Iranian Journal of Veterinary Research, Shiraz University
105
Molecular identification of reovirus in broiler type flocks in
Golestan province, Iran
Mayahi, M.1*; Boroomand, Z.1; Jafari, R. A.1; Khademian, S.2
and Hoseini, H.3
1Department
of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz; Ahvaz, Iran; 2Ph.D.
Student in Poultry Health and Diseases, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran;
3Departmet of Clinical Sciences, Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj, Iran
*Correspondence:
M. Mayahi, Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of
Ahvaz, Ahvaz, Iran. E-mail: mansoormayahi@scu.ac.ir
(Received 9 Jun 2018; revised version 21 Oct 2018; accepted 12 Nov 2018)
Summary
Background: Avian reovirus (ARV) has a global distribution in nature and most clinical signs are found in broiler type chickens.
Aims: This study was conducted to detect and identify reovirus infections from vaccinated breeder chickens and their progenies.
Methods: A total of 20 tissue and blood samples were collected from vaccinated broiler breeders and their progenies with
gastrointestinal or performance problems during peak production. Antibody titers were measured by indirect enzyme-linked
immunosorbent assay (ELISA) tests. RNA extraction from tissue samples was performed and cDNA was prepared and directly used
in the polymerase chain reaction (PCR). Nucleotide sequences were bilaterally determined using internal primers. The analysis of the
nucleotide sequences and their related amino acids was performed by the specialized Molecular Evolutionary Genetics Analysis
software (6th version). Results: The virus variant was detected in two vaccinated broiler breeders and five broiler flocks. The vaccine
strains in breeder flocks included S1133, SS412, 1733, 2408 belonging to genotype 1 from the reovirus phylogenetic tree. Sequence
7 from the isolated reovirus based on the σC revealed that they were different from the reovirus vaccine, and that 6 isolates belonged
to genotype 1 of the phylogenetic tree while 1 isolate belonged to branch 4 of the phylogenetic tree. Conclusion: The study showed
that the new reovirus strain isolated from vaccinated birds differs from common strains used in the vaccines. It is therefore essential
to prevent the effects of the field reovirus on the performance of industrial poultry, by updating and inventing new commercial
vaccines, live and killed, against the reovirus.
Key words: Broiler, Broiler breeder, Phylogenetic tree, Reovirus, σC protein
Introduction
Avian reovirus (ARV) has a global release in nature
and creates a wide range of diseases in different bird
species like poultry, pheasants, turkeys, ducks, geese,
pigeons, quails, hunting birds, and parrots (Jones and
Swane, 2013; Lu, 2015). Avian reoviruses belong to
Orthoreovirus genus of the Reoviridae family with 10
segments of double-stranded RNA (dsRNA) without any
covers, all of which being classified according to their
electrophoretic stimulation into three large (LI-L3),
medium (M1-M3) or small (S1-S4) groups (Mcnulty et
al., 2008; Jones, 2009; Jones and Swane, 2013). The
virus size ranges from 4/0 to 5/2 kbp (Tang and
Huaguang, 2015) and the segmented genome includes
eight structural proteins (λA, λB, λC, µA, µB, σA, σB,
σC) and four non-structural proteins (µNS, P10, P17,
σNS) (Tang and Huaguang, 2015). The virus has 6
branches in the phylogenetic tree (Huang et al., 2015).
Most clinical signs are observed in broiler poultry and
breeder chickens (Lu, 2015). Reovirus infections in
domestic birds have numerous noticeable destructive
economic effects. Clinical signs in young chickens
include tenosynovitis, malabsorption syndrome, runtingstunting syndrome (RSS), digestive problems, immune
system suppression, and secondary infections caused by
a bacterium or a virus (Jones and Swane, 2013; Lu,
2015). Simultaneous infection with other pathogens such
as mycoplasma sinwellia leads to severe immune
deficiency, faintness, weight loss, egg production
reduction, and more particularly, the creation of
slaughterhouse waste (Senties-Cue et al., 2005; Reck,
2013). The findings indicate that the σC protein encoded
by genomic S1 is a cell binding agent and the main
determinant of the antigenicity of ARVs (Hoseini et al.,
2015; Lu, 2015). Genomic S1 segment in the current
ARVs strain has been well identified and evaluated in
poultry viruses (Spackman et al., 2005; Mcnulty et al.,
2008). Avian reoviruses are usually based on molecular
functions especially the nucleotide and the amino acid
sequence determination of the σ protein (Jones and
Swane, 2013; Hoseini et al., 2015). Recent studies based
on the phylogenetic analysis of the σC immunogenic
protein indicate many varieties that are different from the
current cluster of ARVs (Kant et al., 2003). New isolates
seem to be distinct from conventional vaccine strains
(Kant et al., 2003; Hoseini et al., 2015). Recent studies
in Australia showed that there is a potential for genetic
reassortment when the cell is infected with more than
one reovirus, hence the emergence of new variants.
