Genetic and pathogenic characteristics of newly emerging avian reovirus from

infected chickens with clinical arthritis in China

Xiaohui Zhang,∗ Xiangdong Lei,∗ Lifang Ma,† Jiaxin Wu,∗ and Endong Bao∗,†,1
∗
College of Veterinary Medicine, Nanjing Agricultural University, Nanjing 210095, China; and † Tianjin Ruipu
Biotechnology Co. Ltd., Tianjin 300350, China

ABSTRACT In recent years, emerging avian reovirus
(ARV) strains causing viral arthritis have become a
challenge to the worldwide chicken industry, and were
responsible for significant economic losses. In this study,
we characterized emerging variant ARV strains and
examined their genetic relationship and pathogenicity
variation with reference strains. A total of 18 emerging
variant ARV strains were isolated from tendon and cap-
sular synovial fluid of broiler chickens with clinical cases
of arthritis/tenosynovitis at commercial farms in China.
Comparative analysis based on σ C sequence showed
that 4/18 isolates were in the same cluster (Cluster 1) as
vaccine strains (S1133), whereas 14 of 18 isolates were
Key words: avian reovirus, isolation and identification, pathogenicity, chicken
in Clusters 2, 3, and 6. The field isolates shared a rather
low identity (38.1 to 81.9%) with S1133 in Cluster 1,
especially for those from Cluster 6 (38.1 to 67.2%). A
higher ARV isolation rate was observed in chicken em-
bryos (47/61) compared to cell culture (37/61) through
PCR with a detection primer. A total of 3 isolates were
selected to infect specific-pathogen-free (SPF) chickens,
showing that the tested isolates, especially that from
Cluster 6, displayed greater pathogenicity than S1133
strain, characterized by higher incidence. These find-
ings suggest that the virulence of Chinese ARVs has
been increasing rapidly in recent years, and the vaccine
need to be updated correspondingly.
2019 Poultry Science 98:5321–5329
http://dx.doi.org/10.3382/ps/pez319

INTRODUCTION $23,000/per affected flock (28,000 birds/flock) in

Avian reovirus (ARV) is a widespread poultry
pathogen that was first described and isolated in 1959
as the pathogenic agent responsible for tenosynovitis
in young chickens (Olson, 1959). ARV has been
taxonomically classified as a member of the family
Reoviridae, subfamily Spinareovirinae under the genus
Orthoreovirus, according to the viral genome segment
S1 encoding sigma C (σ C) sequence (Attoui, 2011).
Moreover, ARV infections often cause enteric disease,
myocarditis, hepatitis, runting-stunting syndrome,
and immunosuppression in young broilers, with se-
vere arthritis representing the most common clinical
symptom (Gershowitz and Wooley, 1973; Sterner
et al., 1989; Van der Heide, 2000; Songserm et al.,
2002; Clarke and Tyler, 2003; Davis et al., 2013). ARV
infections in domestic poultry result in a general lack
of performance, poor feed conversion, low uniformity of
the affected birds, and secondary infections from other
viruses or bacteria (Van der Heide, 2000; Jones, 2008).
In addition, the cost attributed to ARV infections
in broiler chickens was reported to be approximately

C 2019 Poultry Science Association Inc.
Received December 14, 2018.
Accepted May 23, 2019.
1
Corresponding author: b endong@njau.edu.cn
Pennsylvania, USA (Lu et al., 2015), which is a major
economic loss.
ARV is an icosahedral non-enveloped double-
stranded RNA virus divided into 10 genome segments
based on their electrophoretic mobility (Spandidos and
Graham, 1976; Van der Heide, 2000). The segmented
genome encodes 12 primary translation products, of
which the Sigma C protein (σ C) coded by the S1 seg-
ment is the immunologically dominant structural pro-
tein involved in cell attachment (Ayalew et al., 2017;
Sellers, 2016). Substantial research has been conducted
to characterize the σ C protein at both the molecular
and nucleotide sequence level (Shapouri et al., 1995),
and 6 genotypes have been described for ARV based on
the classification established by Kant et al. (2003) and
Palomino-Tapia et al. (2018).
Although several conventional ARV vaccines have
been used in layer and broiler breeders, an increasing
prevalence of novel ARV strains with antigenic vari-
ants and stronger virulence have been reported in recent
years (Dandár et al., 2013; Lu et al., 2015; Nuñez et al.,
2015; Yi and Lu, 2015; Zhong et al., 2016), suggesting
vaccine failure. Moreover, no recognized ARV vaccina-
tions have been practiced in the commercial poultry
industry, and ARV infections in broiler chickens have
been increasingly detected in China since 2013 and con-
tinue to be observed (Zhong et al., 2016). Furthermore,

