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Investigation
Journal of Veterinary Diagnostic
http://vdi.sagepub.com/content/14/5/377
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DOI: 10.1177/104063870201400503
2002 14: 377 J VET Diagn Invest
Chang-Won Lee, Corrie Brown and Mark W. Jackwood
Examined by in Situ Hybridization with Antisense Digoxigenin-Labeled Universal Riboprobe
Tissue Distribution of Avian Infectious Bronchitis Virus following in Ovo Inoculation of Chicken Embryos

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377
J Vet Diagn Invest 14:377381 (2002)
Tissue distribution of avian infectious bronchitis virus following
in ovo inoculation of chicken embryos examined by in situ
hybridization with antisense digoxigenin-labeled
universal riboprobe
Chang-Won Lee, Corrie Brown, Mark W. Jackwood
Abstract. Chicken embryos were inoculated with 8 different strains of infectious bronchitis virus (IBV)
representing 7 different serotypes at 17 days of embryonation. At 2 and 5 days postinfection (dpi), tissues were
collected for in situ hybridization using an antisense digoxigenin-labeled riboprobe corresponding to the se-
quence of the mRNA coding for the membrane protein. Extensive antigen staining in the cytoplasm of epithelial
cells in the trachea, lung, bursa, and intestine was detected at 2 dpi with all 8 strains of IBV. At 5 dpi, little or
no positive staining was observed in these tissues. However, tubular cells of the kidney showed multifocal
positive staining with the Wolgemuth strain-, Gray strain-, JMK strain-, and Mass41 strain-infected chickens.
No viral RNA was detected in the spleen at any time point. The results demonstrated strict epitheliotropic
nature and wide tissue tropism of strains of IBV in the chicken embryo and the universality of our riboprobe.
In situ hybridization with this probe will be useful for understanding the tissue tropism and the pathogenesis
of IBV in vivo.
Infectious bronchitis (IB) is a worldwide disease of
poultry caused by infectious bronchitis virus (IBV), a
member of the genus coronavirus, in the new order
Nidovirales.
5
Coronaviruses are a common cause of
enteric infection, respiratory infection, or both in ani-
mals and man.
25
The genome is a single-stranded, non-
segmented RNA molecule that has positive polarity.
The approximately 27-kb IBV genome encodes 4
structural proteins, spike (S), envelope (E), membrane
(M), and nucleocapsid protein (NP) in the 5 to 3
direction.
17
The precursor S protein is posttranslation-
ally cleaved into S1 and S2 subunits.
7
Neutralizing and
serotype specic epitopes are associated with the S1
subunit.
6,13,21
IBV is one of the best studied coronavi-
ruses, yet limited information is available on tissue
tropism and virulence of that virus. The IBV causes
an upper-respiratory tract disease in young chickens
characterized by tracheal rales, coughing, sneezing,
gasping, and nasal discharge.
8
However, several strains
of IBV have been identied as nephropathogenic, and
some of these strains cause signicant renal lesions
and high mortality in affected chickens.
1,4,14,26
In situ hybridization (ISH) techniques can be uti-
lized to precisely localize specic nucleic acids within
histologic sections.
2
Detection based on the use of a
high-afnity antidigoxigenin (DIG) antibody conju-
gated to an enzyme was developed in 1987.
12
The
From the Departments of Avian Medicine (Lee, Jackwood) and
Pathology (Brown), College of Veterinary Medicine, The University
of Georgia, Athens, GA 30602.
Received for publication August 21, 2001.
DIG-labeled probes are comparable in sensitivity to
radiolabeled probes, and their use is increasing.
3,9,20,22
This study was undertaken to develop a universal
probe that can detect all serotypes of IBV. An antisense
DIG-labeled RNA probe (riboprobe), complementary
to mRNA of the membrane gene, was constructed, and
the viral distribution of 8 different strains of IBV in
in ovo-infected chicken embryos and hatched chicks
was examined.
Materials and methods
Viruses. Eight different strains of IBV, representing 7 dif-
ferent serotypes that have wide prevalence in the United
States, were used in this study (Table 1). Gray and Wolge-
muth (Gelb J Jr, 1999 AVMA, New Orleans, Louisiana, ab-
stract) strains were characterized previously as nephropath-
ogenic.
1,10
Viruses were propagated in 9- to 11-day-old spe-
cic-pathogen-free embryonating chicken eggs.
a,23
Titrations
were done as previously described.
27
Cross-contamination
among different viruses were examined by conducting re-
verse transcriptase-polymerase chain reaction (RT-PCR) and
restriction fragment length polymorphism (RFLP) following
virus isolation in tracheal tissues as previously de-
scribed.
15,16,18
Viral inoculation and tissue preparation. Chicken eggs
were inoculated on the 17th day of embryonation with 10
4.0
EID
50
/0.2 ml of each strain by the chorioallantoic sac route
as previously described.
24
The following tissues were col-
lected at 2 days postinfection (dpi) and 5 dpi (1 day post-
hatch); trachea, lung, spleen, bursa, kidney, and intestine.