Reoviruses are found everywhere and a large number of
the isolates (over 80%) are non-pathogens (Rosenberger
IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111
Iranian Journal of Veterinary Research, Shiraz University
106
et al., 1989). Horizontal transmission of the virus is one
of the main ways of infecting birds (Robertson et al.,
1989; Mcnulty et al., 2008; Jones and Swane, 2013).
Horizontal transmissions are often carried out through
faecal contamination and can also be transmitted through
the respiratory tract. Reoviruses can also be transmitted
vertically (Mcnulty et al., 2008; Jones and Swane, 2013).
Vaccination of breeder flocks is essential to transfer
breeder antibodies to the progenies and protect the birds
from infection (Kant et al., 2003). However, breeder
vaccination will be effective in protecting the progeny
when there is a homologue between the strains of the
vaccine and the virus circulating in the fields. There have
been recent reports of reovirus infections despite the
vaccination of breeder flocks and their progeny (Jones,
2009; Troxler, 2013; Hoseini et al., 2015; Mayahi et al.,
2015). The present study was conducted to identify
possible reoviruses in vaccinated breeder chicken flocks
and their progenies.
Materials and Methods
Sampling
Initially, a history of target broiler breeder flocks
from the Golestan province was gathered including
information about location, capacity, age, flock
performance, reovirus infection signs and vaccination
programs against reovirus. This information was then
documented in a work sheet (Hoseini et al., 2015).
During peak production season, 5 to 10 cm sections of
duodenum, jejunum, ileum, and rectum of 10 birds of
each of the 20 reovirus-vaccinated broiler breeder flocks
with digestive disorders or performance problems were
collected, and placed in a container consisting of saline
solution. These samples were then slowly mixed and
numbered as one sample. After a 30 min centrifuge
cycle, the supernatant was separated, transferred to a test
tube and immediately sent to the laboratory on dry ice. In
flocks with lameness symptoms, samples was taken from
the tibiotarsal joint and gastrocnemius and finger
retraction muscle tendons, all pooled and numbered as
one sample (Table 1). In addition, blood samples were
collected from the wing vein of the 20 birds of each
breeder broiler flock. Twenty broiler farms that raised
chicks from the same broiler breeder flocks were
investigated at the age of about three weeks, for signs of
gastrointestinal, enteritis problems, growth retardation or
limb problems. Blood samples from the wing vein and
Table 1: Breeder flock features and vaccination programs
1
Breeder, hybrid
flock codes
1-Ross 308
2
3-Hubbardf15
Golestan
3
5-Ross 308
Golestan
4
6-Ross 308
Golestan
5
9-Arbor acres
Golestan
6
11-Arbor acres
Golestan
7
13-Ross 308
Golestan
8
15-Ross 308
Golestan
9
17-Hubbardf15
Golestan
10
19-Ross 308
Golestan
11
21-Ross 308
Mazandaran
12
23-Ross 308
Golestan
13
25-Ross 308
Golestan
14
26-Arbor acres
Golestan
15
27-Ross 308
Golestan
16
29-Hubbardf15
Golestan
17
32-Ross 308
Mazandaran
18
33-Ross 308
Mazandaran
19
34-Ross 308
Mazandaran
20
39-Hubbardf15
Golestan
Number
Site of flocks
Golestan
Vaccination program
A live vaccine (merial) at 12 days and two killed vaccines (ceva)
at weeks 12 and 20
A live vaccine (MSD) at 11 days and a killed vaccine (MSD) at
week 20
A live vaccine (MSD) at 11 days and a killed vaccine (MSD) at
week 20
A live vaccine (MSD) at 12 days and a killed vaccine (ceva) at
week 20
A live vaccine (merial) at 35 days and a killed vaccine (MSD) at
week 20
A live vaccine (MSD) at 30 days and a killed vaccine (MSD) at
week 20
Two live vaccines (merial) at week 1 and 10 and two killed
