Fig.1 Schematic diagram of constructing recombinant strain（PRV-△gI-（p12）-△TK-（CD2v））

Fig.2 Preparation of homologous recombinant fragment and verification of recombination sites A:M:DL 5000 DNA marker. 1:PCR results of p12 gene with both sides donor from pcDNA3.1-PRV-TK-P12. 2:PCR results of CD2v gene with both sides donor from pcDNA3.1-PRV-gI-CD2v. 3 and 4:Blank control（water as template）. B:M:DL 5000 DNA marker. 1:PCR results of TK gene from wild-type PRV. 2:PCR results of TK gene from PRV-∆gI-（p12）-∆TK-（CD2v）. 3:PCR results of gI gene from wild-type PRV. 4:PCR results of gI gene from PRV-∆gI-（p12）-∆TK-（CD2v）. C:M:DL2000 marker. 1:PCR results of CD2v gene with 5' donor from PRV-∆gI-（p12）-∆TK-（CD2v）. 2:PCR results of CD2v gene with 3' donor from PRV-∆gI-（p12）-∆TK-（CD2v）. 3:PCR results of gI gene with 5' Donor from PRV-∆gI-（p12）-∆TK-（CD2v）. 4:PCR results of gI gene with 3' donor from PRV-∆gI-（p12）-∆TK-（CD2v）. D:Sequencing analysis from Fig.2-C

Fig.3 Identification of genetic stability A:PCR detection for genetic stability of recombinant virus. M:DL5000 maker; 1,3 and 5:the PCR result of p12 gene from 10th,20th and 30th Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）recombinant virus,respectively; 2,4 and 6:the PCR result of CD2v gene from 10th,20th and 30th Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）,respectively. B:The expression of CD2v protein in Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）recombinant virus is confirmed by Western blot. Lane M2:Protein marker; C1:control（Vero cells）; C2:Vero cells infected with PRV-Fa; C3:Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）. C:Expression of EGFP and mCherry in PRV-∆gI-（p12）-∆TK-（CD2v）via immunofluorescence analysis

Fig.4 One-step growth curve of recombinant viruses and mice’s survival curve A:Crystal violet staining of Vero cells（mock）. B:Crystal violet staining of Vero cells infected with PRV-Fa virus. C:Crystal violet staining of Vero cells infected with PRV-∆gI-（p12）-∆TK-（CD2v）virus. D:Statistical results of the plaque area. E:Virus sample after the amplification in Vero cells was collected at 12,24,36,and 48 h,and the virus titer was calculated by the Karber method to draw a one-step growth curve. F:Five-week-old ICR mice were injected with viruses into the right hind leg to observe mice’s survival daily（n = 15/each group）. All data were analysed by unpaired t-test（GraphPad Prism 5.0,GraphPad Software,San Diego,CA,USA）,*P<0.05;**P<0.01;***P<0.001;ns（not significant）

Fig.5 Histopathological sections of mice tissues A:HE（Hematoxylin-Eosin）staining of heart. B:HE staining of liver. C:HE staining of spleen,there are a lot of immune cells accumulated in the spleens of PRV-∆gI-（p12）-∆TK-（CD2v）infected group. D:HE（Hematoxylin-Eosin）staining of lung,the lung of PRV-Fa infected group was swollen obviously. E:HE staining of kidney. F:HE staining of brain,the brain of PRV-Fa infected group showed a large number of immune cell accumulated