Slov Vet Res 2018: 55 (4): 213-18 DOI 10.26873/SVR-366-2018 UDC 636.5.09:578.2:616.98:575.86(55) Original Research Article MOLECULAR CHARACTERIZATION AND PHYLOGENETIC ANALYSIS OF AVIAN POX VIRUS ISOLATED FROM PET BIRDS AND COMMERCIAL FLOCKS, IN IRAN Arash Ghalyanchilangeroudi1, Hossein Hosseini2*, Rima Morshed3 department of Microbiology and Immunology, Faculty of Veterinary Medicine, University of Tehran, Tehran, 2Department of Clinical Sciences, Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj, 3Agriculture and Veterinary group, Iran Encyclopedia Compiling Foundation, Ministry of science, research and technology, Tehran, Iran Corresponding author, E-mail: hosseini.ho@gmail.com Abstract: Avian pox (AP) is a viral disease with a wide host range. The aim of the study was molecular identification and characterization of field isolated pox virus from pet birds and commercial flocks in Iran, by polymerase chain reaction (PCR). Scab materials of skin andmucosallesions were collected from five clinicallyaffectedcases. PCR wasusedtoamplify a 578 bpfragment of the poxvirus 4b core protein. In order to determine the genetic relationships among the viruses, this conserved poxvirus genetic region was sequenced and analyzed. The Iranian Avipoxvirus isolates in this study grouped in clade A1 (commercial chicken and turkey flocks) and B1 (canary). Further studies on a larger scale need to be developed to have a better understanding of the molecular characterization of the Iranian APV strains. Key words: avian pox; phylogenetic analysis; molecular characterization; Iran Introduction Avian Pox (AP) is a viral well-known disease in hens, turkeys and many other birds (278 species from 70 families and 23 orders), characterized by cutaneous lesions on the feather-less skin and/or diphtheritic lesions of mucous coats of the upper alimentary and respiratory tract. Moreover, concurrent systemic infection causing high mortality often occurs in canaries. AP lesions, however, may compromise vision, the ability to feed or lead to secondary bacterial or Received: 13 February 2016 Accepted for publication: 19 November 2018 fungal infection leaving wild birds vulnerable to predation.The poxviruses which infect birds belong to the genus Avipoxvirus of the Poxviridae family. Avipoxviruses (APVs) within the family Poxviridae contain nearly 300-kilo base pair (kbp) of double-stranded DNA that replicate in the cytoplasm of infected cells and the members of the genus Avipoxvirus in the subfamily chordopoxvirinae (1). Pox infection usually occurs through the mechanical transmission of the virus to injured skin and also the bite of mosquitoes or mites. The incubation period and duration of APV infection is variable (from a few days to many months), but affected birds with mild lesions frequently recover and this is considered to be the most common 214 A. Ghalyanchilangeroudi, H. Hosseini, R. Morshed situation in wild birds. Its incidence is variable in different areas because of differences in climate, management and hygiene or the practice of regular vaccination. It can cause drops in egg production, or retarded growth in younger birds (2). The conventional laboratory diagnosis of APV is carried out by histopathological examination, electron microscopy, virus isolation on chorioal-lantoic membrane (CAM) of embryonated chicken eggs or cell cultures, and serologic methods (2). The 4b core protein gene (p4b) of APV that encodes the protein with molecular weights of 75.2 kDa is usually chosen for comparative genetic analysis (3,4,5). Also, amplification of the p4b of APV by PCR has often been used as a molecular tool for the detection of APVs (6). PCR in combination with restriction endonuclease enzyme analysis (REA) followed by sequence analysis of the amplified fragments is used for detection, differentiation and molecular characterization of fowl pox virus isolates (7). Even considering the decrease in problems caused by poultry production, avian pox is still a significant pathogen which can have serious effects on wild Galliformes. The incidence of AP in Iran is high in pet birds and also in fewer levels in commercial farms. In recent years, some outbreaks of skin lesions suspected to be avian pox were observed in the backyard poultry in different parts of western areas in