5321
5322 ZHANG ET AL.
from 2013 to 2016, 7 cases (flocks) were confirmed to
be ARV infections by the laboratory of Tianjin Ruipu
Biotechnology Co. Ltd., and 61 positive samples were
identified in 2017 (data not published). Most of the
ARV-positive cases were from sick broilers, which ex-
hibited severe arthritis that involved multiple joints
and tendons of the legs, including the hock and foot
pads.
To date, systematic investigation into the level of
genomic variation and virulence evolution of ARVs in
China is scarce and is limited to 1 publication that ana-
lyzed 11 isolates obtained only in northern China, and it
is unclear when the tested strains were isolated (Zhong
et al., 2016). We hypothesized that the clinical cases
of viral arthritis detected by us in China were closely
associated with the presence of more variant ARVs.
Therefore, this paper describes our recent research
findings regarding the molecular characterization and
pathogenicity of these ARV field strains/variants
from the middle and eastern regions of China in
2017.
MATERIALS AND METHODS
Source and Gross Findings of Poultry
Epidemic Materials
From January to December in 2017, 151 samples, in-
cluding tendon and capsular synovial fluid, from dead
chickens were collected from 151 henneries located in
the middle and eastern regions of China. The sam-
pled chickens primarily exhibited varying degrees of vi-
ral arthritis and were suspected to be infected with
ARV. The tendon tissues and the tarsal joint swabs
were aseptically mixed with 2 mL normal saline and
ground using a high-flux tissue grinder. The super-
natants were obtained after centrifugation at 10,000 ×
g for 5 min, and stored at −80◦ C until further
use.
Through the amplification of the S1 segment of the
σ C gene using RT-PCR and the detection primers, a to-
tal of 61 samples were confirmed to be positive for ARV
infection. Data showed that more infections occurred
during the summer and autumn, the positive samples
were mainly distributed in Shanxi, Inner Mongolia, Fu-
jian, Zhejiang, and Anhui, white-feathered broiler was
the breed most prevalently infected with ARV, and
ARV infection primarily existed in the poultry from 4
to 12 wk old.
Meanwhile, using sequencing primers, we further
identified 18 positive samples out of 68 (including 7
positive samples stored in Ruipu from January 2013 to
December 2016), which were termed HeN130728,
SD150703, RPT-74, HeN170407, LN160607-1,
SD150716, GX150816, SD150806, LN170725-4 P1,
LN170725-4 P2, JS170705-1, JS170730, LN170725-5,
HeN170730-1, HeN170808-1, HeN170730-2, SD170920-
2, and LN170919, respectively.
Cells, Specific-Pathogen-Free Chicken
Embryos, and Bacteria
LMH cells (Leghorn Male Hepatoma; chicken
liver hepatocellular carcinoma cell line) and CEL cells
(primary chick embryo hepatocytes) were provided by
the Animal Disease Detection Center (Tianjin Ruipu
Biotechnology Co., Ltd., Tianjin China), and cul-
tured in DMEM-F12 medium containing 10% fetal
bovine serum and 1% (v/v) penicillin/streptomycin so-
lution. Specific-pathogen-free (SPF) chicken embryos
and chickens were purchased from Beijing Merial Vital
Laboratory Animal Technology Co., Ltd. We obtained
E.coli DH5α-sensitive cells from Dalian TaKaRa Bio
Co., Ltd.
Viral PCR Detection and Genome
Sequencing
PCR: the tendon tissues of chickens suspected to
be infected with ARV were mixed with 2 mL saline,
crushed with a high-throughput tissue fragmenter, and
centrifuged at 10,000 × g to obtain the supernatant.
Total RNA was extracted from the supernatant us-
ing the nucleic acid extraction kit (TaKaRa, Beijing,
China). Reverse transcription was carried out using the
extracted RNA as a template. PCR amplification was
conducted in a 50 μL system and examined by agarose
gel electrophoresis.
Cloning and sequencing of the viral S1 gene: the PCR
products obtained from agarose gel electrophoresis were
retrieved and purified. The PCR products were con-
nected with a PMD18-T vector and ligation products
were transformed into DH5α competent cells. Then,
400 μL liquid LB medium was added and incubated
at 37◦ C for 1 h. Next, 100 μL of the culture was
spread onto an LB plate containing 0.1% (v/v) ampi-
cillin (100 mg/mL) and incubated at 37◦ C overnight.
Bacterial sequencing PCR was performed. The posi-
tive clones were selected from each PCR product for
sequencing at Beijing Genomics Institute (BGI, China).
For detection (297 bp) and sequencing (951 bp), 2 pairs
of primers were designed and synthesized according to
the S1 gene sequence of the classical strain, S1133, at
BGI as follows:
detection primer: ARV1-F:ATGCTGCGTATGCC
TCCCG;
ARV2-R:TCAAACGTCGTATGGCGGAG.
sequencing primer: P1Mod-F: TGATACYSTCVTT
RACTTCGA;
P2Mod-R: ACGGCGCCRCACCTTARGT.
ARV Isolation and Identification
Cell infection: the supernatant of the grinding fluid
described above was filtered through a 0.22 μm filter
and 500 μL of the supernatant was added to a T-25
 LATEST FEATURES OF AVIAN REOVIRUS IN CHINA 5323

