Comparative Immunology, Microbiology and Infectious Diseases 65 (2019) 110–115

Contents lists available at ScienceDirect

Comparative Immunology, Microbiology

and Infectious Diseases
journal homepage: www.elsevier.com/locate/cimid

Genotyping of avian infectious bronchitis virus in Iran: Detection of D274 T

and changing in the genotypes rate
⁎
Arash Ghalyanchilangeroudia, Hossein Hosseinib, , Mohammad Hossein Fallah Mehrabadic,
Seyed Ali Ghafourid, Amir Modiri Hamdana, Zahra Ziafatia, Reza Esmaeelzadeh Dizajia,
Peyman Mohammadia
a
Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran
b
Department of Clinical Sciences, Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Alborz, Iran
c
Department of Poultry Diseases, Razi Vaccine and Serum Research Institute, Agricultural Research, Education and Extension Organization (AREEO), Tehran, Iran
d
Iranian Veterinary Organization, Tehran, Iran

A R T I C LE I N FO A B S T R A C T

Keywords: The coronavirus avian Infectious bronchitis virus (IBV) poses economic threats to poultry farms worldwide,
Avian infectious bronchitis virus affecting the performance of both meat-type and egg-laying birds. To define the evolution of recent IBVs in Iran,
D274 a genetic analysis based on hypervariable nucleotide sequences of S1 gene was carried out. Tracheal swab
Spike samples were collected from 170 Broiler flocks during 2017. Ten tracheal swabs from each flock pooled. From a
Phylogenetic analysis
total number of 170 flocks tested, 84.71% found to be positive. Phylogenetic tree analysis revealed the presence
Iran
of D274 as a first time in Iran. IS/1494/06 was showed to be dominant IBV type circulating in broiler farms with
a significantly higher prevalence than other four genotypes. Considering fluctuations in QX-type prevalence in
recent years, continuous monitoring is necessary to reduce economic consequences in layer and broiler farms.
The findings highlight the importance of using modified vaccination strategies that are adapted to the changing
disease scenario.

1. Introduction which do not confer complete cross-protection against each other

[3–6]. Among a large number of serotypes or variants emerged after the
Avian Infectious bronchitis virus is the prototype gammacoronavirus first documentation of IBV in 1931, Massachusetts 41 (M41) and 793/B
in the family Coronaviridae, order Nidovirales. As its name implies Avian serotypes have been expanded through the world and commercial
infectious bronchitis virus (IBV) is responsible for acute respiratory vaccines are available against both serotypes providing a broad cross-
tract infections in chickens. Laying hens experience reduced produc- protection against many different IBV types when used together [7–9].
tion, eggshell abnormalities, and decreased internal egg quality. In Despite vaccination efforts, novel field IBVs continue to emerge; some
addition to tracheal damage, some strains of IBV can cause renal le- antigenic variants may subsequently become dominant which makes
sions. The positive-sense viral genome 27.6 kb in length encodes at least prevention of IBV infections very challenging [10,11]. Similar to what
ten open reading frames (ORFs) organized as follows: 5′ UTR-1a-1ab-S- has been happening in Iranian poultry farms leading to unceasing IB
3a-3b-E-M-5a-5b-N-3a-3′UTR. ORFs 1a and 1b encode 15 non-struc- outbreaks despite extensive vaccination. In this work, we focus on po-
tural proteins responsible for viral replication and pathogenesis. Four tential IBV variants escaping from vaccine-induced immunity.
structural proteins including the spike glycoprotein (S), small mem-
brane protein (E), membrane glycoprotein (M), and nucleocapsid pro- 2. Material and methods
tein (N) are encoded by mRNAs 2, 3, 4 and 6, respectively [1,2]. The
spike protein is post-translationally cleaved into S1 and S2. The S1 2.1. Sample collection
subunit is the main target of neutralizing antibodies. Evolution in IBV is
essentially associated with the sequences of the S1 glycoprotein, and Tracheal swab samples were collected from 170 broiler flocks re-
the genetic diversity of IBV is principally screened by analysis of the S1 presenting signs of the respiratory complex in 2017. Chickens had a
gene. Mutations in the S1 leads to the emergence of variant serotypes history of vaccination against IBV (Massachusetts + 793/B type

⁎
Corresponding author.
E-mail address: [email protected] (H. Hosseini).

https://doi.org/10.1016/j.cimid.2019.05.011
Received 30 March 2019; Received in revised form 7 May 2019; Accepted 7 May 2019
0147-9571/ © 2019 Elsevier Ltd. All rights reserved.
A. Ghalyanchilangeroudi, et al. Comparative Immunology, Microbiology and Infectious Diseases 65 (2019) 110–115

Fig. 1. Geographical locations of provinces selected for sample collection.

