key: cord-0728994-3l6tjf3t authors: Hajijafari Anaraki, Mozafar; Sheikhi, Nariman; Haghbin Nazarpak, Hadi; Nikbakht Brujeni, Gholamreza title: Molecular characterization of infectious bronchitis virus based on RNA‐dependent RNA polymerase gene date: 2020-05-26 journal: Microbiol Immunol DOI: 10.1111/1348-0421.12825 sha: f6dee5238da8eba56f2ed14b66b17fbc8d0251ec doc_id: 728994 cord_uid: 3l6tjf3t Extensive rate of variations in in spike glycoprotein subunit gene of infectious bronchitis virus (IBV) caused challenges for counting variants for differentiation of infected from vaccinated birds and addressing the variants of unknown significance. The study aimed at investigating the possibility use of RNA‐dependent RNA polymerase gene (RdRp) as a target for molecular characterization of IBV strains in Iran. Samples collected from commercial broiler flocks (n= 52) showing respiratory syndrome. Specific polymerase chain reaction (PCR) primers were designed for a variable region locates in RdRp gene, flanked by highly conserved regions. Reverse transcriptase PCR (RT‐PCR) followed by sequencing and sequence analysis could identified 8 IBV variants in an overall prevalence of 44.2%. Deduced nucleotide and amino acid sequences were compared with published sequences for IBV strains. Due to the long‐distance similarities, the field samples could be discriminated from vaccine strains. Phylogenetic analysis of RdRp gene sequences resulted in clustering the IBV strains related to each area. Using RdRp, as a genetic marker eliminates the challenges arise from the enormous variations that making difficult the discrimination between field and vaccine strains as well as affiliation of certain variants to various geographical areas. This article is protected by copyright. All rights reserved. replicase complex that codes for functional domains such as a papain-like proteinase (PL(pro)), main protease (M(pro)), RNA helicase, and RNA-dependent RNA polymerase (RdRp) (4). The replicase is a multifunctional polyprotein which, after cleavage, produces a number of functional products those are indispensable for viral transcription and replication (5). Mutation and recombination in IBV genome are the main cause of phenotypic variations. Consequently, emergence of newly variant strains is mainly considered as a potential for new pathotypes, serotypes, and protectotypes. Nucleotide sequencing and subsequent genetic analysis indicated a continuous spatio-temporal dynamics of IBV variations. Most of all investigations on the IBV strains conducted based on the S and N protein gene sequences (6-8). It is believed that structural proteins are imperative target parts of the virus particles for immunization and reaching the protective immunity. Therefore, variations in IBV S and N sequences are mostly considered for selecting the best vaccine strains for vaccination programs. However, extensive rate of variations in S1 gene among IBVs poses multiple challenges for clinicians in counting variants for differentiation of infected from vaccinated birds and addressing the variants of unknown significance (VUS)(9). Furthermore, in molecular epidemiology approaches, high polymorphism means that there is a low probability that a new variant could be classified in an identified profile or group. Because of high degree of polymorphisms on S1 sequence, diverse genetic group designations for IBV strains are usually inconsistent with phylogenetic history (10). Therefore, another powerful target for monitoring the virus variations seems to be necessary. As a genetic marker it also must be capable of being used for phylogenetic and epidemiological evolution of IBV variants. Here we suggested the molecular characterization of infectious bronchitis virus based on RNA-dependent RNA polymerase gene. This article is protected by copyright. All rights reserved. Direct sequencing method was performed for the analysis of the amplified RdRp, S1 and N genes cDNA. The PCR products were purified using the purification kit (Bioneer, Seoul, Korea) and each amplicon was separately sequenced in both directions by using specific forward and reverse primers. All procedures were carried out by Sanger sequencing on an ABI 3730 XL automatic DNA sequencer (Macrogen Inc. Seoul, Korea). Sequences were analyzed by BLAST through the national center for biotechnology information website (http://www.ncbi.nlm.nih.gov/, NCBI). Alignments and the amino acids prediction were made using the Bioedit version 7.0.5.3 software package. The partial sequence of RdRp gene belonged to Iranian IBV field strains, aligned with non-redundant sequences from all available strains, retrieved from the GenBank database, were chosen for phylogenetic analysis. The partial sequence of S and N genes which is conventionally used for IBV genotyping (OIE, 2013) was also used for phylogenetic analysis and making comparisons. Phylogenetic analysis was conducted using MEGA6 Beta (http://www.megasoftware.net/mega41.html) and the tree was constructed by the neighbor-joining method. Bootstrapping over 1,000 replicates was done to assess the confidence level of the branch pattern. The RT-PCR with specific primers for RdRp gene of avian IBV resulted in the amplification of a Figure 4) and N nucleotide sequences. The S1 tree consisted of This article is protected by copyright. All rights reserved. three main clusters divided from ancestor node. Serotypes Gray and JMK were separated by a long branch from other groups. Gammacoronavirus, one Chinese strain and two QIA strains segregated with one vaccine strain (4/91). The third cluster contained H120, Ma5, Pakistan, Massachusetts and Chinese strains. The N tree also consisted of three main clusters divided from ancestor node. Iranian strains separated in one cluster containing China, Italy, Canada and 4/91 vaccine strains. Pakistan, Jordan and H120 strains segregated to second cluster and Poland, China and SNU strains in third cluster. There are three groups of avian, mammals and insect circoviruses. Iranian CaCV strains phylogenetically clustered closely with canary circovirus. Porcine and canine circoviruses segregated with human and goat in a two separate clade. Mosquito, panda and hermit crab, which accounts for distinct amino acid sequences.. One branch only contained. Replicase gene of IBV encodes PL (pro), M (pro), RNA helicase and RdRp domains those are important for virus replication. The genotypic characterization assay developed in the current study focused on the variable regions of RdRp gene. This idea came from the observation that IBV S1 is a highly variability-prone region especially due to the lack of RNA polymerase proofreading (6). It is also a shifting viewpoint from the assays targeting the incessant S1 gene variation to the characterization/detection based on the cause or origin of variation which is the RdRp gene. However, this idea must be elaborated by study the numerous variations as well as the association with pathogenicity and virulence. Based on the RT-PCR detection and sequence analysis of IBV RdRp gene, an overall prevalence of field strains estimated as 44.2%. A similar prevalence has been reported for farms with clinical respiratory signs in which varies from 45 % to 54.4 % between districts (11-14). Eight variants of the virus were identified in 23 farms, with the noticeable dominance of M6. The emergence of new IBV variants, that is an ongoing process, is a great challenge for identification, discrimination and characterization of the novel variants. In our experiment identifying the viruses that did not overlap the vaccine strains, stands for the capability of the test for detection of IBV and differentiating infected from vaccinated animals (DIVA). In addition, differentiations made by sequence analysis could be used for further characterizations such as affiliation of certain variants to certain geographical area. This article is protected by copyright. All rights reserved. Comparative Analysis of Complete Genome Sequences of Three Avian Coronaviruses Reveals a Novel Group 3c Coronavirus Infectious Bronchitis Novel genotype of infectious Accepted Article 13 Seroprevalence and genotyping of avian infectious bronchitis virus detected from Iranian unvaccinated backyard chickens Genotyping of avian infectious bronchitis virus in Afghanistan Molecular detection and phylogenetic properties of isolated infectious bronchitis viruses from broilers in Ahvaz, southwest Iran, based on partial sequences of spike gene Detection of Newcastle disease, H9N2 avian influenza, and infectious bronchitis viruses in respiratory diseases in backyard chickens in Ahvaz Comparison of four regions in the replicase gene of heterologous infectious bronchitis virus strains Phylogenetic analysis of a highly