Document Type : Original Article
Authors
1
Department of Animal Medicine (Infectious Diseases), Faculty of Veterinary Medicine, Kafrelsheikh University, 33516, Egypt
2
Epizootic Research Station, Equine Research Institute, The Japanese Racing Association, Tochigi 329-04, Japan.
3
Laboratory of Veterinary Microbiology, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
Abstract
RacL11 and Kentucky D are classical virulent strains of equine hepesvirus-1 (EHV-1) which are worldwide used for vaccine manufacture. These two strains are the parent strains of commercial vaccines. The present study were applied in the Laboratory of Veterinary Microbiology, Faculty of Applied Biological Sciences, Gifu University, ,Japan in 2006. We compared the two strains with the reference strain Ab4P using Long and accurate PCR (LA-PCR) Restriction Fragment Length Polymorphism (RFLP) for the EHV-1 whole genomic DNA using eight different types of restriction enzymes (SacI, SspI, NaeI, ApaI, EcoRI, HincII, BglII, and StuI) which digest the whole genome into 600 fragments. Digests were electrophoresed on an agarose gel of the appropriate concentration. Restriction maps of RacL11 and Kentucky D genomic DNA in comparison to Ab4p revealed no restriction sites differences except in one fragment (Fragment 12) which corresponds to nt 104,320 to 111,287 in the genome sequence of the standard Ab4p strain and includes a part of ORF62, the intergenic region between ORFs 62-63, and a portion of ORF63. and by sequencing we found that the intergenic region between ORFs 62-63 (IGR) revealed that the RacL11 and Kentucky D strains show sequence length difference between all examined strains. The increased length of fragment 12 of RacL11 was found to be due to the presence of 10 copies of 5'-GGCTAGCGCTAACGCTAG-3' while the Kentucky D and Ab4p sequences contained 4 copies and 3 copies, respectively. Using more restriction enzymes for future RFLP comparison is recommended.
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