1, Kurdistan; 2, Ardabil; 3, Qazvin; 4, Golestan; 5, Qom; 6, Semnan; 7, Khurasan Razavi; 8, Chahrmahal-E-Bakhtiari; 9, Kerman.

vaccines). The chicken farms were located in nine different provinces of Korea). Chromatograms were evaluated with CromasPro (CromasPro
Iran including Qazvin, Ardabil, Semnan, Kerman, Khurasan Razavi, Version 1.5). The nucleotide sequences of S1 gene from Afghan IBV
Chahrmahal-E-Bakhtiari, Kurdistan, Qom, and Golestan. Geographical strains obtained in this study were subjected to BLAST (primary geno-
locations of these provinces are shown in Fig. 1. typing and similarity results), aligned and compared with reference
strains downloaded from NCBI GenBank database from foreign
Countries and neighboring States. We remove the similar sequences
2.2. RNA extraction and cDNA synthesis
Sequence homology analysis was performed using MEGA7.0.
Phylogenetic trees were constructed using MEGA7.0 with the neighbor-
Ten tracheal swabs from each flock were pooled. Homogenized
joining algorithm (bootstrap values of 1000) with the Kimura2 para-
pooled samples were submitted for RNA isolation using RNA easy mini
meter model [14]. The nucleotide and amino acid sequences de-
kit (Qiagen). The cDNA was synthesized using a RevertAid First Strand
termined in this study are available in the GenBank under accession
cDNA Synthesis Kit (Thermo Scientific) [12].
number: MH106448-MH106510.

2.3. Real-time PCR for IBV detection and RT-PCR for IBV Genotyping
3. Results
A previously designated Real-time PCR [13] was used for IBV de-
tection targeting 5′ UTR of the IBV genome. IBV positive samples were From a total number of 170 flocks tested, 84.71% found to be po-
submitted to a nested PCR amplification with the aim of genotyping sitive. The phylogenetic analysis revealed detected IBVs were divided
[12]. PCR products were purified using the AccuPrep® PCR purification into five major clusters (Fig. 2). The distribution of prevalence rates in
Kit (Bioneer Co., Korea) and submitted for sequencing (Bioneer Co., each province is depicted in Table 1. IS-1494-like IBV was the most
Korea). common type accounted for 85% of detected strains. The other types
The AccuPrep® PCR purification Kit (Bioneer Co., Korea) was used overall created a lower proportion; including 793/B having a pre-
for purification of the PCR products. Sequencing was performed using valence of 7%, QX, and Mass with prevalence rates of 5% and 2%, re-
ABI 3100 Genetic Analyzer (Applied Biosystems, USA) with the primers spectively, and D274 with the incidence of 1% (Fig. 3)
(Both directions) used in the second step of nested PCR (Bioneer Co., The sequence identity among IS/1494/06 IBV types detected in this

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Table 1
Geographical distribution of IBV strains in different provinces
of Iran during 2017.
Province Positive (%)

Qazvin 93.33
Ardebil 88.00
Semnan 85.00
Kerman 85.00
Khorasan-e-Razavi 82.35
Chahrmahal-E-Bakhtiari 82.35
Qom 82.35
Golestan 82.35
Kurdestan 81.82

Fig. 3. Proportion of each IBV type circulating in Iran from 2017.

work and reference strains was more than 98.86%. Mass IBVs shared
more than 98.28% sequence similarities including some strains com-
pletely identical to the H120 vaccine. 793/B type IBVs in this study
shared 93.29-100% sequence homology with each other, while less
with those detected in the last few years. Sequence identities among QX
IBVs varied between 89 to 100% (Table 2). Figs. 4 and 5 shows com-
parisons of partial S1 sequences of IBV strains detected in this work
with some selected IBV types with 793/B and Mass type respectively
(Figs. 4 and 5).

