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Establishment of drug-resistant HBV small-animal models by hydrodynamic injection.

ABSTRACT: In antiviral therapy of hepatitis B virus (HBV) infection, drug resistance remains a huge obstacle to the long-term effectiveness of nucleoside/tide analogs (NAs). Primary resistance mutation (rtM204V) contributes to lamivudine (LAM)-resistance, and compensatory mutations (rtL180M and rtV173L) restore viral fitness and increase replication efficiency. The evaluation of new anti-viral agents against drug-resistant HBV is limited by the lack of available small-animal models. We established LAM-resistance HBV replication mice models based on clinical LAM-resistant HBV mutants. Double (rtM204V+rtL180M) or triple (rtM204V+rtL180M+rtV173L) lamivudine-resistant mutations were introduced into HBV expression vector, followed by hydrodynamic injection into tail vein of NOD/SCID mice. Viremia was detected on days 5, 9, 13 and 17 and liver HBV DNA was detected on day 17 after injection. The serum and liver HBV DNA levels in LAM-resistant model carrying triple mutations are the highest among the models. Two NAs, LAM and entecavir (ETV), were used to test the availability of the models. LAM and ETV inhibited viral replication on wild-type model. LAM was no longer effective on LAM-resistant models, but ETV retains a strong activity. Therefore, these models can be used to evaluate anti-viral agents against lamivudine-resistance, affording new opportunities to establish other drug-resistant HBV small-animal models.

SUBMITTER: Cheng J

PROVIDER: S-EPMC4629082 | biostudies-literature | 2014 Aug

REPOSITORIES: biostudies-literature

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Establishment of drug-resistant HBV small-animal models by hydrodynamic injection.

Cheng Junjun J Han Yanxing Y Jiang Jian-Dong JD

Acta pharmaceutica Sinica. B 20140714 4

In antiviral therapy of hepatitis B virus (HBV) infection, drug resistance remains a huge obstacle to the long-term effectiveness of nucleoside/tide analogs (NAs). Primary resistance mutation (rtM204V) contributes to lamivudine (LAM)-resistance, and compensatory mutations (rtL180M and rtV173L) restore viral fitness and increase replication efficiency. The evaluation of new anti-viral agents against drug-resistant HBV is limited by the lack of available small-animal models. We established LAM-res ...[more]

PMID: 26579395

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Project description:BackgroundHydrodynamic injection (HI) of hepatitis B virus (HBV) mouse model is an useful tool for HBV related research in vivo. However, only 40% of C57/BL6 mice injected with 10 μg HBV genome contained plasmid (pAAV-HBV1.2), serum HBsAg more than 6 months and none of the BALB/c mice injected with 10 μg pAAV-HBV1.2 plasmid DNA, serum HBsAg positive more than 4 weeks in the previous study.MethodsIn this study, C57/BL6 and BALB/c mice were hydrodynamic injected with different doses of pAAV-HBV1.2 plasmid DNA. HBV related serum markers were detected by ELISA. ALT levels in the serum were measured using full automated biochemistry analyzer. HBcAg positive cells in the liver were detected by immunohistochemical staining. The mRNA levels of IRF3, ISGs including ISG15, OAS, PKR and immune factors including IFNγ, TNFα, TGFβ, IL-6, IL-10, PDL1 in liver of the mice were quantified by qRT-PCR.ResultsThe results showed that the mice injected with 100 μg high-concentration or 1 μg low-concentration of pAAV-HBV1.2 plasmid DNA did not excert dominant influence on HBV persistence. In contrast, injection of 5 μg intermediate-dose of pAAV-HBV1.2 plasmid DNA led to significant prolonged HBsAg expression and HBV persistence in both C57/BL6 (80% of the mice with HBsAg positive more than 6 months) and BALB/c (60% of the mice with HBsAg positive more than 3 months) mice. IFNγ was significant up-regulated in liver of the mice injected with 1 μg or 100 μg pAAV-HBV1.2 plasmid DNA. TNFα was up-regulated significantly in liver of the mice injected with 100 μg pAAV-HBV1.2 plasmid DNA. Moreover, PDL1 was significant up-regulated in liver of the mice injected with 5 μg pAAV-HBV1.2 plasmid DNA.ConclusionIn this paper we demonstrated that, in the HBV HI mouse model, the concentration of injected pAAV-HBV1.2 plasmid DNA contributes to the diverse kinetics of HBsAg and HBeAg in the serum as well as HBcAg expression level in the liver, which then determined the HBV persisternce, while the antiviral factors IFNγ, TNFα as well as immune negative regulatory factor PDL1 play important roles on HBV persistence.

Dataset Information

Establishment of drug-resistant HBV small-animal models by hydrodynamic injection.

Publications

Establishment of drug-resistant HBV small-animal models by hydrodynamic injection.

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