Thursday, December 8, 2016

The Vaccines of the Future: Influenza’s New Opponent

As the days get shorter and shorter, we are not only reminded of the impending winter weather but we are also reminded of the dreaded spread of the flu. The scientific name for the flu is Influenza, which is a RNA virus that often is replicating and infecting the cells in your respiratory tract.  The flu, as there is a yearly occurrence of infection due to slight mutations in the virus strain, has been the focus of lots of research to create effective vaccines to distribute every year to the public. From the extensive research on this process, Influenza has in some senses become a model for creating vaccines that could potentially be used to produce vaccines for different viruses. One issue, however, is that inactive and attenuated virus vaccines for Influenza often have a loss of productivity and efficacy. (1) Nevertheless,  in a recent Science paper titled, Generation of Influenza A Viruses as Live but Replication-Incompetent Virus Vaccines by Si Longlong et al, (2) the authors demonstrate how they were able to generate live but replication incompetent vaccines for Influenza by genetically expanding the genome.  In other words, the author proved that they could make genetic alterations to Influenza that would usefully replicate in a cell line but would be replication incompetent in vivo.

Before we start of the details of the paper, I am sure you are wondering what it means to be a replication incompetent vaccine? A replication incompetent vaccine is a vaccine that is full of virus particles but these particles have had the coding regions for the genes necessary for additional rounds of virion replication and packaging deleted. These viruses are capable of infecting their target cells and delivering their viral payload, but then fail to continue the typical lytic pathway that leads to cell lysis and death. This is rare to have for vaccines in some sense because in order to make a vaccine you need it to replicate itself and have the virion replication but to lack this when it is injected into you.

What makes this paper rare is that they do not remove any of the viral genes they alter the genetics so the replication is dependent on an UAA (unnatural amino acid). This is seen in their Figure 1 A below:

This figure demonstrates how they used plasmids to generate cell lines with premature termination codon Influenza viruses that function in transgenic cells with UAA but do not function in conventional cells. After this they went through the process of checking to make sure the vaccine has everything that is desirable in a vaccine. They found that the replication of their PTC virus did not affect the genetic stable that made it incompetent, and probably most important to any vaccine is how safe it is to use in vivo. For this aspect of the vaccine, the authors did a fantastic job and utilized three animal models. They test the PTC virus on mice, ferrets and guinea pig and found the virus not only to be effective for offering protection against influenza but also to be able to elicit a robust humoral, mucosal and T cell mediated immunity. Both of these can be seen below. The safety of the in vivo is demonstrated in Figure 3A and the robust immune response is displayed in Figure 4A:

Overall, the question always comes to why do we care about these findings? These findings are important because of the potential they hold. It is great that this is very effective vaccine against Influenza but as I mentioned earlier there is already the existence of them.  The intrigue of this paper is really the future directions.  You could take this method of genetically expanding the genome of the virus and making them cell line dependent to maybe create a vaccine for a virus that doesn’t have one yet. You could maybe even apply this to something like HIV, where you introduce the virus to the patient then when they are actually infected the immune response might be so great that it lowers the set point to a point where they don’t develop AIDS. Such generation of PTC virus vaccines can be adapted as long as the virus genome can be manipulated and packed in a cell line. According to a recent Science article the most desirable vaccine is for Ebola. (3) So maybe the next research that should be attempted is use this technique for that purpose. This is a new approach to making vaccines and definitely one you should check out.

Source paper:
1.Si, L. et al. "Generation Of Influenza A Viruses As Live But Replication-Incompetent Virus Vaccines". Science 354.6316 (2016): 1170-1173. Web.

Additional Research:
2. Jang, Yo Han and Baik-Lin Seong. "Principles Underlying Rational Design Of Live Attenuated Influenza Vaccines". Clinical and Experimental Vaccine Research 1.1 (2012): 35. Web.
3. Cohen, J. "Unfilled Vials". Science 351.6268 (2015): 16-19. Web.

All Figures were taken from the paper.

