J Vet Diagn Invest 14:377—381 (2002)
Tissue distribution of avian infectious bronchitis virus following
in ovo inoculation of chicken embryos examined by in situ
hybridization with antisense digoxigenin-labeled
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 DIG-labeled probes are comparable in sensitivity to
poultry caused by infectious bronchitis virus (IBV), a radiolabeled probes, and their use is increasing.3,9,20,22
member of the genus coronavirus, in the new order This study was undertaken to develop a universal
Nidovirales.5 Coronaviruses are a common cause of probe that can detect all serotypes of IBV. An antisense
enteric infection, respiratory infection, or both in ani- DIG-labeled RNA probe (riboprobe), complementary
mals and man.25 The genome is a single-stranded, non- to mRNA of the membrane gene, was constructed, and
segmented RNA molecule that has positive polarity. the viral distribution of 8 different strains of IBV in
The approximately 27-kb IBV genome encodes 4 in ovo-infected chicken embryos and hatched chicks
structural proteins, spike (S), envelope (E), membrane was examined.
(M), and nucleocapsid protein (NP) in the 5 to 3
direction.17 The precursor S protein is posttranslation- Materials and methods
ally cleaved into S1 and S2 subunits.7 Neutralizing and Viruses. Eight different strains of IBV, representing 7 dif-
serotype speciﬁc epitopes are associated with the S1 ferent serotypes that have wide prevalence in the United
subunit.6,13,21 IBV is one of the best studied coronavi- States, were used in this study (Table 1). Gray and Wolge-
ruses, yet limited information is available on tissue muth (Gelb J Jr, 1999 AVMA, New Orleans, Louisiana, ab-
tropism and virulence of that virus. The IBV causes stract) strains were characterized previously as nephropath-
an upper-respiratory tract disease in young chickens ogenic.1,10 Viruses were propagated in 9- to 11-day-old spe-
characterized by tracheal rales, coughing, sneezing, ciﬁc-pathogen-free embryonating chicken eggs.a,23 Titrations
gasping, and nasal discharge.8 However, several strains were done as previously described.27 Cross-contamination
among different viruses were examined by conducting re-
of IBV have been identiﬁed as nephropathogenic, and
verse transcriptase-polymerase chain reaction (RT-PCR) and
some of these strains cause signiﬁcant renal lesions restriction fragment length polymorphism (RFLP) following
and high mortality in affected chickens.1,4,14,26 virus isolation in tracheal tissues as previously de-
In situ hybridization (ISH) techniques can be uti- scribed.15,16,18
lized to precisely localize speciﬁc nucleic acids within Viral inoculation and tissue preparation. Chicken eggs
histologic sections.2 Detection based on the use of a were inoculated on the 17th day of embryonation with 104.0
high-afﬁnity antidigoxigenin (DIG) antibody conju- EID50/0.2 ml of each strain by the chorioallantoic sac route
gated to an enzyme was developed in 1987.12 The as previously described.24 The following tissues were col-
lected at 2 days postinfection (dpi) and 5 dpi (1 day post-
From the Departments of Avian Medicine (Lee, Jackwood) and hatch); trachea, lung, spleen, bursa, kidney, and intestine.
Pathology (Brown), College of Veterinary Medicine, The University Following 24 hr of ﬁxation in 10% neutral buffered forma-
of Georgia, Athens, GA 30602. lin, tissues were placed in phosphate buffered saline (PBS)
Received for publication August 21, 2001. until embedded in parafﬁn. Tissues were routinely processed
378 Lee, Brown, Jackwood
Table 1. List of IBV strains used in this study. there was widespread viral nucleic acid in epithelial
Strain Serotype M gene* Source
cells of trachea, lung, bursa, and intestine at 2 dpi. At
5 dpi, little or no staining was observed in these tis-
ArkDPI Arkansas AF363597 J. Gelb, Jr.† sues. Extensive staining was detected in the tubular
Conn Connecticut AF363598 J. Gelb, Jr.†
CV56b California AF363599 CVDLS‡
cells of the kidney in chickens infected with Wolge-
CWL0470 Georgia 98 AF363600 PDRC§ muth, Gray, JMK, and Mass41 strains at 5 dpi. No
Gray JMK AF363607 J. Gelb, Jr.† staining was observed in the spleen at any time.
JMK JMK AF363608 J. Gelb, Jr.† Other tissues, such as the esophagus, air sac, shell
Mass41 Massachusetts AF363609 ATCC VR-21 gland, and mesentery, were also collected by chance
Wolgemuth Wolgemuth AF363610 J. Gelb, Jr.†
because of their anatomical proximity to the 6 tissues
* GenBank accession number. examined in this study. Extensive viral replication was
† University of Delaware, Newark, Delaware. observed in epithelial cells of the esophagus (Fig. 2a).
‡ California Animal Health and Food Safety Laboratory System,
Multifocal areas of positive staining were also ob-
§ Poultry diagnostic and Research Center, Athens, Georgia. served in the air sac adjacent to the lung, mesentery
American Type Culture Collection, Rockville, Maryland. 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
into parafﬁn, and 3- m sections were placed on positively chicken embryos and hatched chickens.
charged slidesb for in situ hybridization.
