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====== Hepatitis C ======
 

==== Introduction ====


Hepatitis C is a contagious disease caused by the infection of Hepatitis C virus which predominantly attacks the liver cells (hepatocytes). Hepatitis C virus (HCV) is an RNA virus that replicates in the liver, and infects 200 million people worldwide.Infections can only occur in cases where the virus enters the bloodstream and attacks the liver (Canadian Center for Occupational Health and Safety,2013). Infections are caused by the infection due to the Hepatitis C virus (HCV) which is an RNA virus which consists of a single stranded positive sense RNA that is able to mutate efficiently thus avoiding host’s recognition and immune defense. The disease is spread mainly through the exposure to blood containing the virus – sharing needles, unsafe tattooing, piercing and any other activities that include sharing unsterile equipment are common routes of infection. It’s important to note that the risk increases in situations where poor infection control is practiced (Canadian Center for Occupational Health and Safety, 2013). 

<box width classes red| **General Hepatitis C Labelled Virus**>{{ http://liverdoctor.healthdirection.netdna-cdn.com/wp-content/uploads/2012/10/hep-c-virus-labelled1.jpg }}</box| Sourced from LiveDoctor.com>

====Symptoms====


Symptoms can vary greatly as to when they are manifested as it can take up to 10 years for the disease to become symptomatic after the virus enters the bloodstream. Common symptoms of the disease are fever, fatigue, stomach pain, liver pain, nausea, vomiting, discoloration in urine and stool, and jaundice. When the disease progresses to result in advanced liver failure , symptoms such as ascites, edema, esophageal varices leading to fatal internal bleeding can result. Symptoms are particularly manifested in the cases of chronic infection (Centers for Disease Control and Prevention, 2014). Even though, in a small share of people, acute infections due to HCV can be cleared by the body’s immunity, a majority of the population develops chronic Hepatitis C. Even though acute version can become symptomatic as early as within 6 months after exposure to the virus, chronic hepatitis C, which occurs when the virus stays within the body for longer periods of time, most often  causes  lifetime illnesses causing fatal liver complications such as cirrhosis, liver cancers such as hepatocellular carcinoma (Canadian Center for Occupational Health and Safety, 2013).
<box width classes red centre| **Healthy Liver Versus Cirrhotic Liver**>{{ http://giant.gfycat.com/AlarmedOrderlyHamster.gif }}</box| Animated by Brandon Sarjoo (Shepard et al., 2005).>


==== Epidemiology ====

=== Prevalence ===
The WHO has stated that there is a worldwide prevalence of HCV infection. Although HCV infection is prevalent across the globe, there is great geographic variability in its distribution in which some countries are more affected. Africa and Asia are the two continents with the highest prevalence rates (Shepard, Finelli & Alter, 2005). Lower prevalence of HCV infection is found in industrialized areas such as North America, parts of Europe, and Australia. Canada is a populous nation with a relative low rate of 0.8% HCV infection (Shepard, Finelli & Alter, 2005).

=== Trends in Incidence of HCV Infection ===
As mentioned, there are both acute and chronic forms of HCV infection. High percentage of the morbidities due to infection is caused by chronic liver disease in the affected patients. Several models conducted revealed that there was a period of low incidence in the years before 1965, in which only 0-44 people were affected for every 100 000 people tested (Shepard, Finelli & Alter, 2005). However, there was an increasing incidence of HCV infection between the years 1965 and 1980, rendering this time to be considered a transitional period (Shepard, Finelli & Alter, 2005). The 1980s was considered to be a period of high incidence, in which 100-200 people are affected for every 100 000 people tested (Shepard, Finelli & Alter, 2005). A similar trend of increasing incidence in the 1980s was found in countries such as France and Australia.

