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In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors.

ABSTRACT: The paucity of animal models that simulate the replication of the hepatitis B virus (HBV) is an impediment to advancing new anti-viral treatments. The work reported here employed recombinant adeno-associated viruses (AAVs) to model HBV subgenotype A1 and subgenotype D3 replication in vitro and in vivo. Infection with subgenotype A1 is endemic to parts of sub-Saharan Africa, and it is associated with a high risk of hepatocellular carcinoma. Recombinant AAV serotype 2 (AAV2) and 8 (AAV8) vectors bearing greater-than-genome-length sequences of HBV DNA from subgenotype A1 and D3, were produced. Transduced liver-derived cultured cells produced HBV surface antigen and core antigen. Administration of AAV8 carrying HBV subgenotype A1 genome (AAV8-A1) to mice resulted in the sustained production of HBV replication markers over a six-month period, without elevated inflammatory cytokines, expression of interferon response genes or alanine transaminase activity. Markers of replication were generally higher in animals treated with subgenotype D3 genome-bearing AAVs than in those receiving the subgenotype A1-genome-bearing vectors. To validate the use of the AAV8-A1 murine model for anti-HBV drug development, the efficacy of anti-HBV artificial primary-microRNAs was assessed. Significant silencing of HBV markers was observed over a 6-month period after administering AAVs. These data indicate that AAVs conveniently and safely recapitulate the replication of different HBV subgenotypes, and the vectors may be used to assess antivirals' potency.

SUBMITTER: Limani SW

PROVIDER: S-EPMC8618177 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:AimTo molecularly characterize hepatitis B virus (HBV) isolates from Kerala and to relate them to the clinical manifestation of infection.MethodsSera and clinical data were collected from 91 patients diagnosed with chronic HBV infection and HBV-related hepatocellular carcinoma (HCC). HBV from 44 HCC, 22 cirrhotic and 25 chronic hepatitis patients were genotyped by sequencing of the complete S region or by restriction fragment length polymorphism assays. The basic core promoter/precore region was sequenced. The complete surface DNA sequences were assembled and aligned manually, and then compared with the sequences of HBV of genotypes (A-J) from GenBank. The evolutionary history was inferred using the Neighbor-Joining method and the evolutionary distances computed using the Kimura 2-parameter method. Bootstrapping was performed using 1000 replicates. The TaqMan BS-1 probe was used to quantify HBV DNA at a lower detection limit of approximately 20 IU/mL. Continuous variables were compared using an independent Student's t test. The χ² test or Fisher's exact test was used to compare categorical variables. The differences were considered statistically significant at P < 0.05.ResultsIrrespective of disease status, the predominant genotype was A (72%); 95% belonging to subgenotype A1, followed by genotypes D (27%) and C (1%). HCC patients infected with subgenotype A1 were significantly younger than those infected with D. Mutation A1762T/G1764A was significantly associated with HCC in both genotypes A and D. Mutation G1862T was more frequent in subgenotype A1 (P < 0.0001), and in combination with A1762T/G1764A, it was significantly associated with HBV from HCC patients. Mutation C1766T/T1768A was significantly associated with genotype A (P = 0.05) and HCC (P = 0.03). The preS2 start codon M1T/I mutation was unique to genotype A strains (15.6%) from all disease groups and occurred at a higher frequency in isolates from HCC patients (P = 0.076). A higher frequency of preS deletion mutants (33.3%) was observed in genotype A from HCC compared with non-HCC patients, but did not reach statistical significance. The preS2:F22L mutation was found in genotypes A and D.ConclusionKerala is the first Indian state in which subgenotype A1 has been found to predominate in liver disease patients who developed HCC at a relatively young age.

Project description:Viral vectors are engineered virus variants able to deliver nonviral genetic information into cells, usually by the same routes as the parental viruses. For several virus families, replication-competent vectors carrying reporter genes have become invaluable tools for easy and quantitative monitoring of replication and infection, and thus also for identifying antivirals and virus susceptible cells. For hepatitis B virus (HBV), a small enveloped DNA virus causing B-type hepatitis, such vectors are not available because insertions into its tiny 3.2 kb genome almost inevitably affect essential replication elements. HBV replicates by reverse transcription of the pregenomic (pg) RNA which is also required as bicistronic mRNA for the capsid (core) protein and the reverse transcriptase (Pol); their open reading frames (ORFs) overlap by some 150 basepairs. Translation of the downstream Pol ORF does not involve a conventional internal ribosome entry site (IRES). We reasoned that duplicating the overlap region and providing artificial IRES control for translation of both Pol and an in-between inserted transgene might yield a functional tricistronic pgRNA, without interfering with envelope protein expression. As IRESs we used a 22 nucleotide element termed Rbm3 IRES to minimize genome size increase. Model plasmids confirmed its activity even in tricistronic arrangements. Analogous plasmids for complete HBV genomes carrying 399 bp and 720 bp transgenes for blasticidin resistance (BsdR) and humanized Renilla green fluorescent protein (hrGFP) produced core and envelope proteins like wild-type HBV; while the hrGFP vector replicated poorly, the BsdR vector generated around 40% as much replicative DNA as wild-type HBV. Both vectors, however, formed enveloped virions which were infectious for HBV-susceptible HepaRG cells. Because numerous reporter and effector genes with sizes of around 500 bp or less are available, the new HBV vectors should become highly useful tools to better understand, and combat, this important pathogen.

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In Vivo Modelling of Hepatitis B Virus Subgenotype A1 Replication Using Adeno-Associated Viral Vectors.

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