Porcine reproductive and respiratory syndrome virus as a vector: Immunogenicity of green fluorescent protein and porcine circovirus type 2 capsid expressed from dedicated subgenomic RNAs
Porcine reproductive and respiratory syndrome virus as a vector: Immunogenicity of green fluorescent protein and porcine circovirus type 2 capsid expressed from dedicated subgenomic RNAs
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DOI：
10.1016/j.virol.2009.03.036
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发表时间：
2009-06-01
期刊：
影响因子：
3.7
通讯作者：
Yoo, Dongwan
中科院分区：
文献类型：
--
作者：
Pei, Yanlong;Hodgins, Douglas C.;Yoo, Dongwan
猪繁殖与呼吸综合征病毒（PRRSV）是猪繁殖与呼吸综合征的病原体，其特征是母猪晚期流产和仔猪呼吸道疾病。利用北美PRRSV菌株P129的感染性cDNA克隆，对病毒基因组进行了工程改造，使其转录在非结构基因和结构基因之间启动的额外亚基因组RNA。在orf 1b和orf 2a之间插入两个独特的限制性内切位点和ORF6转录调控序列（TRS6）的拷贝，形成一个通用表达载体。增强的绿色荧光蛋白（GFP）基因克隆在独特的位点之间，插入的基因转录在ORF1b上游的TRS2前面，而TRS6拷贝驱动ORF2a/b转录。用该质粒转染细胞后，引发PRRSV感染，获得子代病毒“P129-GFP”。P129-GFP感染的细胞显示荧光，并且插入的基因在至少37次连续体外传代中表型稳定。随后，从猪多系统消耗综合征（PMWS）暴发中恢复的猪圆环病毒2型（PCV2）中克隆一个衣壳(C)蛋白基因，并插入PRRSV感染克隆载体中，生成病毒“P129-PCV”。为了确定重组病毒的免疫原性，分别用P129-WT（野生型）、P129-GFP或P129-PCV2肌内免疫猪。感染后5周，诱导了针对GFP和PCV2衣壳的特异性抗体反应。这是第一个利用PRRSV特异亚基因组rna表达外源基因的报道，并证明了PRRSV作为猪病原体疫苗载体的潜在用途。(c) 2009爱思唯尔公司版权所有。
Porcine reproductive and respiratory syndrome virus (PRRSV) is the causative agent of PRRS, which is characterized by late-term abortions in sows and respiratory disease in young pigs. Using an infectious cDNA clone of North American PRRSV strain P129, the viral genome was engineered to transcribe an additional subgenomic RNA initiating between non-structural and structural genes. Two unique restriction sites and a copy of the transcription regulatory sequence for ORF6 (TRS6) were inserted between ORFs 1b and 2a, yielding a general purpose expression vector. The enhanced green fluorescent protein (GFP) gene was cloned between the unique sites such that the inserted gene was transcribed front TRS2 which was located upstream within ORF1b, while the copy of TRS6 drives ORF2a/b transcription. Upon transfection of cells with this plasmid, PRRSV infection was initiated and progeny virus "P129-GFP" was obtained. Cells infected with P129-GFP showed fluorescence and the inserted gene was phenotypically stable for at least 37 serial ill vitro passages. Subsequently, a capsid (C) protein gene was cloned from porcine circovirus type 2 (PCV2) recovered from an outbreak of porcine multisystemic wasting syndrome (PMWS) and inserted into the PRRSV infectious clone vector, generating virus "P129-PCV". To determine the immunogenicity of the recombinant viruses, pigs were immunized intramuscularly with P129-WT (wild-type), P129-GFP, or P129-PCV2. By 5 weeks post-infection, specific antibody responses to GFP and PCV2 capsid were elicited. This is the first report of foreign gene expression using PRRSV from dedicated subgenomic RNAs and demonstrates the potential use of PRRSV as a vaccine vector for swine pathogens. (c) 2009 Elsevier Inc. All rights reserved.