Abstract
Four DNA fragments (fragments A, B, C and D) covering the whole genome of chicken anemia virus (CAV) were amplified enzymatically by polymerase chain reaction (PCR) using four pairs of oligonucleotide primers. The DNA fragments were amplified from each of nine CAV isolates including eight Malaysian isolates and one European isolate (Cux-1). For all nine CAV isolates, fragment A (1518 bp) was digested with one restriction enzyme, Eco130I (StyI); fragment B (926 bp) with three enzymes, Eco130I (StyI), HpaII and MboI separately; fragment C (675 bp) with also three enzymes, BsuRI (HaeIII), HinfI, and HpaII separately; and the fragment D (552 bp) with one enzyme, EcoRI. Enzyme digested products of different fragments were separated by agarose gel or polyacrylamide gel electrophoresis. Each of the eightenzymatic reactions differentiated at least two isolates except the HpaII digestion of fragment C where no isolate was distinguished. The overall restriction endonuclease (RE) analysis separated four isolates (BL-1, BL- 2, BL-4 and BL-5) in one group and the rest five isolates (SMSC-1, SMSC-2, 3-1, BL-3 and Cux-1) were differentiated from each other and also from the group of four isolates, based on the number of restriction site differences and the fragments generated by different enzymatic digestions. The study revealed that RE analysis could be used to identify and differentiate CAV isolates based on the number of restriction site differences. The study showed that more isolates, even the isolates from the same poultry farm, could be differentiated with proper genomic diversity after RE analysis of more genome fragments compared to that of single genome fragment.SAARC J. Agri., 15(2): 1-18 (2017)
Highlights
We investigated for the first time the molecular differences of the genomes of different Chicken anaemia virus (CAV) isolates by restriction endonuclease analysis of polymerase chain reaction (PCR)-amplified DNA fragments covering the whole CAV genome
The restriction sites found for all enzymes with regard to different PCR amplified fragments of different isolates are shown in table 1
The study revealed that restriction endonuclease analysis could be used to identify and differentiate CAV isolates based on the number of restriction site differences, though these isolates are antigenically indistinguishable
Summary
Chicken anaemia virus (CAV) is an economically important avian pathogen with a worldwide distribution (AboElkhair et al, 2014; De Herdt et al, 2001; Farkas et al, 1998, Ganar et al, 2017; Ledesma et al, 2001; McNulty 1991; Olszewska-Tomczyk et al, 2016; Rehman et al, 2011; Stanislawek and Howell, 1994; Zhou et al, 1996). Studies have been carried out to characterize the virus based on its DNA sequence (Noteborn et al, 1992; Todd et al, 1992; Tham and Stanislawek, 1992) Both Southern analysis and restriction mapping showed only minor differences on the CAV genomes of the field isolates from United States (Noteborn et al, 1992). Genetic variations of different CAV isolates were detected by other workers using RE analysis of PCR-amplified CAV genome fragments (Oluwayelu et al, 2005; Nayabian and Mardani, 2013; van Santen et al, 2001). We investigated for the first time the molecular differences of the genomes of different CAV isolates by restriction endonuclease analysis of PCR-amplified DNA fragments covering the whole CAV genome
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