vaccine (ceva) at weeks 7 and 18
Two live vaccines (merial) at 7 and 27 days and a killed vaccine
(MSD) at week 18
Two live vaccines (merial) at10 and 35 days and a killed vaccine
(ceva) at week 20
A live vaccine (merial) at 12 days and two killed vaccines
(MSD&ceva) at weeks 12 and 20
A live vaccine (MSD) at 12 days and a killed vaccine (MSD) at
week 20
Two live vaccines (merial) at 32 days and week 21 and a killed
vaccine (ceva) at week 20
A live vaccine (merial) at 12 days and a killed vaccine (MSD) at
week 20
Two live vaccines (merial) at week 4 and 8 and a killed vaccine
(ceva) at week12
A live vaccine (MSD) at 41 days and a killed vaccine (ceva) at
week 20
Two live vaccines (merial) at 19 days and week 10 and a killed
vaccine (Biomune) at week 17
A live vaccine (MSD) at 12 days and two killed vaccines
(MSD&ceva) at weeks 12 and 20
A live vaccine (merial) at 12 days and two killed vaccines (ceva)
at weeks 7 and 18
A live vaccine (merial) at 12 days and two killed vaccines (ceva)
at weeks 7 and 18
Two live vaccines (MSD) at 9 and 70 days and two killed
vaccines (MSD) at week 5 and 20
IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111
Age of sampling (week)
Breeder flocks
Progeny
35
2
37
3
33
4
38
2
40
2
33
2
32
3
35
3
28
3
35
3
36
2
29
3
36
2
34
3
30
2
33
3
40
2
36
2
35
2
30
3
Iranian Journal of Veterinary Research, Shiraz University
10 intestine samples (pieces of duodenum, jejunum,
ileum, and rectum) were collected along with samples
from the tibiotarsal joint, gastrocnemius and finger
retraction muscle tendons of 20 birds (Hoseini et al.,
2015).
Experimental method
Serology
The anti-reovirus antibody titer in the blood serum of
broiler chickens and their progeny was measured using
Biocheck Company’s enzyme-linked immunosorbent
assay (ELISA) kit.
Autopsy
Birds with clinical signs were euthanized and autopsy
was carried out to take tissue samples from joints,
tendons, and the gastrointestinal tract (Troxler, 2013).
RT-PCR
Identification of the reovirus
RNA extraction: Homogeneous tissue samples were
centrifuged at 4°C and 3000 rpm for 10 min. The
supernatant solution of the intestinal contents was
transferred to a microtube for RNA extraction. The RNA
extraction from samples was performed using the RNXplus solution (CinnaGen, Tehran, Iran) according to the
manufacturer’s protocol. Isolated RNAs were either used
immediately for the reverse transcription-polymerase
chain reaction (RT-PCR) or stored at -80°C.
Synthesis of cDNA (RT reaction)
After extracting RNA for cDNA synthesis, the
Random Hexamer Primer and Canadian Revert aid first
stand cDNA Synthesis kit were used. These primers are
attached to the RNA nonspecifically and as a result, the
entire RNA was converted to cDNA (Troxler, 2013).
The cDNA was prepared and used in the PCR
reaction immediately. In order to run the PCR reaction,
two pairs of primers were used as Nested-PCR. These
primers are specific to the S1 gene of reovirus and allow
the detection of reovirus with high sensitivity from
clinical specimens (Hoseini et al., 2015). The primer’s
sequence was as follows: S1C 5´ ATT GAA TTC TCT
CTG TTA TCT AAC CTTG3´738 bp, S1D 5´AAG
GAA TTC GTT GAG AAC AGA AGT AGG3´738 bp,
S1E 5´TCT GAA TTC ATC CGC AGC GAA GAG
AGG TG3´324 bp and S1F 5´AGT GAA TTC AGT
ATC GCC GTG CGC AG3´ 342 bp (Tang and
Huaguang, 2015). In all cases, positive and negative
controls were used simultaneously.
The master mix used contained 2 μL PCR buffer
10X, 1 μL MgCl2 (50 mM/μL), 0.25 μL dNTPs (10
mM/μL), 0.25 μL of each 10 mM/μL primer, 6 μL
cDNA, 10 μL distilled water, and the final addition of
0.25 μL Taq DNA polymerase (5 IU/μL). The thermal
programs were similar to those presented in Table 2.