Inactivation of HCV and HIV by microwave: a novel approach for prevention of virus transmission among people who inject drugs

Across the world about 80 million people are estimated to be chronically infected with Hepatitis C virus (HCV). Chronic infection of liver cells, called hepatocytes, can cause serious long-term health risks such as liver degeneration, or cirrhosis, and even cancers, such as hepatocellular carcinoma. HCV death rates have surpassed AIDS related deaths in recent years, motivating researchers to explore novel therapy options, as a vaccine for HCV still does not exist. One population of at risk individuals and infected persons that exists within middle and higher income countries such as the United States, is people who inject drugs. It is estimated that 50-80% of HCV infections in these countries occur in intravenous drug users, which adds up to about 10 million people worldwide. Similarly, because HIV-1 infection is also spread through shared intravenous drug paraphernalia use, it is estimated the 20-30% of HIV-1 infected persons are coinfected with HCV. Because of HIV’s effect on the body’s immune system, coinfected individuals are at a higher risk for the development of the deadly complications of HCV.
In light of recent focus on domestic drug abuse and the heroin epidemic, it is very important that research target new ways to prevent the spread of infection in a growing population of people who inject drugs. While prevention strategies, such as needle or syringe exchange programs and opioid substitution therapy do exist, HCV infection rates have not fully been managed. Previous work by researchers at the Institute of Experimental Virology, in conjunction with many other research institutes, has shown that all genotypes of HCV are temperature sensitive. Using this concept, researchers tested the ability of microwaves, the house hold appliance used to heat foods and liquids, to affect the infectivity of HCV and HIV-1 separately, upon co-infection, and when found on drug paraphernalia.
The main findings of this study show that the infectivity all HCV genotypes is significantly reduced in cell culture, on contaminated syringes, in contaminated syringe filters, and in cultures co-infected with HIV-1. The reduction of infectivity is owed to temperature dependent degradation of the viral envelope and RNA. Researchers exposed the HCV treated solutions to microwave irradiation at intervals of 1, 2, and 3 minutes at various wattages between 90 and 800 W. It was found from analysis of the collected data points that upon reaching a critical temperature of between 56-60 °C, infectivity of the HCV particles was reduced by 90%. This corresponds to 2 minutes of irradiation at a minimum of 360 W. Importantly, it was found that HIV-1 particles also significantly lost infectivity upon being microwaved alone or in the presence of HCV. Syringes contaminated with HIV and HCV were tested with microwave irradiation for 3 minutes at various levels of wattage. The test showed again that at 360 W, both viruses displayed significantly reduced infectivity. Finally, researches tested drug preparation filters, which capable of supporting virus survival for up to two days and are often reused by intravenous drug users. To make the tests more realistic, filters were used to filter actual street heroin before being microwaved. In accordance with the previous findings, irradiation of drug filters for a minimum of 2 minutes at a minimum of 360 W caused significant reduction in viral infectivity.

Stability of HCV and/or HIV-1 after microwave irradiation. Viral titers were significantly decreased in suspensions subject to microwave irradiation above above 360 W for both HCV and HIV-1. 

The purpose of this study was to determine the parameters of a new mode of reducing the spread HCV infection among intravenous drug users, specifically microwave irradiation. This population, which mainly consists of people in middle and upper income countries, is at risk of HCV infection, reinfection after treatment, and coinfection with HIV-1 due to transmission through contaminated syringes and paraphernalia. The research presents a relatively simple solution to prevent infection with a few key advantages, namely ease of procedure, accessibility, and safety both for users and their drugs. Because microwaves are available in most households, and are simple to use they offer an attractive and easy method of ensuring sanitary conditions for intravenous drug use. Because of the high melting temperature of heroin and cocaine, drug users need not fear harming their product while sterilizing their equipment. This method, however, is not without draw back. Because metal cannot be microwaved safely, the needle portion of syringes cannot be decontaminated of HCV or HIV-1. This poses a major drawback for this method as a stand-alone solution for controlling viral spread, however in conjunction with needle exchange programs that are already established, the microwave method could prove very effective as a mode of antiviral therapy. Moving forward, researchers should focus of simple household methods of disinfecting the needles to ensure that people who inject drugs can quickly and safely reduce the infectivity of viral pathogens such as HCV and HIV-1.

Source Paper: Siddharta, A. et al. Inactivation of HCV and HIV by microwave: a novel approach for prevention of virus transmission among people who inject drugs. Sci. Rep. 6, 36619; doi: 10.1038/srep36619 (2016).

