key: cord-1009719-66d8c75g
authors: Seger, Waleed; GhalyanchiLangeroudi, Arash; Karimi, Vahid; Madadgar, Omid; Marandi, Mehdi Vasfi; Hashemzadeh, Masoud
title: Genotyping of infectious bronchitis viruses from broiler farms in Iraq during 2014-2015
date: 2016-02-18
journal: Arch Virol
DOI: 10.1007/s00705-016-2790-2
sha: d29aff541cdafaf123b8c8ccae6689ab888affbb
doc_id: 1009719
cord_uid: 66d8c75g

Infectious bronchitis virus (IBV) is one of the most critical pathogens in the poultry industry, causing serious economic losses in all countries including Iraq. IBV has many genotypes that do not confer any cross-protection. This virus has been genotyped by sequence analysis of the S1 glycoprotein gene. A total of 100 tracheal and kidney tissue specimens from different commercial broiler flocks in the middle and south of Iraq were collected from September 2013 to September 2014. Thirty-two IBV-positive samples were selected from among the total and were further characterized by nested PCR. Phylogenetic analysis revealed that isolates belong to four groups (group I, variant 2 [IS/1494-like]; group II, 793/B-like; group III, QX-like; group IV, DY12-2-like). Sequence analysis revealed nucleotide sequence identities within groups I, II, and III of 99.68 %-100 %, 99.36 %-100 %, and 96.42 %-100 %, respectively. Group I (variant 2) was the dominant IBV genotype. One Chinese-like recombinant virus (DY12-2-like) that had not been reported in the Middle East was detected. In addition, the presence of QX on broiler chicken farms in the area studied was confirmed. This is the first comprehensive study on the genotyping of IBV in Iraq with useful information regarding the molecular epidemiology of IBV. The phylogenetic relationship of the strains with respect to different time sequences and geographical regions displayed complexity and diversity. Further studies are needed and should include the isolation and full-length molecular characterization of IBV in this region.

Coronaviruses are an important group of viruses that cause highly contagious respiratory and enteric diseases in animals and humans [13] . Avian corona viruses can infect a wide range of avian species, particularly those reared in close proximity to domesticated poultry, such as fowl, partridge, geese, pigeons, guinea fowl, teal, ducks, and pea fowl [8] . Infectious bronchitis virus (IBV) causes enormous problems, causing a highly contagious disease in chickens. Respiratory, reproductive, digestive, and renal infections are the primary clinically important types of IBV infections in domestic chickens [15] . IBV belongs to the genus Gammacoronavirus, along with other avian coronaviruses. It is an enveloped, positive-stranded RNA virus with a genome of about 27 kb containing 5' and 3' untranslated regions (UTRs) and a poly(A) tail [5] . A major part of the genome is composed of two overlapping open reading frames (ORFs), 1a and 1b, which are translated into large polyproteins 1a and 1ab, respectively, through a ribosomal frameshift mechanism. The primary structural proteins spike (S), envelope (E), membrane (M), and nucleocapsid (N) are encoded in the remaining part of the RNA genome [20, 24] . The glycoprotein S consists of two subunits (S1 and S2) and contains a wide variety of antigenic determinants that may induce the production of specific neutralizing antibodies. Mutations within this genomic region may result in the emergence of new viral variants [19] . In recent years, genotyping has been performed using S1 gene sequencing [17] , which has become the procedure of choice for differentiating between vaccine and field viruses.

More than 20 IBV serotypes have been recognized worldwide [30] . IB still causes serious problems in the Iraqi poultry industry due to the inability of vaccines to provide cross-protection between different genotypes. Due to the limited network of poultry diagnostic laboratories in Iraq, differential diagnosis is can only be done based on clinical signs and gross lesions. The characterization of IBV has raised additional problems in terms of both epidemiology and control. Although IBV on the poultry farms in Iraq (with H120 and 4/91 strains) is presently controlled by inactivated and live attenuated vaccines, outbreaks of IB have nevertheless been observed on broiler farms [1, 21] . Thus, the spread of viral respiratory diseases has become the most commonly reported condition in commercial broiler flocks in Iraq; however, there have been no studies on the detection and genotyping of these viruses using molecular techniques and sequencing. In this study, genotyping of IBV isolates from broiler farms in the south and middle of Iraq was carried out based on partial S1 sequencing.