Finally, the PCR products were separated in 2% agarose
gel containing safe stain (CinnaGen, Tehran, Iran), using
an ultraviolet trans-illuminator.
107
Table 2: Reverse transcription-polymerase chain reaction
thermal program of reovirus
Phase
Early denaturation
Denaturation
Annealing
Elongation
Final elongation
Temperature (°C)
Time
94
94
53
72
72
3 min
10 s
15 s
20 s
15 min
Determination of nucleotide sequences and
phylogenetic analysis
In order to determine the sequence, PCR products of
the identified positive samples were purified using a
Roche commercial kit. The determination of nucleotide
sequences using internal primers was bilaterally
conducted by BioNeer Co., South Korea. Analysis of the
nucleotide sequences and their related amino acids was
conducted using the specialized Molecular Evolutionary
Genetics Analysis Software (6th version).
Nucleotide sequences of the S1 gene of these isolates
were compared with each other and with the number of
different reoviruses in the GeneBank. Multiple alignment
was then performed using the CLUSTAL W method for
all nucleotide sequences. The nucleotide sequences were
then evaluated based on the codes equivalent to the
corresponding amino acids. The amino acid sequences
were then investigated along with those of the
nucleotides. Phylogenetic analysis based on the
nucleotide sequence and the amino acid was performed
by the neighbor joining (NJ) method using the TamuraNEI model. The validity and accuracy of the
phylogenetic tree were then evaluated using the
bootstrapping method and 1000 replications. All analyses
were performed with the Pro Molecular Evolutionary
Genetics Analysis software (6th version).
Results
Despite being vaccinated, some clinical symptoms
like lameness and gastrointestinal problems were
observed in the broiler breeder flocks. Moreover, through
a molecular study and PCR test, in two cases of broiler
breeder flocks (codes 13 and 29), positive reovirus
variants were reported. In 25% of the broiler flocks
(codes 4, 7, 14, 30, and 40) originating from the broiler
breeder flocks vaccinated against reovirus, the PCR test
was positive for the virus variant (Table 3, Fig. 1). In
reovirus-positive flocks, clinical symptoms like lameness
and lack of growth were observed and in the autopsy,
swelling and rupture of gastrocnemius tendons were
found. The sequence of the isolates of code samples (4,
14, 29, 30, and 40) was determined and classified in the
phylogenetic tree (Figs. 2 and 3). Samples 7 and 1 were
related to the gastrointestinal tract and tendon,
respectively. Table 4 shows the degree of similarity of
the isolates of this study with the vaccine strain.
In the serologic test, the highest antibodies titer were
measured in the breeder flocks which received live and
killed vaccines twice. In the progeny, the maternal
IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111
Iranian Journal of Veterinary Research, Shiraz University
108
Table 3: ELISA test and RT-PCR results
Breeder, hybrid
flock codes
Tissue
sample
Clinical
features
PCR
result
Mean Ab
titre
CV
(%)
Broiler
flock codes
Tissue
sample
PCR
result
MAS/VA
Mean Ab
titre
CV
(%)
1
1-Ross 308
MAS/VA
-/-
8321
38
10
gut
-
MAS
2496
46
2
3-Hubbardf15
MAS/VA
-/-
7739
27
2
MAS/VA
3224
51
5-Ross 308
MAS/VA
-/-
7500