==== Modes of Transmission ====
  * 1) Blood transfusions
  * 2) Use of injection drugs
  * 3) Unsafe therapeutic injections


=== 1) Blood Transfusions ===
Transfusion of blood remains as one of the most effective methods of transmitting HCV infection.  However, within the past 4 decades, several actions have been undertaken to reduce the progression of transfusion-related HCV infection. For example, most developed countries have adopted a donor system of volunteer-basis. A study conducted in the US revealed a 3-fold decrease in the incidence of post-transfusion HCV infection as soon as the proportion of paid blood-donors decreased from 91% to a staggering 4% (Shepard, Finelli & Alter, 2005). Contrarily, in developing countries, because most blood donations are non-voluntary and often compensated donors, transfusion is a major source of HCV transmission (Shepard, Finelli & Alter, 2005). Many facilities also conduct screening of the blood donations, actively testing for signs of liver disease and HIV (Shepard, Finelli & Alter, 2005).

=== 2) Use of Injection Drugs ===
In the current developed world, the use of injection drugs is a primary transmission mode of HCV infection. Primarily among middle-aged individuals, injection drugs accounts for 68-80% of existing infections (Shepard, Finelli & Alter, 2005). The notion that HCV infections occur quickly after the initiation of injecting behavior further exacerbates the development of HCV infections. Having a fewer number of sharing partners is extremely important to limit HCV transmission. It is also important to reduce drug sharing and control preparation practices, such as injecting drugs using a syringe filled with drugs, which were initially mixed in another person’s syringe (Shepard, Finelli & Alter, 2005).


=== 3) Unsafe Therapeutic Injections ===
In many countries, which are not yet developed, there may be an inadequate source of sterile equipment, such as sterile syringes. Non-professionals may also administer injections in a non-medical setting, which increases the risk of HCV infection (Shepard, Finelli & Alter, 2005). In addition, although many medications can be orally ingested, a greater number of individuals in developing countries are instead delivering medication through injection, increasing the likelihood of HCV infection (Shepard, Finelli & Alter, 2005). Therefore, several contaminated injections over a lifetime can trigger a collective risk of developing HCV infection.


==== Diagnosis ====
 
Due to largely asymptomatic nature in early period of infection, early diagnosis is difficult. Commonly, anti- hepatitis C virus antibodies in serological test can be indicative of exposure to the virus. In order to confirm diagnosis, those tested positive for the anti-hep C antibodies will need to undergo a test for HCV RNA, as a minority will usually spontaneously clear the infection from their systems while retaining the antibodies. In addition, it’s important to identify the genotype of the virus that caused the Hep C in each given patient in order to tailor the treatments accordingly (World Health Organization, 2014). 

=== Mode of Infection ===

The hepatitis C virus particles called virions bind to the cell surface receptors on the surface of hepatocytes which are then pinched into the cytoplasm as vesicles. These vesicles release a complex of viral RNA and proteins into the cytoplasm of the cell. These RNAs are translated into proteins by the ribosomes of the cell while a copy of the RNA serves as a template for new RNA production. The copies of RNA thus made combines with the protein components to form vesicles of complete virions that are then released from the cell via its secretory pathway to infect other hepatocytes (The Rockefeller University, 2014).

<box width classes red centre| **Video of HCV trasnmission**><html>
<object width="560" height="315"><param name="movie" value="//www.youtube.com/v/tqVmXtOgZ58?version=3&amp;hl=en_US"></param><param name="allowFullScreen" value="true"></param><param name="allowscriptaccess" value="always"></param><embed src="//www.youtube.com/v/tqVmXtOgZ58?version=3&amp;hl=en_US" type="application/x-shockwave-flash" width="560" height="315" allowscriptaccess="always" allowfullscreen="true"></embed></object>
</html></box| Sourced from: https://www.youtube.com/watch?v=ZJ0wQ4sTAwQ>

===== Immunology of HCV =====

==== Background Information ====

The biological defense mechanism of higher organisms, including humans, is generally divided into innate immunity and adaptive immunity (Dranoff, 2004). In the adaptive immune response, gene rearrangement by T cells and B cells enables the establishment of a defense mechanism of high specification against “the molecular microstructure of a foreign substance,” and this mechanism is immunologically memorized. Adaptive immunity takes a few days to activate and thus the innate immunity is employed first. Innate immunity has existed as a biological defense mechanism from the earliest stages of evolution: for example, insects have only innate immunity as a defense mechanism. Cells involved in innate immunity include macrophages, neutrophils, NK cells, NKT cells, and γδT cells. Important humoral factors include complements, lectins, and interferons (IFNs) (Biron, 2001). However primitive innate immunity may seem, it may be the key to preventing HCV. 