Iran. Generally, the number of reports concerning incidence and characterization of avian pox viruses in Iran is very low. Gholami-Ahangaran et al. performed a survey on 328 backyard poultries with suspected signs of avian pox virus infection. Their results showed 217 and 265 out of 328 samples were positive for avian pox virus on histopathological and PCR examination, respectively (8). In the study of Fasaei et al. (2014), Avipoxvirus specific DNA was detected in all 10 different isolates from chicken, canary and mynah that were collected from Tehran province (3). The aim of this study was a characterization of AFPv isolates from canary, chicken and turkey flocks by PCR. Materials and methods Sampling Samples (cutaneous scrub and caseous lesions) were collected from different species with characteristic clinical signs (5 samples from chicken, canary, turkey) had been submitted to PCR Veterinary Diagnostic Laboratory (Tehran, Iran), during 2012- 2014. The data of samples are available in table 1. DNA extraction DNA was extracted from the skin or pulmonary lesions of the clinical cases and lyophilized vaccines (as a positive control) by QIAamp DNAMini Kit (Qiagen) following the manufacturer's guidelines. DNA samples were stored at -20°C until analysis. Amplification of 4b Gene The AVP-specific PCR was performed using primer pairs described based on FPV 4b core protein (P4b) gene sequence of Fowl pox virus strain HP444 previously (9). The sequence of the primers was as follows: forward primer: 5'-CAGCAG-GTGCTAAACAACAA and reverse primer: 5'- CG-GTAGCTTAACGCCGAATA. PCR consisted of 25 pl reaction containing1.5 units of Taq DNA polymerase, 1.5 mM MgCl2, 200pM of each deoxynu-cleoside triphosphate, 6 pmol of each primer, DNA extracted from clinical samples and nuclease free water up to 25 pl. Amplification was performed after initial denaturation for 2 min at 94°C for 35 cycles and consisted of 1 min denaturation at 94°C, 1 min annealing at 60°C, and 1 min extension at 72°C. In this study, live fowl pox vaccine (Razi Vaccine and Serum Research Institute, Iran) was provided and used as positive control.Negative control includes all the reagents without a template. Sequencing and Phylogenetic analysis The positive PCR products were analyzed by electrophoresis on a 1% agarose gel and visualized bye GelRed ™ (Biotium , USA) staining and ultraviolet transillumination. The PCR products were purified by the PCR AccuPrep® PCR Purification Kit (Bioneer Co., Korea) and purified PCR products were sent for sequencing (Source Bioscience, UK) with PCR primers for in a forward and in a reverse direction. Sequencing reactions were run on an ABI Prism 310 Genetic Analyzer. The sequence results were downloaded and analyzed using Chromas (Technelysium Pry Ltd., Australia). Phylogenetic analysis was carried out by analyzing the data obtained here with those of other sequences of FPVs Molecular characterization and phylogenetic analysis of avian pox virus isolated from pet birds and commercial flocks, in Iran 215 from the GenBank database. The phylogenetic analysis was performed with the MEGA5 (Phylogeny Inference Package) software, version5. Distance-based neighbor-joining trees were constructed using the Tamura-Nei model (10). The robustness of the phylogenetic trees was assessed by 1,000 bootstrap replicates. Bootstrap values lower than 50 were omitted. The FPV sequences tested in this study were deposited in GenBank under accession numbers KT003286 -KT003290. Results Because of the highly conserved nature of the analyzed genes, nucleotide sequences rather than amino acid sequences were used to determine divergence. Clades and sub clades have been named according to previous APV phylogenetic studies based on the P4b (11). The strains were placed in A1 and B1 sub clades. The homology between isolates was 67.3%-100% (Table 3). Table 1: Description of fowl pox virus strains investigated in this study Strain name Nam in Tree Species Type of Lesion Year Province Accession No. IR/Canary poxvirus/ H364/12 CP/H364/12 Canary Cutaneous 2012 Alborz KT003286 IR/Canary poxvirus/ H913/14 CP/H913/14 Canary Cutaneous 2014 Kurdistan KT003287 IR/Fowl pox virus/H252/12 FP/H252/12 Chicken Caseous 2012 Isfahan KT003288 IR/Fowl pox virus/H756/13 FP/H756/13 Chicken Caseous 2013 Tehran