45
4
-/+
MAS/VA
6016
32
4
6-Ross 308
MAS/VA
-/-
10700
23
20
Tendon
& gut
Tendon
& gut
gut
-/-
3
-
MAS
2996
41
5
9-Arbor acres
MAS/VA
-/-
8300
34
7
gut
+
MAS
16709
110
6
11-Arbor acres
MAS/VA
-/-
22352
32
12
MAS/VA
3751
36
13-Ross 308
MAS/VA
+
9707
29
14
-/+
MAS/VA
2890
63
8
9
15-Ross 308
17-Hubbardf15
MAS/VA
MAS/VA
-/-
8420
9638
32
48
16
18
-
MAS
MAS
2216
2641
91
30
10
19-Ross 308
MAS/VA
-/-
11643
33
22
MAS/VA
7034
47
21-Ross 308
MAS/VA
-/-
14845
47
8
Tendon
& gut
gut
-/-
11
-
MAS
2924
31
12
23-Ross 308
MAS/VA
-/-
11906
35
24
MAS/VA
5988
74
25-Ross 308
26-Arbor acres
VA
MAS/VA
-/-
7120
10954
40
38
38
28
Tendon
& gut
gut
gut
-/-
13
14
-
MAS
MAS
270
1971
91
44
15
27-Ross 308
Tendon
Tendon
& gut
Tendon
& gut
Tendon
& gut
Tendon
& gut
Tendon
Tendon
& gut
gut
Tendon
& gut
Tendon
& gut
gut
gut
-/-
7
Tendon
& gut
Tendon
& gut
Tendon
& gut
Tendon
& gut
Tendon
& gut
Tendon
& gut
gut
MAS
-
13255
33
31
-/-
MAS/VA
3438
56
16
29-Hubbard f15
MAS/VA
-/+
6854
35
30
+
MAS
4220
50
17
32-Ross 308
MAS/VA
-/-
10086
44
35
gut
-
MAS
1561
59
18
33-Ross 308
MAS/VA
-/-
9611
38
36
gut
-
MAS
2607
60
19
34-Ross 308
MAS/VA
-/-
3065
45
37
gut
-
MAS
2240
56
20
39-Hubbard f15
Tendon
& gut
Tendon
& gut
Tendon
& gut
Tendon
& gut
Tendon
& gut
Tendon
& gut
gut
MAS/VA
-/-
6481
44
40
Tendon
& gut
+/+
MAS/VA
512
128
Number
The sample related to the gastrointestinal tract and malabsorption (MAS: Mal absorption syndrome), the sample related to joint
problems (VA: Viral arthritis), positive sample (+), negative sample (-), first or second positive or negative sample (+/-), Coefficient
of variation: CV, and Ab: Antibody
Fig. 1: The 324 base pair RT-PCR products. M: DNA marker
(100 bp), Lines 1-2 and 4-6: Positive samples, Line 3: Negative
sample, No. 7: Positive control, and Line 8: Negative controls
IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111
Fig. 2: The phylogenetic tree based on the sequence of amino
acids in Iranian poultry along with isolates of the poultry virus
whose sequences existed in the GeneBank. The isolates related
to the present study are marked with a bold circle
Iranian Journal of Veterinary Research, Shiraz University
109
Table 4: Similarity of this study’s isolates with vaccine strains based on amino-acid sequences
Number
1
2
3
4
5
6
7
8
Strain
Accession number
% Identity with vaccine strain S1133
Phylogeny phylum
Farm codes
IR/kH1996.71/17
IR/KH2003.50/17
IR/KH1996.70/17
IR/KH1996.45/17
IR/KH1996.11/17
IR/KH1996.74/17
IRK/H1996.76/17
IR/H1996.31/17
MG922798
MG922799
MG922800
MG922801
MG922802
MG922803
MG922804
MG922805
81
81
81
81
81
79.5
81
57.5
1
1
1
1
1
1
1
4
40
40
29
29
4
29
30
14
IR is related to the isolated country, code 1996 belongs to gastrointestinal samples, code 2003 belongs to tendon samples, numbers
are related to the samples, and 17 is related to the year of isolation
Fig. 3: The phylogenetic tree was based on the sequence of
amino acids in Iranian poultry along with isolates of the poultry
virus whose sequences existed in the GeneBank. Branch
spacing represents the degree of sequence difference. The
isolates related to the present study are marked by a bold circle
antibody derived from the breeder flock was detectable.
In the infected flocks, however, the level of antibody in
the ELISA test increased (Table 3).