==== Innate Immunity of HCV ====
 

The discovery of Toll-Like Receptors (TLRs) in 1996 has led the shift in immunology research back to focusing on the innate ascpects of immunity. TLRs are specific to structures regarding microbes like LPS, flagellin, double stranded RNA and CpG DNA (Akira et al., 2006). The discovery of TLRs was important because it showed that, at the molecular level, a living body recognizes the entrance of pathogens as “pathogen-associated molecular patterns” (PAMPs). This has shown that the innate immune system discriminates between self and not- self and recognizes abnormality via a mechanism that is different from the gene rearrangement of the adaptive immune system. TLRs are expressed by immune cells, which include macrophages, monocytes, dendritic cells and B cells. Recognition of ligands by cognate TLRs expressed on cells that are effectors of the innate immune response leads to the rapid activation of inflammation and microbicidal pathways. Thus stimulation of correct TLR regulated pathways can activate innate immune responses to target HCV. One recognized pathway for HCV for innate immunity is that hepatocytes sense HCV dsRNA structures via pattern recognition receptors RIG-I and TLR3 and will bind PAMP RNA and signals antiviral interferon-stimulated genes that limit infection (Saito et al, 2008). Binding of interferons or IFNs to the receptor ectodomain results in recruitment of JAK1 and TYK2 kinases to the cytoplasmic tails (Bartenschlager 2013). This kinase actiation leads to phosphorylation of STAT1 and STAT2 to ultimately lead to stimulation of multiple interferon-stimulated genes or ISGs (Saito et al, 2008). 

==== Natural Killer Cells and Their Impact on HCV ====


NK cells are inhibited in normal “self cells” because of their naturally occurring MHC Class I expression. But in the case of non-self these MHC class I are not expressed. The expression of NK-cell receptors and their ligands changes dynamically during the infection of HCV. The expression kinetics of these molecules might have a close connection with the process. The modification of the expression of these molecules by drugs or cytokines might lead to the development of cancer immunotherapy based on a new perspective. There are great expectations for further progress of research in this field. One thing we do know is that NK cells and their activation lead to dendritic cell (DC)activation which will spur the adaptive immune response. 			

==== Adaptive Immune Response to HCV ====
 
			
While the innate immune response attempts to block the HCV virus via a cellular means dominated by the induction of viral IFNs, ISGs, and proinflammatory cytokines and chemokines, we also know that it begins the activation of the adaptive immune response.The adaptive immune response against HCV includes interaction of antibodies, CD4+ T Cells, and CD8+ T cells. Sometimes there can be a successful adaptive immune response and clear the HCV entirely. This is done by neutralizing antibodies that block the transmission by blocking viral binding, entry or post-entry steps (Bowen et al., 2005). CD4+ T cells recognize HCV antigens presented by HLA class II alleles via their T cell receptor (TCR). This recognition step is crucial in a successful adaptive immune response as they provide help to CD8+ T cells. CD4+ T cells are therefore known as “T-helper cells”. These developed HCV-specific CD8+ T cells recognize the viral antigens presented on the HLA class I molecules and act non-cytolytically by secreting antiviral cytokines such as IFN-γ as well as perforin secretion. The CD8+ T cell response is shown to occur 6-8 weeks after infection (Bowden et al., 2005). In an unsuccessful immune response usually occurs in patients with acute persistent infection. In which case, the CD8+ T cell responses are weak and target only a small number of viral epitopes. HCV is an RNA virus as discussed with a high replication rate and it’s RNA polymerase NS5B does not proofread it’s transcription. Consequently, multiple viral variants are created and can circulate through a single patient and can completely evade the immune system responses (Bartenschlager 2013). Another way HCV evades the immune system is through lipoproteins (mainly HDL) transport which prevents the antibodies and immune system from getting access to it (Bowden et al., 2005). 
							