Following 24 hr of xation in 10% neutral buffered forma-
lin, tissues were placed in phosphate buffered saline (PBS)
until embedded in parafn. Tissues were routinely processed
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378 Lee, Brown, Jackwood
Table 1. List of IBV strains used in this study.
Strain Serotype M gene* Source
ArkDPI
Conn
CV56b
CWL0470
Arkansas
Connecticut
California
Georgia 98
AF363597
AF363598
AF363599
AF363600
J. Gelb, Jr.
J. Gelb, Jr.
CVDLS
PDRC
Gray
JMK
Mass41
Wolgemuth
JMK
JMK
Massachusetts
Wolgemuth
AF363607
AF363608
AF363609
AF363610
J. Gelb, Jr.
J. Gelb, Jr.
ATCC VR-21
J. Gelb, Jr.
* GenBank accession number.
University of Delaware, Newark, Delaware.
California Animal Health and Food Safety Laboratory System,
Fresno, California.
Poultry diagnostic and Research Center, Athens, Georgia.
American Type Culture Collection, Rockville, Maryland.
into parafn, and 3-m sections were placed on positively
charged slides
b
for in situ hybridization.
Preparation of universal riboprobe. A universal ribo-
probe was created from the 452-bp carboxyl terminal region
of the membrane gene from the CV56b strain. The 452-bp
gene was inserted into the pCR4-TOPO (pCR4-CAVm484)
vector.
c
This region shares more than 95% sequence simi-
larity with other IBVs, including the 8 strains used in this
study. In vitro transcription was performed using T3-RNA
polymerase
d
with DIG-labeled deoxynucleotides (NTPs)
d
to
make antisense RNA complementary to mRNA. The con-
centration of DIG-labeled riboprobes was determined by dot-
blot comparison with a known standard DIG-labeled RNA
d
.
In situ hybridization. Tissue sections were deparafnized,
rehydrated, and digested with 30 g/ml proteinase K for 15
min at 37 C in a humid chamber. Hybridization occurred
overnight at 42 C with approximately 20 ng of riboprobe in
hybridization solution: 5 sodium chloride, sodium nitrate
(SSC), 50% formamide, 5% blocking reagent,
d
1% N-lau-
roylsarcosine, and 0.02% sodium dodecyl sulfate (SDS).
Slides were washed once in 2 SSC with 1% SDS for 30
min at 50 C, once in 1 SSC with 0.1% SDS for 30 min at
50 C, 3 times in 1 SSC for 10 min at room temperature,
and once in 0.1 SSC for 15 min at room temperature. Then
the slides were incubated with a 1:300 dilution of alkaline
phosphatase-labeled goat-anti-DIG antibody
d
in 1% sheep
serum for 2 hr at 37 C. The bound probes were visualized
by the addition of the chromogen/substrate nitroblue tetra-
zolium and 5 bromo-4 chloro-3-indolylphosphate (NBT/
BCIP)
d
in 100 mM Tris-Cl, pH 9.5, 100 mM NaCl, and 50
mM MgCl
2
with 5 mM Levamisol
e
for 45 min at room tem-
perature. Slides were lightly counterstained with Mayer he-
matoxylin and coverslipped.
f
Results
Using the universal riboprobe, viral nucleic acid
from 8 different strains of IBV in several tissues was
detected (Fig. 1). Staining was localized to the cyto-
plasm of epithelial cells. The extents of viral replica-
tion detected by in situ hybridization with 8 different
strains are summarized in Table 2. With all 8 strains,
there was widespread viral nucleic acid in epithelial
cells of trachea, lung, bursa, and intestine at 2 dpi. At
5 dpi, little or no staining was observed in these tis-
sues. Extensive staining was detected in the tubular
cells of the kidney in chickens infected with Wolge-
muth, Gray, JMK, and Mass41 strains at 5 dpi. No
staining was observed in the spleen at any time.
Other tissues, such as the esophagus, air sac, shell
gland, and mesentery, were also collected by chance
because of their anatomical proximity to the 6 tissues
examined in this study. Extensive viral replication was
observed in epithelial cells of the esophagus (Fig. 2a).
Multifocal areas of positive staining were also ob-
served in the air sac adjacent to the lung, mesentery
adjacent to the spleen and kidney, and the shell gland
near the bursa (Fig. 2b, 2c, 2d). No positive staining
was observed with control tissue from uninfected
chicken embryos and hatched chickens.
Discussion
In this study, the ISH technique was successfully
applied to detect viral RNA in formalin-xed parafn-
embedded IBV-infected tissues using a digoxigenin-
labeled riboprobe. Using this universal riboprobe, viral
replication in tissues infected with 8 different IBV
strains representing 7 different serotypes was delin-
eated. All of the strains were isolated in the United
States and have been or are becoming a major concern
in the eld. Viral RNA could be detected at 2 days
after infection in epithelial cells of trachea, lung, in-
testine, and bursa. In chickens, virus has been routinely
isolated from trachea, lung, and cecal tonsils but not
bursa. Extensive viral replication in bursa of in ovo-
infected embryos may be caused by the dissemination
of virus through the digestive and cloacal route as well
as the respiratory tract. At 5 dpi, the extent of staining
was variable, depending on strains. Usually, little or
no staining was observed in these tissues compared
with the staining at 2 dpi. The kinetics of viral repli-
cation in the chicken embryo is variable, depending
on the embryo adaptation of strains of IBV. The max-
imum virus titer can be observed within 24 hours with
highly embryo-adapted strains.
