Isolation And Identification Of Porcine Pseudorabies Virus FZJX Strain And Cloning And Sequencing Of The TK Gene

In this study, a virus strain was isolated from the lung of a dead pig in a farm in the suburbs of Fuzhou. With related experiments, the virus was identified as Pseudorabies virus and thymidine kinase (TK) gene of the strain was cloned and sequenced.There was no obvious Cytopathogenic effect (CPE) when the virus was inoculated with the Marc-145 cells in the first generation. The obvious CPE of infected cells, such as shrunk, aggregated, dissolved and detached from the culture system accompanied with lots of polykaryocytes, appeared in the second generation and the time was about 48 hours. The time of CPE had significantly shortened after several generations and that generally maintained approximately 20 hours. Electronic microscope features of the virions were round or elliptic, 150-180nm in diameter, enclosed by a membranous structure with radical tubers. There were capsules in the cell nuclear. Immature nucleocapsids gemmated in the cytoplasm vesicula and on the epicyte. In the hemagglutination, the virus did not react with the erythrocytes of rooster and rat but did with the erythrocytes of mouse with the hemagglutination titler of 1:64. When the virus was inoculated in the allantocherion, we could observe that white variolas emerged in the allantocherion, the allantocherion was dropsical and congestive, the cranium of the dead embryo was protuberant, and the embryo was full of hemorrhage. When the virus was inoculated in the allantoic cavity, we could observe that the allantocherion and the embryo showed similar symptoms, while the white variolas didn't emerge in the allantocherion. The adult rabbits and the mouses inoculated with the virus died after 59 hours and 96 hours, respectively. They both had nerval symptoms without the pruritus and the worry. The piglet inoculated with the virus also showed the nerval symptoms, with hyperpyrexia to 41℃. The piglet was convalescent after being inoculated for 10 days and did not show nerval symptoms. After convalescence, the piglet was not sensitive to the virus.The total DNA of the virus and the cells was extracted from the cells infected by the virus, and a pair of specific oligonucleotide primers were designed, based on the PRV TK gene sequence in the GenBank.Through the polymerase chain reaction (PCR) technology, a gene about 917 bp was amplified successfully. The result coincided with our expectancy and proved that the virus should be PRV on the nucleinic acid level. From all the results above, we could conclude that the virus isolated from the farm was PRV, named as FZJX strain.Through cloning and sequencing the TK gene, we found that there was an ORF of 897bp which coded a protein of 298 amino acids in the 917bp PCR product. With the DNAStar software, we compared the differences of the TK gene sequences between FZJX strain and FJFZ strain, Ea strain, SH strain, Min-A strain, LA strain, Korea strain, USA strain and Spain strain in the GenBank, deduced the corresponding amino acid sequences, and analyzed the sequence distances and the phylogenetic tree. The results of sequence distances analysis showed that the homology of TK gene sequences between FZJX strain and USA strain, Ea strain, FJFZ strain, Korea strain, LA strain, Min-A strain, SH strain and Spain strain was 93.3%, 99.7%, 99.3%, 99.4%, 99.8%, 98.3%, 98.5% and 99%, respectively. The homology of deduced amino acid sequences was 84%, 100%, 99.7%, 99.3%, 100%, 98.7%, 54.7% and 98.7%, respectively. The results of the phylogenetic tree analysis indicated that the variation of TK gene was relatively regional, while the variation of deduced amino acids was not regional.