RESEARCH ARTICLE

Kang et al., Journal of General Virology 2018;99:512–524

DOI 10.1099/jgv.0.001025

Chicken astrovirus as an aetiological agent of runting-stunting

syndrome in broiler chickens
Kyung-il Kang,† Erich Linnemann, Alan H. Icard, Vijay Durairaj,‡ Egbert Mundt§ and Holly S. Sellers*

Abstract
Despite descriptions of runting-stunting syndrome (RSS) in broiler chickens dating back over 40 years, the aetiology has not
yet been described. A novel chicken astrovirus (CkAstV) was isolated in an LMH liver cell line from the intestines of
chickens affected with RSS. Clinical RSS is characterized by retarded growth and cystic crypt lesions in the small intestine.
In 1-day-old broiler chickens infected with the CkAstV isolate, virus was only detected in the intestinal epithelial cells during
the first few days after infection. Notably, the preferred host cells are the crypt epithelial cells following initial replication in
the villous epithelial cells, thus implying viral preference for immature intestinal cells. Nevertheless, the CkAstV isolate did
not induce remarkable pathological changes, despite the presence of the virus in situ. Serial chicken-to-chicken passages of
the virus induced increased virulence, as displayed by decreased weight gain and the presence of cystic lesions in the small
intestine reproducing clinical RSS in chickens. The analysis of the full-length genome sequences from the isolated CkAstV
and the CkAstV from the bird-to-bird passages showed >99 % similarity. The data obtained in this study suggest that the
CkAstV isolate is capable of inducing RSS following serial bird-to-bird passages in broilers and is as an aetiological agent of
the disease.

INTRODUCTION have been detected in diseased and healthy flocks [9–15].

Astroviruses are small, round, non-enveloped viruses with a
positive-sense, single-stranded RNA genome, and belong to
the family Astroviridae. The virus particles – 28–30 nm in
diameter with a star-like shape – can be observed by elec-
tron microscopy [1]. Astroviruses have been isolated from
faeces in a wide variety of animals, including humans,
and are primarily associated with gastroenteritis in young
individuals [2]. However, extraintestinal diseases such as
neuroinvasive astrovirus-induced encephalitis have been
reported in humans and animals [3–7]. Nephritis in chick-
ens and a fatal hepatitis in ducklings are also associated with
astroviruses [8, 9]. The astroviruses isolated from birds
belong to the genus Avastrovirus, which includes viruses
isolated from chickens, turkeys and ducks. In chickens, sev-
eral astroviruses, namely chicken astrovirus (CkAstV) and
avian nephritis virus type 1 (ANV-1), ANV-2 and ANV-3
The characterization of astroviruses is obstructed by the fact
that they are rarely isolated or adapted to cell culture sys-
tems. Baxendale and Mebatsion [16] previously reported
the isolation of CkAstVs in cell cultures, but the sequence
data for these isolates are very limited. The infection of
CkAstV may cause mild clinical signs consisting of diar-
rhoea and maldigestion [16] or severe clinical signs and
death after infection [11], highlighting the the isolates’ wide
range of virulence.
The general genome organization of astroviruses (5¢-non-
coding region, ORF1a/ORF1b, ORF2, 3¢-noncoding region
and poly A tail) is consistent for all published full-length
sequences. ORF 1a encodes for a protease containing the
protease 3C motif, and ORF 1b encodes for the RNA-
dependent RNA polymerase (RdRp) [17]. A ( 1) frameshift
between ORF1a and ORF1b leads to the synthesis of an

Received 18 August 2017; Accepted 30 January 2018

Author affiliation: Poultry Diagnostic and Research Center, College of Veterinary Medicine, University of Georgia, Athens, Georgia, USA.
*Correspondence: Holly S. Sellers, hsellers@uga.edu
Keywords: runting stunting syndrome; RSS; chicken astrovirus; cystic enteropathy.
Abbreviations: ANV, avian nephritis virus; CkAstV, chicken astrovirus; CkAstV-cc, chicken astrovirus isolated in LMH cells; CkAstV-Ckp5, chicken
astrovirus following the fifth passage in chickens; CkAstV-gut, chicken astrovirus present in the intestine of RSS-infected chickens; CPE, cytopathic
effect; IFA, indirect immunofluorescence assay; ISH, in situ hybridization; rab-anti-CkAstV-gut, serum from rabbit immunized by recombinant chicken
astrovirus capsid protein; RSS, runting-stunting syndrome.
†Present address: National Poultry Research Center, U.S. Department of Agriculture, Athens, Georgia, USA.
‡Present address: Boehringer Ingelheim Vetmedica, Inc., Ames, Iowa, USA.
§Present address: Boehringer Ingelheim, Veterinary Research Center, GmbH Co. KG, Bemeroder Straße 31, 30559 Hannover, Germany.
The GenBank accession number for the full length genome sequence of the CkAstV isolate (CkAstV-cc) is KX397575. The GenBank accession number
for the full length genome sequence of the CkAstV after five passages in chickens (CkAstV-Ckp5) is KX397576.
One supplementary table and two supplementary figures are available with the online version of this article.