8
Multifocal staining was observed in kidneys from
chickens infected with 4 of the strains: Wolgemuth,
Gray, JMK, and Mass41 strains. Virus replication in
the kidney of chicken embryos infected with Gray and
Wolgemuth strains was expected because those 2
strains were known to be nephropathogenic in vivo. In
addition, Mass41 has been isolated from the kidneys.
14
Because the JMK strain does not replicate in the kid-
ney of experimentally infected chickens, this indicates
either a difference in virus propagation in chicken em-
bryos or potential nephrotropism of this strain. Im-
mature immune function or difference in cell receptors
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379 In ovo tissue distribution of IBV
Figure 1. In situ hybridization using universal riboprobe. Positive staining can be observed as a dark area in the cytoplasm of epithelial
cells. A, Trachea (20) 2 days postinfection with the JMK strain. B, Bursa (20) 2 days postinfection with the CV56b strain. C, Intestine
(20) 2 days postinfection with the CWL0470 strain. D, Lung (40) 5 days postinfection with the Wolgemuth strain. E, Kidney (40) 5
days postinfection with the JMK strain. F, Kidney (40) 5 days postinfection with the Gray strain. G, Kidney (40) 5 days postinfection
with the Mass41 strain. H, Kidney (20) 5 days postinfection with the Wolgemuth strain. All the sections were counterstained with Mayer
hematoxylin.
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380 Lee, Brown, Jackwood
Table 2. Extent of viral replication detected by in situ hybridization.*
Strain
Trachea
2 dpi 5 dpi
Lung
2 dpi 5 dpi
Bursa
2 dpi 5 dpi
Intestine
2 dpi 5 dpi
Kidney
2 dpi 5 dpi
Spleen
2 dpi 5 dpi
ArkDPI
CV56b
Conn
CWL470

Gray
JMK
Mass41
Wolgemuth

* 5 positive cells per high-power eld (400); 1 and 5 positive cells per high-power eld; no positive cells.
Figure 2. In situ hybridization using universal riboprobe. Positive staining can be observed as a dark area in the cytoplasm of epithelial
cells. A, Esophagus (20) 2 days postinfection with the Gray strain. B, Shell gland near bursa (40) 5 days postinfection with the JMK
strain. C, Mesentery near kidney (20) 2 days postinfection with the ArkDPI strain. D, Air sac around lung (20) 2 days postinfection
with the Conn strain. All the sections were counterstained with Mayer hematoxylin.
in chicken embryos should not be overlooked in in-
terpreting this result.
In addition to the tissues described above, we fur-
ther identied positively stained epithelial cells in the
air sac, mesentery, shell gland, and esophagus with
certain strains of IBV. Virus replication in the epithe-
lial cells of air sacs has also been demonstrated by
other researchers (Nauwynck H, Pensaert M, 1988,
Abstract In: Proc 1st Int Symp Infect Bronchitis,
Rauischholzhausen, Germany, pp. 113119). In a pre-
vious study,
19
maximum virus isolations were obtained
from the esophagus of chickens infected with an en-
teric isolate of IBV. Extensive staining in the epithelial
cells of the esophagus in our results conrms that IBV
actually multiplies in this tissue. Detection of IBV in
the mesentery and shell gland has been reported pre-
viously.
10
Considering the close proximity of these tis-
sues with visceral organs, these may provide a route
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381 In ovo tissue distribution of IBV
of virus dissemination throughout the chicken embryo.
The results clearly show the strict epitheliotropic na-
ture of IBV. No positive staining in the spleen at any
time further conrms this fact. The receptor on the cell
surface for IBV is known to be 2,3-linked N-acetyl-
neuraminic acid.
11
However, binding of IBV to this
molecule in itself is not likely the basis of viral cell
tropism because sialic acid is a common cell surface
carbohydrate residue. Thus, an additional, more epi-
thelial cell type-specic molecule is probably required
for IBV infection.
The main advantage of the ISH technique is topo-
logic assessment of nucleic acid localization. Seen
against the backdrop of histologic change, ISH can
help dene the role that the virus plays in tissue dam-
age. In addition, ISH can be useful for retrospective
analysis because nucleic acid is indenitely stable in
parafn-embedded tissue.
Sources and manufacturers
a. Charles Rivers Spafas, North Franklin, CT.
b. Fisher Scientic, Pittsburgh, PA.
c. Invitrogen, San Diego, CA.
d. Roche, Indianapolis, IN.
e. ICN, Irvine, CA.
f. Permount, Fisher Scientic, Pittsburg, PA.
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