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经典猪瘟病毒LOM株感染性cDNA克隆的构建与鉴定

Establishment and characterization of an infectious cDNA clone of a classical swine fever virus LOM strain.

作者信息

Park Gil-Soon, Lim Seong-In, Hong Seung-Ho, Song Jae-Young

机构信息

DNA Analysis Division, National Forensic Service, Seoul 158-707, Korea.

出版信息

J Vet Sci. 2012 Mar;13(1):81-91. doi: 10.4142/jvs.2012.13.1.81.

Abstract

Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.

摘要

经典猪瘟病毒(CSFV)可在猪群中引发一种具有高度传染性的疾病,在全球范围内造成重大经济影响。CSFV毒株LOM是1956年从日本分离出的宫城低毒株的减毒病毒。通过逆转录聚合酶链反应(RT-PCR)获得了代表CSFV毒株LOM基因组的八个DNA片段。这些片段用于确定完整的核苷酸序列并构建全长cDNA克隆,该克隆被称为Flc-LOM。与亲本序列相比,重组克隆(Flc-LOM)的序列分析显示存在八个突变,导致两个氨基酸替换。LOM和Flc-LOM的RNA转录本在PK-15细胞中均具有直接感染性。拯救出的Flc-LOM病毒在细胞中的生长速度比亲本病毒LOM慢。用Flc-LOM进行肌肉注射免疫在猪中是安全且具有高度免疫原性的;35天后未观察到临床症状或病毒传播给哨兵动物。感染后14天检测到CSFV特异性中和抗体。在用强毒CSFV毒株SW03攻击后,用Flc-LOM免疫的猪显示出完全受到保护。因此,我们新建立的CSFV感染性克隆Flc-LOM可作为候选疫苗。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/1cba/3317462/9eabcd34f728/jvs-13-81-g001.jpg

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