DOI: 10.5061/dryad.s7h44j167
title: Supplementary Information for: Sequence diversity and evolution of Infectious Bursal Disease Virus IBDV in Iraq

description: Background and Aim: Infectious Bursal Disease (IBD) is a highly infectious
 disease which causes huge economic losses to the poultry industry due to
 the direct impact of the illness and indirect consequences such as
 decreasing the general immunity of the flock, leaving it naive to other
 diseases. In Iraq, IBD is highly prevalent despite vaccination programs,
 yet studies on sequence diversity of the causative virus are still rare.
 Methods: A sample from Bursa of Fabricius from an IBD outbreak in a flock
 in the city of Najaf in Iraq was smeared on an FTA card. Amplicons of
 targeted regions in VP1 and VP2 genes were generated and sequenced.
 Sequences were then compared with other local and global sequences
 downloaded from GenBank repositories. Sequence alignment and DNA sequence
 analyses were achieved using MUSCLE, UGENE and MEGAx software. The
 molecular clock and sequence evolutionary analyses were applied using
 MEGAx tools. Results: The strain sequenced in this study belongs to a very
 virulent Infectious Bursal Disease Virus (vvIBDV) as the DNA and
 phylogenetic analysis of VP1 and VP2 gene sequences showed a mutual
 clustering with similar sequences belonging to vvIBDV genogroup 3.
 Analyses of the hyper variable region of VP2 gene (hvVP2) of IBDV isolates
 from Iraq indicates a presence of sequence diversity. Interestingly, the
 two vaccine strains Ventri IBDV Plus and ABIC MB71 that showed the highest
 sequence similarity to the local isolates in the hvVP2 region are not used
 in vaccination routine against IBDV in Iraq. Conclusion: Sequences of
 vvIBDV in Iraq are diverse. Remarkably, some of the available vaccine
 strains show high sequence similarity with local strains in Iraq; however,
 they are not included in the routine vaccination programs. Analysis of
 more samples involving more geographical regions is needed to draw a
 detailed map of antigenic diversity of IBDV in Iraq. Please ensure that
 the sequence data has been uploaded to a structured repository, e.g.
 NCBI/ENA. Details can then be included in the data availability statement
 after the conclusion. Please include details in the Software availability
 statement. I have added the format for you at the end of this paper.
description: <b>Sample
 collection </b> A newly died bird
 from an outbreak of IBDV from a flock in An-Najaf province, was
 post-mortem examined and the enlarged Bursa of Fabricius was incised and
 examined. <b>RNA extraction </b> Bursa contents was sampled on (FTA) card (Whatman<sup>®</sup> FTA<sup>®</sup> card technology) with four sample areas per card containing cell wall lytic enzymes, protein denaturing agents and inhibit the nucleases effects on nucleic acids (Ali <i>et al.</i>, 2017). RNA samples on the FTA card were then sent to AniCon<sup>®</sup> Labor GmbH (Muehlenstraße 13a 49685 Hoeltinghausen, Germany), where the extraction of the IBDV RNA from FTA card was performed using Kylt<sup>®</sup> RNA/DNA Purification Kit according to the manufacturer’s protocol. <b>Multiple sequence alignment and sequence manipulation</b> The IBDV VP1 targeted region obtained in this study was aligned with other similar sequences downloaded from GenBank repositories using MUSCLE (Edgar and Edgar, 2004), alignments were manually edited, gaps removed and the percentage of pairwise sequence similarity matrices (Extended data were generated using the UGENE pipeline version 35.1 (Okonechnikov <i>et al.</i>, 2012). This was also conducted for the hvVP2 nucleotide sequences. <b>Inferring the evolutionary history and time tree</b> The evolutionary history was obtained by the neighbour-joining (NJ) method (Saitou and Nei, 1987), with 1000 bootstrap replicates (Felsenstein, 1985), while the evolutionary pairwise distances (Extended data) were calculated by Maximum Composite Likelihood Method (MCL) using Tamura-Nei model ( Tamura and Nei, 1993). Heuristic search of initial tree was obtained automatically by applying Neighbour-Joining and BioNJ to a matrix of pairwise distances (Extended data) estimated by the MCL method, then the topology was selected with superior log likelihood value. For hvVP2 sequences of Iraq and vaccine strains, a time-tree to the NJ phylogenetic tree was inferred using the RelTime method (Tamura <i>et al.</i>, 2012; Tamura, Tao and Kumar, 2018). The time-tree was estimated using 31 correction restraints and all ambiguous positions were removed for each sequence to give a final dataset of 309 sites. Molecular phylogenetic analysis was also performed using the maximum-likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993). All DNA sequence analyses and evolutionary inferences were performed using MEGAx software version 10.1.8 (Kumar <i>et al.</i>, 2018) and the plotting of datasheets to the phylogenetic tree was achieved by iTOL web application (Letunic and Bork, 2019).