4. Discussion

Despite vaccination efforts, novel field IBVs continue to emerge in

many parts of the world, leading to the dominance of some antigenic
variants that makes prevention of IBV infections very challenging
[10,15]. Two independent studies have been dedicated to investigating
the IBV epidemiology in Iran. The first one was carried out by Najafi
et al. between 2014 and 2015[16], the second was done during
2015–17 [12]. Comparison of their results with the result obtained in
this study showed the growing trend in IS/1494/06 IBV prevalence
Fig. 2. Neighbor-joining phylogenetic tree based on partial S1 gene sequences (Fig.6).
of Iranian IBVs and selected reference strains. The percentages of replicate trees The circulation of such a high rate of IS/1494/06 genotype un-
in which the associated taxa clustered together in the bootstrap test (1000 re- related to the used vaccine strains declaring they originated from sev-
plicates) are shown next to the branches IBVs obtained in this study are marked eral sources. It is one of the most prevalent IBV genotypes in Middle
with black squares; other Iranian IBVs detected in recent years are shown by East countries such as Kuwait, Lebanon, Oman, Saudi Arabia [17]. High
black diamonds. sequence similarities among IS/1494/06 IBVs in Iran and those

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Table 2
Nucleotide homology between representatives from each distinct cluster and selected reference strains for partial S1 sequences.
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26

1 Iran/IS-1494/UT-GL29/2017
2 Iran/IS-1494/UT-GL30/2017 100
3 Iran/IS-1494/UT-GL28/2017 100 100
4 Iran/IS-1494/UT-GL22/2017 100 100 100
5 Iran/IS-1494/UT-GL37/2017 99.425 99.43 99.425 99.425
6 Iran/Mass/UT-GL50/2017 77.011 77.01 77.011 77.011 77
7 Iran/Mass/UT-GL51/2017 78.736 78.74 78.736 78.736 78.2 98.3
8 Iran/D274/UT-GL48/2017 82.184 82.18 82.184 82.184 81.6 75.3 77.01
9 Iran/793-B/UT-GL55/2017 81.034 81.03 81.034 81.034 80.5 69.5 71.26 81
10 Iran/QX/UT-GL53/2017 82.184 82.18 82.184 82.184 81.6 73.6 75.29 78.7 82.184
11 Iran/QX/UT-GL54/2017 82.184 82.18 82.184 82.184 81.6 73.6 75.29 78.7 82.184 100

113
12 Iran/QX/UT-GL52/2017 75.862 75.86 75.862 75.862 75.3 67.2 68.97 71.8 74.138 91.38 91.4
13 Iran/793-B/UT-GL57/2017 78.736 78.74 78.736 78.736 78.2 68.4 70.11 81 97.701 79.89 79.9 71.8
14 Iraq/QX/SGK21(KU143898) 83.908 83.91 83.908 83.908 83.3 74.7 76.44 79.9 82.759 98.28 98.3 89.7 80.46
15 Iran/793-B/UT-IVO108(KT583579) 78.736 78.74 78.736 78.736 78.2 68.4 70.11 81 97.701 79.89 79.9 71.8 100 80.46
16 IR-1_UTIVO-117(KT583581) 86.207 86.21 86.207 86.207 86.8 71.3 72.41 77 78.161 79.89 79.9 72.4 75.86 81.61 75.9
17 Iran/IS-1494/UT-MG161/2017 100 100 100 100 99.4 77 78.74 82.2 81.034 82.18 82.2 75.9 78.74 83.91 78.7 86.2
18 Iran/793-B/UT-MG5/2015 80.46 80.46 80.46 80.46 79.9 70.1 71.84 82.2 95.977 82.18 82.2 74.1 94.83 82.76 94.8 78.2 80.5
19 Iran/QX/UT-MG37/2016 83.908 83.91 83.908 83.908 83.3 74.7 76.44 79.9 82.759 98.28 98.3 89.7 80.46 100 80.5 81.6 83.9 82.8
20 Iran/Mass/UT-MG5_-2015 80.46 80.46 80.46 80.46 79.9 96.6 98.28 78.7 72.989 75.86 75.9 69.5 71.84 77.01 71.8 74.1 80.5 73.6 77
21 4/91Vaccine(KF377577) 81.034 81.03 81.034 81.034 80.5 69.5 71.26 81 100 82.18 82.2 74.1 97.7 82.76 97.7 78.2 81 96 82.8 73
22 H120(JN600610) 78.736 78.74 78.736 78.736 78.2 98.3 100 77 71.264 75.29 75.3 69 70.11 76.44 70.1 72.4 78.7 71.8 76.4 98.3 71.3
23 D3896(X52084) 82.759 82.76 82.759 82.759 82.2 75.3 77.01 95.4 79.885 79.31 79.3 74.1 79.89 80.46 79.9 78.7 82.8 80.5 80.5 78.7 79.9 77
24 IS/1494/06(HM131453) 99.425 99.43 99.425 99.425 98.9 77 78.74 82.8 81.609 82.18 82.2 75.9 79.31 83.91 79.3 86.2 99.4 81 83.9 80.5 81.6 78.7 82.8
25 QX(AF193423) 83.908 83.91 83.908 83.908 83.3 74.7 76.44 80.5 83.333 97.7 97.7 89.1 81.03 99.43 81 81.6 83.9 83.3 99.4 77 83.3 76.4 81 83.9
26 Q1(AF286302) 81.609 81.61 81.609 81.609 81 72.4 74.14 79.3 79.31 78.16 78.2 73 77.01 79.31 77 79.9 81.6 79.3 79.3 75.9 79.3 74.1 79.9 82.2 79.9
Comparative Immunology, Microbiology and Infectious Diseases 65 (2019) 110–115
A. Ghalyanchilangeroudi, et al. Comparative Immunology, Microbiology and Infectious Diseases 65 (2019) 110–115

