key: cord-1037001-hhnzl3ij authors: Kalokhoran, Ali Yousefzadeh; Ghalyanchilangeroudi, Arash; Hosseini, Hossein; Madadgar, Omid; Karimi, Vahid; Hashemzadeh, Masoud; Hesari, Parvaneh; Zabihi Petroudi, Mohammad Taha; Najafi, Hamideh title: Co-circulation of three clusters of 793/B-like avian infectious bronchitis virus genotypes in Iranian chicken flocks date: 2017-07-08 journal: Arch Virol DOI: 10.1007/s00705-017-3473-3 sha: 320c565a295cff68b0fc1849dfb196a9f99371b2 doc_id: 1037001 cord_uid: hhnzl3ij Avian infectious bronchitis (IB) is an acute and highly contagious viral disease causing severe economic losses in the poultry industry. The 793/B IB virus is an important infectious bronchitis virus (IBV) genotype currently circulating in several countries, including Iran. One hundred confirmed IBV samples (between 2014 and 2015; from 15 provinces in Iran) were selected for genotyping based on S1 sequencing. After phylogenetic analysis, it was found that 30% of the IBV isolates belonged to the 793/B genotype. Results showed that the Iranian 793/B-like IBV isolates could be divided in to three clusters: 4/91-like (50%), 1/96-like (40%), and IB88-like (10%). The sequence similarity between Iranian 793/B-like IBV isolates is 87.69%–100%. The highest identity is between the 4/91 and IB88 clusters (96.38%), and the lowest similarity is between the 1/96 and IB88 clusters (87.62%). This study provides a comprehensive analysis of 793/B-type IBV in Iran and characterization of IBV molecular epidemiology in the country. Avian infectious bronchitis virus (IBV) is a gamma-coronavirus which causes a highly contagious respiratory disease of economic importance in chickens that is prevalent throughout the world [13] . A variety of different IBV strains have been reported in chickens worldwide, with pathology ranging from mild respiratory symptoms to severe kidney and oviduct diseases [31] . The coronaviruses are enveloped and contain a single-stranded, positive-sense RNA genome of 28-32 kb, which is un-segmented, 5'capped and 3'-polyadenylated [5] . The genome encodes multiple proteins including structural proteins (E), nucleoprotein (N), spike (S), and membrane (M) [8] . The first known strain of the 793/B serotype, also known as 4/91 and CR88 was isolated in France in 1985 [11] . This serotype may also have entered the United Kingdom in the winter of 1990/91, where it was sometimes associated with deep pectoral muscle myopathy in addition to the more usual manifestations of IB [11] . Subsequently, it was discovered that this serotype has been present in most European countries. Following this, 793/B-type viruses were detected in several Asian countries, including Japan [21] , India [30] , Iran [32] , and Iraq [26] . The first report of IBV isolation from Iranian chicken flocks was in 1994 [29] . IBV strains isolated in Iran were classified into seven distinct phylogenetic groups: Massachusetts, 793/B, IS/1494, IS/720, QX, IR-1, and IR-2 [19, 22] . Currently, the major control measures for IB in Iran are vaccination with live attenuated IB vaccines (Massachusetts serotypes), such as H120, Ma5 and 793/B strains, including 4/91 (Intervet; from 2006), IB88 (Merial; from 2007), and iBird (Ceva; from 2016). As several types of 793-like IBV vaccines are used in Iran and the 793/B is a prevalent genotype in Iran the aim of this study was to generate more detailed information on the distribution of 793/B-like IBVs in Iran. One hundred confirmed IBV samples (Ghalyanchilab, Department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran), which were collected from chickens (broiler) suspected of disease between 2014 and 2015 (from 15 provinces) were selected for the study. Also, several 793/B-type IBV vaccines (iBird (1/96), IB88, and 4/91) were selected as positive controls. The presence of IBV was confirmed using a 5' UTR realtime reverse-transcription polymerase chain reaction [RT-PCR] [6] . Viral RNA was extracted from samples using the Cinapure RNA extraction kit (Sinaclon Co., Iran), following the manufacturer's instructions. For the reverse transcription (RT) reaction, random hexamers were used as described previously [24] . Nested PCR was then performed using spike gene primers that were designed to amplify a 390-bp fragment of the gene (a partial segment), as described previously [24, 33] . Sequencing was performed with the primers (both directions) used in the second step of nested PCR (Bioneer Co., Korea). Sequences were initially analyzed in Chromas PRO to confirm good quality read data had been obtained. We then performed NCBI Blast on the results and only 793/B-positive samples were subsequently considered for continued bioinformatics analysis. To determine phylogenetic relationships between the isolates, the deduced amino acid sequence for the hyper-variable region of the S gene sequence obtained in this study was compared with sequences from our lab and with corresponding regions from representative sequences available in GenBank. Alignment and comparison of amino acid sequences were performed using Clustal W in MEGA 7.0 [14] . Phylogenetic trees were constructed using MEGA7.0 with the neighbor-joining algorithm (bootstrap values of 1000) and the Kimura2 parameter model [20] . (Nucleotide sequences used in this study were submitted to the NCBI database with the accession numbers: KX702136-KX702181.) We detected 793/B-like IBV isolates in 30% of our IBV samples. The results showed that Iranian IBV 793/B-like genotypes could be divided in to three clusters: 4/91-like, 1/96-like, and IB88-like (Figure 1 ). Fifty percent of the IBV 793/B-like viruses belonged to the 4/91-like cluster, 40% were placed in the 1/96like cluster and 10% belonged to the IB88-like cluster. The average sequence similarity of the Iranian isolates was 87.62 -100%. The highest homology was between 4/91like and IB88-like clusters (96.38%), and the