Genomic Sequencing And Sequence Analysis Of A Field Pseudorabies Virus Qihe547 Strain

Porcine pseudorabies(PR) is caused by Pseudorabies virus(PRV). PR is one of the most serious porcine infectious disease which results in great economic losses in swine industry. It is prevalent all over the world. Since 1990 s, due to the application of PRV g E-deleted vaccine in most swine herds and the method of identification diseased aniamls from immunized animals, PR had been effectively controlled in China. However, since late2011, many swine farms vaccinated with Bartha-K61 in China outbreaked PR. Meanwhile,many swine farms vaccinated Bartha-K61 in Shandong province also occurred PR. The clinical symptoms are characterized as neural symptoms in the piglets and miscarriage in pregnant sows. Five field PRV strains were isolated from different areas in Shandong province. In this study, we amplified and sequenced one strain named Qihe547 isolated from swine herd in Qihe area.1. Isolation and identification of field PRV strainsFrom December 2014 to March 2015, lesion tissues were collected from five swine herds vaccinated with Bartha-K61 vaccine that have emerged a serious disease which was suspected of occurring PR. We first detected the pathogen infection of these lesion tissues using Quantitative Real-time RT-PCR/PCR. We identified the pathogen as PRV, not hog cholera virus, porcine reproductive and respiratory syndrome virus or porcine circovirus.The supernatant of grinded tissue samples were filtered to infect PK15 cells and the typical cytopathogenic effects of PRV appeared post infection 8 hours. After purification, the isolates were propagated in PK15 cells and identified by PCR using g E gene specific primers. The detection results showed that all isolates are PRV positive. We named these isolates Qihe547, SDYS1, SDYS2, SDFX, SDJN. The rabbits infected with PRV isolates appeared classical symptoms of PR and died finally. The titter of Qihe547 isolate in PK15 cells was up to 10-7.0/0.1ml.2. The complete genome sequencing of Qihe547 strainIn this study, we designed more than 200 pair of primers to amplify the fragments by PCR. After successfully sequencing and removing the repetitive sequences, we joined sequences of all fragments together. Finally, we obtained the complete sequence of Qihe547 and located genes sites. The full length is 143404 bp.3. The genetic variation analysis of Qihe547 strainTo analyze the genetic variation of PRV Qihe547, we performed the comparison between Qihe547 and the reference PRV strains from Gen Bank. The comparison results based on complete sequences of PRV strains showed that, compared with Chinese PRV strains, Qihe547 shared lower nucleotide identity with foreign PRV strains. Phylogenetic analysis based on complete sequences of PRV strains indicated Chinese PRV strains belong to an independent branch in evolutionary tree. Meanwhile, we found that Qihe547 belonged to genotype?. We also conducted variation analysis, potential glycosylation sites and antigenic sites analysis. The analysis results show that glycoprotein(g)B, g C, g D gene varies greatly. g B have three amino acid deletion(75SPG77) and one insertion(94G); g C have seven successive amino acid insertion(63AAASTPA69); g D have two amino acid insertion(278RP279).Compared with Bartha strain, the differences of potential antigenic sites and O-glycosylation sites of glycoprotein(g)B, g C, g D of Qihe547 mainly existed in the dominant antigenic epitope regions of g B(aa59- aa126), the extracellular region of g C(aa23- aa453)and g D. These differences might result in antigenic drift of the isolate and contribute to PRV evade the host immune response. PRV structural protein genes and nonstructural protein genes have different variations. Among these genes, gene regulation and replication related genes vary greater. We performed analysis on repeat sequences of PRV coding regions and non-coding regions. There are differences in the distribution in coding regions and non-coding regions and these may affect gene expression, transicription and protein function. The variation analysis contribute to the prevention and control of PR and provide the foundation for the study of PRV vaccines.