==== HCV Immunopathogenesis ====


The immune system of the infected individual is quick to recognize HCV and attempt to eliminate it. The rate rate of mutation as the virus replicates, however, ensure that an “evolved” variation of the HCV will always be able to elude the immune response. As a result, this hide and seek as the HCV mutates makes it hard to develop a vaccine.The adaptive immune response plays a central role in the outcome of hepatitis C virus (HCV) infection. Indeed, spontaneous viral clearance is associated with an early neutralizing antibody response as well as vigorous and sustained HCV-specific CD4+ and CD8+ T cell responses. In persistent HCV infection, however, all three components of the antiviral adaptive immune response fail due to different viral evasion strategies.

<box width classes red centre| **Overview of HCV Immune Response**> {{http://i.cubeupload.com/Hwr2GU.jpg?610}}</box| Image referenced from Shepard et al., 2005>

 ===== Past Treatments: Interferon and Ribavirin ===== 
Hepatitis C viral infection primarily targets humans and our primate, the chimpanzee. Specifically, HCV mainly attacks the hepatocytes – liver cells—of the body. Because there is no effective vaccine for HVC or an effective antiviral strategy, drugs are used to treat HVC infection. One such drug that is used to treat HCV infection is the interferon, IFN-α (Tan, Pause, Shi & Sonenberg, 2002). IFN-α is a glycoprotein, which occurs naturally in the body, and possesses antiviral and immunomodulatory properties (Tan, Pause, Shi & Sonenberg, 2002). As of now, it remains as one of the only drug that is capable of sustaining the clearance of HCV infection, while improving the structure of liver tissues. However, this treatment method only achieves a sustained virological response (SVR) in 15% of the patients treated (Tan, Pause, Shi & Sonenberg, 2002). In addition, the therapy of IFN-α alone causes unfavorable side effects such as flu-like symptoms. It is also considered to cause leukopaenia, a disorder involving the decrease in leukocytes, as well as thrombocytopaenia, a condition that involves a decreased number of thrombocytes circulating in the blood (Tan, Pause, Shi & Sonenberg, 2002).
 
A more effective therapy to treat HCV infection is to administer a combination of drugs. Combining ribavirin, an active synthetic guanosine analogue, with IFN- α has been found to be much more successful than treatment with IFN-α alone (Tan, Pause, Shi & Sonenberg, 2002). This dual therapy yields a SVR in approximately 35-40% of the patients treated (Tan, Pause, Shi & Sonenberg, 2002). Ribavirin is used because of its ability to prevent the activity of inosine monophosphate dehydrogenase (IMPDH), an enzyme that normally works to catalyze a rate limiting step in the biosynthesis of GTP. This decreases the available pool of GTP levels, and thus implicitly preventing viral RNA synthesis. Although this combination therapy, consisting of IFN-α and ribavirin, has been proven to be more effective, there are still many patients who do not reach a SVR. This therapy also correlates with higher risk of haemolytic anaemia, caused by the build-up of ribavirin triphosphates in erythrocytes (red blood cells) (Tan, Pause, Shi & Sonenberg, 2002).

