Molecular Basis For The Changes Of Biological Characteristic Of High-temperature Passaging Pseudorabies Virus

Since 2011,an outbreak of porcine pesudorabies disease coused by a variant PRV strain has spread to many vaccinate pig farms and resulted in considerable economic losses in China.The traditional vaccine strains such as Bartha-K61 did not provide complete immuno-protection against the emerging variant PRV infection.In the previous study,a PRV variant JS-2012 strain was isolated from piglets with suspected PRV infection in Jiangsu Province,China in 2012.The PRV variant was serially passaged in Vero cells at 40?C and an attenuated virus?JS-2012-F120 strain?was obtained after 120times passaging.Challenge experiments showed that JS-2012-F120 could not cause any clinical symptoms in the sucking piglets,and some biological characteristics such as plaque morphology and virus titer were significantly different from JS-2012.In this study,the biological characteristics of JS-2012 and its derivatives at different passages?The 25th,45th,50th,71th,91th,110th and 120th passages virus?were compared by plaque assay,viral growth abilities and challenge experiment in a mice model.The titers were gradually increased after culturing at the Vero cells.The titer of JS-2012-F120 was up to 10^9.0 TCID₅₀/ml,which was100-fold higher than that of JS-2012.However,the plaque size of the JS-2012-F50 was 50%of the parental virus,while the JS-2012-F120 could reach to 95%.Furthmore,the virulence to mice was gradually decreased as passage number increased.The morbidity and mortality was 100%and 50%in JS-2012-inoculated group respectivly,while the morbidity was 30%for JS-2012-F120and no mice died throughout the experiment.The coding sequences of the viruses at different passages were obtained by high-throughput sequencing and sanger sequencing method to explain the biological changes of the attenuated pseudorabies virus at a genetic level.Compared with JS-2012,six amino acids mutation were found in four proteins?IE180,UL18,UL44 and UL50?,two frameshift mutations were found in the UL16 and UL46 genes,and a 2307nt continous deletion was found in the gE and gI genes.The UL16gene had a cytosine insertion at 952nt,which leads to the following 13 amino acids mutation at the C end.The UL46 gene had a 29 nt insertion at position 1796,which lead to 130 amino acids mutations at the C end.The deletion of 2307bp of gE-US2 gene might be the main factor of virulence attanuation and plaque reduction.Furthmore,the virus attenuation might be associated with the frameshift of the UL16 and UL46 genes.The amino acid mutation?G90D?in gC protein might faciliate virus transmission between cells.All these results were very helpful to understand the pathogenic mechanism and growth characteristics of pseudorabies virus.In this study,3'RACE,Western-blot,and IFA methods were used to verify the UL16 and UL46genes'frameshift mutations at transcriptional and translational level.It was confirmed that the frameshift mutation did not affect the expression of these genes.Frameshift mutation is a very rare phenomenone since the genome structure of PRV is very stable.These results were helpful to deeply understand the functions of UL16 and UL46 genes of PRV.The key antigenic epitope regions of gE of Pseudorabies virus?PRV?was expressed in E.coli for the preparation of monoclonal antibody?MAb?against gE protein.The purified recombinant protein was used to immunize 6 week old BALB/c female mice.Four strains of hybridoma cell lines?2E5,5C3,5B2,and 6E6?stably producing anti-gE protein MAb were obtained using the hybridoma technique.The four MAbs was able to react with PRV variant strain?JS-2012?,but not with the gE deleted vaccine virus Bartha-K61 strain by Indirect immunofluorescence assays?IFA?analysis.Western-Blot results showed that 5C3 and 2E5 were able to react with PRV?JS-2012?but 5B2 and 6E6 were not able to react with PRV?JS-2012?.These specific monoclonal antibodies might provide a good tool for the study of the PRV and the establishment of PR diagnosis method.