Other Resources:
 & Global epidemiology and genotype distribution of the hepatitis C virus infectionJournal of hepatology 61, S45–S57, doi: 10.1016/j.jhep.2014.07.027 (2014).
2.  et al. Recommendations for the management of hepatitis C virus infection among people who inject drugsClinical infectious diseases: an official publication of the Infectious Diseases Society of America 57 Suppl 2, S129–S137 (2013).
3. &  Prevention, treatment and care of hepatitis C virus infection among people who inject drugsThe International journal on drug policy 26 Suppl 1, S22–S26 (2015).
4. et al. Global epidemiology of hepatitis B and hepatitis C in people who inject drugs: results of systematic reviewsLancet 378, 571–583, doi: 10.1016/S0140-6736(11)61097-0 (2011).
5. et al. Sharing of drug preparation equipment as a risk factor for hepatitis CAmerican journal of public health 91, 42–46 (2001).
6. &  Comparison of deaths related to Hepatitis C and AIDS in ScotlandJournal of viral hepatitis 14, 870–874 (2007).

Figures were taken directly from source paper.

A new class of hepatitis B and D virus entry inhibitors, proanthocyanidin and its analogs, that directly act on the viral large surface proteins.

World wide, over 240 million people are infected with Hepatitis B (HBV), a liver cell infecting virus capable of causing degeneration, such as cirrhosis, and cancers such as hepatocellular carcinoma. Unlike HIV or Hepatitis C infections, which carry the genetic code for about 10 viral proteins, HBV only carries four genes, making it very difficult to target with drug treatment. Two broad categories of antiviral treatment do exist for HBV infections, nucleoside analogs, which block the viruses ability to translate its genes in to proteins and replicate, and interferon’s, which amplify the body’s immune response to the virus presence. Unfortunately, these treatment options both have drawbacks like causing the development of drug resistant viruses and intolerable side effects for the infected patient. Because combining antiviral treatments has been shown to be very effective in managing infections such as HIV, it is important today for researchers to identify safe molecules with novel antiviral inhibition modes. Recently, researchers at the National Institute of Infectious disease in Tokyo, Japan have found that a chemical called proanthocyanidin has shown strong potential in inhibiting Hepatitis B and Hepatitis D infection both alone and in conjunction with a commonly used nucleoside analog. 
            Proanthocyanidin, referred to here as PAC, is a chemical found in grape seed extract that is made up of multiple flavonoid units. Through screening many molecules, researchers were able to identify PAC for its ability to inhibit HBV infection. After identification of its inhibitory quality, researchers pressed on to answer some important questions regarding PAC’s mode of inhibition. First, researchers studied what step of the viral replication cycle PAC disturbed, then what host-virus interaction it affected, whether PAC interacted with liver hepatocytes or HBV virions, which specific region of the host or virus receptor PAC interacted with, and finally how PAC performed as antiviral therapy when combined with a nucleoside analog. The results they found offered a very promising outlook for PAC as a non-toxic antiviral treatment option that works through a novel mechanism.
The LHB membrane protein found on the surface of Hepatitis B. This protein is responsible for binding to the NCTP receptor on hepatocytes and facilitating viral entry. 
            Through numerous chemical tests performed in cell cultures that mimicked the human liver, researchers deduced that PAC and related analogs interact with a specific 7 amino acid sections in the preS1 region of a large protein found on the virus envelop surface. This interaction prevented the viral protein LHB from binding to hepatocyte receptors, thus preventing viral entry necessary for productive infection. Researchers first determined that PAC prevented virus entry by treating cell cultures with PAC before infection and after infection. The results showed that only cells treated before introduction of virus avoided infection. By inferring the method of entry inhibition from previously identified, yet toxic antiviral molecules, the researchers were able to design a chemical test to specifically monitor the interaction of the LHB preS1 domain and its cell receptor counterpart, a sodium dependent bile uptake protein (NTCP) found on the liver cell surface. The introduction of PAC to this system prevented the specific interaction between LHB and NTCP. Researchers questioned which side of the LHB-NTCP interaction PAC was inhibiting. They were able to answer this question by infecting culture cells previously treated with PAC and infecting culture cells with viral particles previously treated with PAC. The results showed that viral particles treated with PAC before being introduced to cells showed limited capacity to infect, whereas cells treated with PAC were still susceptible to infection. Finally, by introducing mutations to the preS1 domain of the viral protein LHB responsible for receptor binding and virus particle entry, researchers were able to locate PAC-preS1 interactions to amino acids 9-16. Taken together this study showed that PAC works as a viral-entry-inhibiting-molecule that prevents the preS1 domain, specifically amino acids 9-16, of envelop protein LHB from binding to the NTCP receptor found on hepatocytes, and thus prevents viral particles from entering cells and infecting them.
            As an antiviral treatment, PAC and its analogs have been shown to safely inhibit viral infection in both HBV and HDV, and to work effectively in conjunction with nucleoside analogs to manage infection. In tests with tenofovir, a nucleoside analog used to treat HBV, PAC was shown to inhibit viral production and spread of infection without any toxic effect for cells. This result has important implications for multidrug treatment, as PAC works to affect viral particles through a novel inhibition pathway. PAC is also a naturally occurring molecule that is already found in supplements of grape seed extract at concentrations high enough to exhibit its antiviral properties. This is a very significant finding for the millions of people currently infected with HBV looking to manage infection and people at risk of contracting an HBV infection as it is readily available, safe to ingest daily, and cost effective as it does not require chemical synthesis. While this work is very promising, PAC has not been confirmed to effectively interact with virus cells in live humans. This means that the most important research that needs to be carried out in the future is studies of the effectiveness of PAC and its derivatives at inhibiting viral infection and spread in live organisms rather than cell culture.