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ORFla/lb fusion polyprotein [18, 19]. Recently, we reported
the complete genome sequence of a new CkAstV amplified
directly from the intestinal contents of runting-stunting
syndrome (RSS)-affected chickens (referred to from this
point as CkAstV-gut) (Table S1, available in the online
version of this article). Sequence analysis of the RSS
CkAstV-gut revealed low similarity with other known avian
astroviruses. Furthermore, the genome analysis of the
CkAstV-gut proposed a different translation mechanism,
which suggests that the initiation for the RdRp may occur at
the existing start codon of ORF1b [12]. ORF 2 is the coding
sequence for the capsid protein and is likely translated from
a subgenomic messenger RNA [19, 20]. The amino acid (aa)
sequences of the capsid protein are relatively diverse
between astroviruses [14].
RSS is an infectious enteric disease of young broiler chickens
that is associated with a mild to moderate diarrhoea
that results in retarded growth of affected birds. While its
description dates back over 40 years, the aetiology has not
been reported to date. In affected birds, dilated or cystic
crypts are consistently observed in the crypts of Lieberkühn
of the small intestine with other microscopic lesions [21–
24]; thus cystic enteropathy is a hallmark lesion of the
disease. Based on the induction of clinical signs and micro-
scopic lesions following the administration of filtered intes-
tinal content [23, 24], a viral aetiology is almost certain. To
date, several viruses have been described as possible aetio-
logical agents for RSS: rotavirus [21], astrovirus [16, 25],
reovirus [26] and parvovirus [27, 28]. However, the disease
has not been reproduced with any of the viruses isolated.
The involvement of astroviruses in enteric diseases in mam-
mals and birds has been frequently described, but little is
known about the disease mechanism. In our previously
described vaccination experiments, the data supported a
novel CkAstV as an RSS aetiology. We reported that
the vaccination of broiler breeders with the recombinant
capsid protein of CkAstV-gut mitigated the outcome of RSS
in the offspring, likely due to the presence of CkAstV-
specific antibodies [24]. In addition, we demonstrated the
presence of CkAstV, as well as ANV-1 and ANV-2, in
broiler chickens affected by RSS [25].
There is a critical need to identify the causal agent of RSS to
understand the disease mechanism and, more importantly,
develop preventive interventions. The aim of these studies
was to investigate the aetiological role of CkAstV in RSS.
Here we describe the pathogenicity of a newly isolated
CkAstV (CkAstV-cc) in commercial broiler chickens and
present data to support its role as an aetiological agent of
the disease.
RESULTS
Isolation of a new CkAstV in LMH cells
A CkAstV-specific antiserum (rab-anti-CkAstV-gut) was
generated in an SPF rabbit immunized with a recombinant
capsid protein of CkAstV-gut that was reported in a
513
previous study [24]. Verification of the specific reactivity
was confirmed by Western blot and an immunofluorescence
assay. Western blot analysis with rab-anti-CkAstV-gut only
recognized the capsid protein, from the CkAstV-infected
LMH cells, of the appropriate size and calculated
an apparent molecular weight of 98 kDa. The size of the
detected protein correlated with the recombinant capsid
protein from the baculovirus-infected Sf9 cells (Fig. 1a).
The isolation of CkAstV from an intestinal filtrate of RSS-
positive chickens was initially attempted in several cell lines
(MDCK, DF1, QM7, Vero, LMH and Sf9). Among the cell
lines tested, only inoculated LMH cells showed a cytopathic
effect (CPE) beginning at passage 3. The CPE in the third
passage became visible 72 h after inoculation of the subse-
quent passage material and by 120 h p.i. detached small and
round cells were predominant, representing 100 % CPE
(Fig. 1b). Only infected LMH cells were positive for CkAstV
antigen by the immunofluorescence assay (IFA) using the
rab-anti-CkAstV-gut serum (Fig. 1c). In order to determine
whether the CPE observed in the cells was a result of infec-
tion by a chicken astrovirus, alpha and beta virus neutraliza-
tion tests were performed. Using a constant volume of
serum (alpha test), 100 µl of the original cell culture super-
natant was diluted from 10 1 down to 10 8 and incubated
with either the rab-anti-CkAstV-gut serum or with serum
obtained from the same rabbit prior to immunization. CPE
was observed in all flasks with virus incubated with pre-
immune serum at dilutions of 10 6 or below. In contrast, no
cytopathic effect was observed in virus incubated with rab-
anti-CkAstV-gut serum in dilutions down to the 10 4 dilu-
tion and this was confirmed by the absence of staining in an
indirect immunofluorescence assay. In the beta virus neu-
tralization assay, the diluted rab-anti-CkAStV-gut serum
neutralized 100 TCID50 of the CkAstV-cc down to a dilu-
tion of 2 13, which was confirmed by IFA. Furthermore,
PCRs and RT-PCRs specific for common avian viruses were
also performed to evaluate the cell culture for the presence
of extraneous avian viruses. Only the primers specific for
CkAstV revealed a PCR fragment of the appropriate size,
indicating that CkAstV was isolated in the cell culture (here-
after referred to as CkAstV-cc) (Table S1).
The replication kinetics of CkAstV-cc were investigated
(Fig. 1d). One hour after the virus was added to the cell cul-
ture (time point 0 h after infection), the virus was primarily
cell-associated, whereas only a few infectious virus particles
were present in the supernatant. Twelve hours later, the
viral eclipse was observed. One replication cycle appeared
complete between 12 and 24 h after infection. The analysis
of time points at 24, 48, 72 and 96 h post-inoculation
revealed a constant increase in viral titres in both cells and
supernatants. Approximately 10-fold of the newly produced
viruses remained cell-associated.
CkAstV replicated in the crypt epithelial cells
The pathogenesis of CkAstV-cc in the intestine was
investigated in day-old chickens at 6, 12, 18, 24, 48, 72
and 120 h post-infection (Fig. 2). RSS clinical signs were
 Kang et al., Journal of General Virology 2018;99:512–524

Fig. 1. CkAstV-specific antiserum and the isolation and characterization of a CkAstV in LMH cell culture. (a) Western blot analysis of
protein samples of (i) LMH cells either infected with CkAstV-cc (CkAstV) or not (cont); (ii) Sf9 cells infected (BacV) with a recombinant
baculovirus encoding for the capsid protein of a CkAstV-gut [24] or uninfected Sf9 cells (cont). The membranes were incubated with
either the rabbit serum rab-anti-CkAstV-gut (dilution 1 : 10 000) or the HRP-conjugated anti 6 His monoclonal antibody (dilution
1 : 5000). A ladder (M) for protein molecular weights (Bionexus 20 kDa dual-colour prestained protein marker) was indicated on the left
part of the gel. (b) Unifected LMH cells at 3 days p.i (left panel) and LMH cells infected with CkAstV-cc at 3 days p.i. (right panel) are
detached with small and round cells present in the media (400). (c) LMH cells were infected with CkAstV-cc and indirect immunofluo-
rescence was performed 24 h after infection using rab-anti-CkAstV-gut serum and goat-anti rabbit FITC-conjugated antibodies. (d) LMH
cells were infected with 100 TCID50/well of CkAstV-cc. The virus titres of the supernatant and the cells were determined individually at
the indicated time points after infection [hours post-infection (p.i.)]. The data represent the average of three independent studies and
the standard deviation is shown as bars at each time point. The grey dotted line represents the lowest detection limit of the TCID50
assay.

