Soleimanjahi H, Motamedi Sedeh F, Jalilian A R, Mahravani H
Virology Department, Faculty of Medical Science, Tarbiat Modares University, Tehran, Iran.
Folia Biol (Praha). 2013;59(2):93-8.
The foot and mouth disease virus (FMDV) causes a vesicular and contagious disease of cloven-hoofed animals. In this study, the virus was isolated from vesicles of the infected cattle using cell culture and serotyped by ELISA test. The extracted RNA from the infected cells was reverse transcribed and amplified using VP1 gene-specific primer pairs by means of one-step RT-PCR. The purified VP1 gene was sub-cloned into the uniqe KpnI and BamHI cloning sites of the pcDNA3.1+ vector. The DH5α strain of E. coli was transformed by the vector. The sequences of sub-cloned FMDV type O/IRN/2007 VP1 were aligned with FMDV type O/UKG/2001 VP1 using MegAlign software. Nucleotide sequence comparisons were made using the BLAST software available from the NCBI website. The amino acid sequences of three sub-cloned FMDV type O/IRN/2007 VP1 were also aligned with three other similar sequences using MegAlign software. Nineteen of the most similar VP1 nucleotide sequences (by BLASTN program), FMDV O/IRN/2007 VP1 sequence, twenty isolates of FMDV-O VP1 in Iran and eight topotypes of FMDV type O were aligned by Mega5 to create a FMDV-O VP1-based sequence similarity tree. The nucleotide sequence comparison indicated that FMDV O/ IRN/2007 VP1 had the greatest nucleotide sequence similarity to the VP1 gene of FMDV O1/Manisa/Turkey/69 (99%), FMDV O1/Manisa/Netherlands (98%) and FMDV O1/Manisa/iso87/Turkey (98%). It was also observed that the highest identity between FMDV O/IRN/2007 VP1 sequence and other nucleotide sequences of FMDV type O VP1 genes isolated in Iran during 1997-2004 was about 91%.
口蹄疫病毒(FMDV)可引发偶蹄动物的水疱性传染病。在本研究中,利用细胞培养从感染牛的水疱中分离出该病毒,并通过ELISA试验进行血清分型。从感染细胞中提取的RNA经反转录,使用VP1基因特异性引物对通过一步法RT-PCR进行扩增。将纯化的VP1基因亚克隆到pcDNA3.1+载体独特的KpnI和BamHI克隆位点。用该载体转化大肠杆菌DH5α菌株。使用MegAlign软件将亚克隆的口蹄疫病毒O/IRN/2007型VP1序列与口蹄疫病毒O/UKG/2001型VP1序列进行比对。使用NCBI网站提供的BLAST软件进行核苷酸序列比较。还使用MegAlign软件将三个亚克隆的口蹄疫病毒O/IRN/2007型VP1的氨基酸序列与其他三个相似序列进行比对。通过Mega5将19个最相似的VP1核苷酸序列(通过BLASTN程序)、口蹄疫病毒O/IRN/2007 VP1序列、伊朗的20株口蹄疫病毒O型VP1以及口蹄疫病毒O型的8个拓扑型进行比对,构建基于口蹄疫病毒O型VP1的序列相似性树。核苷酸序列比较表明,口蹄疫病毒O/IRN/2007 VP1与口蹄疫病毒O1/马尼萨/土耳其/69(99%)、口蹄疫病毒O1/马尼萨/荷兰(98%)和口蹄疫病毒O1/马尼萨/iso87/土耳其(98%)的VP1基因具有最高的核苷酸序列相似性。还观察到,口蹄疫病毒O/IRN/2007 VP1序列与1997 - 2004年期间在伊朗分离的口蹄疫病毒O型VP1基因的其他核苷酸序列之间的最高同一性约为91%。