Discussion
Evaluation of lameness and growth retardation in
broiler flocks showed a high prevalence of reovirus in
industrial poultry farms (Mayahi et al., 2015). In the
past, good protection was provided by vaccination in the
breeder flocks due to the similarity of the vaccine virus
and the circulating virus. In the broiler flocks, on the
other hand, this protection was due to breeder antibody
transmission. Recently, however, genetic changes in the
virus have reduced vaccine effectiveness. The σC gene is
the main cause of cellular connection and the induction
of neutralizing antibodies and is considered to be the
variable segment of the virus. The most important way to
control the activity of the virus in poultry farms is to
vaccinate the breeder flocks with an effective vaccine to
protect the vertical transmission of the virus as well as
the progeny through the breeder’s antibody against the
circulating virus. The sequencing of the σC gene of S1
segment of reovirus helps differentiate the reovirus
vaccine virus from the circulating reovirus virus. The
present study was hence conducted to identify possible
reoviruses in the vaccinated breeder flocks and their
progenies. Bokaie et al. (2008) investigated the
seroprevalence of reovirus infections in broiler chicken
flocks of Tehran on 582 serum broiler chickens
originating from non-vaccinated breeders using the
ELISA test. Of the total samples, 572 serum samples
were positive with a serum prevalence of 98.3%
(Spackman et al., 2005). Hedayati and Shojadoust (2012)
examined the reovirus of tenosinovitis in birds from
Iranian breeder flocks using RT-PCR and restriction
fragment length polymorphism (RFLP) methods. The
analysis of the segments obtained from the enzymatic
digestion of PCR products in all positive samples
indicated that the enzymatic digestion patterns of the
samples were consistent with the standard digestive
pattern of each S1133 vaccine. Mayahi et al. (2015)
reported the occurrence of reovirus in broiler chicks
originating from vaccinated broiler breeders in Golestan
province. According to their clinical study, signs of
lameness were detected from the second week in a
number of rooster chicks of broiler farms, and the
presence of reovirus in the gastrointestinal tract and
gastrocnemius tendons of the infected chicks was
confirmed using RT-PCR. In the present study, the
investigation of the phylogenetic tree based on the σC
segment showed that most isolates belonged to branch
one, and one isolate belonged to branch 4 of the tree.
Vaccine strains such as S1133 and 1733 belonged to
branch one of the phylogenetic tree, but the isolates
studied in this research were different from the vaccine
strains. Most closely related to the isolates were German
GEL06 97M and GEL12 98M isolates which were
identified in 1997 and 1998 with malabsorption
symptoms from branch one of the phylogenetic tree. A
single case of one isolate from broiler flocks and growth
retardation syndrome belonged to branch 4 and showed
the most similarity with the isolate of the American AVS
B in branch four of the phylogenetic tree. In the study
IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111
110
conducted by Hoseini et al. (2015), cases of
tenosynovitis and lameness in broiler flocks were
observed despite the vaccination of breeder flocks. In all
cases, hock joint inflammation, flexor digital tendon,
swelling of the foot and subsequently poor performance
of the flocks were observed. In the serological study, all
affected flocks were found to have high levels of
antibodies in the ELISA test. S1 segment sequences that
codify the σC gene were examined. After being separated
from the first broiler flock in the Ardehal area in central
Iran, this isolate was named Ardehal variant.
Investigations showed that the Ardehal variant belonged
to branch one of the phylogenetic tree but differed from
the strains of the vaccine which also belonged to branch
one of the phylogenetic tree. Broiler breeder flocks in
Iran are protected against reovirus by live and killed
reovirus vaccine. Thus, chicks of these breeders are also
expected to be protected against reoviruses. However,
recent cases of prevalence of the reovirus in broiler
flocks indicate that vaccination of breeder flocks with
current vaccines is not enough to provide immunity to
chicks. Tang et al. (2015) reported severe clinical disease
and economic loss in Pennsylvanian turkey breeder
flocks since 2011. The study of molecular identification
of the 114 cu gene of field isolates from 2011 to 2014
indicated that the isolated reovirus included only 21.93%
of the 114 farm isolates of a genotypic branch (genotypic
branch1) such as the vaccine strain (S1133, 1733, 2408).
The remaining 78.7% of the strains belonged to a
different genotypic branch of the vaccine (Branch 2, 3, 4,
5, and 6) and indicated the emergence of new strains of
reovirus. In this report, the emerging genotype six was
reported for the first time (Lu, 2015). Troxler (2013)
reported a severe economic loss in French broiler breeder
farms due to reovirus infection despite routine
vaccinations in broiler farms. Clinical signs included
lameness, growth retardation, and lack of uniformity in
flocks. Observations of autopsy and bacteriological and
serologic tests confirmed the presence of tenosynovitis
caused by reovirus infections. Moreover, sequencing the
cσ gene and the phylogenetic evaluation of the RT-PCR
product determined the new genotype of the reovirus.