 ===== Potential New Therapy: Direct Antiviral Agents (DAA) ===== 
It has been proven that inhibiting the activity of inosine monophosphate dehydrogenase (IMPDH) is a major necessity in the treatment of HCV infection (Tan, Pause, Shi & Sonenberg, 2002). Therefore, several new inhibitors are in the process of being developed to potentially treat HCV infection, and are presently in clinical trials. One such compound is VX-497, an oral inhibitor of IMPDH, and is currently in phase II of clinical trials. VX-497 is a direct antiviral agent and plays a vital role in lymphocyte migration and proliferation during an immune system response (Tan, Pause, Shi & Sonenberg, 2002). This displays its potential to cease virus-like proliferation as well as liver inflammation.
 
Because HCV infection particularly attacks the liver cells of the body, it is also important in finding potential ways to reverse the effects of liver damage. A pilot study conducted revealed that interleukin-10 (IL-10), which is a cytokine involved in the down-regulation of pro-inflammatory responses and modulating fibrogenesis of hepatocytes, gradually reduced the extent of liver fibrosis (Nelson, Lauwers, Lau & Davis, 2000). IL-10 also worked to dramatically improve the tissue structure of hepatocytes in patients suffering from chronic HepC, in which the mono therapy of IFN-α or the combination therapy of ribavirin and IFN-α were ineffective (Nelson, Lauwers, Lau & Davis, 2000).



====== USP18 ======
When HCV infection occurs, the host innate immune response detects infected cells by cell surface receptors located on the infected cell. Infected cells will release cytokines including interferon alpha and beta. Interferon alpha/beta stimulate receptors of nearby cells inducing the JAK/STAT pathway which further induce transcription factors that travel to the nucleus of the cell. These elements will promote the transcription of ISGs(Interferon stimulated genes). Upregulation of ISGs promote antiviral activity to battle HCV and other types of viruses. ISG15 is one of the most abundant types of ISGs and is unregulated in presence of exogenous/endogenous Interferon, as well as in the presence of bacterial/viral infections. The ISG 15 exists either in free form extra-cellularly, or within the cell. In free-form, ISG 15 protein can act as a cytokine and activate certain types of immune response cells to induce other types of antiviral interferons. However intracellular ISG 15 protein conjugates itself to many proteins which regulate a wide variety cellular functions including cell cycle regulation, immune response regulation, and protein translation. Another key ISG is the Ubiquitin-like Specific Protease 18(USP18) which specifically targets ISG15-conjugated proteins. USP18 cleaves ISG15 from its protein. Both USP18 and ISG15 are complementary, and regulate each other. However, the mechanism by which they regulate each other must be further elucidated. USP18 just like ISG 15 can also modulate interferon response (McGilvary et al., 2001).

====Significance====
The USP18/ISG15 activity have an important role in HCV infection. Patients with HCV are generally prescribed exogenous IFN alpha to promote the stimulation of the JAK/STAT pathway to ultimately upregulate more ISGs for antiviral activity. Patients with already highly expressed levels of USP18 and ISG15 did not treat to subsequent IFNa-based therapeutics. It is believed that pre-activation of USP18/ISG15 pathway in hepatocytes led to high ISG15 and USP18 expression which not only promotes HCV production, but also inhibits IFN-alpha treatment. This is possibly due to the patient already sensitized to IFN alpha.  The mechanism of pre-activation of the USP18/ISG15 pathway is not fully understood. However, in some mice studies (McGilvary et al., 2001), knockout of the USP18 gene prior to IFN treatment results in the up regulation of ISG 15 protein and increased sensitivity to IFN alpha treatment resulting in antiviral activity and prolonged survival of the mice. Researchers believe the antiviral activity comes from the conjugation activity of ISG15. It is possible ISG15 conjugates itself to viral proteins which disrupt the life cycle of the virus, while other researchers believe it has no direct effect on the virus.Treatments could be designed for humans which block USP18 and allow for ISGylation to occur to fight off the virus. Modulating host immune function through the ISG15/USP18 pathway may serve as a better tool to fight HCV rather than the actual virus itself as the HCV is well known to mutate rapidly in the face of antiviral agents. This would provide a more definitive way to fight the HCV (McGilvary et al., 2001).

