| Pseudorabies virus,(PRV), belongs to herpesvirdae α- herpes virus subfamily. A variety of domestic and wild animals can be infected by PRV, and pig were the most suspected animals. In addition, pig was the natural host and reservior of PRV. PRV could cause huge economic losses and was listed it as Category 2 diseases by Chinese government.Recently, the prevalence of PR was effectively controlled due to the widespread using of PRV vaccine, including attenuated vaccine, inactivated vaccine and genetically engineered vaccine. Some countries have eradicated PR by vaccination and the endemic of PR was also controlled in China before 2010. However, the outbreak of PR in vaccinated pig flocks were still reported by Tong Guang-zhi, Tian Zhijun, Li Xue Wu and Chen Huanchun etc since 2011 in Jilin, Heilongjiang, Henan and Shandong provinces. The infected boars showed fever, scrotal inflammation,It showed significant phenomenon that sow repeated with infertility or produced low earners, still birth or miscarriage after mating, While piglets displayed diarrhea, associated with neurological symptoms and high mortality. Zhang Qing zhan, Zhao Hongyuan, Pan Xiao and other scholars have isolated PRV from vaccinated pig flocks in Henan, Heilongjiang, Jilin, Jiangsu and other provinces., and these isolated viruses were proved to be virulent mutant PRV strains., These results suggested that the available vaccine could not completely protect pigs against PR, and the threat of PR outbreaks still exist.Recently, the frequency of PR outbreaks present a increase trend in Shaanxi province. To study the prevalence of PR in Shaanxi province, 12 large-scale pig farms were choosed to conduct sample collection and PRV detection. Subsequently, a PRV was isolated form one pig farm that suspected to suffer PRV infection. And the g E gene was amplified and then used to perform phylogenetic analysis. The main results are as follow:1. The survey of PRV infection at scale pig farms in Shaanxi province. Twelve typical vaccinated pig farms were selected to collect blood for serum prepare. Totally seventy eight serum sample were collected and these sample were tested by PCR for PRV nucleotide and ELISA for antibody against g E protein. Results showed that four pig farms were PCR positive, suggested that the PRV positive farms rate was 33.33%. Fourteen serum samples were PRV nucleotide positive, accounting for 17.95% of the total detected serum samples. Five pig farms were antibody against g E protein positive, accounting for 41.46% of the pig farms. Seventeen serum samples were antibody against g E protein positive, accounting for 21.79% of the investigated samples.2. PR diagnosis and PRV isolation and identification. In October 2015, an suspected PR outbreak was appeared in one pig farm in Shaanxi provinces. Then the blood, brain, spinal cord, liver and spleen of the piglet were collected. These samples were detected by PCR, ELISA and challenge experiment for laboratory diagnosis. Finally, it was identified as PRV infection. BHK-21 cells were used for PRV isolation, and the cytopathic effect was observed after six consecutive passages. The TCID50 of isolated virus titer was 10-5.386/0.1m L. To further confirm the result, we performed PCR and inoculated the virus into rabbit and embryo chicken eggs. The isolated PRV was designed as SX-10-2015.3. Amplification and sequence analysis of PRV SX-10-2015 strain g E gene. To further study genetic evolutionary status of PRV SX-10-2015 strain, g E gene was amplified and sequenced, and then the amplified g E gene was compared with g E gene of the popular PRV strains, WG strain, as well as domestic and foreign classical PRV sequences and phylogenetic tree was constructed. Results showed that the genetic distance between isolates SX-10-2015 and domestic PRV pandemic strain GD-4-2013 strains, JN and WZ genetic strains was close, belongs to the same branch,and the distance with foreign Kaplan, Ni A3 and 00V7 was far, belong to different branches. The homology of SX-10-2015 strain amino acid of g E gene with that of GD-4-2013 strain and He N1 strain were 99.7% and 99.3%, respectively. While the homology of SX-10-2015 strain amino acid of g E gene with Ea and FA PR vaccine strains g E amino acid were 98.8% and 98.8%.According to the above results, we concluded that the PRV positive rate of pig farms and pigs were relatively high in the Shaanxi Province.The nucleotide and amino acid sequences of SX-10-2015 g E genes were highly homology to PRV isolated and reported in recent years. Their phylogenetic position was also closer. While its amino acid homology with domestic vaccine strain Ea and FA homology was also as high as 98.8%. So further study are required to confirm whether the vaccine can provide protection against PRV SX-10-2015 strain. |
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