ACTA LABORATORIUM ANIMALIS SCIENTIA SINICA ELISA. HBsAg HBeAg HBsAb HBeAb HBcAb PCR HBV DNA. raav8
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1 ACTA LABORATORIUM ANIMALIS SCIENTIA SINICA June 2013 Vol. 21 No. 3 檵 檵檵檵檵檵檵檵檵檵 8 HBV 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 Doi /j. issn 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 China 2. Institute of Transfusion Medicine Academy of Military Medical Sciences Beijing Corresponding author SUN Shi-hui 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 raav HBV into the liver of tree shrews. Methods Serum and liver tissues were collected at indicated times after i. v. injection of raav 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 raav 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 raav HBV injection. The copies of HBV DNA in liver reached 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 raav 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 raav 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. 2012ZX Tel shsun916@ yahoo. com. cn
2 Acta Lab Anim Sci Sin June 2013 Vol. 21. No 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 hepatocellular carcinoma HCC 1 HBV SYXK ~ 28 40% ~ 60% HBV 10% HBV QIAGEN ALT 5-10 Beckman CX HBV 1. 2 HBV 1. 3 HBV 1. 3 HBV HBV 2% 11 AAV8 250 μl /100 g raav8-1. 3HBV ayw vg /ml AAV HBV vg /100μL / HBV ayw 8 raav8 raav d HBV d % ELISA HBsAg HBeAg HBsAb HBeAb HBcAb ALT HBsAg HBeAg HBsAb HBeAb HBcAb PCR HBV DNA A 450 nm 1 CUTOFF = ~ 140 g 5 14h ~ 8 00 HBV 3R HBV HBV 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 A A CUTOFF A CUTOFF
3 Acta Lab Anim Sci Sin June 2013 Vol. 21. No PCR HE DNA DNA raav HBV PBS DNA 1. 9 DNA HBV Graphpad Prism5 HBV PCR one-way ANOVA Dunnett μl Taq 0. 2 μl UNG μl DNA 2 μl PCR 37 2 min 94 1 min 95 5 s s 40 PCR HBV DNA 2. 1 HBV 1. 6 ALT raav8-1. 3HBV A HBeAg 4h 8000 r /min 10 Beckman CX5 B HBsAb HBeAb HbcAb 14 ~ ALT 25 d HBV HBsAg HBcAg 1. 8 raav8-1. 3HBV 55 4% P < raav8-1. 3HBV 3 d HBsAg 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
4 Acta Lab Anim Sci Sin June 2013 Vol. 21. No 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 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 raav HBV 6d HBV 3 d 55 d HBV 3 A-C raav HBV ALT raav HBV ALT ALT raav HBV HBV HBV HBV 1 3 HBV HBV DNA 13 2 HBV 4 Fig. 4 ALT Assay of serum ALT in the tree shrews
5 Acta Lab Anim Sci Sin June 2013 Vol. 21. No. 3 HBV d AAV 100 AAV 8 AAV8 AAV HBV HBV HBV HBsAg -HBs HBV DNA HBsAg / HBcAg Southern HBV DNA vg / mouse raav8 18 HBV % HBV AAV8 HBV raav ml 87 /96 raav % HBV RNA RNA mirna Dane HBsAg 4 raav8 HBV raav8 HBV HBV 90% 56% GFP AAV8 1 HBsAg 1 dalt 35 ~ 40 IU /L2 ~ 3 d 80 % 20 5 d HBV 20 d phbv 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
6 Acta Lab Anim Sci Sin June 2013 Vol. 21. No HBsAb 23 HBV HBV HBV HBsAg 3 HBV 24 AAV8 HBV raav8 HBV % HBV M 1. 3 HBV ayw 8 raav8 raav8-1. 3HBV HBV HBV HBV DNA HBV raav Don Ganem M. Prince. Hepatitis B virus infection-natural history and clinical consequences J. New Eng J M J Liang L Li Y et al. Breeding of tree shrews in laboratory J. 4. Sichuan J Zool J Walter E Keist R Niederost B et al. Hepatitis B virus infec- 5. tion of tupaia hepatocytes in vitro and in vivo J. Hepatology J Yan H Zhong G Xu G et al. Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus J. elife e DOI /elife Ren ST Nassal M. Hepatitis B virus HBV virion and covalently closed circular DNA formation in primary Tupaia hepatocytes and human hepatoma cell lines upon HBV genome transduction with replication-defective adenovirus vectors J. J. virol J Baumert FT Yang C Schurmann P et al. Hepatitis B virus mutations associated with fulminant hepatitis induce apoptosis in primary tupaia hepatocytes J. Hepatology J J Pan QC Zhang JX. Natural history and clinical consequences of hepatitis B virus infection J. Int J Med Sci HBV J J Barbon CM Ziegler RJ Li C et al. AAV8-mediated hepatic expression of acid sphingomyelinase corrects the metabolic defect in the visceral organs of a mouse model of Niemann-Pick disease J. Mol Thera Wang LL Calcedo R Timothy C et al. Sustained correction of disease in naive and AAV2-pretreated hemophilia B dogs AAV2 /8-mediated liver-directed gene therapy J. Blood J Su JJ Yan RQ et al. Adult tree shrews experimentally infected with hepatitis B virus J. Chn J Pathol HBV J J l l J
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