2013 6 21 3 ACTA LABORATORIUM ANIMALIS SCIENTIA SINICA June 2013 Vol. 21 No. 3 檵 檵檵檵檵檵檵檵檵檵 8 HBV 1 1 1 1 1 1 2 1 1 1. 100071 2. 100850 8 1. 3 HBV 1. 3HBV ayw HBV 1. 3 HBV 8 recombinant adeno-associated virus 8 raav8-1. 3HBV ELISA HBsAg HBeAg HBsAb HBeAb HBcAb PCR HBV DNA ALT HBV 1 ~ 2 30 d 55 d HBV DNA 10 4 ~ 10 5 HBV DNA ALT raav8 HBV HBV raav8 8 HBV Q95-33 A 1005-4847 2013 03-0036-06 Doi 10. 3969 /j. issn. 1005-4847. 2013. 03. 008 Establishment of a tree shrew model of acute hepatitis B virus infection by transduction with a recombinant adeno-associated virus 8 carrying 1. 3 copies of HBV genome ZENG Yang 1 WU Xiao-hong 1 HU Jing-ya 1 LIU Chen-feng 1 YU Hong 1 GUO Yan 1 ZHOU Yong 2 SUN Shi-hui 1 ZHOU Yu-sen 1 1. Institute of Microbiology and Epidemiology Academy of Military Medical Sciences Beijing 100071 China 2. Institute of Transfusion Medicine Academy of Military Medical Sciences Beijing 100850 Corresponding author SUN Shi-hui E-mail shsun916@ yahoo. com. cn Abstract Objective To establish a tree shrew model of acute hepatitis B virus infection by injection of a recombinant adeno-associated virus 8 vector carrying 1. 3 copies of HBV genome ayw subtype raav8-1. 3 HBV into the liver of tree shrews. Methods Serum and liver tissues were collected at indicated times after i. v. injection of raav8-1. 3 HBV into the tree shrews. The HBsAg BeAg HBsAb HBeAb HBcAb ALT and HBV virus load were examined by ELISA and real-time PCR respectively. The expression of HBcAg and pathological changes in the liver were also observed after the raav8-1. 3 HBV infection. Results Markers of serum HBV were all positive 2 weeks after and HBcAg-positive hepatocytes were even detected in the liver 55 days after raav8-1. 3 HBV injection. The copies of HBV DNA in liver reached 10 4-10 5 at 55 days after raav8-1. 3HBV injection. Serum HBV DNA could be detected for over one month. Mild pathological changes with elevated ALT were observed after raav8-1. 3 HBV injection. Conclusions A tree shrew model of a- cute HBV infection has been successfully established with the recombinant adeno-associated virus 8 carrying 1. 3 copies of HBV genome raav8-1. 3 HBV. Our study provides a novel and useful small animal model for the research of immunopathologic mechanism as well as the evaluation of vaccine and anti-hbv agents. 2012ZX10004502 Tel. 010-66948457 E-mail shsun916@ yahoo. com. cn
2013 6 21 3 Acta Lab Anim Sci Sin June 2013 Vol. 21. No. 3 37 Key words Tree shrew Hepatitis B virus HBV Recombinant adeno-associated virus 8 raav8 Acute hepatitis B virus Infection Animal model hepatitis B virus HBV4 SCXK 2011-0004 hepatocellular carcinoma HCC 1 HBV SYXK 2007-004 3. 5 24 ~ 28 40% ~ 60% HBV 10% 1. 2 2 HBV QIAGEN ALT 5-10 Beckman CX5 1. 1 HBV 1. 2 HBV 1. 3 HBV 1. 3 HBV HBV 2% 11 AAV8 250 μl /100 g raav8-1. 3HBV ayw 1 12 10 12 vg /ml 5 10 11 AAV8 1. 3 HBV vg /100μL / 0 1 3 6 14 25 31 38 1. 3 HBV 3 6 2 ayw 8 raav8 raav8-2 55 d 5 1. 3HBV 2 3 0 1 3 6 14 25 31 38 d - 80 3 6 55 4% ELISA HBsAg HBeAg HBsAb HBeAb HBcAb ALT HBsAg HBeAg HBsAb HBeAb HBcAb PCR HBV DNA A 450 nm 1 CUTOFF = 1 1. 1 9 130 ~ 140 g 5 14h 18 00 ~ 8 00 HBV 3R HBV HBV 1. 2 3 HBV raav8-1. 3HBV ayw HBV 2% HBV 4% HBsAg HBeAg HBsAb HBeAb HbcAb 4 HBV HCV HDV QIAGEN DNA 1. 4 HBV ELISA PBS 0 1 3 6 14 25 31 38 1 4 A 2. 1 0. 05 0. 05 2 A CUTOFF A CUTOFF