Samples were collected from 100 broiler chicken farms in the south and middle (five governorates) of Iraq (Al-Kut, Al-Najaf, Thi-Qar, Al-Muthanna and Al-Basrah; 20 farms per governorate; Fig. 1 ) from September 2014 to December 2015. The samples (trachea and kidney) were taken from chickens that showed clinical signs suggesting IB (respiratory problems such as gasping, sneezing and bronchial rales, and nephritis lesions such as enlargement, and congestion in kidneys). The samples were collected with aseptic technique and frozen at -70°C. The details of positive samples are shown in Table 1 .

RNA was extracted from tissue samples using CinnaPure RNA Extraction Kit (SinaClone, Iran). For cDNA synthesis, 1 lL (0.2 lg) of random hexamer primer (SinaClon, Iran) was added to 5 lL of extracted RNA, and the mixture was heated at 65°C for 5 minutes. Fourteen lL of cDNA master mix containing 7.25 lL of DEPC-treated water (SinaClon, Iran), 2 lL of dNTP mix (SinaClon, Iran), 0.25 lL of RiboLock RNase Inhibitor (Thermo Fisher Scientific, USA), 0.5 lL of Revert Aid Reverse Transcriptase (Thermo Fisher Scientific, USA), and 4 lL of 5X RT reaction buffer was added to each tube, resulting in a final volume of 20 lL. Then, the mixture was incubated at 25°C for 5 min, 42°C for 60 min, 95°C for 5 min, and 4°C for 1 min. The cDNA was stored at -20°C until use. Sampling, RNA extraction, and cDNA synthesis were conducted in Iraq, and the remainder of the work was carried out in Iran.

Real-time PCR for IBV detection based on the 5 0 UTR was chosen for this study. The amplification was performed by using an amplification kit (Bioneer, South Korea) with the forward primer 5 0 GCTTTTGAGCCTAGCGTT3 0 , reverse primer 5 0 GCCATGTTGTCACTGTCTATTG3 0 and TaqMan Ò dual-labeled probe FAM-CACCACCAGAAC CTGTCACCTC-BHQ1 as described by Callison et al. [7] .

Nested PCR was performed using spike gene primers that were designed to amplify a *390-bp fragment of the gene [16] . First-round amplification (494 bp) was performed in a final volume of 20 lL containing 2 ll of distilled water, 13 ll of SinaClon 2 9 PCR master mix (SinaClon, Iran), 2 ll of SX1 (5 0 CACCTAGAGGTTTGYTWGCATG3 0 ) and SX2 (5 0 TCCACCTCTATAAACACCYTTAC3 0 ) primers (10 lM), and 3 ll of cDNA . The amplification was performed with a 35-cycle thermal profile (94°C for 2 min, 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 72°C for 10 min). In the second round of the nested PCR, SX3 (5 0 TAATACTGGYAATTTTTCAGATGG3 0 ) and SX4 (5 0 AATACAGATTGCTTACAACCACC3 0 ) primers were used. The second round of amplification was performed in a volume of 20 lL (4.5 lL of distilled water, 13 lL of SinaClon 2 9 PCR master mix (SinaClon, Iran), 2 lL of SX3 and SX4 primers (10 lM), and 0.5 lL of the first-round PCR product). The reaction was carried out under the same cycling conditions. The PCR product was analyzed by electrophoresis on a 1.5 % agarose gel and visualized under UV light.

An AccuPrep Ò PCR Purification Kit (Bioneer Co., Korea) was used for the purification of the PCR products. Sequencing was performed with the primers (both directions) that were used in the second step of nested PCR (Bioneer Co., Korea). Chromatograms were evaluated with CromasPro (CromasPro Version 1.5). A phylogenetic tree was constructed by the neighbor-joining method, using MEGA 5.1 software, and each tree was produced using a consensus of 1000 bootstrap replicates [31] . The nucleotide sequences of a partial segment of the S1 gene were compared with several S1 sequences from GenBank, including H120 ( Table 3 ). The S1 gene sequences of the IBVs were submitted to the NCBI GenBank database with the accession numbers KU143883-KU143914.

Real-time PCR for IBV detection (IBV incidence rate)

Real-time PCR for IBV detection based on the 5 0 UTR was used in this study. Of the 100 samples tested, 32 were positive for IBV by real-time PCR. The number of positive samples from each governorate is presented together with the sample size from each governorate. The total incidence rate for the middle and south Iraqi governorates was 32 %, where the highest incidence rate of IBV in broiler chickens was detected in Al-Najaf governorate 10/20 (50 %), while the lowest rate 4/20 (20 %) was detected in Al-Muthana governorate ( Table 2) .