assessed by evaluation of body weight gain/retardation,
presence of cystic crypt lesions and replication of the
virus in the intestine. The average body weights of chick-
ens infected with CkAstV-cc were similar to those of
the uninfected controls except at 72 h p.i. (P<0.05), which
was insufficient to claim a difference during the entire
course of the study (Fig. 2a). The occurrence of obvious
cystic crypt lesions was absent in the chickens, even
though mildly dilated crypts were observed and counted
in the CkAstV group (one bird with two lesions) and in
the negative control group birds (two birds with one
lesion each). However, staining for CkAstV by ISH was
only observed in the intestines of the CkAstV-cc-infected
birds (Fig. 2b, c). The location dynamics of CkAstV RNA
during the course of the experiments were more
interesting. During the first 12 h p.i., viral RNA was
exclusively detected in the villous epithelial cells. The
location of CkAstV staining migrated from the villous
epithelial cells to the crypt epithelial cells at 18 h p.i.
onwards, based on ISH staining. Within 48 h after infec-
tion, viral RNA was predominantly observed in the crypt
epithelial cells and even in the attenuated epithelial cells
in a dilated crypt (Fig. 2c). The specific infection of
CkAstV-cc was verified by ISH using other riboprobes
(ANV-1 and ANV-2). The ANV-1 and ANV-2 nucleic
acids were not detected in the tissues by ISH (data not
shown), again supporting the contention that only
CkAstV-cc is likely present in the inoculated material. No
signals were detected in the control birds at any of the
time points.

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Fig. 2. Dynamics of CkAstV-cc in the duodenum of chickens during the first few days of infection. Forty 1-day-old broiler chickens
were infected with either a CkAstV-cc (CkAstV) or were not infected (control). (a) Five birds from each group were euthanized at the
given time after infection [hours post-infection (p.i.)] and were weighed. (b) The number of birds with CkAstV RNA as detected by ISH in
the duodenal loop was determined from CkAstV-cc-infected birds. The location of the ISH signals was differentiated between the intes-
tinal villi and the crypt. (c) The presence of ISH signals was documented as an example for representative time points after infection.
The inlet at 72 h p.i. represented a haematoxylin and eosin (H and E)-stained section and showed a lesion in the crypt of the duodenum.
Bar, 50 µm.

CkAstV replicated for the first few days after
infection in the absence of RSS signs
To further evaluate the ability of CkAstV-cc to reproduce
the clinical RSS disease, day-old chickens were infected
orally with CkAstV-cc and evaluated at days 5 and 12 after
infection. Day-old chicks inoculated orally with an RSS fil-
trate served as positive RSS controls, as previously described
in an RSS challenge model [25], and day-old chicks inocu-
lated with cell culture media served as negative controls
(control) (Fig. 3).
The manifestation of clinical RSS was consistently repro-
duced in the positive RSS controls as we described previ-
ously [25]. The body weight retardation in the positive RSS
control group was evident during the study until day 12 p.i.,
while the cystic crypt count peaked at around day 5 p.i. The
ISH staining for CkAstV was clearly detected in the positive
RSS group at day 5 d p.i.; however, the ISH signals were
remarkably reduced by day 12 p.i. Unlike the positive RSS
control group, the body weight retardation was not signifi-
cantly reduced in the CkAstV-cc group at either day 5
or day 12 p.i. compared to the negative controls (Fig. 3a).
Even though a few minimally dilated cystic crypts were
present in the CkAstV-cc, neither the number nor the
abnormal shape of the cystic crypt differed from those of
the negative control group (Fig. 3b). The ISH staining for
CkAstV was only detected in the CkAstV-cc and positive
RSS groups at day 5 d p.i. (Fig. 3c). The majority of the ISH
signals were located in the crypt epithelial cells in both
infected groups (i.e. CkAstV-cc and RSS) (Fig. 3d). None-
theless, no link between the replication of CkAstV-cc and
clinical signs with microscopic lesions consistent with RSS
was obvious at this time point. Additional experiments were
necessary to further evaluate the aetiological role of
CkAstV-cc in RSS.
Bird-to-bird passage experiment I: RSS was
reproduced by CkAstV-cc via a serial passage in
chickens
Based on the possibility that the CkAstV-cc was attenuated
during the cell culture adaptation, a serial bird-to-bird pas-
sage of virus in broiler chickens was conducted to evaluate
whether a change in pathogenicity of CkAstV-cc would
occur during a serial passage. The CkAstV-cc was orally
administered in day-old broiler chickens for the first pas-
sage, from which a filtered (0.22 µm) intestinal homogenate
(10 % w/v in cell culture media) was prepared for consecu-
tive passages conducted in the same manner. For all bird-

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Fig. 3. Infection of broiler chickens with CkAstV-cc resulted in a short period of viral replication in the absence of the RSS signs. Ten
1-day-old broiler chickens were infected with either intestinal content of RSS-affected chickens (RSS) or an astrovirus isolated in cell
culture (CkAstV-cc). One group of chickens was inoculated with sterile cell culture media and served as a negative control. The body
weight (a), the number of cystic lesions in the duodenal loop (b) and the presence of viral RNA as detected by in situ hybridization (ISH)
(c and d) were determined from four to five chickens each at days 5 and 12 post-infection (p.i.). The number of positive birds/number
of birds examined is included in (b) and (c). The ISH score was based on the following scale: 0, no signals; 1, five signals per high-
power field; 2, five to fifteen signals per high-power field; 3, >fifteen signals per high-power field. (d) The presence of the CkAstV-cc
RNA was documented in the crypt epithelial cells of a chicken on day 5 after infection by ISH. Bar, 100 µm.