The Hidden Threat: HIV Latency and How to Find it

Human Immunodeficiency Virus also known as HIV has been at the center of public view and of virus based research since it was discovered. This focus has most been driven by the fact that as HIV infection continues in a majority of cases it leads to acquired immune deficiency syndrome (AIDS). AIDS is a terrifying occurrence where your immune system is basically gone and you are extremely susceptible to opportunistic infections.  These infections then lead to a drastic reduction of your life span and in many cases an unpleasant death. One of the major issues with HIV is that the virus is able to become latent in cells.  Latency is when a virus inserts its genome into the genome of the host cell in areas that are not being actively expressed. By doing this the virus is effectively able to hide from the hosts immune system and at a later time become active and produce viral particles. A very recent paper in Nature, specifically the structural and molecular biology section, titled Position Effects Influence HIV Latency Reversal by Heng-Chang Chen et al (1), attempts to address the idea of HIV latency through the creation of a new method to study this effect.

HIV in modern day is treated by antiretroviral therapy (ART), which suppresses HIV’s ability to replicated. The issue is, however, that this viruses that have already gain latency in cells are not target by this type of therapy or even the hosts own immune system. Thus, in recent years’ new research has develop around the idea of treating a patient with drugs that reactivate latent viruses to make them susceptible to clearance by the immune system. (2) By combining these two methods researchers believe that the treatment of HIV can become much more effective.  Research in  past has not provided very much information on how latency is establish or how viruses actually come out of latency other than the hand waving idea of stress.  Previous data has had shown that the virus preferentially integrates into active genes and gene rich chromosomes. (3,4) This leads to the idea that something about the insertion site matters and thus the reactivation agent used would also need to be specific and not only based on the idea that a histone deacetylase inhibitor will do the trick.
The new method that the authors create is called Barcoded HIV ensembles (B-HIVE) and allows for the mapping of individual HIV proviruses in the chromosome while also tracking each of the virus’s transcriptional activity. In a very broad in general way (see paper for more information), B-HIVE works by assigning a unique 20 nucleotide barcode to individual HIV genomes, then these barcodes can provide the location in the host genome. These barcodes are also then paired with a green fluorescent protein gene so that cells that have the inserted barcode can be separated through fluorescent sorting. The viral expression levels are determined by looking at the ratio of barcode abundance of the RT-PCR of the RNA pool and the PCR of the DNA pool. All of this can be seen below in figure 1:

From this method, the authors were able to find two really interesting results. First,  they found that the expression of HIV is strongest close to endogenous enhancers. (1) This is a really important finding because this demonstrates a possible mechanism for how the infection works. HIV might have, if it is going to be active and not latent, a signal that helps it search and ingrate near the host’s enhancers. By having this specificity, the virus is able to increase its productivity. This finding leads to an idea of future directs as one could possibly investigate the molecular interaction that HIV is experiencing around the enchanter regions. The second major finding is that the authors found that the latency-reversing agents only effect or reactivate at distinct locations. (1) This means that the one general drug approach that is currently being utilized is not as effective as it could be. Thus, the authors propose that patients should be treated with a drug cocktail to revert as many latent viruses as possible. This also provides an area of future direction for identify what these drugs are that will cause reactivation. One note to make on this when the treat the cell lines with the two reactivating drugs then removed them the expression of the viruses decreased. This hints that there might be a mechanism that is actively silencing the virus to maintain its latency.  If latency was more of a switch, then once reactivated it would continue to have expression even after the drugs had been removed. This is yet another area where research can begin. This effect can be seen below in Figure 5C:


Overall, this method helped immensely to understand HIV latency.

So why do we care about this? Like any good research, you need to start broad then begin to work your way down into the details and that is exactly what the authors did here. Little to nothing was known about the positional effects on HIV latency and really not too much was understood about the latency itself so the authors set out to fix this. They started broad and by doing this they create a lot of areas where new research can be completed. It is also important to note that from these new areas of research new targets for treatment can be found. These will then prove to help the lives of many individuals. Although the authors of this paper might not get the millions of dollars or fame from find a drug that cures HIV, it is important that the scientific community pays due respect to the process and not only the results. They potentially made the process possible and thus deserve to be appreciated for it.

Source Paper:
1.Chen, Heng-Chang et al. "Position Effects Influence HIV Latency Reversal". Nature Structural & Molecular Biology (2016): n. pag. Web.

Additional Sources:
2. Deeks, S.G. HIV: shock and kill. Nature 487, 439–440 (2012).
3. Schröder, A.R.W. et al. HIV-1 integration in the human genome favors active genes and local hotspots. Cell 110, 521–529 (2002).
4.Lewinski, M.K. et al. Genome-wide analysis of chromosomal features repressing human immunodeficiency virus transcription. J. Virol. 79, 6610–6619 (2005).

All images taken from source paper.

It’s Time for Infections: How Viruses and Circadian Rhythms Interact

Have you ever wondered how time works? Now I don’t mean the societal creation of time that lets me know I am writing this post at 1:00 am, I mean biological time.  For example, how does my body know to make certain proteins or how does it know when make me tired for sleep? In other words, have you ever thought about the existence of circadian rhythms in humans. In a recent paper in Proceedings of the National Academy of Sciences titled, Cell Autonomous Regulation of Herpes and Influenza Virus Infection by the Circadian Clock by Rachel Edgar et al., (1) the authors do just this in terms of viruses. They investigate how the natural cycling and timing of biological processes in your body can influence the interaction and effects of virus.

Before we enter into more detail about what the paper demonstrates, I first wanted to briefly talk about what a circadian clock is and why they are so important. A circadian clock in its simplest form is a twenty-four hour cycle during which our genes vary expression level and transcriptional activity to coordinated our complete physiology. In a metaphoric sense, circadian clocks are like a symphony. In a symphony, there are all the different sections playing their own special music. The individual music is fine but when you put them together you can create a beautiful work of art. This is the clocks. They are what brings each individual body process together to make us the humans we are. They are also not to be taken lightly as they have been linked to overall fitness, many aspects of human health and disease such as the sleep/wake cycle, and many immune functions. (2,3) Thus, the disruption of these clocks can be disastrous. 

The authors of this paper realized the essential nature of these processes so they set out to understand how the clock can affect viral replication, viral dissemination and most importantly how the viruses affect the clocks. For all of their research they looked at the clock gene transcription factor Bmal1 and its mRNA levels as the representation of the circadian cycling in mouse and cell culture experiments.  They then complete a myriad of bioluminescent experiments to understand the change in expression level activity (a luciferase assay) and the localization of the infected cells. It is a relatively simple paper in this sense as the authors only use a luciferase assay under different experimental conditions to collect all of their data. 

They first hypothesize that the time of day when the infection occurred would influence the viral replication and found this to be true. Amazingly, mice that were infected with herpesvirus at the onset of resting phase (the beginning of the day) exhibited a 10-fold higher viral replication then mice infected just before their active phase (10 hours later, right before the night). (1)  Bmal1 transcription activity cycles so that during the day it is not active but as night approach transcription and translation begin. This is scene below in Figure 1A from the paper:

They then found that acute infections were enhanced in arrhythmic mice with Bmal1 knocked-down and that the spread of infection was greater spatial. Overall, they conclude that the entire kinetic profile of infection depends on the circadian phase that the virus encounters.