description: This DATSETNAMEreadme.txt file was generated on 2021-03-13 by Ali
 Hadi Abbas <br> GENERAL INFORMATION
 Title of Dataset: Extended dataset of Sequence
 diversity and evolution of infectious bursal disease virus (IBDV) in
 Iraq Author Information Principal
 Investigator Contact Information<br>         Name: Ali Hadi
 Abbas<br>         Institution: Department of Microbiology, Faculty
 of Veterinary Medicine, University of Kufa, Iraq<br>       
  Address: University of Kufa, Najaf, Iraq<br>         Email:
 alih.abbas@uokufedu.iq Associate or Co-investigator
 Contact Information<br>         Name: Haider Ali AL saegh<br>
         Institution: Department of Pathology and Poultry Diseases, Faculty
 of Veterinary Medicine, University of Kufa<br>         Address:
 University of Kufa, Najaf, Iraq<br>         Email: -
 haidera.alsaigh@uokufa.edu.iq Alternate Contact
 Information<br>         Name: Furkan Sabbar ALaraji<br>       
  Institution: Department of Pathology and Poultry Diseases, Faculty of
 Veterinary Medicine, University of Kufa<br>         Address:
 University of Kufa, Najaf, Iraq<br>         Email:
 furkans.alaraji@uokufa.edu.iq 
 Date of data collection (single date, range, approximate date):
 2019-01-07 Geographic location of data collection:
 32.0174395, 43648665, Najaf, Iraq. Information about
 funding sources that supported the collection of the data:<br> The
 authors did not receive any funding to carry out this project.
 SHARING/ACCESS INFORMATION 
 Licenses/restrictions placed on the data: free
 access. Links to publications that cite or use the
 data: Links to other publicly accessible locations of
 the data: 
 The new sequences isolated in this study, were deposited in
 GenBank repositories: GenBank:
 Infectious bursal disease virus isolate IqKufa 01 VP2 gene, partial cds.
 Accession number: MW020533.1.
 https://www.ncbnlm.nih.gov/nuccore/MW020533.1.
 GenBank: Infectious bursal disease virus isolate IqKufa 01 VP1
 gene, partial cds. Accession number: MW020534.1.
 https://www.ncbi.nlm.nih.gov/nuccore/MW020534.1. 
 Links/relationships to
 ancillary data sets: Was data derived from another
 source? Yes 
 If yes, list source(s): Most of the
 data were retrieved from GenBank repositories, NCBI: 
 https://www.ncbi.nlm.nih.gov/nuccore/?term=vp2%20infectious%20bursal%20disease<br> 2 https://www.ncbi.nlm.nih.gov/nuccore/?term=vp1+infectious+bursal+disease The extended data file (Table 1) contains the detailed information of GenBank accession numbers of sequences used in generating these data files. Recommended citation for this dataset:<br> Please cite this dataset in relation to distribution of infectious burial disease in broiler chicken in Iraq. DATA & FILE OVERVIEW File List: Table1_accesson_numbers_of_VP1_and_VP2_sequences_used_in_this_study. (GenBank accessions of VP1 and VP2 sequences used in this study.) TextFileToShow_connection_iTol_VP2. (Connections datasets allow the drawing of straight or curved lines between any two nodes in the tree. Width, colour, and opacity can be set for each line.) TextFileToShow_GRADIENT_iTol_VP2. (In gradient datasets, each ID is associated to a single numeric value which is converted to a coloured box based on the gradient defined.) IraqiVP2_pairwiseDistance.csv. (Pairwise distance of VP2 aligned region among strains isolated from Iraq).<br> •    IBVD_VP1_NCBI_Hits_OurVP1.txt. (DNA sequences of all VP1 sequences used in this study). ABIC_and VentriVaccin_IraqV2_DNA_percentSimilrity_matrix.csv. (hvVP2 sequence similarity percentage of two vaccine strains compared to the Iraqi isolates). all_VP2_DNA_sequencesUsed.txt. (DNA sequences of all VP2 sequences used in this study). Relationship between files, if important: The row parental datasets are: IBVD_VP1_NCBI_Hits_OurVP1.txt. all_VP2_DNA_sequencesUsed.txt TextFileToShow_connection_iTol_VP2. TextFileToShow_GRADIENT_iTol_VP ABIC_and VentriVaccin_IraqV2_DNA_percentSimilrity_matrix.csv. TextFileToShow_GRADIENT_iTol_VP2. IraqiVP2_pairwiseDistance.csv. Additional related data collected that was not included in the current data package: Are there multiple versions of the dataset? no METHODOLOGICAL INFORMATION Description of methods used for collection/generation of data: Main data used in this study were collected from GenBank repositories and accession numbers of entries are provided in extended data Table 1. The method used for identifying and genotyping-based classification carried out in this study, have been previously adopted by: doi: 10.1007/s00705-017-3500-4. https://patentimages.storage.googleapis.com/3d/e5/37/910756ed03cc58/WO2005017488A2.pdf Methods for processing the data: Multiple sequence alignment and sequence