cm2 cell bottle where a monolayer of LMH or CEL cells
had been cultured in advance. The cells were incubated
at 37◦ C with 5% CO2 for 30 min. The liquid contain-
ing the virus was discarded and DMEM/F12 containing
2% fetal bovine serum was added to maintain cell vi-
ability. The status of the cells was observed daily for
5 D. The cells were harvested and cryopreserved when
lesions were present in 75% cells, or continued to be cul-
tured blindly to the third generation if no lesions were
present.
Fluorescent antibody testing: LMH cells were used
to perform fluorescent antibody tests. A monoclonal
antibody against σ C, which was produced in the lab-
oratory of Ruipu, was used as the primary antibody,
and fluorescein isothiocyanate-conjugated anti-mouse
IgG (Sigma-Aldrich, St. Louis, MO) was used as the
secondary antibody. The fluorescence signal was ob-
served under a fluorescent inverted microscope (Olym-
pus, Tokyo, Japan).
The products of the cell culture and virus-containing
liquid from the chicken embryos were further identified
by RT-PCR with detection primer and gel electrophore-
sis according to the methods described above.
Pathogenicity Testing on Chicken Embryos
and SPF Chickens
Chicken embryo infection: The supernatant of the
grinding fluid obtained from ARV-positive tissues was
filtered through a 0.22 micron filter. SPF chicken
embryos aged approximately 9 to 10 D old were
inoculated with 0.2 mL of the supernatant through
the chorioallantoic membrane, and incubated in an
incubator at 37◦ C. The chicken embryos were observed
daily for 120 h, and any dead chicken embryos found
within 24 h post-inoculation (p.i.) were discarded.
The allantoic fluid and chorioallantoic membrane were
collected from both the live and dead chicken embryos
after 24 h p.i. The supernatant was collected after
grinding and centrifuging the samples as described
above, and stored at −80◦ C until future use.
SPF chicken infection: The TCID50 of 3 ARV isolates
were determined in LMH cells using the Reed−Muench
method (Wu et al., 2017). Virus-containing liquid col-
lected from cell cultures was inoculated into 28-day-old
SPF chickens using 0.1 mL per chook by footpad punc-
ture. Each strain was administered to 20 chickens fed
in separate isolators. Morbidity and mortality were ob-
served every 2 D for 14 D. On day 7 post-infection, 2
chickens from each group were humanely sacrificed by
decapitation and the footpads were dissected to observe
any pathological changes. The details are presented in
Table 1.
All experimental protocols concerning the handling
of chickens were performed in accordance with the
guidelines approved by the Institutional Animal Care
and Use Committee (IACUC), and the requirements
of the experimental animal ethics guidelines of the
Ethics Committee at laboratory animal center of Nan-
jing Agricultural University, and every effort was made
to minimize any stress experienced by the animals.
Histopathological Examination
Tendon, liver, spleen, lung, and trachea samples from
the tested chickens were collected and fixed in 10% for-
malin. The fixed specimens were embedded in paraffin,
after which serial sections (5-μm-thick) were cut from
the paraffin-embedded blocks and mounted onto glass
slides. Following dewaxing with xylene and hydration
with different concentrations of alcohol, the sections
were stained with hematoxylin and eosin (H&E) for