To carry out IBV genotyping, all 32 positive samples were subjected to amplification of a portion of the S1 gene for sequencing. All samples that were positive in real-time PCR were positive in nested PCR. Four genotypes were detected after sequencing. The percentage of IS/1494, 793/B, QX, and DY12-2 genotypes in five Iraqi governorates was 46.87 %, 40.62 %, 9.37 % and 3.12 %, respectively ( Table 2 and Fig. 2 ). The QX genotype was detected in Al-Kut and Al-Najaf governorates, while the DY12-2 genotype was only detected in Al-Najaf governorate. IS/1494 and 793/B genotypes were detected in all governorates ( Table 2) . 

The nucleotide sequences of the S1 gene from 32 Iraqi IBV isolates obtained in this study were aligned and compared with those of previously identified isolates from Iraq, neighboring countries, and worldwide reference IBV strains, as shown in Table 3 . The analysis revealed that all sequences obtained in this study were genetically different. Phylogenetic analysis of the 32 strains (Fig. 2) revealed that Iraqi IBV strains could be classified into four genetic groups or genotypes: group I, variant 2 IS/1494-like viruses, including 15 field isolates (46.87 %); group II, 793/Blike viruses, including 13 field strains (40.62 %); group III, QX-like viruses, including three field strains (9.37); and caused by bacteria and mycoplasmas have already been detected on broiler farms located in the central and southern parts of Iraq. However, the molecular detection studies (infection rate and genotyping) of respiratory viral diseases such as IB in broilers in Iraq are limited (personal communication with veterinary organizations). We conducted the first study to determine the infection rate and IBV genotypes on broiler farms, using molecular and phylogenetic techniques. The approved vaccine strains used in Iraq are based on serotypes Ma5, H120, and 793/B [21] . IB still causes serious problems in the Iraqi poultry industry due to the inability of the vaccines to provide cross-protection between different genotypes [1] . The genotyping of IBV is necessary not only for understanding virus evolution but also for effective modification of the vaccination programs [10] . In this study, the infection rate of IBV was 32 %, which indicated widespread distribution of IBV in the southern and middle parts of Iraq. The highest infection rate of IBV was observed in Al-Najaf (50 %) governorate, while the lowest (20 %) was observed in Al-Muthana (Fig. 3) . [32] . The infection rate was lower than the rate previously reported on commercial farms within neighboring countries: 58.8 % and 42.8 % in Jordan and Iran, respectively [2] . Even where there were no poultry farms in Iraq, the infection rate was as high as 32 %. This may be attributed to poor biosecurity on the farms, resulting in the spread of infection between and within the flocks [22] . Phylogenetic analysis showed that the Iraqi isolates clustered into four genetic groups (group I, variant 2 (IS/ 1494)-like; group II, 793/B-like; group III, QX-like; and group IV, DY12-2-like). Variant 2 (IS/1494-like) viruses were the dominant IBV genotype infecting broiler chickens, with an overall prevalence of 46.87 % in IBVpositive samples. They shared high nucleotide sequence identity with IBV isolates from Iran, Israel, Egypt, Turkey, and Kurdistan. The isolation of IS/1494/06 (EU780077) IBV, one of two Israeli variant strains, was first reported by Meier and Mahar in Israel [23] . IBV variants have been recognized in Iraq since 2012 (Iraqi Veterinary Directorate/Ministry of Agriculture). The present work is similar to another study in which three IBV genotypes were found in Slemani-Kurdistan, Iraq, including group A (very similar to Iranian isolates), group B (closely related to Chinese isolates) and group C (similar to IS/1494 and Egypt/Beni-Seuf/01 isolates) [26] . These findings are in agreement with those of Kanan et al., who reported that IS/1494/06 IBV was the most common genotype in Egypt (2010), Lebanon (2010-2012), Jordan (2011-2012), Kuwait (2012), and Oman (2013) [12] . The current study is similar to another study on an IS/1494 strain in Turkey [18] .