to-bird passages, birds were evaluated on day 5 p.i. For pas-
sages 1–5, hatch mates inoculated with cell culture media
served as negative controls (Table 1). We conducted an
additional passage (passage 6) that included the intestinal
homogenate passage from the negative control birds. We
consistently reproduced clinical signs and microscopic
lesions consistent with the RSS in CkAstV-cc-passaged
birds. Clinical signs and lesions consistent with RSS were
absent in negative control birds regardless of the inoculation
material. The results are summarized in Fig. S1.
The differences in body weights between the negative con-
trol chickens and the CkAstV-cc-passaged chickens
ranged from no difference to minimal difference until pas-
sage 3. However, the passage 4 body weights were signifi-
cantly different (6 %) at P<0.05 compared to the negative
controls and became even more discrepant during passage 5
(17 % difference) and passage 6 (14 % difference) at
P<0.001. The number of chickens with cystic crypt lesions
also gradually increased from 2 out of 8 in the first passage,
to 4 out of 10 at the third to sixth passages (Table 1). The
lesion counts also increased from 2 to 3 in the first and sec-
ond passages up to 6 to 10 in subsequent passages. Further-
more, the cystic crypt lesions observed in the later passages
correlated with those observed in clinical RSS disease
(Fig. 4). The intestinal contents from the infected chickens
at each passage were positive for CkAstV by RT-PCR, while
the negative controls remained negative. From each passage,
the CkAstV titre from pooled intestinal contents was deter-
mined in LMH cell cultures using indirect immunofluores-
cent staining. The titre of CkAstV-cc used for the first
passage inoculation was 106.3 TCID50 ml 1. The titres of the
intestinal contents in sequential passages ranged between
103.5 TCID50 ml 1 (passage 2) to 105.0 TCID50 ml 1 (pas-
sage 4 and 6), thus supporting the continuous passage of
infectious CkAstV. In addition, the CkAstV RNA was
detected by ISH in the intestines of CkAstV-infected chick-
ens at each passage (Table 1).
Bird-to-bird passage experiment II: the induced
pathogenicity of CkAstV-cc via bird-to-bird passage
was unaffected by the intestinal flora
To determine what effect, if any, the other components in
the normal intestinal environment (most likely normal bac-
terial flora) had, the intestinal material for both CkAstV-cc
and control groups was prepared separately as an unfiltered
and a 0.22 µm filtrate and then passaged in parallel.
The body weight comparisons at each passage are summa-
rized in Fig. 5(a).

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Table 1. RSS signs in broiler chickens during a serial passage of CkAstV

Group Pass 1 Pass 2 Pass 3 Pass 4 Pass 5 Pass 6

Body weight Control 83.3±7.8* 81.3±8.1 86.8±7.5 101.4±10.4 96.8±8.4 107.1±10.3

CkAstV 85.2±3.8 76.3±5.4 81.6±7.7 93.6±8.9 80.0±5.8 92.1±8.2
% difference +2† 6 6 8 17 14
P value 0.5630 0.1500 0.1430 0.0444 0.0001 0.0021
Cystic crypt Control 0 (0/8)‡ 0 (0/8) 0 (0/10) 0 (0/9) 0 (0/10) 0 (0/10)
CkAstV 3 (2/8) 2 (2/9) 8 (4/10) 10 (4/10) 6 (4/10) 6 (4/10)
ISH Control 0/8§ 0/8 0/10 0/9 0/10 0/10
CkAstV 7/8 7/9 6/10 6/10 6/10 10/10
Titre Control Negative Negative Negative Negative Negative Negative
CkAstV 4.5|| 3.5 3.8 5.0 4.3 5.0

*Average body weight (g)±SD.

†[(Control-CkAstV)/control] 100.
‡Total lesion count (no. of birds with lesions/no. of birds examined).
§No. of birds with positive ISH/no. of birds examined.
||log10 TCID50 ml 1.

The average body weight of the chickens infected with
CkAstV-cc was low even at the first passage (P<0.05). Dur-
ing the second chicken passage, the unfiltered negative con-
trol group had a lower body weight compared to the filtered
counterpart, which was unusual and later determined to be
the result of a mechanical issue with the isolation unit. No
virus was detected from either of the control groups. After
the mechanical issue was resolved, the passages continued.
Clinical signs of RSS signs were observed in the remaining
passages of the filtered and unfiltered groups. Body weight
suppression in the CkAstV-cc passage groups was consis-
tent through all of the experiments, regardless of whether or
not the intestinal contents were filtered, and thus correlated
with findings observed in bird-to-bird passage experiment I.
No significant differences were observed between the fil-
tered and unfiltered intestines in the CkAstV-cc groups,
supporting the contention that the findings in the CkAstV-
cc-passed groups were unrelated to any extraneous agents
within the intestines.
The presence of CkAstV nucleic acid was detected by RT-
PCR only in the CkAstV-passaged groups at each passage.
The viral titres ranged between 104.5 TCID50 ml 1 and
105.75 TCID50 ml 1, regardless of filtration (Fig. 5b). Fur-
thermore, the intestines from CkAstV passage 5 were posi-
tive for CkAstV by ISH (10 out of 10) but negative for
ANV-1 (0 out of 10) and ANV-2 (0 out of 10), suggesting
the absence of other known astroviruses.
Analysis of the genetic variation of the CkAstV
after serial passage in chickens
Our previously published RNA genome sequence of the
new CkAstV-gut that was directly amplified from the intes-
tinal contents of RSS-affected chickens (CkAstV-gut) [12]
served as the reference for the full-length sequence determi-
nation of the virus isolated in cell culture (CkAstV-cc) and
the virus sequence following the fifth passage in chickens
(CkAstV-Ckp5) from the bird-to-bird passage experiment I
(Table S1). The CkAstV-cc and CkAstV-Ckp5 nucleotide
sequences were the same length (7499 nucleotides without
the poly-A tail sequence), and both were 21 nucleotides
shorter than the genome sequence of CkAstV-gut (Fig. 6a).
The overall homology of the nucleotide sequences between
CkAstV-gut and CkAstV-cc was 85 %. The aa homology
between CkAstV-gut and CkAstV-cc was 96.9 % in ORF1a,
98.3 % in ORF1b and 84.8 % in ORF2, given total lengths of
1139 aa, 519 aa and 743 aa, respectively. The majority of the
amino acid changes within the ORF2 encoding for the cap-
sid protein occurred in the C-terminal third of the protein
(Fig. 6b). The genetic variation between CkAstV-cc and
CkAstV-Ckp5 was also analysed. Only a few nucleotide
changes occurred during the bird passages and this resulted
in an almost identical nucleotide sequence with a homology
of 99.8 % (Fig. 6c). The comparison of the aa sequences
revealed three aa changes in ORF1a (A6V, V45A and S50T)
and one aa change in ORF2 (F371Y), which indicated a sta-
ble virus during transmission from chicken-to-chicken.
Interestingly, two of the three aa changes observed in
ORF1a (V45A and S50T) and the one aa change in ORF2
(F371Y) involved the amino acids present in the aa
sequence of the CkAstV-Gut (Fig. 6d).
DISCUSSION
Chicken astroviruses have a worldwide distribution. It has
been reported that the virus is detected in 96 % of broiler
flocks with growth problems [29]. However, commercial
broiler chickens and parent flocks also have a high preva-
lence of the virus, regardless of the disease condition [30,
31]. The mechanism for the pathogenicity of CkAstVs is
not known, but factors such as co-infections with other
viruses, age of the host, virus concentration, strain of virus
and maternal antibodies may play a role in the disease [32].

