Isolation And Identification Of Porcine Pseudorabies Virus FZ Strain And Cloning Of The GD Gene Of The Strain

A virus strain was isolated from a pig farm suspected of Pseudorabies disease in Fuzhou, and Carring biology characteristic analysis and Cloning and Sequencing the gD gene of the strain. The virus doesn' t induced Cytopathogenic effect(CPE) after innoculated in Vero cell line in the primary generation. The cell starts to induce Cytopathogenic effect (CPE) after 24 hours from 3rd generation.Affected cell shrunk, aggregated,and detached from the culture system in 16h approximately. The virus was continually inoculated onto MDCK cell and induces typical Cytopathogenic effect(CPE). After the selection of phagocyte, we gain the rarefied virus clone and multiplicate the virus to other experiments.We manufacture tissueultra thin slice after the virus infected the MDCK cell. The irregular circular(110-180nm) and enveloped virus could be seen under the transmission electron microscope. Tissue culture infectious dose(TCID⁵⁰ ) in MDCK cell line transfected with isolated virus was 10^6．604 /0．1ml. With the method of Reed-Muench' s, we performed neutralizing test.lt was proved that the virus could he neutralized by PRV positive serum,and the titer of the virus strain is 1:77．Furthermore, we have detected viral protein synthesis in infected cells after 8 hours with immunofluorescence microscopy using PRV-specific antibodies. The virus was sensitive to ether, heat, trypsin, and maintaining stably between pH5．0-9．0, and it does not condense red cell of the chicken,big mouse. Pigs were generally insensitive to the virus infection and did not develop disease symptoms. However, antibody against this virus was detected after 25 days of innoculation, and virus particles were isolated from the lungs of young pigs. Importantly, innoculation of adult rabbits and mice with the virus resulted in typical clinical symptoms of Pseudorabies. The virus infected 9-12 days chicken embryos, after 86 hours, developed clinical symptoms characteristic of Pseudorabies virus (PRV) infection, including pockmark formation, whole body hemorrhage, and skull breakout, etc. The isolated virus is proved the Pseudorabies Virus, and named as PRV FZ.A pair of oligonucleotide primers were designed based on the PRV gD gene sequence in GenBank(AJ271966) .The DNA extracted from virus of the infected MDCK cell was subjected to polymerase chain reaction(PCR) analysis and the sequence have 1579 bases constituing the gene coding the envolope glycoprotein D, and include a ORF of 1203bp and code a protein of 400 amino acids. Contained EcoRâ… and Hindlâ…¡sites in the upstream and downstream of gD gene respectively, Cloned the amplified gD on the vector PCAGGS to construct recombinant plasmid PCA-gD. PCA-gD is transformed to the DH5a, and selected from the resistance bacterium colony. We gain successfully gD gene by PCR Analysis and electrophoresis experiment. Nucleotide acid and amino comparison of envelope glycoprotein gene D utilizing MegAlign program. The sequencing result showed that the nucleotide homo logy of PRV FZ strain gD gene with Min A, Fa,La,Kaplan,Ea strain is 99．8%,99．6%,99．4%,99．2% and 99．1%. The liomology of deduced amino acids of PRV FZ strain with above 5 strains is 99．5%,99．0%,99．3%,97．8% and 98．5% respectively. It indicated that the gD gene of different PRV strains shares highly conserved.