The second group consisted of 793/B-like viruses that were detected in different Iraqi governorates at the high prevalence of 40.62 %. The present study revealed the presence of 793/B on the broiler farms. The IBV strains of the 793/B serotype were first identified in France in 1985, followed by Great Britain in 1991. Subsequently, these strains spread to other countries in Europe, Asia (Particularly to Iran, Turkey), and North America and are some of the most common IBV serotypes in some countries [9] . Our result were in line with those of Mahmood et al., who conducted the first study of identification and genotyping of IBV isolates and indicated that the 4/91-like virus is circulating on vaccinated broiler farms of the Kurdistan region of Iraq [21] . The detection of 793/B in commercial flocks was previously reported in Iran, Jordan, and Israel [23, 27, 29] . However, because all flocks have been vaccinated, we concluded that the detected viruses probably belonged to the vaccine strains. Therefore, the full-length S1 gene of the isolates should be determined to differentiate field and vaccine isolates. In this study, another important IBV variant (QX-like) known to cause respiratory, renal, and reproductive problems [12] was detected with a prevalence of 9.37 %. In 1996, QX was first described and identified in China, after which the prevalence of the so-called QX-like IBV genotype was reported, and it became one of the most dominant genotypes in many countries [28] . The genotype, designated as QX IBV, has spread from Asia, where it was described for the first time in 1996 in China, and spread to Europe and recently to the southern part of the African continent [4] . The QX-like genotype was also previously detected in central Iraq by the Iraqi veterinary directorate (official report). This finding was in line with another study in the Kurdistan region of Iraq, which showed that the phylogenetic aspects of the Kurdistan viruses were closely related (98.9 %) to the QX strain reported in China from 2009 to 2010 [4] . QX-type virus was isolated in Israel in 2004 [4] . The PCR Lab/06/2012 (JX477827) strain was c Fig. 3 Phylogenetic tree of 32 Iraqi isolates and 24 reference strains of infectious bronchitis virus based on the nucleotide sequences of the S1 gene. The phylogenetic tree was constructed, using MEGA version 5, by the neighbor-joining method with 1000 bootstrap replicates (bootstrap values are shown on the tree). The isolates from this study are indicated by black circles. White dots indicate Iranian reference strains, and black triangles indicate Iraqi reference strains isolated in Iran, which is genetically (99.36 %-99.68 %) related to strains of the present study [6] . This finding revealed that the QX strain has been widely transferred from other countries (probably Iran) to Iraq. Although no information is currently available about the introduction of the QX strain from China into other countries, it has been hypothesized that wild birds may be the source of introduction based on the evidence that IBV may replicate in members of the order Anseriformes [25] . It is of interest that that DYI2-2-like IBV genotype was detected in the present study. This is the first report of this genotype in Iraq, and it has not yet been reported in other countries in the Middle East. The virus appeared following the recombination of CK/CH/GD/LZ09 and TA09 IBV in China and is currently a circulating IBV genotype in China [11] . It is also fair to say that the origin of this virus and the means by which it was introduced to Iraq are not clear. Based on the persistence of the DY12-2 like genotype in China, it could be predicted that this genotype might spread in Iraq and the Middle East in the future. In this survey, some IBV genotypes, such as IS/720, Massachusetts, D274, and Q1, were not detected. These IBV genotypes were detected previously and reported in Iraq, Iran, the United Arab Emirates and Saudi Arabia [3, 12, 14, 21] .

New serotypes or variant strains may emerge because of only a few changes in the amino acid sequence of the S1 protein. These changes could be due to immunological pressure caused by the extensive use of vaccines, recombination as the outcome of mixed infections, or a decrease in the prevalence of dominant serotypes as a result of vaccination, allowing other field strains to emerge [25] . Not much is known about the mode of spread of IBV between the countries in the Middle East. However, crossborder movements of poultry and poultry-related products are likely to be important factors [21] .

In summary, our study demonstrated a high rate of infection with IBV (32%) in central and southern Iraq. This is the first report indicating the presence of an IBV DY12-2like strain in the Middle East. It is an updated and comprehensive study of genotyping of IBV in Iraq and completes the IBV puzzle in the region. Phylogenetic analysis showed that the detected strains are closely related to other IBV strains infecting broilers, pullets, and layers in the region. Finally, it could be suggested that 1) work be done on whole-genome sequencing, 2) cross-protection studies be conducted for designing the best vaccination program in the Iraqi poultry industry, 3) molecular surveillance in other governorates in Iraq be continued, and 4) the pathogenesis of different Iraqi IBV isolates be studied.

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Acknowledgments The authors gratefully acknowledge Dr. Hamideh Najafi, Dr. Iraj Ashrafi, and Mr. Behrooz Asadi for their extensive technical support.

Funding Financial support for this study was provided by the Ministry of Higher Education and Scientific Research, Iraq, University of Basrah (7.30.4603), and the Research Council of the University of Tehran under Grant No. 28692-6-2.

Ethical approval This article does not contain any studies of animals performed by any of the authors.