Cloning and sequencing of genome segment 10 of African horesickness VIRUS TYPE 9

Abstract: African horsesickness virus, type 9 Plum Island isolate, (AHSV, T9 PI)
was propagated on VERO cells till reaching 70 %CPE. The double stranded (ds) RNA genome was extracted using the Phenol Chloroform Method, (FAVALORO et al 1980), cDNA of S10 was obtained by applying the Reverse Transcryptase (RT) reaction. The exponential amplification of S10 cDNA from the dsRNA genome was achieved through performing the Polymerase Chain Reaction (PCR) using 2 primers designed in accordance with the published sequence of the cognate gene. Ligation of S 10 to the PCR TM11 vector as well as transformation of the “One Shot” competent cells (NVaF) with such a plasmid was carried out following the instructions of the TA cloning Manual (Invitrogen). The assertiveness of the proper ligation and transformation was assessed by applying the minilysate preparation protocol and checked up in 1.2 % agarose gel electrophoresis for the right size of S10 gene (~700 bp) For obtaining an adequate amount of plasmid BNA needed for sequence analysis of S10, PEG plasmid preparation protocol was performed. The nucleotide sequence of S10 was determined by using version 2.0-DNA. Sequencing Kit based on the strategy of the dideoxy Termination Reaction (SANGER et al 1977). This study proved that there is a high degree of homonology in the nucleotide Sequence of AHSV-T9 PI, S10 and the published sequence of the cognate gene so that it could be of diagnostic value. It is also a preliminary step for further investigations for the role of, S10 gene in AHS virus replication as well as its pathogenesis. This work was accomplished in Plum Island laboratories (USA) and the associated authors shared in.
Publication year 1996
Pages 13-21
Availability location معهد بحوث الامصال واللقاحات البيطرية
Availability number
Organization Name
City assiut
serial title Assiut veterinary medical jornal
ISSN 1012-5973
Author(s) from ARC
Agris Categories Animal diseases
Proposed Agrovoc Horesickness virus;
Publication Type Conference/Workshop
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