Southern blots from Sapphire confirm presence of cccDNA in Hepatitis B study

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Imaging Publication Spotlight

In a recent publication published in PLOS from Pennsylvania State University College of Medicine, Hong et al tested the potential for using woodchucks (also referred to as groundhogs) as animal models for Hepatitis B virus (HBV) using Southern blot results obtained from an Azure Sapphire Biomolecular Imager. HBV is a deadly pathogen affecting worldwide every year. It can cause severe liver disease, which results in over 800,000 deaths from HBV infection every year. The treatment options available rarely cure the disease. This opens the need for new therapeutic options; however, their development is limited due to a lack of testing methods, including available animal models. 

Since the release of this publication, the Azure Sapphire has been succeeded by the new Azure Sapphire FL, which was designed to be the flexible choice in bringing precise quantitation of nucleic acids and proteins.

Using woodchucks as HBV models

The authors investigated the potential cellular mechanisms responsible for species tropism by comparing woodchuck cells to human cells. HBV has strict species and cell tropism that favors human hepatic cells, and the mechanisms behind this are not clearly understood. Researchers  are looking to develop new animal models. The first step is to understand the factors that contribute to HBV host tropism.

According to Lucifora, it is rare for current HBV therapies to see cures in chronic HBV patients because of the refractory nature of the stable closed circular DNA (cccDNA). The methods of blocking cccDNA formation and eliminating existing cccDNA pools are crucial for curing patients2.

Figure 1 from Hong et al 2022. Woodchuck cell lines were evaluated for cccDNA as an indication of HBV infection susceptibility. The Azure Sapphire Biomedical Imager was used for imaging cccDNA in Southern blots.
Figure 1 from Hong et al 2022. Woodchuck cell lines were evaluated for cccDNA as an indication of HBV infection susceptibility. The Azure Sapphire Biomedical Imager was used for imaging cccDNA in Southern blots.

Research methods using Southern blots

Hong et al looked at the ability of both hepatic and non-hepatic woodchuck cells to support HBV infection. The authors transfected cells with an HBV replicon to bypass the entry steps since HBV does not naturally infect woodchuck cells. Through a variety of assays, they found that woodchuck hepatic cell lines could indeed support HBV replication. One measure of HBV infection is to look for the presence of viral covalently cccDNA.

To confirm the presence of cccDNA, the researchers imaged Southern blots using an Azure Sapphire Biomolecular Imager. This cccDNA detection method was used throughout their research to confirm HBV infection.

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The HBV entry receptor for human cells is huNTCP. After expressing huNTCP on two different woodchuck cell lines, WC3 and WCH-17, the authors exposed the cells to HBV in cell culture. Interestingly, only WCH-17 cells were able to support the HBV infection. To try and understand which factors could contribute to this difference, transcriptome analysis of the two cell lines was performed.

Since WCH-17 cells supported HBV replication when the HBV replicon was inserted into cells after the entry steps, the genes revealed with the transcriptome analysis suggests that the issue is somewhere in the process of HBV binding to the surface receptor, undergoing the initial entry steps and before nuclear import of relaxed circular DNA (rcDNA). This is common in cccDNA formation.

The new Sapphire FL is the ultimate biomolecular imager for flexibility. With customizable and user-changeable laser and filter modules, the Sapphire FL easily adapts to a lab’s changing needs and advancing research.

The research findings

Having an acceptable animal model will allow for the investigation and development of new therapeutic treatment options for those infected with HBV. This study provides new insights into the mechanisms of HBV tropism and supports the potential to develop an HBV-susceptible woodchuck model.

Background on woodchuck hepatitis virus

Woodchucks are naturally infected with woodchuck hepatitis virus (WHV). While woodchucks are not susceptible to HBV infection, WHV is similar to HBV and can be used as a model to better understand which host-specific factors affect HBV infection.

In addition to Southern blots, the Sapphire also provides a variety of other features, including multichannel fluorescence, white light imaging of tissues and gels, and fluorescent imaging of tissue culture plates. To learn more about the Sapphire and the ways Azure Biosystems can support your research, click here.

SOURCES

  1. Hong et. al. PLoS Pathog. 2022 Jun; 18(6): e1010633. Published online 2022 Jun 17. doi: 10.1371/journal.ppat.1010633

  2. Lucifora, J. & Protzer, U. Attacking hepatitis B virus cccDNA-The holy grail to hepatitis B cureJ. Hepatol. 64, S41–S48 (2016).