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Fig. 4. Serial passage of a CkAstV-cc in broiler chickens induced RSS-like microscopic lesions. Ten 1-day-old broiler chickens were
infected with the CkAstV-cc isolate (CkAstV). The filtered intestinal content from the initial CkAstV-cc-infected chickens was serially
passaged until passage 6. At day 5 after infection, the chickens were sacrificed and haematoxylin and eosin (H and E)-stained tissue
sections from the duodenum were examined. Representatives of the cystic crypts in the duodenal mucosa from CkAstV-cc passages 1,
3 and 6 are shown. For comparison, typical cystic lesions are shown from an experimental infection of RSS in chickens. Bar, 200 µm.

The CkAstV-cc reported in this study was isolated and eval-
uated on the basis of the genetic information generated in
our previous findings, which supports there being a new
CkAstV as the aetiology of RSS [12, 24, 25]. The primary
isolation of avian viruses in an in vitro system, such as
embryonated eggs or cell culture, depends on many factors
and is not predictable. Among the cell types evaluated, the
CkAstV-cc only infected, and subsequently replicated in,
one cell line, the liver-derived LMH cell. The propagation of
a CkAstV in LMH cells has been described previously [11,
16] and it seemed to be a suitable host cell for the isolation
of CkAstV from intestinal contents. Trypsin was unneces-
sary for the isolation of the CkAstV-cc described here, as
reported for other astroviruses [33]. However, trypsin has
been used for the isolation of many astroviruses from
bovine, swine and human origin samples [33–35].
The replication kinetics of the CkAstV-cc in LMH cells
revealed a cell association. This would be an important fac-
tor to consider for possible vaccine production.
The capsid protein is the most hypervariable region of the
virus and determines its antigenicity [14, 36]. The majority
of aa changes are located in the 3¢ end of the protein
sequence, while the N-terminal region has a high homology.
Our rab-anti-CkAstV-gut antiserum targets the specific
capsid protein [12, 24], thus giving a high sensitivity and
specificity to the virus isolation assay in conjunction with
other molecular assays. The nucleotide and amino acid
sequence comparison of the CkAstV-cc with the CkAstV-
gut genome sequence obtained directly from RSS-affected
chickens revealed that the two viruses are genetically
different, especially in the capsid protein gene. While the
neutralizing antibody-inducing epitopes for CkAstVs are
unknown, neutralization of the CkAstV-cc with the rab-
anti-CkAstV-gut serum suggests that the viruses are anti-
genically related, despite genetic variation.
The dynamics of virus replication in chickens revealed that
villous epithelial cells are initially susceptible to the CkAstV
infection, but later become refractory, while the virus is able
to spread and replicate in the crypts of Lieberkühn. Consid-
ering that crypt epithelial cells actively proliferate and
migrate toward the villi, the CkAstV may favour less mature
cells of the intestine. Furthermore, the replication of the
CkAstV-cc involved a short and self-limited infection for
only a few days after infection, as also observed in clinical
RSS disease [25].
The virus titres obtained through the serial bird passages
revealed a relatively wide variation between the passed
intestinal contents in the bird-to-bird passage experiment I,
although it was relatively uniform in experiment II. The
comparison of the total virus concentration in infected
chicks was not precise, but rather served as an estimate,
since there was variation in the volume and tissue location
between the intestinal samples collected during necropsy.
However, none of the virus titres in the passaged intestinal
contents exceeded the CkAstV titre used for the first-
passage infection.
While the presence of a quasi-species of CkAstVs in the
original CkAstV-gut-positive content cannot be ruled out,
the genetic similarity of the full-length genomes of

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 Kang et al., Journal of General Virology 2018;99:512–524

Fig. 5. Reproduction of RSS by CkAstV-cc bird-to-bird passage was independent of the passage of the intestinal flora in commercial
broiler chickens. For the first passage (pass 1) the chickens were inoculated with either cell culture medium or with CkAstV-cc. For
the subsequent passages (2 to 5), 4 groups of 10 birds were included. The first two passage groups received 0.22 µm filtered and unfil-
tered intestinal content (cont filt, cont unfilt) from chickens that had initially been inoculated with cell culture medium. The remaining
two groups were used to pass the filtered and unfiltered intestinal content from chickens that had initially been infected with CkAstV-
cc (CkAstV filt, CkAstV unfilt). The average body weights and standard deviations of each group at day 5 after inoculation are shown.
Significant differences between the groups were indicated by asterisks above the bars. *, P<0.05; **, P<0.01. (b) The virus titres were
determined from each passage material in the LMH cells.

CkAstV-cc and CkAstV-Ckp5 was extremely high
(99.8 %) and indicates the successful infection and passage
of CkAstV-cc through young broiler chickens resulting in
clinical RSS disease when passaged from bird-to-bird. The
detection of replicating CkAstVs in the cystic crypts fol-
lowing the infection and bird-to-bird passage of CkAstV-
cc provides for the first time evidence for the association
of a virus with the hallmark lesion of RSS. The data
obtained from the unfiltered intestinal content passage
(bird-to-bird passage experiment II) also support CkAstV-
cc having a role in RSS via serial passage by excluding
other factors resident in the normal chicken intestine,
especially bacterial microflora. The mechanism for the
reproduction of clinical RSS via bird-to-bird passage of
CkAstV-cc is not known at this time.
Whether CkAstV-cc was attenuated via genetic modifica-
tions during virus passage in cell culture, as reported for a
human astrovirus isolate [37], remains unclear. Further
evaluation of the possible role or influence of deletions and
aa changes observed between the CkAstV-cc and CkAstV-
gut and pathogenicity needs to occur. Interestingly, during
the serial passage of CkAstV-cc in broiler chickens,
the amino acid sequences reverted from the sequence
observed in the CkAstV-Ckp5 to those observed in the
astrovirus sequences obtained from the intestinal samples
(CkAstV-gut) (ORF 1a, V45A, S50T; ORF2, F371Y). How-
ever, since these were almost the only aa changes observed
during serial passages of CkAstV-cc in chickens, it will be
important to determine whether aa changes influence the
disease outcome. To this end, a reverse genetics system
needs to be established for CkAstV, as previously described
[9] for ANV-1. This would allow the direct examination of
the aa differences observed and improve our biological
understanding of CkAstV.
In addition to the characteristic viral pathogenesis, success-
ful reproduction of RSS by serial passage of CkAstV-cc in
birds was reported, as evidenced by the results obtained
from two separate experiments. This report presents strong
evidence for the first time that CkAstV-cc is a causal agent
of RSS, which may provide a fundamental understanding of
RSS in chickens as well as astroviral enteritis in other
species.