lowest similarity was found between the 1/96-like and IB88-like clusters (87.62%). The sequence similarities within each cluster (4/91-like, 1/96-like, and IB88-like) were 98.87-100%, 95.3-100% and 96.48-100%, respectively (Table 1 ). Phylogenetic analysis revealed that the first 793/B IBV genotype submitted to GenBank (VM/113654) in 2000 is located within the 1/96-like cluster ( Figure 1 ). Considering the presence of our isolates within this cluster and the fact that the 1/96 vaccine has only recently been imported into the country (after this study), it can be concluded that the Iranian 1/96-like isolates are likely field viruses. Viruses that grouped in the 1/96-like cluster were close to isolated made in France, Brazil, Poland and Spain. Viruses grouped in the 4/91 cluster have a close relationship with viruses isolated in the Ukraine, India, and Poland. The 4/91 IBV type was first identified in the UK in 1990/1991 but was retrospectively found to have been present in France since 1985 [9] . Antibodies reactive with the 4/91 type have been detected in chickens in Europe, Asia and Africa [12] . The first isolation of IBV in Iranian chicken flocks was reported by Aghakhan et al (1994) , which was confirmed to be the Massachusetts serotype [1] . Vasfi Marandi and Bozorgmehri Fard (2001) [19] . In a study performed by Najafi et al (2015), 793/Blike viruses had a total prevalence of 21%, ranking second among IBV types in Iranian chicken flocks [22] . In Israel, Massachusetts was the only type detected for many years until the 793/B type of IBV (Israel/793/B/variant 1/96) was identified in 1996 [13] . Similarly, a total of 100 tissue specimens from different commercial broiler flocks in Iraq were collected from 2013 to 2014. The prevalence rate of 793/B-like IBV isolates was 40.62%, second only behind1494-like IBV [26] . Ganapathy et al (2015) reported the IBV genotypes circulating in seven Middle East countries between 2009 and 2014. The prominence of 793/B (43.66%) was not surprising given that 793/B vaccine strains are widely used in the Middle East. Sequence analysis demonstrated that the majority of 793/B (67.13%) strains were closely related to vaccine strains, based on high homology (99-100%). After 2012, the 793/B field strain started to show distinct clustering, when compared to strains from earlier years. Indeed 793/B prevalence was 85.2% in UAE, 63.7% in Oman, 43.2% in Saudi Arabia, 16.8% in Egypt and 13% in Lebanon; however, it was not detected in Jordan or Kuwait [17] . In a separate study, two hundred and five samples were collected from broilers and layer chicken farms from all over Egypt in 2012. Sixty-four percent of suspected farms were positive for IBV, by real time RT-PCR. Thirteen IBV-positive samples were selected for further isolation and characterization. Only two isolates had a close relationship with CR/88121 and 4/91 viruses, with identities of 95% and 96%, respectively [28] . [33] . Ten IBV isolates collected from commercial chickens in Italy in 1999 were also characterized. Phylogenetic analysis showed five field viruses clustered together with 793/B-type strains, having 91.3-98.5% nucleotide identity within the group [4] . The phylogenetic analysis of IBV isolates from 1997 to 2006 revealed that the 793/B type is present in Poland. One isolate (PL-338-04-, isolated in 2004) grouped in the same cluster as the 4/91 vaccine while others, which were isolated in 1997 and 1999, grouped in another cluster [15] . Twenty-six IBVs isolated in Spain between 1992 and 2005 were also molecularly characterized. Genotype I represented 13 out of the 26 field isolates (isolated between 1992 and 2000) and grouped with 4/91 isolates [14] . Due to the use of a specific vaccine (4/91; Intervet) in the Iranian poultry industry, these strains might be related to the vaccine strain. In addition, 10% of Iranian 793/B IBV isolates were related to the IB88 strain. Interestingly, a significant difference in the detection rate between 4/91 and IB88 groups indicates that the 4/91 strain is more stable than IB88 in chicks. This data may reflect the re-isolation of vaccine strains. It is also possible that these live vaccine strains could have given rise to these genetically-altered field isolates. This is agreement with data from Franzo et al that detected 793/B IBV on farms that was derived from two in-use 793/B vaccines. Their evidence showed that the 793/B type became undetectable after the withdrawal of the two 793/B vaccines on several hundred Italian farms; they concluded that 793/B vaccine use is no longer required for 793/B virus control [16] . Finally, 40% of the 793/B IBV isolates were similar to the 1/96-like virus. Since in the period of sampling, iBird (1/96) was not approved for use in Iran, and, the first reported Iranian 793/B IBVs are located within this cluster, our underlying hypothesis and theory is that our 1/96-like IBV viruses are 793/B field isolates. This means that around 40% of 793/B type IBV strains should be considered as field IBV strains. This study examined the molecular epidemiology of the 793/B IBV genotype and studied the molecular dynamics of IBV in Iran and the surrounding region. This will help researchers to differentiate the origin of 793/B like viruses in the future. Of note, full genome sequencing of the different IBV isolates helped us to gather more information about the circulating 793/B IBV type. 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Finally, thanks so much Dr.Mehdi Mirsalimi for providing us the sequences of two of the first Iranian 703/B genotype IBV isolates (IR-10-2000, IR-13-2000) that were sent for sequencing. Funding This study was conducted under a grant from the research council, University of Tehran and CEVA Sante animal, Iran Branch. Ethical approval This article does not contain any studies with animals performed by any of the authors.