Gancz AY, Kistler AL, Greninger AL, Farnoushi Y, Mechani S, Perl
S, et al. Experimental induction of proventricular dilatation disease
in cockatiels (Nymphicus hollandicus) inoculated with brain homo-
genates containing avian bornavirus 4. Virol J. 2009;6:100. DOI:
27. Gough RE, Drury SE, Harcourt-Brown NH. Virus-like particles as-
sociated with macaw wasting disease. Vet Rec. 1996;139:24.
28. Rossi G, Crosta L, Pesaro S. Parrot proventricular dilatation disease.
Vet Rec. 2008;163:310.
29. Ludwig H, Kraft W, Kao M, Gosztonyi G, Dahme E, Krey H. Bor-
navirus infection (Borna disease) in naturally and experimentally
infected animals: its signifi cance for research and practice. Tierarztl
30. Stitz L, Bilzer T, Richt JA, Rott R. Pathogenesis of Borna disease.
Arch Virol Suppl. 1993;7:135–51.
31. Briese T, Hornig M, Lipkin WI. Bornavirus immunopathogenesis
in rodents: models for human neurological diseases. J Neurovirol.
1999;5:604–12. DOI: 10.3109/13550289909021289
Address for correspondence: Ian Tizard, Schubot Exotic Bird Health
Center, Veterinary Pathobiology, Texas A&M University, 4467 TAMU,
College Station, TX 77843-4467, USA; email: firstname.lastname@example.org
Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 3, March 2010 479