It is important to note that ISG15/USP18 pathway is multi-faceted. In the presence of certain viruses, the ISG15/USP18 pathway serves to create an antiviral environment. However, in some cases, it is possible that the ISG15/USP18 pathway is in fact, vital for the replication of other types of viruses. This makes the ISG15/USP18 pathway very hard for researchers to understand the role of both of these ISGs (McGilvary et al., 2001).

  
<box width classes red centre| **USP18 KO Mice**> {{http://i.cubeupload.com/BgXSH3.jpg?610}}</box| Xue-Zhong et al., 2001>

====== Presentation Slides ======
<html><iframe src="https://docs.google.com/file/d/0B6CW8D38xrsPR3hCMGhLUzc2QW8/preview" width="1000" height="600"></iframe></html>

 ====== References ====== 

Ma, X. Z., Bartczak, A., Zhang, J., He, W., Shalev, I., Smil, D., ... & McGilvray, I. (2014). Protein ISGylation delays but does not overcome coronavirus proliferation in a model of fulminant hepatitis. Journal of virology, JVI-03801.

Canadian Center for Occupational Health and Safety. (2013). Hepatitis C. Retrieved 10/21, 2014, from http://www.ccohs.ca.libaccess.lib.mcmaster.ca/oshanswers/diseases/hepatitis_c.html

Centers for Disease Control and Prevention. (2014). Hepatitis C information for the public. Retrieved 10/20, 2014, from http://www.cdc.gov.libaccess.lib.mcmaster.ca/hepatitis/c/cfaq.htm

The Rockefeller University.Hepatitis C life-cycle. Retrieved 10/20, 2014, from http://www.rockefeller.edu/pubinfo/hepc.html

World Health Organization. (2014). Hepatitis C. Retrieved 10/20, 2014, from http://www.who.int.libaccess.lib.mcmaster.ca/mediacentre/factsheets/fs164/en/

Dranoff, G. (2004). Cytokines in cancer pathogenesis and cancer therapy. Nature Reviews Cancer, 4(1), 11-22.

Biron, C.A. (2001). Interferons alpha and beta as immune regulators—a new look. Immunity, 6(1), 661–664.

Akira, S., Uematsu, S., & Takeuchi, O. (2006). Pathogen recognition and innate immunity. Cell, 124(4), 783-801.

Saito, T., Owen, D. M., Jiang, F., Marcotrigiano, J., & Gale Jr, M. (2008). Innate immunity induced by composition-dependent RIG-I recognition of hepatitis C virus RNA. Nature, 454(7203), 523-527.


				

Bauer, S., Groh, V., Wu, J., Steinle, A., Phillips, J.H., Lanier, L.L., Spies, T. (1999). Activation of NK cells and T cells by NKG2D, a receptor for stress-inducible MICA. Science, 285: 727–729. 

Szabo, G., & Dolganiuc, A. (2006). HCV immunopathogenesis: virus-induced strategies against host immunity. Clinics in liver disease, 10(4), 753-771.				
			
Bartenschlager, R. (2013). Hepatitis C virus from molecular virology to antiviral therapy. Berlin: Springer.		

Bowen, D. G., & Walker, C. M. (2005). Adaptive immune responses in acute and chronic hepatitis C virus infection. Nature, 436(7053), 946-952.

Nelson, D. R., Lauwers, G. Y., Lau, J. Y. N. & Davis, G. (2000). L.Interleukin 10 treatment reduces fibrosis in patients with chronic hepatitis C: a pilot trial of interferon nonresponders. Gastroenterology, 118(4), 655–660.

Shepard, C.W., Finelli, L., & Alter, M.J. (2005). Global epidemiology of hepatitis C virus infection. Lancet Infect Dis, 5, 558-67.

Tan, S.L., Pause, A., Shi, Y. & Sonenberg, N. (2002). Hepatitis C therapeutics: current status and emerging strategies. Nature Reviews Drug Discovery, 1, 867-881. doi:10.1038/nrd937
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