Sequence Analysis Of GE,gC,gD And TK Gene Among Pseudorabies Virus Strains

Pseudorabies virus(PRV) causes pseudorabies(PR), which results in severe economic losses in porcine production. A wide range of mammals are able to be infected by PRV, but only pig is able to survive in an acute infection and represent the natural reservoir for the virus. Before 2011, in consequence of the application of PRV gene-deleted vaccine and differential diagnosis method of gE-ELISA, PR has been effectively under control in China.However, since 2011, PR occurred in many pig farms that have been vaccinated Bartha-K61 in Shan dong Province. This research consists of two parts as follows:Part 1. Isolation and identification of 12 PRV strains in Shandong provinceIn December 2013 to May 2014, lesion tissues were collected in twelve pig farms that have emerged a serious disease that appears to be PR symptoms. These farms had been vaccinated with Bartha-K61 vaccine. Lesion tissues were grinded and filtered to infect BHK-21. Virus were propagated in BHK-21 for six times and the fifth and sixth generation were detected by Quantitative Real-time PCR and the virus solution of sixth generation were inoculated to rabbits.TCID50 of 12 PRV strains were detected. Quantitative Real-time PCR test results were PRV positive. The infected rabits began to dead and presented a typical clinical symptoms of PR after 24 hours. TCID50 of the 12 strains were 10-7.1/0.1ml to10-9.5/0.1ml. These results show that 12 PRV strains were isolated in Shandong Province.Part 2. Sequence analysis of gE, gC, gD and TK gene of 12 PRV strains isolated in Shan dong Province.In this study, gE, gC, gD and TK gene of the 12 PRV strains isolated in Shandong province were amplified by PCR, sequenced and analyzed. The sequence analysis of gE gene showed that the 12 strains shared nucleotide and amino acid identities of 99.9%~100% and99.7%~100%, respectively. Compared with Asian strains, the 12 strains shared lower nucleotide and amino acid identities with Euramerican strains. Phylogenetic analysis of gE amino acid indicated that all the 52 PRV(including 12 strains isolated in this study and 40 reference strains) were clustered into two genotypes: all the Asian strains belonged to genotype I(GI), and all the Euramerican strains belonged to genotype II(G II). Discrepancies can be seen between two genotypes at sites of 58, 105, 148, 178, 180, 214, 215, 470, 500, 505,518, 522 and 569. These discrepancies can be regarded as genetic marker to distinguish the two genotypes.The gC sequence analysis showed that the 12 strains both shared nucleotide and amino acid identities of nucleotide and amino acid of 99.8%~100% and 99.6%~100%, respectively.Phylogenetic analysis showed that most China strains isolated after 2011 belonged to a relatively independent branch. The gD sequence analysis showed that the nucleotide and amino acid homologies between the 12 PRV isolates both were 99.8%~100%. Compared with the Euramerican strains, the 12 strains shared higher nucleotide and amino acid identities with the domestic strains. Phylogenetic analysis showed that the gD gene of all the 28 PRV strains were divided into two genotypes: all the domestic strains belonged to genotype I(GI), and all the Euramerican strains belonged to genotype II(GII). Two consensus mutants had been found between domestic strains and Euramerican strains at the site of 340 and 370. However,no significant changes have been found in the TK gene of PRV.