MOLECULAR BASIS OF VIRULENCE IN INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (IHNV) USING A REVERSE GENETICS APPROACH
MOLECULAR BASIS OF VIRULENCE IN INFECTIOUS HEMATOPOIETIC NECROSIS VIRUS (IHNV) USING A REVERSE GENETICS APPROACH
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Date
2009
Authors
Ammayappan, Arun
Advisor
Vakharia, Vikram N
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Abstract
Infectious hematopoietic necrosis virus (IHNV) is a pathogen of major economic importance to the aquaculture industry. The long-term goal of our work is to develop a safe and effective recombinant IHNV vaccine and possibly use IHNV as a virus vector to express foreign genes. To achieve this goal, the complete genome of IHNV 220-90 virulent strain was sequenced and characterized. Subsequently, a full-length cDNA clone of IHNV was generated by constructing the full length cDNA clone, between the cytomegalovirus (CMV) promoter and the autocatalytic hammerhead and hepatitis delta virus ribozymes. Transfection of a full-length plasmid, along with the supporting plasmids resulted in the recovery of infectious rIHNV-220-90. Characterization of the rIHNV-220-90 showed that its growth characteristics in tissue culture were comparable to those of the parental virus.
The possible role of IHNV proteins in virulence was explored to some extent. For this, the entire genome of attenuated virus (IHNV-61) was sequenced and compared with its virulent strain. The comparative sequencing analysis studies revealed that majority of differences were located in the glycoprotein gene. The M and G genes, and the trailer region between virulent and attenuated viruses were exchanged; recombinant chimeric viruses were recovered and studied for their pathogenicity in rainbow trout. The results obtained from in vivo studies indicate that the glycoprotein plays a major role in IHNV virulence in fish, whereas the M gene and trailer region play a negligible role in virulence of IHNV. The potential of rIHNV to serve as a viral vector was explored by expressing the VP2 protein of IPNV and hemagglutinin-estrase (HE) protein of ISAV. The recovered rIHNV-VP2 and rIHNV-HE viruses stably expressed the VP2 and HE proteins respectively for at least five serial passages and showed characteristics comparable to that of the parental virus, except that there was a one-log reduction in the virus titer. These results demonstrated that the established reverse genetics system can be utilized effectively to examine the molecular determinants of virulence, pathogenesis, and new approaches for vaccine development.