title: A vaccinia-virus-free reverse genetics system for infectious hematopoietic necrosis virus.
abstract: Reverse genetics system is a powerful tool to study the function of a particular gene. The currently available reverse genetics system for Novirhabdovirus is based on vaccinia-driven T7 RNA polymerase expression. An improved system for the recovery of infectious hematopoietic necrosis virus (IHNV) was developed which utilizes cellular RNA polymerase II machinery for transcription. A full-length cDNA clone of IHNV, flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences, was assembled in an expression plasmid under the control of a cytomegalovirus promoter. Transfection of this full-length plasmid along with the supporting plasmids (N, P, NV and L) into epithelioma papulosum cyprini cells resulted in the recovery of a viable recombinant IHNV (rIHNV). The authenticity of rIHNV was confirmed by the presence of restriction sites introduced artificially in the genome. A recombinant IHNV expressing a foreign gene - enhanced green fluorescent protein - was also recovered. The recovered IHNVs showed similar growth characteristics as the parental IHNV in cell cultures. Challenge of susceptible rainbow trout with wild-type IHNV and rIHNV induced clinical disease signs indistinguishable from the parental strain and produced mortality. Thus, a vaccinia-virus-free reverse genetics system described for IHNV is applicable for the recovery of any Novirhabdovirus, which will facilitate studies on viral pathogenesis and the design of new generation of viral vectored vaccines.
authors.affiliation: Center of Marine Biotechnology, University of Maryland Biotechnology Institute, Baltimore, 701 East Pratt Street, Baltimore, MD 21202-3101, USA. ammayapp@umbi.umd.edu
authors.forename: Arun
authors.initials: A
authors.lastname: Ammayappan
authors.forename: Scott E
authors.initials: SE
authors.lastname: Lapatra
authors.forename: Vikram N
authors.initials: VN
authors.lastname: Vakharia
mesh_terms: D000818:Animals; D002460:Cell Line; D003587:Cytomegalovirus / Q000235:genetics; D005393:Fish Diseases / Q000473:pathology / Q000821:virology*; D017930:Genes, Reporter; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics; D003698:Hepatitis Delta Virus / Q000235:genetics; D029142:Infectious hematopoietic necrosis virus / Q000235:genetics* / Q000472:pathogenicity*; D017686:Oncorhynchus mykiss; D011401:Promoter Regions, Genetic; D016337:RNA, Catalytic / Q000235:genetics; D018353:Rhabdoviridae Infections / Q000473:pathology / Q000662:veterinary* / Q000821:virology; D014616:Vaccinia virus / Q000235:genetics*; D014773:Virology / Q000379:methods*; D014774:Virulence
chemical_list: D016337:RNA, Catalytic; C485184:enhanced green fluorescent protein; C427204:hammerhead ribozyme; D049452:Green Fluorescent Proteins