Generation and characterization of a stable cell line persistently replicating and secreting the human hepatitis delta virus

Abstract

Human hepatitis delta virus (HDV) causes the most severe form of viral hepatitis. Approximately 15–25 million people are chronically infected with HDV. As a satellite virus of the human hepatitis B virus (HBV), HDV uses the HBV-encoded envelope proteins for egress from and de novo entry into hepatocytes. So far, in vitro production of HDV particles is restricted to co-transfection of cells with HDV/HBV encoding cDNAs. This approach has several limitations. In this study, we established HuH7-END cells, which continuously secrete infectious HDV virions. The cell line was generated through stepwise stable integration of the cDNA of the HDV antigenome, the genes for the HBV envelope proteins and the HBV/HDV receptor NTCP. We found that HuH7-END cells release infectious HDV particles up to 400 million copies/milliliter and support virus spread to co-cultured cells. Due to the expression of NTCP, HuH7-END cells are also susceptible to de novo HDV entry. Virus production is stable for >16 passages and can be scaled up for preparation of large HDV virus stocks. Finally, HuH7-END cells are suitable for screening of antiviral drugs targeting HDV replication. In summary, the HuH7-END cell line provides a novel tool to study HDV replication in vitro .

Interplay between Hepatitis D Virus and the Interferon Response

“…Although these integrates are unable to produce infectious HBV because they consist of incomplete HBV DNA, they produce HBV mRNAs encoding HBsAg. This cccDNA-independent production of HBsAg is sufficient to support HDV assembly and secretion in an infected cell [ 50 , 51 , 52 , 53 ]. Notably, such integrated HBV DNA may also serve as an important source of HBsAg in chronically infected patients [ 75 ] and provide considerable replication space for HDV in the livers of infected patients.…”

“…This is mainly due to the lack of a suitable HBV/HDV co-infection system and the rarity of “true” co-infection at the single cell level. In response, hepatoma cell lines stably expressing NTCP and the three HBV envelope proteins under endogenous promoter control have been constructed recently [ 52 , 53 ]. Such models mimic the possible replication of HDV in HBsAg-expressing hepatocytes after the integration of double-strand linear HBV DNA [ 50 ], as reported frequently in preferentially hepatitis B e antigen (HBeAg)-negative patients [ 51 , 75 ].…”

Microarray Data Mining and Preliminary Bioinformatics Analysis of Hepatitis D Virus-Associated Hepatocellular Carcinoma

“…Approximately 292 million people worldwide are chronically infected with HBV, which causes liver injury that can progress to cirrhosis, resulting in HCC, liver failure, and eventually death [ 5 , 6 ]. The HDV is known as the satellite of HBV and affects 15–20 million people in the world [ 7 ]. Concurrent HBV and HDV infections significantly increase both the incidence and mortality of HCC among patients with chronic hepatitis B (CHB) [ 8 ].…”

New insights into HDV persistence: The role of interferon response and implications for upcoming novel therapies

“…The three J o u r n a l P r e -p r o o f membrane-embedded HBV envelope proteins small (S-), middle (M-) and large (L-), collectively termed HBsAg, are encoded by the covalently closed circular DNA (cccDNA) in HBV replicating hepatocytes and the integrated HBV DNA in chromosomes. [21][22][23][24][25] The HDV replication cycle is depicted in Fig. 1.…”

“…Application of newly developed cell culture models, e.g. cell lines expressing both, the HDV receptor and the HBV envelope thereby supporting the full HDV life cycle, 23,24 will be helpful to better understand to what extend these HDV-specific countermeasures contribute to the surveillance of immune recognition. 103 Of note, the sensitivity of HDV RNA quantification assay at that time was low 104 and therefore rates of serum RNA negativation were probably overestimated.…”