Preparation of universal riboprobe. A universal ribo- Discussion
probe was created from the 452-bp carboxyl terminal region
of the membrane gene from the CV56b strain. The 452-bp In this study, the ISH technique was successfully
gene was inserted into the pCR4-TOPO (pCR4-CAVm484) applied to detect viral RNA in formalin-ﬁxed parafﬁn-
vector.c This region shares more than 95% sequence simi- embedded IBV-infected tissues using a digoxigenin-
larity with other IBVs, including the 8 strains used in this labeled riboprobe. Using this universal riboprobe, viral
study. In vitro transcription was performed using T3-RNA replication in tissues infected with 8 different IBV
polymerased with DIG-labeled deoxynucleotides (NTPs)d to strains representing 7 different serotypes was delin-
make antisense RNA complementary to mRNA. The con- eated. All of the strains were isolated in the United
centration of DIG-labeled riboprobes was determined by dot- States and have been or are becoming a major concern
blot comparison with a known standard DIG-labeled RNAd. in the ﬁeld. Viral RNA could be detected at 2 days
In situ hybridization. Tissue sections were deparafﬁnized,
after infection in epithelial cells of trachea, lung, in-
rehydrated, and digested with 30 g/ml proteinase K for 15
min at 37 C in a humid chamber. Hybridization occurred
testine, and bursa. In chickens, virus has been routinely
overnight at 42 C with approximately 20 ng of riboprobe in isolated from trachea, lung, and cecal tonsils but not
hybridization solution: 5 sodium chloride, sodium nitrate bursa. Extensive viral replication in bursa of in ovo-
(SSC), 50% formamide, 5% blocking reagent,d 1% N-lau- infected embryos may be caused by the dissemination
roylsarcosine, and 0.02% sodium dodecyl sulfate (SDS). of virus through the digestive and cloacal route as well
Slides were washed once in 2 SSC with 1% SDS for 30 as the respiratory tract. At 5 dpi, the extent of staining
min at 50 C, once in 1 SSC with 0.1% SDS for 30 min at was variable, depending on strains. Usually, little or
50 C, 3 times in 1 SSC for 10 min at room temperature, no staining was observed in these tissues compared
and once in 0.1 SSC for 15 min at room temperature. Then with the staining at 2 dpi. The kinetics of viral repli-
the slides were incubated with a 1:300 dilution of alkaline cation in the chicken embryo is variable, depending
phosphatase-labeled goat-anti-DIG antibodyd in 1% sheep
on the embryo adaptation of strains of IBV. The max-
serum for 2 hr at 37 C. The bound probes were visualized
by the addition of the chromogen/substrate nitroblue tetra-
imum virus titer can be observed within 24 hours with
zolium and 5 bromo-4 chloro-3-indolylphosphate (NBT/ highly embryo-adapted strains.8
BCIP)d in 100 mM Tris-Cl, pH 9.5, 100 mM NaCl, and 50 Multifocal staining was observed in kidneys from
mM MgCl2 with 5 mM Levamisole for 45 min at room tem- chickens infected with 4 of the strains: Wolgemuth,
perature. Slides were lightly counterstained with Mayer he- Gray, JMK, and Mass41 strains. Virus replication in
matoxylin and coverslipped.f the kidney of chicken embryos infected with Gray and
Wolgemuth strains was expected because those 2
Results strains were known to be nephropathogenic in vivo. In
Using the universal riboprobe, viral nucleic acid addition, Mass41 has been isolated from the kidneys.14
from 8 different strains of IBV in several tissues was Because the JMK strain does not replicate in the kid-
detected (Fig. 1). Staining was localized to the cyto- ney of experimentally infected chickens, this indicates
plasm of epithelial cells. The extents of viral replica- either a difference in virus propagation in chicken em-
tion detected by in situ hybridization with 8 different bryos or potential nephrotropism of this strain. Im-
strains are summarized in Table 2. With all 8 strains, mature immune function or difference in cell receptors
In ovo tissue distribution of IBV 379
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
380 Lee, Brown, Jackwood
Table 2. Extent of viral replication detected by in situ hybridization.*
Trachea Lung Bursa Intestine Kidney Spleen
Strain 2 dpi 5 dpi 2 dpi 5 dpi 2 dpi 5 dpi 2 dpi 5 dpi 2 dpi 5 dpi 2 dpi 5 dpi
* 5 positive cells per high-power ﬁeld (400 ); 1 and 5 positive cells per high-power ﬁeld; no positive cells.
in chicken embryos should not be overlooked in in- Rauischholzhausen, Germany, pp. 113–119). In a pre-
terpreting this result. vious study,19 maximum virus isolations were obtained
In addition to the tissues described above, we fur- from the esophagus of chickens infected with an en-
ther identiﬁed positively stained epithelial cells in the teric isolate of IBV. Extensive staining in the epithelial
air sac, mesentery, shell gland, and esophagus with cells of the esophagus in our results conﬁrms that IBV
certain strains of IBV. Virus replication in the epithe- actually multiplies in this tissue. Detection of IBV in
lial cells of air sacs has also been demonstrated by the mesentery and shell gland has been reported pre-
other researchers (Nauwynck H, Pensaert M, 1988, viously.10 Considering the close proximity of these tis-
Abstract In: Proc 1st Int Symp Infect Bronchitis, sues with visceral organs, these may provide a route
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 ovo tissue distribution of IBV 381
of virus dissemination throughout the chicken embryo. mentally infected pigs, as demonstrated immunohistochemically
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