Today I would like to discuss in part with you an article published in the journal Nature: Scientific Reports titled "Transgenic hepatitis B: a new model of HBV infection". Because it will give me the opportunity to talk a bit about transgenesis with you all again (which is the real reason for this post... mua ha ha). I think this is an important topic that needs to be discussed ad nauseum as the very concept is foreign to people, and perhaps there are some misconceptions.

Transgenic, it's a word that drives fear into many of you. I am sure you associate it with food, GMO foods to be specific. However this technology has applications outside of that, and is actually very useful in the laboratory setting for manipulating organisms to be better for study. You are familiar with transgenic 'GloFish' as well right?

These little guys

GloFish are transgenic zebra fish which have been genetically engineered to include genes which result in the production of various fluorescent proteins (like GFP (Green Color, comes from a jellyfish, or dsRED (Red Color, comes from a coral note coral is an animal, the reef is where they live, coral are not the fancy structures!). When you shine a UV light on these fish, the fluorescent proteins... glow! These fish serve as a good example to think about when first trying to understand transgenesis as it is a pretty harmless display of what the technology can do. Take a gene for something (in this case a glowing protein) from an organism and produce the protein which the gene encodes for in a different organism.

I am sure many of you are not comfortable with this technology being used in our foods, attributing a whole host of nefarious activities to its employment there. Lets move away from that for the time being, and discuss a different aspect of transgenesis. The study of viruses. For that lets discuss Hepatitis B.

Hepatitis B

Hepatitis, like the name implies is a disease of the liver (derived from Hepar meaning liver and itis meaning inflammation), and Hepatitis B is one such virus that can cause this liver inflammation disease. Hepatitis B is a blood borne illness, meaning that exposure to an infected persons blood (or other bodily secretions) is how it is transmitted from person to person and it was very common in the past among IV drug users as they would share needles, resulting in easy blood contact. [2].

The disease itself can manifest in two forms, an acute or chronic. Chronic infections usually occur from infections occurring at birth, while those infected later in life do not usually develop chronic Hep B.[3]

There is a vaccine which is fairly effective at providing protection against Hepatitis B infection, however this vaccine is only 95% effective, and for those exposed at birth the likleyhood of developing the chronic is very high (>90%). This results in a lot of people still having this disease, over 350 million worldwide![4], [5].

To make matters worse the available treatments are not cures, they just attempt to manage the amount of the virus in the infected people. However they are not particularly effective for many people. So new sorts of treatments are badly needed, and in order to develop those treatments we can fall back on our deep understanding of how this virus infects and grows right?

Wrong, unfortunately. Science just doesn't understand all that much about the lifecycle of hepatitis B, nor all of the ways it interacts with cellular proteins for growth. No understanding, nothing to target to potentially treat the infection. The authors of the article we are discussing today turned to glofish technology (GFP, or that green fluorescent protein) and generation of a transgenic hepatitis B virus to better get a handle on how it grows and interacts with cells during its life. In addition to generating this GFP glowing virus, they also made a second transgenic version with am antibiotic resistance gene (for a different means of selection).

Challenges In Getting The Fluorescent Gene Into Hepatitis B

For a lot of organisms, genes in genomes are organized sequentially, one gene begins, then ends and then the next gene begins downstream. However for hepatitis B the organization is more complicated, and genes overlap with one another ( meaning that the start of one gene is actually INSIDE another gene sequence), the mechanism biologically how this works isn't really important for you to understand however if you are interested it is because of overlapping reading frames (I put a link to what a reading frame is so dive on in). Suffice it to say, this complicates adding a transgenic gene into the genome. Nevertheless, careful analysis of the genome revealed a small location where an insertion was possible, and the authors placed their GFP/antibiotic resistance gene(s) there.

The virus genome was packaged into a circular plasmid to allow it to be expressed in mammalian liver cells (that are grown in flasks, they weren't infecting actual livers, just cells). (Plasmids are just small DNA constructs that allow for genetic information to get into and pass between cells, they have all the necessary instructions for producing proteins once inside, or in this case the whole virus).

HBV Infected HepG2 Cells

Here (image to the left) the authors were using an antibody to detect whether or not cells (they were using HepG2) cells here were getting infected with their hepatitis B construct. To the left are unstained cells, while to the right the red color comes from the antibody stain used to detect the infected cells (why they didn't just use the fluorescence of the GFP I do not know).

Using Cells That Lack Certain Proteins To See What Hepatitis B needs to grow

The authors, having seen that their virus construct works and can result in cells getting infected then wanted to see if they could identify some cellular proteins that were essential for the viruses survival. To do this they used cells that were lacking a variety of proteins, and then singly allowed for various ones to be re-expressed. And then exposed those cells to the ones they created earlier that were already infected with the virus. They wanted to see what cells could then also get infected and what ones couldn't.

Gene UQCR10 Was Identified As Essential For Hepatitis B Infection

There analysis found that the gene UQCR10 was important, this gene encodes for a protein called Ubiquinol-Cytochrome C Reductase, which is a component of the metabolic pathways of cellular mitocondria. Odd that this is a required protein for infection since the cells themselves can survive with out it. An interesting observation, and one which allows for a whole host of new research.

What Was The Point In Discussing This

The authors of the article I was discussing go into great detail about the potential reasoning why this protein they identified plays a role, but that is not the true reason for this blog. The point I want to stress is that this finding would not have been possible with out the transgenesis. This technology opens up doors in the lab that otherwise would remain closed, and presents new and novel methods to gain better understanding of how cells, viruses, bacteria and a whole host of other biological phenomena work and interact with one another.

Will the protein identified in the study briefly discussed above lead to a novel cure for Hepatitis B? That isn't known, it might or it might not, but with out the technology to make transgenic organisms, viruses, proteins etc the potential for this sort of discovery would not be there. You may not be comfortable with transgenic food, but I hope with repeated exposure that perhaps you can find at least a bit more of a relaxed attitude to the technology as a whole. The potential for advancement that is derived from these sorts of experiments are great, have been involved in the discovery of a variety of treatments in the past and will likely be for a plethora in the future.

I Leave You With A Youtube Video Of Glo-Fish

These little guys are quite pretty!

Sources

Extra Image Sourcing

Glo-Fish

All Non Cited Images Are From Pixabay.com or Flickr.com And Are Available Under Creative Commons Licenses

Any Gifs Are From Giphy.com and Are Also Available for Use Under Creative Commons Licences

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Better Understanding The Workings Of Hepatitis B With A Transgenic Virus