38 2013 6 21 3 Acta Lab Anim Sci Sin June 2013 Vol. 21. No. 3 1. 5 PCR 5 2 3 HE DNA DNA raav8-1. 3 HBV PBS 0 1 3 6 14 25 31 38 47 55 1 2 DNA 1. 9 DNA HBV Graphpad Prism5 HBV PCR one-way ANOVA Dunnett 17. 8 μl Taq 0. 2 μl UNG 0. 03 μl DNA 2 μl PCR 37 2 min 94 1 min 95 5 s 60 30 s 40 PCR HBV DNA 2. 1 HBV 1. 6 ALT raav8-1. 3HBV 0 1 3 6 14 25 31 40 55 14 1A HBeAg 4h 8000 r /min 10 Beckman CX5 B HBsAb HBeAb HbcAb 14 ~ ALT 25 d HBV 1. 7 1 HBsAg HBcAg 1. 8 raav8-1. 3HBV 55 4% P < 0. 01 2 raav8-1. 3HBV 3 d HBsAg 14 25 1 1 Tab. 1 A HBsAg B HBeAg 1 HBsAg HBeAg Note A Assay of HBsAg B Assay of HBeAg Fig. 1 Assay of HBsAg and HBeAg in serum of the tree shrews HBV / Detection indexes Assay of serological detection index in serum of the tree shrews positive /total raav8-1. 3HBV Days post raav8-1. 3HBV infection 0 d 1 d 3 d 6 d 14 d 25 d 31 d 38 d HBsAg 0 /9 0 /9 9 /9 7 /7 0 /5 0 /5 0 /5 0 /5 HBeAg 0 /9 0 /9 4 /9 5 /7 5 /5 0 /5 0 /5 0 /5 HBsAb 0 /9 0 /9 0 /9 0 /7 1 /5 3 /5 1 /5 1 /5 HBeAb 0 /9 0 /9 0 /9 0 /7 2 /5 4 /5 5 /5 4 /5 HBcAb 0 /9 1 /9 0 /9 7 /7 5 /5 5 /5 5 /5 5 /5
2013 6 21 3 Acta Lab Anim Sci Sin June 2013 Vol. 21. No. 3 39 2. 2 PCR raav8-1. 3HBV 55 5 HBV DNA DNA PCR raav8-1. 3HBV HBV DNA 2B 5 DNA PCR HBV HBV DNA 10 4 ~ 10 5 NO. 2 ~ NO. 6 HBV NO. 1 3 10 9 HBV DNA 0 h 2 A A PCR HBV DNA B PCR HBV DNA 1 2 ~ 6 raav8-1. 3HBV 55 d 2 PCR HBV DNA Note A HBV DNA copies in tree shrews serum B HBV DNA copies in tree shrews liver. No. 1 Control No. 2 ~ 6 Tree shrews injected with raav8-1. 3HBV. Fig. 2 Copies of HBV DNA in the liver and serum of tree shrews by real-time PCR 2. 3 HBV raav8-1. 3 HBV 6d HBV 3 d 55 d HBV 3 A-C raav8-1. 3 HBV 3 2. 4 ALT raav8-1. 3 HBV 0 1 3 6 14 25 31 40 55 ALT ALT 1 14 55 4 2. 5 raav8-1. 3 HBV HBV 3 6 55 5 HBV HBV 1 3 HBV HBV DNA 13 2 HBV 4 Fig. 4 ALT Assay of serum ALT in the tree shrews
40 2013 6 21 3 Acta Lab Anim Sci Sin June 2013 Vol. 21. No. 3 HBV 14 50 d AAV 100 AAV 8 AAV8 AAV HBV HBV HBV HBsAg -HBs HBV DNA HBsAg / HBcAg Southern HBV DNA 7. 2 10 11 vg / mouse raav8 18 HBV 15 100% HBV 19 1987 AAV8 HBV raav8 0. 5 ml 87 /96 raav8 90. 63% HBV RNA RNA mirna Dane HBsAg 4 raav8 HBV raav8 HBV 233 16-17 3 HBV 90% 56% GFP AAV8 1 HBsAg 1 dalt 35 ~ 40 IU /L2 ~ 3 d 80 % 20 5 d HBV 20 d phbv1. 2 1 HBsAg AAV8 ALT 1 ~ 3 d 3 d HBV AAV8 1 ALT 1. 3 HBV 1. 1 HBV 1. 2 HBV 2. 0 HBV 10 ~ 100 HBV HBsAg HBeAg HBV HBV HBV 21 2 HBV HBV Eike 22 HBV HBV HBsAg AVV8 HBV 10 2 ~ 3
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