Table 1. The clinical manifestation of ARV-infected chickens.

Clinical manifestation

Genotype
Strain clusters 1D 3D 5D 7D 10 D 12 D 14 D
Blank control / Normal Normal Normal Normal Normal Normal Normal
S1133 1 Foot and Foot and joint Minor joint swelling; Most clinical Minor foot Minor foot No obvious
joint swelling swelling in half foot swelling and signs were swelling in 8 swelling in 5 clinical signs
in 4 chickens of the chickens ulceration in half of relieved chickens chickens
the chickens
RPT-74 1 Joint swelling Right foot Right foot swelling Right foot Foot and Foot swelling Foot swelling
in 1 chicken swelling in most in most chickens, swelling in most joint swelling in 8 chickens in 5 chickens
chickens, joint joint swelling in chickens, joint in 10
swelling in some some chickens swelling and chickens
chickens hemorrhage, and
foot scab in
some chickens
GX150816 3 Normal Minor foot Minor foot swelling Foot swelling in Minor foot Minor foot No obvious
swelling in 2 in 7 chickens 10 chickens swelling in 9 swelling in 5 clinical signs
chickens chickens chickens
SD150806 6 Foot swelling Foot swelling in Foot swelling in Foot swelling in Minor foot Minor foot Minor foot
in 2 chickens most chickens; most chickens, most chickens, swelling in 11 swelling in 9 swelling in 5
paralysis and ulceration in few foot scabs in chickens chickens chickens
lameness in 2 chicken, paralysis some chickens
chickens, some and lameness in 4
joint swelling chickens
5324 ZHANG ET AL.
5 min and 1 min, respectively, and sealed with neutral
balsam. The slices were examined by light microscopy
(CX41; Olympus, Tokyo, Japan).
RESULTS
Genotyping Clusters of ARV Field Strains in
China
Construction of phylogenetic trees using the
MEGA6.0 software and an analysis of the conserva-
tion of the σ C gene S1 segment sequences revealed
that the 18 ARV field strains isolated in this study
were grouped into 4 genotype clusters, or genotypes
(Figure 1). Out of the 18 field strains in these clusters,
the strains isolated from 2013 to 2016 were primarily
located in Clusters 1 to 3, i.e., in addition to RPT-
74, HeN130728 from 2013 and SD150703 from 2015
in cluster 1, the same as the standard ARV commer-
cial vaccine strains (S1133, 1733, and 2048); SD150716
and GX150816 from 2015 were located in Cluster 3;
and LN160607-1 from 2016 was located in Cluster 2.
HeN170407 from 2017 belonged to Cluster 2. In particu-
lar, 11 ARV field strains (SD170920-2, LN170725-4 P1,
LN170725-4 P2, LN170725-5, HeN170808-1, LN170919,
JS170705-1, HeN170730-1, JS170730, and HeN170730-
2 were all from 2017; only SD150806 was from 2015)
were classified to genotype Cluster 6, or genotype 6 in
this study. These strains were novel and distinct from
all previously published ARV reference strains.
Homology Analysis of Gene Sequences
The homology of the sequencing results was an-
alyzed using MegAlign software (Madison, WI, de-
tails not shown). The comparison results among the
present strains demonstrated that, compared to with
S1133 strains, the homology of HeN130728, SD150703,
RPT-74, and HeN170407 in Cluster 1 were 81.9%,
71.5%, 65%, and 63.3%, respectively; the homology
of LN160607-1 in Cluster 2 was 46.3%; the homol-
ogy of SD150716 and GX150816 in Cluster 3 were
48.4% and 48.4%, respectively; and the homology of