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 Kang et al., Journal of General Virology 2018;99:512–524

Fig. 6. Comparison of genomic variation during virus isolation and bird-to-bird passage. The genomic architectures of the CkAstVs
were compared. (a) The nucleotide size difference of the nonstructural polyprotein (ORF1a), the RNA-dependent RNA polymerase
(ORF1b) and the capsid protein (ORF2) of the recently described full-length sequence of a CkAstV from the intestine of RSS-affected
chickens (CkAstV-gut) (NCBI GenBank accession number JF414802) and a CkAstV that has been isolated in cell culture (CkAstV-cc)
(GenBank number KX397575), and the same virus following five passages in chickens (CkAstV-Ckp5) (GenBank number KX397576)
were described. The terminal untranslated regions (UTRs) and each ORF are distinguished and indicated by the size (not drawn to
scale). (b) The amino acid sequences were compared between CkAstV-gut (JF414802) and CkAstV-cc (KX397575). The positions of
the amino acid differences are marked in black and the positions of deletions are marked in red on the grey line, which indicates the
open reading frame backbone for the nonstructural polyprotein (ORF1a), the RNA-dependent RNA polymerase (ORF1b) and the capsid
protein (ORF2). (c) The amino acid sequences of CkAstV-cc (KX397575) and the same virus following five passages in chickens
(CkAstV-Ckp5, KX397576) were compared. (d) The amino acid sequence of CkAstV-cc (KX397575) and CkAstV-Ckp5 (KX397576) are
shown in comparison with the sequences of CkAstV-Gut (JF414802) in parallel. The numbering is in accordance with the amino acid
sequence of CkAstV-gut (JF414802).

METHODS containing DMEM-4.5 and sedimented at 700 g at 4  C for

Antiserum specific for a new CkAstV
The recombinant capsid protein of a CkAstV expressed in a
baculovirus system was purified [24] and used for
the immunization of a rabbit at the Polyclonal Antibody
Production Service facility (University of Georgia, Athens,
GA, USA) and subsequently identified as rab-anti-CkAstV-
gut serum.
To test the reactivity of rab-anti-CkAstV-gut serum, West-
ern blot analysis was performed. LMH cells grown in T25
tissue culture flasks were infected with CkAstV-cc at
a multiplicity of infection (m.o.i.) of 0.01. Three days after
infection, the cells were trypsinized, resuspended in serum-
5 min. In addition, Sf9 cells were infected with the recombi-
nant baculovirus encoding for the capsid protein of a
CkAstV [24]. Five days after infection, the cells were
scraped into the medium and pelleted as described above.
After a PBS washing, the cell pellet was resuspended in
300 µl of 2 Laemmli buffer [38]. The lysate was heated at
95  C for 2 min and centrifuged for 5 min at 13 000 g before
the supernatants were collected. The rest of the steps were
conducted as described by Sellers et al. [24]. Briefly, the
transferred membrane was incubated with rab-anti-
CkAstV-gut serum or an HRP-conjugated anti-6 His-tag
monoclonal antibody (Genscript, Piscataway, NJ, USA).
Antibody binding was detected with goat anti-rabbit