manipulation: The IBDV VP1 targeted region obtained in this study was aligned with other similar sequences downloaded from GenBank repositories using MUSCLE (Edgar and Edgar, 2004), alignments were manually edited, gaps were removed and the percentage of pairwise sequence similarity matrices (Extended data) were generated using the UGENE pipeline version 35.1 (Okonechnikov et al., 2012). This was also conducted for the hvVP2 nucleotide sequences. Inferring the evolutionary history and time tree: The evolutionary history was obtained by the neighbour-joining (NJ) method (Saitou and Nei, 1987), with 1000 bootstrap replicates (Felsenstein, 1985), while the evolutionary pairwise distances (Extended data) were calculated by Maximum Composite Likelihood Method (MCL) using Tamura-Nei model ( Tamura and Nei, 1993). Heuristic search of initial tree was obtained automatically by applying Neighbour-Joining and BioNJ to a matrix of pairwise distances (Extended data) estimated by the MCL method, then the topology was selected with superior log likelihood value. For hvVP2 sequences of Iraq and vaccine strains, a time-tree to the NJ phylogenetic tree was inferred using the RelTime method (Tamura et al., 2012; Tamura, Tao and Kumar, 2018). The time-tree was estimated using 31 correction restraints and all ambiguous positions were removed for each sequence to give a final dataset of 309 sites. Molecular phylogenetic analysis was also performed using the maximum-likelihood method based on the Tamura-Nei model (Tamura and Nei, 1993). All DNA sequence analyses and evolutionary inferences were performed using MEGAx software version 10.1.8 (Kumar et al., 2018) and the plotting of datasheets to the phylogenetic tree was achieved by iTOL web application (Letunic and Bork, 2019). Instrument- or software-specific information needed to interpret the data: UGENE software available from: http://ugene.net/download.html Source code available from: https://github.com/ugeneunipro/ugene/tree/master/src Archived source code at time of publication: version 35.1 (Okonechnikov et al., 2012). MEGAx software available from: https://www.megasoftware.net Source code available from: https://www.megasoftware.net/download_form. Archived source code at time of publication: version 10.1.8 (Kumar et al., 2018). iTOL web application software available from: https://itol.embl.de/itol.cgi Source code available from: https://github.com/albertyw/itolapi Archived source code at time of publication: version 5.0 ( Letunic and Bork, 2019). Standards and calibration information, if appropriate: Environmental/experimental conditions: Describe any quality-assurance procedures performed on the data: BLAST search on locally retrieved sequence to get the sequence relationship to globally, available sequences. People involved with sample collection, processing, analysis and/or submission: Haider Ali AL saegh and Furkan Sabbar ALaraji were involved in field trips and diagnosis of clinically, affected flocks and collection of Bursa of Fabricuos sample. ALli Hadi Abbas is responsible for sequence data submission and data analyses carried out in this study. DATA-SPECIFIC INFORMATION FOR: [IraqiVP2_pairwiseDistance] Number of variables: 22 Number of cases/rows: 22 Variable List: Pairwise distance of VP2 aligned region among strains isolated from Iraqi. Including self comparison =0. Missing data codes: Specialised formats or other abbreviations used: Abbreviations used for variable are the sample name embedded in GenBank accessions. DATA-SPECIFIC INFORMATION FOR: [ABIC_andVentriVaccin_IraqV2_DNA_percentSimilrity_matrix] Number of variables: 2 Number of cases/rows: 16 Variable List:<br> hvVP2 sequence similarity percentage of two vaccine strains compared to all Iraqi isolates: Sequence name Percent of sequence similarity to KJ547670_Ventri_IBDV_Plus_vacc2014 vaccine Percent of sequence similarity to AF457103_ABIC_MB71_vacc_2001 vaccine IqKufa_Najaf2019    96%    96% LC498531_Diyala2018    96%    96% MF142534_422Iraq_2017    96%    96% JX974583_Sulaim2012    96%    96% MN464103_Diyala_2018    96%    96% MN480452_Erbil2019    96%    96% MH676061_Sulaim2018    97%    97% MH676060_Sulaim2018    97%    96% MH676059_Sulaim2018    97%    97% JX974582_Sulaim2012    97%    96% KC352669_kurdis2012    97%    96% KC352668_kurdis2012    97%    96% KC352659_kurdis2012    97%    96% KC352660_kurdis2012    97%    96% MF142535_423Iraq_2017    98%    98% KC352666_kurdis2012    98%    98% Missing data codes: Specialised formats or other abbreviations used: Abbreviations used for variable are the sample name embedded in GenBank accessions. The authors declared that no grants were involved in supporting this work.