SD150806, LN170725-4 P1, LN170725-4 P2, JS170705-
1, JS170730, LN170725-5, HeN170730-1, HeN170808-1,
HeN170730-2, SD170920-2, and LN170919 in Cluster 6
was 46.8%, 38.1%, 39.5%, 48.6%, 48.5%, 49.8%, 49.6%,
67.2%, 48.9%, 48.5%, 47.9%, 49.6%, and 48.7%, respec-
tively. Compared within the same group, the homology
between the isolates in Cluster 1 was 63.9 to 99.4%; the
homology between the isolates in Cluster 6 was 42 to
99.8%.
Viral Isolation and Identification
The virus was inoculated into LMH and CEL cells
with the supernatant from the grinding fluid of the
samples initially identified to be positive, and the
pathogenic features of both of the tested cells were ob-
served. Normal LMH cells were star-like or polygonal,
with a homogeneous cytoplasm (Figure 2A-a). Follow-
ing infection with ARV, giant or “bloom-like”, cyto-
pathic effects (CPEs) were observed in the LMH cell
cultures (Figure 2A-b and c). The CPE varied dur-
ing the different incubation periods. The early CPE
(Figure 2A-b) was characterized by an cellular initial
aggregation at 24 h p.i.; the late CPE was observed
3 to 5 D p.i. within 2 to 3 serial cell passages, and
are characterized by the formation of syncytium clus-
ters suspended in the medium (Figure 2A-c). Primary
chicken embryo hepatocytes (CEL cells) were used to
further identify the CPE of ARV infection and similar
appearances, especially syncytial suspension, were also
observed in the medium (data not shown).
Preliminary identification with immunostaining
demonstrated that the negative control cells (LMH
normal cell cultures) exhibit no fluorescent signals
(Figure 2B-a), whereas detectable fluorescent signals
were observed in the ARV-infected LMH cells with
pathological damage (Figure 2B-b). RT-PCR identifi-
cation for the ARV virus was further performed, and
the result of gel electrophoresis showed that only 37
ARV strains were isolated (Figure 2C), compared with
the initial 61 positive samples.
The Pathogenicity of ARV Isolates to the
SPF Chicken Embryos
After 72 h of infection, ARV induced hyperemia and
hemorrhage of the epidermal vessels of the SPF chicken
embryos. From 7 to 8 D p.i., ARV infection resulted
in the death of SPF chicken embryos, and the entire
embryo body appeared dark red due to systemic hem-
orrhage, which was characteristic of ARV infections
(Figure 3A). RT-PCR identification for the ARV virus
showed that 47 ARV strains were isolated, compared
with the initial 61 positive samples.
Pathogenicity of Representative ARV
Isolates in SPF Chickens
To investigate the pathogenicity of the ARV field
strains in chickens, 3 ARV isolates (RPT-74, GX150816,
and SD150806) and the classical strain, S1133, were se-
lected in the present study. First, 4 isolates were in-
oculated into LMH cells to determine their TCID50,
which were 106.2 /0.1 mL, 106.1 /0.1 mL, 106.1 /0.1 mL,
and 106.5 /0.1 mL, respectively. Next, 4 ARV strains
were inoculated into 28-day-old SPF chickens to test
their pathogenicity on chickens.
ARV-infected chicken flocks suffered from severe
lameness and splay-leg due to tenosynovitis spanning
the intertarsal joints and the plantar metatarsal
region. The presence of major gross pathologic lesions
included marked swelling, edema, hemorrhage in the
tendons and tendon sheaths, and pale yellow mucus
 LATEST FEATURES OF AVIAN REOVIRUS IN CHINA 5325