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 Kang et al., Journal of General Virology 2018;99:512–524
HRP-conjugated antibodies (Sigma-Aldrich, St Louis, MO,
USA) followed by visualization with a chemiluminescent
substrate. Immobilon Western (Millipore, Billerica, MA)
and Gel Logic 2200 (Carestream Health, New Haven,
CT, USA) were used.
Cells and virus isolation
The following cell lines were evaluated for primary isolation
of a CkAstV: Madin–Darby canine kidney cells
(MDCK; CRL-2285, ATCC, Manassas, VA, USA), DF1
(chicken fibroblastcell line, CRL-12203, ATCC), Vero cells
(African green monkey kidney cells, CRL-1587; ATCC),
LMH (chicken hepatocellular carcinoma epithelial cell line,
CRL-2117, ATCC), QM-7 [quail muscle cell line; RIE 466;
Collection of Cell Lines in Veterinary Medicine (CCLV),
Insel Riems, Germany) and Sf9 (Spodoptera frugiperda,
Invitrogen, Carlsbad, CA, USA). The cells listed above,
excluding QM-7 and Sf9, were grown in Dulbecco’s modi-
fied Eagle’s medium with 4.5 g l 1 glucose (DMEM-4.5;
Thermo Scientific, Waltham, MA, USA) supplemented with
10 % foetal bovine serum (FBS; Mediatech, Manassas,
VA, USA). The QM-7 cells were used in a mixture of equal
parts of minimal essential medium (MEM; Invitrogen,
Carlsbad, CA, USA) with Earle’s balanced salt solution and
MEM with Hanks’ balanced salt solution (Invitrogen, Carls-
bad, CA, USA), supplemented with 10 % FBS. LMH cells
were cultivated in a humidified incubator at 39  C with 5 %
CO2. The insect cell line Sf9 was cultivated in serum-free
SFX-Insect medium (Thermo Scientific, Waltham,
MA, USA) at 28  C. The other cells were cultivated in a
humidified incubator at 37  C with 5 % CO2.
The material used for virus isolation was the same as that
used for the infection experiments previously described [24]
for chickens affected with RSS. The resulting supernatant
obtained after full treatment of RSS intestinal contents was
filtered through a 0.45 micron syringe filter followed by fil-
tration through a 0.22 micron syringe filter (Whatman,
Florham Park, NJ, USA). Since the presence of reoviruses
and rotaviruses was expected in the intestine of chickens,
the filtered material was incubated with chicken reovirus
(ck-reovirus) and chicken rotavirus (ck-rotavirus) antisera
(Charles River SPAFAS, Wilmington, MA, USA) in a 1 : 1 : 1
ratio for 60 min at 37  C to neutralize the respective viruses.
One millilitre of the final material was used for passage in
cell cultures (MDCK, DF1, QM7, Vero, LMH and Sf9)
propagated in T25 cell culture flasks grown to 80 % conflu-
ency and incubated for 5 days. The inoculated cells were
examined daily for the presence of a cytopathic effect (CPE)
as compared to appropriate negative control cells. The cell
culture supernatant was collected following centrifugation
at 2000 g for 10 min. One millilitre from each passage of
each cell line was used for a subsequent passage up to pas-
sage 4. Cell cultures grown in 24-well tissue culture plates
were inoculated with a 1 : 100 dilution of the material
obtained from passage 4. At several time points after inocu-
lation (24, 48, 72, 96 h), cells were fixed with ethanol, air
dried, incubated with a 1 : 300 dilution of rab-anti-CkAstV-
521
gut serum and a 1 : 300 dilution of goat anti-rabbit FITC-
conjugated antibodies (Jackson Immunoresearch, West
Grove, PA, USA), and then overlaid with 50 µl of an anti-
fading solution containing 1.25 % (w/v) 1,4-diazabicyclo
[2.2.2]octane (DABCO, Sigma-Aldrich, St Louis, MO, USA)
in PBS. Uninfected cells were used as negative controls. The
immunofluorescence was evaluated using a Carl Zeiss Axio-
vert 40 CFL inverted microscope.
The CkAstV (CkAstV-cc) was evaluated for the presence of
extraneous viral agents by PCRs and RT-PCRs specific for
avian reovirus, avian rotavirus, infectious bursal disease
virus, Newcastle disease virus, avian encephalomyelitis
virus, infectious bronchitis virus, avian adenovirus, reticu-
loendotheliosis virus, chicken anaemia virus, Marek’s dis-
ease virus, infectious laryngotracheitis virus and fowlpox
virus, with appropriate positive controls being employed for
each virus. The investigations were performed at the diag-
nostic virology laboratory at the Poultry Diagnostic and
Research Center (College of Veterinary Medicine, Univer-
sity of Georgia, Athens, GA, USA) using primer pairs
designed to detect a broad range of subtypes within each
virus analysed. In addition, RT-PCR previously described
for the amplification of ANV-1, ANV-2 and CkAstV or
PCR for a chicken parvovirus were conducted as previously
described [25].
A fifth passage of CkAstV-cc in a T175 tissue culture flask
containing LMH cells was frozen at 80  C and thawed,
and then centrifuged at 2000 g for 10 min. The supernatant
was filtered through a 450 nm syringe filter, aliquoted and
stored at 80  C, and served as inoculation material for sub-
sequent experiments.
Titration and growth kinetics of virus
One hundred microlitres of 10-fold serially diluted virus
was added into each of four wells of a 96-well
tissue culture plate, along with 100 µl of the LMH cell sus-
pension. The 96-well tissue-culture plate was incubated for
3 days at 39  C. The tissue cultures were evaluated by immu-
nofluorescence for the detection of specific viruses as
described above. The viral titre (TCID50 ml 1) was calcu-
lated using the method of Reed and Muench [39].
The replication kinetics were evaluated in LMH cells grown
in multiple 24-well tissue culture plates and infected with
the virus at an m.o.i. of 0.01. To this end, the cells were
infected with 250 µl of virus-containing medium and incu-
bated for 60 min at 39  C. Next, the cells were rinsed once
with serum-containing medium and finally overlaid with
1 ml cell culture medium. At several time points during
incubation, the virus-containing supernatant was taken
from one plate, and 1 ml of cell culture medium was added.
Then the cell culture plates were frozen and thawed three
times. Both the cell culture supernatants and the medium
obtained after the freeze/thaw cycles were stored at 80  C,
and the TCID50 for each sample was determined for the
ratio between the cell-associated virus and the virus released
into the supernatant.
 Kang et al., Journal of General Virology 2018;99:512–524
Virus neutralization experiments were performed in LMH
cells. One hundred microlitres of 10-fold serial dilutions of
the supernatant down to 10 8 was incubated for 60 min at
37  C in an alpha virus neutralization experiment with
either the rab-anti-CkAstV-gut serum or with serum
obtained from the same rabbit prior to immunization. Each
sample was inoculated into a T25 tissue culture flask con-
taining LMH cells and incubated for 5 days followed by two
subsequent passages. Using the rab-anti-CkAstV-gut serum,
an indirect immunofluorescence assay was subsequently
performed. In addition, serial twofold dilutions of rab-anti-
CkAstV-gut serum or serum obtained from the same rabbit
prior to immunization were made down to the 2 15 dilution
and incubated with 100 TCID50 CkAstV-cc for 60 min at
37  C in a beta virus neutralization assay.
Animal experiments
All chicken experiments described were performed in
HEPA-filtered Horsfall–Bauer isolation units under forced
air positive pressure. On arrival from a commercial hatch-