The virus was not neutralized by monovalent serums of
the vaccinated chickens in the neutralization test. It was
concluded that these reovirus isolates were serologically
and genetically distinct from conventional reoviruses
used in commercial vaccines and could not, therefore,
provide protection and prevention against disease. Kort
et al. (2013) identified, classified, and determined the
genotype of the Tunisian strains of bird reovirus using
the RT-PCR test, which was performed on both σC and
σB genes, and subsequent RFLP tests to better identify
Tunisian isolates. The replicated segment in the RT-PCR
test showed 15 Tunisian strains with lengths of 738 and
540 bp for σC and σB. Using Acil and Msel enzymes in
the RFLP test showed that all isolates could be clearly
distinct from the vaccine strain. Tang and Huaguang
(2015) determined genotypes of the reovirus isolates in
Pennsylvania. The reovirus strain (Reo/PA/Broiler/05682
/12) was isolated from broiler breeders suffering from
IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111
Iranian Journal of Veterinary Research, Shiraz University
tenosynovitis and severe joint infections, all reovirus
genomes being sequenced by RT-PCR and rapid
amplification of cDNA ends (RACE) methods. The size
of the complete isolated reovirus was 23494 bp and
contained C+G 50%. A phylogenetic study of the
nucleotide sequence of 10 genome segments in
Pennsylvania showed moderate to highly noticeable
differences in the nucleotide sequence of the strain used
in the reovirus S1133 vaccine and 138 isolates. This
genomic data indicated that the new Pennsylvania field
strain is a reovirus that produces tenosynovitis and
differs from the common strain used in the vaccine.
In the present study of broiler breeder flocks, despite
their good performance, some clinical signs such as
growth retardation, lameness, and gastrointestinal
problems were observed. Vaccinating the broiler breeder
flock helped prevent clinical symptoms; however, as
shown in codes 13 and 29, both breeder and broiler
flocks (codes 14 and 30) showed severe cases of growth
retardation. In the molecular investigation of other
breeder flocks, the virus was not detected despite clinical
signs. In some broiler breeder flocks reovirus was not
detected but the virus was detected in their progenies.
Despite that the virus was not detected in some broiler
breeder flocks, their progenies (codes 4, 7, and 40)
showed clinical signs of growth retardation,
gastrointestinal problems, and lameness. Environmental
contamination seems to play a major role in developing
infections in growing broiler flocks and due to the
heterologous nature of the some reovirus and vaccine
strains, the breeder’s antibody does not have the ability
to protect and prevent infection.
Comparison of the nucleotide sequence σC of 8 virus
isolates with the standard virus suggests the emergence
of a variant in the circulating virus that does not show
enough vaccine immunity against the reovirus. Genetic
rearrangement is a noticeable characteristic of the
reovirus (Troxler, 2013), and in the σC gene, segment S1
appears more often than other gene segments. In addition
to the horizontal transmission of the reovirus that is very
common among the flocks, vertical transfer is observed
in codes 13, 14 and 29, 30. Regarding the breeder flock
(code 13) with the isolate belonging to branch one of
reovirus and the broiler flock (code 14) with the isolate
belonging to branch four of the reovirus, the possibility
of vertical transmission of the virus is rejected. However,
regarding the breeder flock (code 29) and the broiler
flock (code 30), while both isolates belonged to the same
branch, there is a high possibility of the vertical
transmission of the reovirus. On the other hand, in cases
such as code 7, in spite of the detection of severe
lameness in the flock, a segment of the bird’s intestine
was removed and examined, indicating the traceability of
the reovirus in the tissues as well as the body’s capability
to clear the virus. Moreover, it was shown that the
gastrointestinal tract was the main reovirus replication
location. This genomic data suggests that the new field
strain of reovirus in Iran has been responsible for the
development of tenosynovitis and growth retardation
problems.
Iranian Journal of Veterinary Research, Shiraz University
The study showed that the new reovirus strain
isolated from vaccinated birds was different from
common strains used in the vaccines and that it is
essential to prevent the effects of the field reovirus on the
performance of industrial poultry by updating and
making new commercial vaccines, live and killed,
against the reovirus. It is necessary, therefore, to design
further experiments to show this.
Acknowledgement
This manuscript was extracted from a dissertation
project approved by vice-chancellery for research at
Shahid Chamran University of Ahvaz.
Conflict of interest
The author(s) declared no potential conflicts of
interest with respect to the research, authorship, and/or
publication of this article.
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IJVR, 2019, Vol. 20, No. 2, Ser. No. 67, Pages 105-111