Figure 1. Genotyping clusters of 18 ARV isolates phylogenetic trees was constructed using MEGA6.0 software and based on the conservation
of the σ C gene S1 segment sequences. Branch lengths are proportional to the evolutionary distances between sequences. The scales represent
nucleotide substitutions per position.

was observed in foot pads of sick chickens (Figure 3B).
Disease onset typically occurred 1 to 3 D p.i. in broiler
flocks. Microscopically (Figure 3C), inflammation was
observed in the tendons and liver, and the predominant
cell types were lymphocytes and plasma cells, which
were distributed in the interstitial tissue of the tendons
and portal area of the affected livers. Disordered
tendons and a reduction of lymphocytes in the spleen
were also observed in the infected chickens. There was
no obvious pathological injury observed in the lung
or trachea (Data not shown). The morbidity of the
ARV-infected birds in a group was approximately 50%
5326 ZHANG ET AL.

Figure 2. ARV isolation and identification. A: LMH (Leghorn Male Hepatoma) cell lesion following ARV inoculation (× 100). a. Normal
LMH cells. b. During the early stage of infection, the LMH cells began to aggregate. c. During the late stage of infection, syncytial bodies formed,
and cellular clusters were suspended in the medium. B: Fluorescence detection of ARV in LMH cells (× 100). a. No signal was detected in the
normal LMH cells. b. Obvious fluorescence was observed in the ARV-infected cells. C: The RT-PCR results of ARV-infected LMH cells. Lane M
is the DNA marker; Lanes 1 to 37 are the isolated strains; “+” represents a positive control and was approximately 297 bp in size.

for S1133 and GX150816, and almost 100% for RPT-74
and SD150806, respectively. Moreover, the severity of
the symptoms in chickens infected with the SD150806
strain was higher than that of the chickens infected with
the RPT-74 strain. Additionally, the mortality in all of
the tested cases was 0%. In general, compared with the
S1133 strains, the GX150816 strains were equivalently
virulent, the RPT-74 strains were stronger, and the
SD150806 strains were the most virulent (Table 1).
DISCUSSION
Significant economic losses in poultry husbandry due
to ARV infections emphasize the importance of con-
tinuously studying the prevalence, genetic characteris-
tics, and evolution of pathogenicity of the newly emerg-
ing ARV isolates (Lu et al., 2015). However, limited
information for emerging isolates in China has been
found, compared to the abundant genetic data for ARV
strains from other countries (Yi and Lu, 2015). Al-
though ARV strains have been reported to cause dis-
ease (e.g., viral arthritis, tenosynovitis, and malabsorp-
tion syndrome) (Bányai et al., 2011), not all clinical
symptoms frequently occur in chickens infected with
these viruses. All samples in the present study were
obtained from chickens from the middle and eastern re-
gions of China (e.g., Inner Mongolia, Liaoning, Hebei,
Shandong, Shanxi, Jiangsu, Zhejiang, Anhui, Hunan,
Jiangxi, and Fujian) which showed obvious symptoms
of avian arthritis. This study greatly improved the
probability to obtain valuable isolates in order to under-
stand the current situation regarding the pathogenic-
ity and evolutionary characteristics of ARV strains in
China.
The σ C protein is the most variable protein in ARV
and recognized as the only viral protein conferring viral
type-specific neutralizing immunity (Lu et al., 2015;
Sellers, 2016; Ayalew et al., 2017; Palomino-Tapia
et al., 2018. Consequently, we focused our molecular
characterization on partial sequences of the σ C gene.
It should be noted that the S1 gene region encoding
σ C sequence is highly variable and some false negative
 LATEST FEATURES OF AVIAN REOVIRUS IN CHINA 5327

