Virus Isolation And Vector Construction Of Koi Herpesvirus (KHV-hlj)

A new continuous cell line (KF-h) from caudal fin of koi, Cyprinus carpio koi, was developed and sub-cultured more than100passages since the present study was initiated.Cell line KF-h predominantly consisted of short fibroblast-like cells and grew well in Dulbecco's modified Eagle medium (M199) supplemented with10%fetal bovine serum (FBS). Chromosome analysis revealed that56%of Outbreaks of koi herpesvirus (KHV) infection in carp are still a serious problem worldwide. Koi herpesvirus [KHV or cyprinid herpesvirus3(CyHV-3)] infections in carp are a serious problem worldwide. Since1988, KHV outbroke in various countries and caused serious economic loss therefore KHV became one of quarantine important projects. KHV was first isolated and confirmed in1998from diseased common carps and koi in Israel and the USA. Acomparison of virus characteristics reveals that KHV is a distinct virus species from two well-known piscine herpesviruses:cyprinid herpesvirus (CyHV-1) and channel catfish herpesvirus (IcHV-1). At the molecular level, multiple sequence alignment analysis indicates that CyHV-3belongs to a new species of cyprinid herpesvirus (the third cyprinid herpesvirus, CyHV-3) of the family Herpesvirus,。KHV has become a globally distributed pathogen, and has been considered one of the most important causative agents to koi and common carps worldwide.Through the experiments of the chromosome number, karyotype and DNA content, the cell line of the koi's tail fin established in this study demonstrates that there is no obvious difference between the somatic cells and culture cells; the chromosome number and the DNA content are in accordance with the proportionate relationship; and the established cell line of the koi's tail fin, named as KF-h, has formed the stable heredity.When the nephrocyte suspension of the carp with the typical symptoms is inoculated to the KF-h cell monolayer through the observation of electron microscope, PCR amplification, sequence analysis and the animal regression experiment, the appearance of the typical KHV CPE phenomenon will indicate that the KHV virus has been separated successfully and named as KHV-hlj.The KHV-hlj strain is made into the cell inactivated vaccine through inactivation, and after the carp is injected and immersed with the immunocyte inactivated vaccine, the detection and virus attacking test towards the leukocyte phagocytic activity and the lymphocyte transformation ability of the immunized carp have initially proven that the carp has the impeccable immune competence, and the non-specific immunity, cellular immunity and humoral immunity can be improved remarkably with vaccination; this test also proves that this preparation method can effectively keep the pathogenic antigen of the virus; the antigen of the virus can effectively stimulate the carp to produce the strong immune reaction, which finally leads to the strong immune protection created by the immunized carp. As for the cell inactivated vaccine of the KHV-hlj strain, the bacterial strain is collected from the cell line with the reasonable formula, easy operation and moderate cost, so it is suitable for the volume production, which enables the application and dissemination of the vaccine to become possible. The immune result of the vaccine demonstrates that if the carp accepts the vaccination by means of injection instead of immersion, the higher agglomerate antibody titer and the immune protection rate will be achieved.The experiment has augmented the mature peptide of the KHV-hlj envelope glycoprotein D gene, and the sequence determination has indicated that gD gene has the high genetic stability, with the nucleic acid xequence homology reaching99.64%; besides, the sequence of the main functional zone doesn't change and make no difference to the function and structure of gD protein. The overall length of the envelope glycoprotein coding gene of KHV is1179bp, and it is coded with US6gene; the amino-terminal signal peptides of393amino acids and25hydrophobic amino acids have been coded, and the mature peptide only has368amino acids, with the transmembrane region located in the place of No.341-363amino acids. The KHV-gD protein belongs to the transmembrane glycoprotein, with only a little part inside the cell, and the transmembrane spiral area of this protein is located at No.4-25of N end and No.341-363of C end, the part between which belongs to the envelope region. The cloned KHV-gD polypeptide has removed the transmembrane region of the first25amino acids of N end (signal peptide) and become the mature peptide of this protein, and its combined scores of the transmembrane helix at C end has increased from2217to2777. When analyzing the sequence of the cloned KHV-gD mature peptide, we can find that there are368amino acids in total with the molecular weight as40.63kD and the isoelecric point p1as7.15, including34basic amino acids,35acidic amino acids,130hydrophobic amino acids and92polar amino acids.This study has constructed the Pichia pastoris expression vector pPIC9K-KHV-gD which contains the complete mature peptide gene of KHV-gD. When further expressing the KHV-gD protein in the saccharomycetes, we can ensure the integrity of the KHV-gD immunogenicity, so as to lay the foundation for studying the diagnostic kit of the recombinant protein and develop the vaccine of the genetic engineering. Besides, the expression of KHV-gD protein in the Pichia pastoris also provides the advantage for researching the structure and function of KHV-gD protein.Because KHV causes severe financial losses in the common carp and koi culture industries worldwide, it is a useful subject for applied science. Safe and efficacious vaccines adapted to mass vaccination of carp and efficient diagnostic methods need to be developed. Several aspects of KHV make it also useful for fundamental science. These aspects are its large genome, the relationship between KHV infectivity and temperature, and the low similarity between KHV genes and the genes of other members of the order Herpesvirales that have been studied. Further studies are needed to identify the roles of KHV genes in viral entry, egress, and disease pathogenesis.