Supplementary Information for: Sequence diversity and evolution of Infectious Bursal Disease Virus IBDV in Iraq

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.

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Methods
Sample collection

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.

RNA extraction

Bursa contents was sampled on (FTA) card (Whatman® FTA® 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 et al., 2017).

RNA samples on the FTA card were then sent to AniCon® Labor GmbH (Muehlenstraße 13a 49685 Hoeltinghausen, Germany), where the extraction of the IBDV RNA from FTA card was performed using Kylt® RNA/DNA Purification Kit according to the manufacturer’s protocol.

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 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).