Figure 3. The pathogenicity of ARV isolates on specific-pathogen-free (SPF) chicken embryos and chickens A: SPF chicken embryo disease
following ARV inoculation. a. Normal chicken embryos. b. chicken embryos infected with ARV for 72 h. c. chicken embryos infected with ARV
for 7 D. B: Clinical symptoms and necropsy lesions of chickens with virulent ARV infection. a. Control chickens. b. ARV-infected chickens with
fluffy feathers and depression. c. ARV-infected chickens were paralytic and could not stand up. d. Swollen joints in ARV-infected chickens (∗).
e. The feet of the control chickens (∗). f. The right footpad of ARV-infected chicken was swollen (∗). g. Swelling and crusting were found on the
backs of the ARV-infected chicken feet (∗). h. Pale yellow mucus was observed in the dissected footpads of the infected chickens. C. Pathological
changes to the tendons, liver, and spleen of the tested chickens. H.E. staining: (× 400). a. Control tendon. b. ARV-infection tendon characterized
by disordered ligament fibers, interstitial inflammatory cell infiltration, aggregation of macrophage foci (∗). c. Control liver. d. ARV-infected
livers were characterized by the focal infiltration of inflammatory cells (primarily monocytes, including plasma cells) around the central vein (∗).
e. Control spleen. f. ARV-infected spleens characterized by blurred boundaries between the red pulp and white pulp, reduction of lymphocytes,
and visible plasma cells (∗).