The most influential finding of the paper is that the virus is able to alter the expression levels of the clock genes to benefit its own functioning. In figure 5 D, seen below, the authors demonstrate that the infection of Herpes Simplex Virus 1 cause the up regulation of Bmal1 for a longer period of time. The paper also concludes that all these effects occur with Influenza A to demonstrate that this is not only herpes viruses.

After all of their findings, I just have one major critique that leads to what I believe should be the next step after this experiment. Looking at the Bmal1 expression was a great way to start but circadian rhythms are influenced by a lot more genes than just this one. Thus, I would propose that they do the exact same experiments but look for expression of other clock genes. There are also a lot of downstream interactions that occur with the products of Bmal1 so an investigation into how these might change could also even up providing so key information on how the system works as a whole.

Now these findings are all very interesting but why do we care? Well first off, it is important to understand how viruses function so we can work on ways to treat them. For example, by knowing that the herpesvirus highjacks the circadian clock gene Bmal1 we could work on developing a drug that would inhibit this interaction. This would not be a cure for anything but it would help to minimize the amount of virus so that your immune system could possibly be more effective in clear the virus and inhibiting latency. The other really interesting possibility is creative a drug that keeps our circadian rhythms the exact same.  In the paper they demonstrate that arrhythmic cycling increase infection and it is known that Bmal1 expression undergoes seasonal variation to be lowest in the winter months.(4) This is possibly why Influenza and other virus are known to have more infection during the winter, so by making a daily supplement that regulates your Bmal1 expression levels you could stave off more infections. 
In the end this paper found some novel findings to demonstrate how research that is done in today’s world is amazing at find molecular interactions but it is also very important to look at the whole system. Isolated findings on how viruses function are great but it isn’t until you are able to understand the role that finding plays in the larger picture that we can move forward with our understanding of the natural world.

Source Paper:
1.Edgar, Rachel S. et al. "Cell Autonomous Regulation Of Herpes And Influenza Virus Infection By The Circadian Clock". Proceedings of the National Academy of Sciences 113.36 (2016): 10085-10090. Web.

Additional Research:
2. Scheiermann C, Kunisaki Y, Frenette PS (2013) Circadian control of the immune system. Nat Rev Immunol 13(3):190–198.
3.Curtis AM, Bellet MM, Sassone-Corsi P, O’Neill LA (2014) Circadian clock proteins and immunity. Immunity 40(2):178–186.
4.Dowell SF (2001) Seasonal variation in host susceptibility and cycles of certain infectious diseases. Emerg Infect Dis 7(3):369–374
All Figures are from source paper.

Reovirus: a cure for liver cancer
Hepatocyte carcinoma is one of the most common cancer types on a global scale. It is responsible for approximately one million deaths each year making it a major cause of cancer related deaths. The development of Hepatocyte carcinoma is associated with chronic infections with Hepatitis C virus.  Owing to the development of an inflammatory environment in the liver, hepatitis C virus (HCV)-infected patients are at a high risk of developing Hepatocyte carcinoma (HCC). This connection creates a possibility to prevent Hepatocyte carcinoma through techniques used to neutralize Hepatitis C virus infections.
It has been known that a majority of Hepatocyte carcinoma development is associated with infections with related viruses such as HBV and HCV. Therefore, as mentioned above, techniques to prevent viral infections can be useful by reducing Hepatocyte carcinoma. A primary article, published earlier this year explores the use of mammalian orthoreovirus superinfection as a treatment for liver cancer through host cell immune response. Mammalian orthoreovirus (or reovirus) is a double stranded DNA virus with segmented genome. This virus is known to have very little pathogenic effects on human beings which makes it a good candidate for viral therapy. The authors of this primary article mentioned above take advantage of this property and use it as an agent against HCC development.
Having confirmed the ability of mammalian reovirus to infect HCC cells that metastasized, the authors infected normal liver cells samples containing both enriched hepatocytes and liver mononuclear cells (LMCs). In order to do so, the cells were fractionated into two groups containing each cell type present and reovirus was introduced to the fractionated samples. It was seen that the reovirus was able to infect the non cancerous cells without causing any cytopathic effect. Furthermore, the researchers observed expressions of cellular cytokine responses upon reovirus infection of both cancerous and non cancerous liver cells. This response against reovirus infection is key for the uses of reovirus in the prevention of HCV infection and the associated Hepatocyte carcinoma.
After learning the ability of reovirus to infect hepatocytes and LMCs, the authors went on to examine the anti-HCC functions of reovirus infection. Immunocompromised mice bearing xenografts of HUH7 and HUH7-JFH1 (HCV containing HUH7 cells) cells were used. Injections of reovirus to these tumors were able to significantly slow the growth of the tumor down. Reduced expression of HCV protein 5A was also found in these tumor cells suggesting a connection between the anti-HCC and anti-HCV effects of reovirus. In addition, the authors also found reovirus injection was associated with necrosis of the immunocompromised cells and caused the anti-HCC effects through toxicity of the cells. This caused severe reactions in the mice used and mandated early sacrifice. To avoid this, the researchers infected immunocompetent mice and infected them with UV-inactivated reoviruses and repeated the experiments. Results from these experiments showed reovirus infections still halted tumor growth. The fact that the cells were immunocompetent and the reovirus was inactivated rules out lytic anti-tumor action which, in turn, suggests that there is some sort of cell response to the infections with reovirus that inhibits tumor growth.