KT003289 IR/Turkey pox virus/ H335/12 TP/H335/12 Turkey Cutaneous 2012 Alborz KT003290 Table 2: Details of poxvirus sequences obtained from GenBank Isolate name Abbreviation (Tree) Host Country Accession Number Clades Fowlpox virus isolate FPV-VR250 FP/FPV-VR250 Chicken Norway AY453172 A1 Fowlpox virusNobilis Variole W (Intervet) FP/ Nobilis Variole W Chicken United Kingdom AM050379 A1 Fowlpox Mild (Websters; Fort Dodge) FP/Websters Mild Chicken United Kingdom AM050378 A1 Avipoxvirus isolate GB 134/01 TP/ GB 134 Turkey Germany AY530304 A1 FWPVD Diftosec CT (Merial) FP/ Diftosec CT Chicken United Kingdom AM050380 A1 pigeonpox PGPV TP-2 PP/ TP-2 Pigeon Germany AY530303 A2 Avipoxvirus isolate CVL 2/11/66 TP/ CVL 66 Turkey United Kingdom AM050387 A2 Avipoxvirus isolate CVL 10/12/98 TP/ CVL 98 Turkey United Kingdom AM050388 A2 Avipoxvirus HNPV/NZL/2002 PP/ HNPV Pigeon New Zealand HQ701713 A3 AvipoxvirusCVL 353/87 AP/CVL 87 Albatross United Kingdom AM050392 A3 Falconpox FLPV GB362-02 FLP/ GB362-02 Falcon Germany AY530306 A4 Canarypox virus isolate CP10IR CP/CP10IR Canary Iran KC193679 B1 Canarypox virus isolate Yazd1 CP/Yazd1 Canary Iran KF673397 B1 Canarypox virus strain AT_Canarypox/1839/2009 CP/1839 Canary Austria GU108510 B1 anarypox virus isolate D98-11133 CP/ D98-11133 Canary Canada GQ487567 B1 Avipoxvirus CVL SP/ CVL Sparrow United Kingdom AM05038 B1 Pigeonpox PPV-B7 PP/PPV-B7 Pigeon Norway AY453177 B2 Parrot pox 364/89 PP/364/89 Parrot United Kingdom AM050383 C Avipoxvirus isolate APIII AP/ APIII Agapornis Germany AY530311 C 214 A. Ghalyanchilangeroudi, H. Hosseini, R. Morshed Figure 1: Phylogenetic tree of 578 bp nucleotide sequences of the 4b core protein gene of APV isolated in this study (marked with a black circle), reference APV sequences. The tree was obtained by the neighbour-joining method. Bootstrap testing of phylogeny was performed with 1000 replications and values equal to or greater than 70 are indicated on the branches (as a percentage).The length of each bar indicates the amount of evolution along the horizontal branches as measured by substitution per site. APV clades A-C and sub clades are labelled. You could find the details of viruses in tables 1 &2 Table 3: Percentage of 4b core protein sequence identity of APV isolated in this study and some selected APV isolates from GenBank 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 1 CP/GHPCRLAB.1 2 CP/GHPCRLAB.2 99.04 3 FP/GHPCRLAB.3 70.26 70.91 4 FP/GHPCRLAB.4 70.67 71.32 99.52 5 TP/GHPCRLAB.5 71.02 71.66 99.28 99.76 6 TP/GB134 70.67 71.32 99.52 100.00 99.76 7 FP/WebstersMild 70.67 71.32 99.52 100.00 99.76 100.00 8 FP/NobilisVarioleW 70.67 71.32 99.52 100.00 99.76 100.00 100.00 9 TP/CVL98 70.18 70.53 89.44 90.00 89.73 90.00 90.00 90.00 10 FP/DiftosecCT 70.67 71.32 99.52 100.00 99.76 100.00 100.00 100.00 90.00 11 FP/FPV-VR250 70.67 71.32 99.52 100.00 99.76 100.00 100.00 100.00 90.00 100.00 12 PP/TP-2 70.18 70.53 89.44 90.00 89.73 90.00 90.00 90.00 100.00 90.00 90.00 13 TP/CVL66 70.18 70.53 89.44 90.00 89.73 90.00 90.00 90.00 100.00 90.00 90.00 100.00 14 AP/CVL87 69.11 69.37 90.54 91.09 90.82 91.09 91.09 91.09 97.58 91.09 91.09 97.58 97.58 15 PP/HNPV 69.11 69.37 90.54 91.09 90.82 91.09 91.09 91.09 97.58 91.09 91.09 97.58 97.58 100.00 16 FLP/GB362-02 70.91 71.99 86.28 86.86 86.58 86.86 86.86 86.86 87.65 86.86 86.86 87.65 87.65 87.10 87.10 17 SparrowCVL 99.28 99.76 71.28 71.69 72.03 71.69 71.69 71.69 70.91 71.69 71.69 70.91 70.91 69.76 69.76 71.62 18 CP/D98-11133 99.28 99.76 71.28 71.69 72.03 71.69 71.69 71.69 70.91 71.69 71.69 70.91 70.91 69.76 69.76 71.62 100.00 19 CP/1839 99.28 99.76 71.28 71.69 72.03 71.69 71.69 71.69 70.91 71.69 71.69 70.91 70.91 69.76 69.76 71.62 100.00 100.00 20 CP/Yazd1 99.04 99.52 71.28 71.69 72.03 71.69 71.69 71.69 70.91 71.69 71.69 70.91 70.91 69.76 69.76 71.62 99.76 99.76 99.76 21 CP/CP10IR 93.28 93.54 64.31 64.77 65.13 64.77 64.77 64.77 63.98 64.77 64.77 63.98 63.98 62.72 62.72 64.27 93.80 93.80 93.80 93.54 22 PP/PPV-B7 81.26 82.15 73.29 73.69 73.36 73.69 73.69 73.69 75.15 73.69 73.69 75.15 75.15 75.15 75.15 75.88 82.15 82.15 82.15 81.83 75.12 23 PP/364_89 71.66 71.62 70.77 71.16 71.14 71.16 71.16 71.16 69.95 71.16 71.16 69.95 69.95 69.26 69.26 67.17 71.99 71.99 71.99 71.62 65.17 72.37 99.52 24 AP/APIII 70.91 70.87 70.39 70.80 70.77 70.80 70.80 