results could occur (Tang and Lu, 2016). According to
Kant et al. (2003), ARV isolates from Europe during
the years 1980 to 2000 can be divided into 5 genotyping
clusters. Then in a study analyzing 17 isolates of ARV,
sigma C was found to evolve into 6 distinct lineages
(Liu et al., 2003). The genetic variation of sigma C
sequences was revealed up to 50% diversity among
28 Israeli isolates (Goldenberg et al., 2010), similar to
the distance of ARV from turkey reovirus (Day et al.,
2007). These studies implied that the variation of ARV
was accelerating, characterized by the appearance of
Clusters 6. Our results of the phylogenetic analysis
indicated that all of the isolated ARV strains were
genetically different and grouped into 4 genotype
5328 ZHANG ET AL.
clusters of 6. A total of 14 of the 18 isolates, in Clusters
2, 3, and 6, were field variants and distinct from the
standard ARV vaccine strains (S1133, 1733, and 2408)
grouped in Cluster 1. More importantly, 11 out of the
14 field strains were in Cluster 6 in this study, and 10
out of 11 strains were isolated in 2017. Compared with
Cluster 1, the new isolates in Cluster 6 exhibited rather
high genetic diversity (up to a difference of 32.8 to
61.9%). The remaining field isolates (4/18) were clas-
sified into Cluster 1, but also exhibited a low identity
with commercial vaccine viruses (63.3 to 81.9%). Re-
search into the infectious bronchitis virus, another RNA
poultry virus, has found that low cross-protectivity
occurred when amino acid differences between the
challenge and vaccine strains reached or exceeded 5%
(Cavanagh, 2007). Commercial or autogenous vaccines
prepared from ARV sharing a low level of identity
with a prevalent field strain even grouped in the
same cluster, cannot confer sufficient cross-protection
(Palomino-Tapia et al., 2018). This indicates that the
variants characterized in this study, including those
in Cluster 1, Cluster 6, and other clusters, can easily
evade the immunity generated by commercial vaccines
(S1133 strain). This likely represents the recently
observed immune failure associated with traditional
vaccine administration and substantially increases the
difficulty and complexity of effective ARV infection
prevention and control. It may be more effective to
produce a vaccine based on local representative viruses
or their sigma C as a subunit vaccine, for each separate
phylogenetic cluster (Goldenberg et al., 2010).
ARV can be isolated and cultured in various avian
cells or chicken embryos (Zhong et al., 2016). In the
present study, both LMH and CEL cells were used to
isolate and identify ARV, and CPEs were detected in 2
cell culture passages following infection with the ARV
isolates. During the initial stage of ARV infection,
the 2 types of tested monolayer cells fused gradually
into several larger cell clusters, around which there
was an open area without any cells. During the later
stages, the cell clusters formed several syncytium that
were suspended in the culture medium. The CPEs of
LMH infected with ARV were similar to those of the
infected CEL cells. The CPE types were consistent
with the study of Troxler et al. (2013). In consideration
of a mixed infection with Fowl adenovirus in the
study of Troxler et al. (2013), a signal of virulence
evolution for ARV in the present study is the shorten
cell culture passages for CPE emerging. ARV can
also be inoculated into chicken embryos through the
yolk sac or chorioallantoic membrane and replicate
in the infected embryos. In this study, following the
inoculation of ARV into 9 to 10 D old SPF chicken
embryos through the chorioallantoic membrane, the
embryos died at 7 to 8 D p.i. The pathological changes
in the chicken embryos were characterized by the thick-
ened chorioallantoic membrane with an obvious white
fleck in the thickened area, and light red or deep red
chicken embryos caused by hemorrhage. In the present
study, in both the cells and chicken embryos, RT-PCR
with sequencing primers was used to further identify
the isolates. The RT-PCR results with detection
primer also demonstrated that the number of isolates
obtained from the chicken embryos was greater than
that obtained from the cell culture, illustrating that
ARV proliferation was better in the chicken embryos
compared to the cell culture. The reasons maybe
associated with the preference for living tissue of the
ARV or not full adaptation in cells culture in vivo.
The representatives from Clusters 1, 3, and 6 (RPT-
74, GX150816, and SD150806) and the vaccine strain,
S1133, were selected to study viral pathogenicity. Vi-
ral inoculation through the footpads induced typical
symptoms of viral arthritis. The gross pathological le-
sions included marked swelling, edema, hemorrhages,
and serous exudate between the tendons observed in
the present study were similar to those caused by ARV
described in the literature (Lu et al., 2015). Histolog-
ical lesions seen in the present study were also sim-
ilar to those observed in another investigation about
ARV, demonstrating mixed inflammatory infiltrate in
the tendon sheaths and liver (Souza et al., 2018). All
lesions were very similar but with variable intensity be-
cause of different virulent. Compared with the S1133
strains, from the point of incidence and severity, the
GX150816 strains from Cluster 3 were equivalently vir-
ulent, the virulence of RPT-74 from Cluster 1 strains
were stronger, and the SD150806 strains from Cluster
6 possessed the highest level of pathogenicity on the
tested chickens. Despite both belonging to Cluster 1,
the virulence of RPT-74 was greater than that of S1133,
illustrating that alterations to the σ C gene/protein
identity might contribute to changes in ARV virulence
(Shih et al., 2004; Lin et al., 2011; Zhang et al., 2015).
GX150816 strains from Cluster 3 shared equal virulence
with S1133, implying that σ C was not the only factor to
influence ARV virulence. Other ARV proteins (e.g., λB
and σ B) have been found to be involved in the virulence
and neutralization of homologous viruses (Yang et al.,
2010; Tang et al., 2016). SD150806 strains from Cluster
6 shared 46.8% homology with the σ C gene in S1133,
but showed strongest virulence in chicken, indicating
that substantial variation of the gene sequence was re-
sponsible for the explosion of ARV virulence (Jones,
2013; Sellers, 2016).
In conclusion, 18 ARV field strains were successfully
isolated and identified. Further analysis revealed that
the isolated strains, especially from 2017, displayed high
variation of the σ C gene and increased mortality com-
pared to the reference strain, S1133. The findings of this
study suggest that the Chinese ARVs exhibit higher ge-
netic diversity, and its pathogenicity has been increas-
ing in recent years. Therefore, since controlling disease
associated with ARV infection is becoming more dif-
ficult, novel measures are required. The present study
was a systemic basic investigation, did not involve the
antigen variation and mechanism of immunologic es-
cape in novel isolates, which would be a key for the
development of new effective vaccine and arranged into
my future study.
 LATEST FEATURES OF AVIAN REOVIRUS IN CHINA
ACKNOWLEDGMENTS
The present study was funded by the funds for Re-
search and Development from Tianjin Ruipu Biotech-
nology Co. Ltd., China, the National Natural Science
Foundation of China (31672520), the National Natural
Science Foundation Youth Funding Project of China
(31802157), the Postdoctoral Science Foundation of the
Jiangsu Province (2018K206C), and the National Natu-
ral Science Foundation Youth Funding Project of China
(31602027).
CONFLICT OF INTEREST
There is no conflict of interest.
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