ery, the birds were weighed and allocated evenly by weight
into each experiment group and housed in isolators with ad
libitum access to feed and water. On necropsy, the chickens
were humanely euthanized in accordance with the policies
of the Institutional Animal Care and Use Committee (AUP
A2010 6-098 and A2011 11-031-R2). Each bird was
weighed, and the duodenal loop was harvested, fixed in neu-
tral buffered formalin and examined for microscopic lesions
by in situ hybridization.
A short-term study was performed to monitor the replica-
tion of the virus isolated in cell culture (CkAstV-cc), weight
gain retardation and microscopic lesions in the duodenal
loop during earlier time periods. Forty 1-day-old, chickens
were inoculated orally with 300 µl of CkAstV at 106.3
TCID50 ml 1. An equal number of hatch mates were inocu-
lated with 300 µl of cell culture media to serve as controls.
Five chickens from each group were randomly selected at 6,
12, 18, 24, 48, 72, 96 and 120 h p.i.
To determine the pathogenic potential of the fifth passage
of the CkAstV-cc, 38 1-day-old chickens were distributed
into 3 experimental groups: CkAstV, RSS and negative con-
trol. The CkAstV (n=14) group was inoculated orally with
300 µl of CkAstV-cc and the chickens in the negative con-
trol group (n=15) were inoculated with the LMH cell culture
media. The RSS (n=9) group was inoculated orally with
300 µl of the RSS intestine homogenate [24, 25] to serve as
the RSS positive control. The titre of CkAstV in the RSS
material was 105.0 TCID50 ml 1. Five birds at 5 days p.i. and
all of the remaining birds at 12 days p.i. were examined. At
each time point, chickens were evaluated as described
above.
Bird-to-bird passage experiments in chickens I
and II
Initially, 1-day-old commercial broiler chickens were inocu-
lated orally with CkAstV-cc as described above. On day
5 p.i., the small intestines of each group were harvested and
522
processed using the same method that was employed for
RSS material [24]. Filtered homogenates from CkAstV-cc
chicken passages 1 through 5 were inoculated orally into
chickens to make the next passage (bird-to-bird passage
experiment I). The hatch mates at each passage were inocu-
lated with cell culture media to serve as negative controls.
The virus recovered from the fifth chicken passage was
identified as CkAstV-Ckp5.
To determine whether the serial passage of unfiltered intes-
tinal material from the animal would induce differences in
RSS reproduction, another set of passage experiments was
conducted that included unfiltered intestinal materials
from the control and virus-infected groups in parallel (pas-
sage II).
RT-PCR
Homogenates from the duodenal loops of each group were
incubated at 95  C for 10 min and subsequently used for
RNA extraction with the Qiagen RNeasy Plus mini kit (Qia-
gen, Valencia, CA, USA). The SuperScript One-Step RT-
PCR with PlatinumTaq DNA polymerase (Invitrogen,
Carlsbad, CA, USA) was used following the manufacturer’s
instructions. Primers were designed to amplify a 428 nt
cDNA of the capsid protein coding region using oligonu-
cleotides ASTCAP-DIAFP (GATAAGGCTG GGCCGCA-
GAAGAAGAGG) and ASTCAP-DIARP (ACAAA
TTTAACAACACACC GCTG), which were delineated
from the CkAstV sequence described in a previous study
(NCBI GenBank accession number JF414802). The ampli-
fied products were separated on a 1.5 % agarose gel. For
each animal experiment, RT-PCR was performed in 1-day-
old hatchmates to confirm the absence of CkAstV RNA and
in birds following each experiment to monitor the presence
or absence of the virus.
Microscopic lesions and in situ hybridization (ISH)
A portion of the descending and ascending loops of the
duodenum were prepared for microscopic evaluation and
ISH as described previously [25]. The crypt of Lieberkühn
in the duodenum was evaluated using H and E-stained
slides, and the number of cystic lesions per section was
counted in both loops of the duodenal sections. The RNA
probe for the CkAstV was generated and the ISH was con-
ducted as previously described [25]. The locations of
the ISH signals and microscopic lesions were compared for
each tissue using consecutively cut tissue sections.
Genome sequences
For the determination of the full-length sequences before
and after passage in chickens, both the cell culture superna-
tant from the infected LMH cells (CkAstV-cc) and filtered
intestinal material obtained after five consecutive passages
of CkAstV in chickens (CkAstV-Ckp5) were used. The cell
culture supernatant (CkAstV-cc) and the RSS intestinal
material (CkAstV-Ckp5) were centrifuged, filtered and
chloroform-treated, as described above, and used for RNA
isolation using the High Pure RNA isolation kit (Roche,
Diagnostics GmbH, Mannheim, Germany).
 Kang et al., Journal of General Virology 2018;99:512–524
On the basis of the sequence for a new CkAstV obtained
directly from intestine samples of RSS-affected chickens
(CkAstV-gut) (NCBI GenBank accession number
JF414802), several pairs of oligonucleotides were delineated
to amplify approximately 800 bp cDNA fragments with
each pair. The primer sequences can be obtained from the
corresponding author upon request. RT-PCR was per-
formed using the SuperScript One-Step RT-PCR with Plati-
num Taq (Invitrogen, Carlsbad, CA, USA). The extreme
5¢ end of the virus genome was determined using the 5¢
RACE System, version 2.0 (Invitrogen, Carlsbad, CA, USA)
as previously described [12]. The cDNA fragments obtained
were separated on a 1 % agarose gel, purified using the QIA-
quick gel extraction kit (Qiagen, Hilden, Germany) and
cloned into the pCR2.1 Topo TA plasmid using the Topo
TA cloning kit (Invitrogen). Three recombinant plasmids
for each cDNA fragment were sequenced using the BigDye
Terminator v3.1 cycle sequencing kit (Applied Biosystems,
Foster City, CA, USA) in both directions to obtain sixfold
coverage for each nucleotide.
The full-length genomic nucleotide sequences of the
CkAstV isolate (CkAstV-cc) and the five consecutive pas-
sages of CkAstV in chickens (CkAstV-Ckp5) were deposited
in GenBank under accession numbers KX397575 and
KX397576, respectively.
Multiple alignment and sequence analysis
Sequence data were analysed using the DNAStar Lasergene
8 software package (DNASTAR Inc, Madison, WI, USA) for
multiple alignments and in silico translation.
Data analysis
The mean body weights between groups were compared by
SigmaStat (SigmaStat for Windows, Jandel Scientific, San
Rafael, CA, USA). The differences between two groups were
examined by t-test, and the results for more than two
groups were analysed using one-way analysis of variance
(ANOVA) followed by Fisher’s least significant
difference (LSD) test for all pairwise multiple comparisons.
Funding information
This study was supported by the Office of Vice President for Research
(University of Georgia), the Georgia Poultry Federation, the Georgia
Research Alliance and the US Poultry and Egg Association, project
#627.
Conflicts of interest
The authors declare that there are no conflicts of interest.
Ethical statement
All animal studies were approved by the University of Georgia Institu-
tional Animal Care and Use Committee (AUP A2010 6-098 and A2011
11-031-R2).
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