Sequence Analysis Of Infectious Hematopoietic Necrosis Virus Strain SX1704 And Establishment Of GiCK Cell Line

Infectious hematopoietic necrosis virus(IHNV)is a nonsegmented negative-strand RNA virus that can horizontally and vertically infect cold-water fish such as rainbow trout and steelhead,the high pathogenicity and high mortality of the virus have caused serious damage to China's trout culture industry.IHNV belongs to the family Rhabdoviruses genus Novirhabdovirus.Since the first discovery of IHNV virus in Liaoning in 1985,the epidemics of IHNV have been reported in many areas in China including Beijing,Hebei,Shandong,Gansu,Sichuan,Jilin,Xinjiang.The infection of IHNV has also become a serious hidden danger to the rainbow trout seedlings.Therefore,identification and analysis of the distribution and genetic evolution of IHNV Chinese strains have far-reaching significance to prevention and risk management of IHNV infection.In this research,rainbow trout samples were collected from a fishery in Ningshan Shaanxi,and IHNV was identified as positive.Gene segments were cloned by RT-PCR after searching for IHNV sequences in GenBank and designing amplification primers.Recombinant plasmids were constructed by inserting the amplified sequences into pTriEx-1.1 or T vector,and subjected to sequencing analysis.The six genes of IHNV: N,P,M,G,NV,L and non-coding regions between genes were cloned by Step-by-step amplification.The IHNV strain identified from rainbow trout was named SX1704,and the genome sequence was submitted to GenBank(accession number: MH629974).The SX1704 strain has a genome with full length of 10802 bp,which sequentially encodes six proteins from 3' to 5': Nucleocapsid protein �Phosphoprotein-Matrixprotein-Surfaceglycoprotein-Nonstructural protein-Viral polymerase protein.Taking CH20101008 as a reference sequence,the genome has 78 nucleic acid point mutations,24 of the mutations lead to amino acid changes.The strain SX1704 has high genetic identity to the IHNV Chinese strain CH20101008(accession number: KJ421216)and HLJ-09(accession number: JX649101),and the similarity reaches up to 99.3%.The phylogenetic trees were constructed for the complete sequence and six genes of SX1704.Phylogenetic analysis showed that IHNV-SX1704 strain was closely related to Korean strains and the Japanese strains.Based on G genotyping,SX1704 belongs to the J Nagano subtype of J genotype.The G protein of IHNV is a viral envelope protein,a structural protein for host recognizing and a main target for vaccine design.In this research,recombinant plasmid pQBD-G was constructed by cloning the IHNV-G gene into a membrane protein expression vector pQBD,and the recombinant plasmid was co-transfected with linearized bacmid qBac-III to recovery the recombinant baculovirus rAcMNPV-G.Western blot assay showed that G protein was successfully expressed in insect cells using baculovirus surface displaying system.The baculovirus expressing IHNV-G could be used as an immunogen in further studies for the prevention of IHNV infection.In this thesis,a novel cell line was established from the kidney of Carassius auratus gibelio and named GiCK.PCR analysis of mitochondrial 16 S ribosomal RNA(rRNA)was done for the verification of the origin of the cells.PCR results revealed that CyHV-2 could not replicate in GiCK cells over 3 passages.Transduction of the constructed recombinant virus rAcMNPV-GFP into GiCK cells confirmed that the recombinant virus could enter the gibel carp cells.It remains to be investigated whether the GiCK can be developed as a highly permissive cell line for CyHV-2 replication in vitro or not.The establishment of GiCK cell line provides a valuable tool for further research on the infection and pathogenesis of CyHV-2.