title: 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.
abstract: 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.
authors.affiliation: Department of Aquatic Life Medicine, Pukyong National University, Nam-gu 599-1, Busan, 608-737, South Korea.
authors.forename: Min Sun
authors.initials: MS
authors.lastname: Kim
authors.affiliation: Department of Aquatic Life Medicine, Pukyong National University, Nam-gu 599-1, Busan, 608-737, South Korea.
authors.forename: Su Jin
authors.initials: SJ
authors.lastname: Lee
authors.affiliation: Department of Marine Bio-Materials & Aquaculture, Pukyong National University, Busan, 608-737, South Korea.
authors.forename: Dong Soo
authors.initials: DS
authors.lastname: Kim
authors.affiliation: Department of Aquatic Life Medicine, Pukyong National University, Nam-gu 599-1, Busan, 608-737, South Korea. khkim@pknu.ac.kr.
authors.forename: Ki Hong
authors.initials: KH
authors.lastname: Kim
mesh_terms: D000818:Animals; D005393:Fish Diseases / Q000401:mortality / Q000821:virology; D005432:Flounder / Q000821:virology; D015967:Gene Expression Regulation, Viral; D059646:Genome Size; D016679:Genome, Viral; D049452:Green Fluorescent Proteins / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics*; D029141:Novirhabdovirus / Q000235:genetics / Q000254:growth & development* / Q000472:pathogenicity*; D014774:Virulence; D014779:Virus Replication; D000097573:Red Fluorescent Protein
chemical_list: D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins; D000097573:Red Fluorescent Protein
keywords: In vivo virulence; Lengthened genome; Reverse genetics; Two foreign proteins; Viral growth; Viral hemorrhagic septicemia virus