Figure 1: Tumor growth is hindered by single injection with reovirus.
The next step in this study was to understand this anti-tumor response of the cells. An increase in natural killer (NK) cells were observed when the tumors were injected with reovirus. This gave a hint on the response mechanism the cells used. Previous studies have shown that type 1 interferon induces these cell types as a response to foreign body infection. To confirm this is the case the authors injected the tumors with reovirus supplemented with type 1 interferon blocking antibody. This resulted in a significant decrease of NK cell count and, subsequently, tumor growth. It was also observed that this reovirus injection technique is ineffective in tumors of SCID cells that had compromised cellular response systems. Thus, reovirus induced anti-HCC immunity is dependent upon type 1 interferon, which in turn activates NK cells, enabling them to recognise and kill tumour targets. These anti-HCC and anti-HCV effects of reovirus infection were observed both in vivo and in vitro experiments.

Figure 2: Increase in NK cells (stained brown) is observed upon reovirus infection.
Then, the authors tried to see if the antiviral and anticancer effects of reovirus extended beyond HCV and associated Hepatocyte carcinoma. PLC/PRF/5 cells (HCC cells containing integrated HBV), and Daudi cells, which are derived from an Epstein–Barr virus (EBV)-positive Burkitt’s lymphoma, were infected with reovirus to see if infection resulted in antiviral responses which prevented the associated cancers. HBV surface antigen (HBsAg) was used to monitor HBV replication in cells. When PLC/PRF/5 cells were infected with reoviruses a significant decrease in HBsAg was observed. Moreover, reovirus infection of Daudi cells led to a significant reduction of EBV early antigen (EA)-positive cells. These results show the ability of reovirus as an antiviral and anticancer agent is not limited to HCV and HCC which was remarkable. This study also revealed that this broad antiviral effect was not seen when other typically oncolytic viruses such as HSV 1, Vaccinia virus and Edmonston strain of Measles virus which sets reoviruses apart and creates a great deal of interest in reovirus studies.

Samson, Adel, Matthew J. Bentham, Karen Scott, Gerard Nuovo, Abigail Bloy, Elizabeth Appleton, Robert A. Adair, Rajiv Dave, Adam Peckham-Cooper, Giles Toogood, Seishi Nagamori, Matthew Coffey, Richard Vile, Kevin Harrington, Peter Selby, Fiona Errington-Mais, Alan Melcher, and Stephen Griffin. "Oncolytic Reovirus as a Combined Antiviral and Anti-tumor Agent for the Treatment of Liver Cancer." Gut Online First(2016). Web.

Grassi et. al.. "Hepatitis C Virus Relies on Lipoproteins for Its Life Cycle." World Journal of Gastroenterology 22.6 (2016). Web.

Wirth et. al. "The Impact of the Revolution in Hepatitis C Treatment on Hepatocellular Carcinoma." Annals of Oncology : Official Journal of the European Society for Medical Oncology. U.S. National Library of Medicine, 18 May 2016. Web. 06 Dec. 2016.