Fig. 4. Percent identity of partial S1 gene sequences of some IBVs from the current study to H120 vaccine strain.

Fig. 5. Percent identity of partial S1 gene sequences of some IBVs from the current study to 793/B vaccine strain.

probably currently the IB variant of most concern for breeder/ layer

flocks. At the stage that the QX live vaccines are not registered in Iran.
D274, a Dutch strain was first isolated in 1979, reported to be ser-
ologically related to both A and B serotypes [22,23]. Strains D207 and
D3896 considered belonging to the same serotype as D274 [24]. D207
was isolated in samples collected between 1981 and 1983 in Britain
[25]. D274 isolates were recovered from vaccinated and especially non-
vaccinated broilers over a 10-year period from 1986 to 1995 in Belgium
[26]. It was identified in Egypt (Egypt/D274/D/89) [27]. D274 was
detected in Sweden, England and Russia in 1995, 1996 and 1998, re-
spectively [28]. Several reports have been confirmed D274 presence in
Western Europe from 2002 to 2006 [19,29,30]. D274 was not detected
in the Middle East countries before 2005, its first report from Jordan
[31]. After that in a six-year investigation of the dynamics of IBV, D274
was detected in Oman, Saudi Arabia, and the United Arab Emirates,
Fig. 6. Prevalence of different types IBV. regarding the sequencing data, 62.50% of them identified as field and
not vaccine strain[17]. D274 has been detected once in Iran using
circulating in our neighboring countries including Israel, Iraq, Turkey serological assays (personal communication); the present study reports
[16,18], may explain the virus origin. It is not yet known how IS/1494/ the first molecular detection of D274 genotype. Since D274 is not in-
06 spread among Middle East countries; one may suspect to the role of cluded in any vaccine strains used in Iran, it is probably originated from
wild birds in virus transmission [19], another possible reason is intense neighboring countries. In addition to biosecurity practices, vaccination
trade among Middle East countries and illegal movement of animals is usually needed to control IB. Selecting the proper vaccination sche-
across borders. With much lower prevalence, 793/B was the second dule will be more complicated considering the existence of many IBV
most predominant IBV type in this study. The 793/B serotype is one of variants. It has been shown that vaccination with two antigenically
IBV types used in our national vaccination program. As the method distinct live-attenuated vaccines such as Mass and 793/B can result in a
used in the present study could not differentiate field and vaccine broad cross-protection against many different IBV types [15]. Awad
strains, 793/B IBV type was expected to the 793/B vaccine strain use et al. showed that administering combined live H120 and CR88 vac-
(Fig. 6). In breeder and laying hens QX-type causes delayed the onset of cines simultaneously at day-old followed by the CR88 vaccine at 14 d-
production, poor peak in egg production, a high percentage of false- old gave more than 94% tracheal ciliary protection IS/1494/06. The
layers and poor quality of eggs [20]. The QX prevalence obtained in the other vaccination program, H120 at day-old followed by CR88 at 14 d-
present study was 5% which could reflect the efficacy of vaccination old, the tracheal ciliary protection conferred was 80 percent from IS/
[21]. Although the prevalence is relatively low, the 2 to 10% fluctua- 1494/06-like [32]. Our experiment in which combination of H120 and
tion in its prevalence within a few last years and also increase the ge- 793/B provided better protection than using homologous H120-H120
netic distance between field and vaccine strains are worrying, since it is vaccination strategy, while it was not still completely protect against

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