Generation of Recombinant Viral Hemorrhagic Septicemia Virus (rVHSV) Expressing Two Foreign Proteins and Effect of Lengthened Viral Genome on Viral Growth and In Vivo Virulence

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• 作者：Min Sun Kim ; Su Jin Lee ; Dong Soo Kim ; Ki Hong Kim
• 关键词：Viral hemorrhagic septicemia virus ; Reverse genetics ; Two foreign proteins ; Lengthened genome ; Viral growth ; In vivo virulence
• 刊名：Molecular Biotechnology
• 出版年：2016
• 出版时间：April 2016
• 年：2016
• 卷：58
• 期：4
• 页码：280-286
• 全文大小：1,049 KB
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• 作者单位：Min Sun Kim (1)
  Su Jin Lee (1)
  Dong Soo Kim (2)
  Ki Hong Kim (1)
  
  1. Department of Aquatic Life Medicine, Pukyong National University, Nam-gu 599-1, Busan, 608-737, South Korea
  2. Department of Marine Bio-Materials & Aquaculture, Pukyong National University, Busan, 608-737, South Korea
  
• 刊物主题：Biotechnology; Biochemistry, general; Cell Biology; Protein Science; Biological Techniques; Human Genetics;
• 出版者：Springer US
• ISSN：1559-0305

文摘

In this study, a new recombinant VHSV (rVHSV-Arfp-Bgfp) was generated by insertion of a red fluorescent protein (RFP) gene between N and P genes, a green fluorescent protein (GFP) gene between P and M genes of VHSV genome, the expression of each heterologous gene in infected cells, and effects of the lengthened recombinant VHSV’s genome on the replication ability and in vivo virulence to olive flounder (Paralichthys olivaceus) fingerlings were compared with previously generated rVHSVs (rVHSV-wild, rVHSV-Arfp, and rVHSV-Brfp). The expression of RFP and GFP in cells infected with rVHSV-Arfp-Bgfp was verified through fluorescent microscopy and FACS analysis. In the viral growth analysis, rVHSV-Arfp and rVHSV-Brfp showed significantly lower viral titers than rVHSV-wild, and the replication of rVHSV-Arfp-Bgfp was significantly decreased compared to that of even rVHSV-Arfp or rVHSV-Brfp. These results suggest that the genome length is a critical factor for the determination of rVHSVs replication efficiency. In the in vivo virulence experiment, the cumulative mortalities of olive flounder fingerlings infected with each rVHSV were inversely proportional to the length of the viral genome, suggesting that decreased viral growth rate due to the lengthened viral genome is accompanied with the decrease of in vivo virulence of rVHSVs. Recombinant viruses expressing multiple foreign antigens can be used for the development of combined vaccines. However, as the present rVHSV-Arfp-Bgfp still possesses an ability to kill hosts (although very weakened), researches on the producing more attenuated viruses or propagation-deficient replicon particles are needed to solve safety-related problems.