70.80 69.57 70.80 70.80 69.57 69.57 68.87 68.87 66.36 71.25 71.25 71.25 70.87 65.17 72.37 Molecular characterization and phylogenetic analysis of avian pox virus isolated from pet birds and commercial flocks, in Iran 215 Discussion Avian pox viruses have been isolated from a wide range of avian species including commercial poultry, wild and pet birds. Poxvirus infection is suspected when proliferative skin and/or oral and tracheal lesions are observed. In such cases, a diagnosis is made by histopathology examination of the lesions. Fowl pox vaccine is used in Iranian poultry industry in layer and breeder farms. We don't have any specific vaccine for pet birds. Canary pox has been known as the disease that can result in high losses in a short time, as a re-emerging disease that has not been present during recent years in canary flocks in Iran (12). Most of our knowledge about the situation of APVs in Iran was obtained from very few reports concerning the epidemiology and the infection biology of the virus. In this study, APVs in clinical cases of affected commercial chickens, turkeys, and canary were identified and characterized by molecular methods to determine the etiology of APV in Iran, 2012. As for the molecular biological analysis, gene P4b amplification products of the expected size were obtained for all the strains of this study, thus confirming that PCR is an extremely valuable diagnostic method for APV infections. Phylogenetic relationships of Avipoxviruses have been analyzed based on the gene corresponding to vaccinia virus (VACV) P4b (fpv167, VACV A3L), indicating that all Avipoxvirus strains cluster into 3 major clades, namely, A (Fowl pox (FWPV)-like), B (Canary pox (CNPV)-like) and C (Psittacine). Clade A can be further divided into seven sub clades (A1-A7) and Clade B is comprised of three sub clades (B1-B3) (14). Based on the phylogenetic analysis of four conserved regions, the viruses characterized from Iranian columbiformes cluster into two groups. The viruses from turkey and commercial chickens grouped in sub clade A1 and the viruses from canary grouped in sub clade B1. Conversely, it has also been shown that the same viruses can infect different birds. Therefore, in this study as well as in others, APVs from the same species of bird are classified in different sub clades (4,13,14). Fasaei et al (2014) in phylogenetic analysis of Avipoxvirus strains isolated from different bird species in Iran showed that a similarity of 71-100% with the other sequences in the GenBank but they didn't submit their sequences in GenBank and didn't determine the clade of isolates (3). The research which did by Gholami-Ahangaran (2014) on avian pox of backyard poultry in Iran indicated that 66.1% and 80.7% of samples were positive for avian pox virus on histopathological and PCR examination, respectively (8). The data presented in this research provide novel insights into the molecular characterization of avian pox viruses collected from the broad host range outbreaks in different geographical parts of Iran on particular period time. 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MOLEKULARNA KARAKTERIZACIJA IN FILOGENETSKA ANALIZA AVIARNEGA VIRUSA POX, IZOLIRANEGA PRI PETIH VRSTAH PTIC V KOMERICIALNIH JATAH V IRANU A. Ghalyanchilangeroudi, H. Hosseini, R. Morshed Povzetek: Osepnice ptic (AP, iz angl. avian pox) so virusna bolezen, ki lahko okužijo veliko različnih vrst ptic. Cilj predstavljene raziskave je bila molekularna identifikacija in karakterizacija izoliranih virusov pox, pridobljenih iz krast ljubiteljskih ptic in farmskih ptic v Iranu, s pomočjo metode PCR. Vzorci krast s področja sprememb na koži in sluznicah je bil zbran pri petih pacientih s kliničnimi znaki bolezni. S pomočjo metode PCR je bil pomnožen 578 baznih parov dolg odsek gena poxvirusa 4b. To ohranjeno območje poxvirusa je pomembno za določitev genskih razmerij med virusi, zato je bilo določeno zaporedje DNK za nadaljne analize. Iranska izolata Avipoxvirusa objavljena v tej študiji sta bila razvrščena v razred A1 (komercialne piščančje in puranje jate) in B1 (kanarčki). Za boljše razumevanje molekularne karakterizacije iranskih sevov virusa AP bo potrebno opraviti nadaljnje študije. Ključne besede: osepnice ptic; filogenetska analiza; molekularna karakterizacija; Iran