Serological, Genotyping and Sequencing of Viral Hepatitis - Case Study Example

Serological, Genotyping, and Sequencing of Viral Hepatitis Hepatitis B background Hepatitis B is a viral infectious disease that attacks the liver. Hepatitis B Virus (HBV), the virus causing hepatitis B, infects the liver causing either chronic or acute disease. The infections may range from mild illness lasting a few days or weeks to lifelong illness. Researchers have shown that over 350 million people live with chronic hepatitis throughout the world (Sanchez, 2007). Most of these individuals live with asymptomatic hepatitis B. up to 25% of adults chronically infected die of hepatocellular carcinoma or cirrhosis as a consequence of the infection (Hedson, 2009). Being an infection caused by virus, the best approach to minimize the chances of infection is through vaccinations and observation of infection control measures. Once infected, one may go for various treatment options. These include the use of anti-viral drugs, interferon, and where necessary liver transplants (Williams, 2014; Rumenapf and Strauss, 2004). Hepatitis B is sometimes spread via contact with semen, blood, or any other body fluids of an infected person. Therefore, the virus may be spread through unprotected sex, sharing of syringes, needs, piercing tools, and injection equipment. However, Hepatitis B can be passed at birth if the mother is infected with the virus (Mayer, 2011). As hinted above, Hepatitis B may be chronic or acute. The acute condition occurs soon after the infection with the causal virus. Remarkably, acute Hepatitis B does not necessarily lead to chronic Hepatitis B. The chronic condition occurs whenever HBV remains in one’s body over a long period (Mayer, 2011). Chronic Hepatitis B often leads to long life illness and eventual death in some cases. Common conditions resulting from the invasion by HBV include liver cancer, liver cirrhosis, and liver failure (Vargas, 2008). Screening for associated diseases and determining the immune state of people is done by detecting hepatitis B surface antigen-specific antibodies (HBsAb), hepatitis B surface antigen (HBsAg), and hepatitis B core antibodies (HBc, Presence of anti-HBsAg IgG in a person’s body indicates the level of immunity (Sanchez, 2007; Anwar and August, 2006). The same is used to determine whether one needs vaccination or not. A commonly used test is immunoassays done on sophisticated analyzers by highly skillful technologists. Performing these immunoassays is at times impractical depending on the availability of necessary laboratory facilities (Williams, 2014). Such resources may not be available in undeveloped and some developing countries. Technologists required for the analysis are also difficult to find. Signs and symptoms of Hepatitis B often resemble those of flu. They include loss of appetite, fever, joint pain, low energy, cramping, jaundice, nausea, and vomiting. Notably though, in about 30 percent of Hepatitis B infections, no signs are noticed. It is necessary to conduct frequent tests to know ones status. The largest percentage of infected people recovers from the disease without any complications (Hedson, 2009; Rumenapf and Strauss, 2004). About 5 to 10% of infected cases become chronic carriers mainly because they show no symptoms. They, thus, do not seek timely medication. The worst bit of it is that such individuals may transmit the virus to many other people unknowingly (Vargas, 2008; Hedson, 2009). Knowledge of risk factors is necessary for people to avoid falling victims to hepatitis B. Behaviors such as unprotected sex, contact sports, sharing of piercing tools, toothbrushes, and drug injection equipment (Mayer, 2011). Hepatitis C background Hepatitis C virus (HCV), being blood borne may cause multi-system infection is now a major health concern throughout the world is the virus behind hepatitis C. the virus causing Hepatitis C is currently a main public health issue throughout the world. More than 170 million people have the infection. HCV is a single stranded RNA positive virus belonging to the Flaviviridae family (Nwosuh and Nwosuh, 2011). The usual course of the Hepatitis C infection shows variations among individuals and depends on a number of factors. Among the common routes of contracting HCV infection include history of blood transfusion, hemodialysis, tattooing, exposure to non-disposable needle, intravenous drug abuse, and frequent dental operations. About 20-30 percent of people who contract the virus clear it from their body systems in the course of the acute phase (Nwankiti and Ndako, 2010). The rest often develop chronic hepatitis that progresses to hepatocellular carcinoma and cirrhosis within a 20 to 30 years period (Agada, 2009). Researchers have revealed that HCV infection is an independent risk factor often linked with type-2 diabetes mellitus by multivariate psychoanalysis. Further, researchers interested in the field have shown that family history, age limit of over 57 years, and body mass index above 25 kg/m2 as well as previous interferon treatment are independent factors associated with the development of type 2 DM among patients with chronic hepatitis C (Echeonwu, 2012; Guangyu and Wei, 2013). After careful adjustments on DM confounding factors, researchers found that people with HCV infection were 3 times likely to develop type 2 diabetes mellitus compared to those without the infection. These findings were derived from a sample of people aged above 40 years (Nwosuh and Nwosuh, 2011). Hepatitis C is largely a precipitator factor for diabetes though mainly for people risk factors. Patients with infected with hepatitis C virus appear more prone to type two diabetes. In fact, such patients are up to 5 times more likely to develop the type 2 DM compared to those without the infection. Research has shown a 2 to 10 fold increment in diabetes cases worldwide among HCV patients when compared to liver disease control cases (Nwankiti and Ndako, 2010). It is not clear why some patients especially those with HCV infection develop diabetes. The pathogenic mechanism causing diabetes mellitus in people with HCV are not fully explored yet. Impaired insulin secretion or Insulin resistance plays a crucial role towards the development of diabetes mellitus (Agada, 2009). With that, it is tempting to contemplate that HCV infection can easily trigger autoimmune mechanism against pancreatic beta cells responsible for producing insulin. This is specifically in the case of vulnerable persons (Echeonwu, 2012). Approximately 75 percent of people with acute hepatitis C develop chronic infection. Researchers have revealed that about twenty percent of those with chronic hepatitis C ultimately develop cirrhosis. The process may take between 10 and 20 years. Liver failure has been a major reason for liver transplant in the US and the world at large. Notably, DM and the hepatitis C virus are known to trigger each other (Nwosuh and Nwosuh, 2011). Which of the two predisposes the other, however, remains an unexplored aspect. Although explored, genetic predisposition to DM may be activated by HCV. It is solely important to establish the HCV status of people feared to be genetically predisposed in order to suppress their chances of developing diabetes (Echeonwu, 2012). HBV DNA Background Hepatitis B Virus (HBV) DNA (HBV DNA) is a genetic material carrying the blueprint of the hepatitis B virus. The level of HBV DNA in a person’s blood shows how fast the virus is replicating in the patients liver. Laboratories often measure the quantity of HBV DNA in a one milliliter of blood (Dane DS, 2005). The test gives patient’s viral load. High level of HBV DNA called copies or particles, often ranging between 100,000 and one billion per milliliter show that the level of viral replication in the liver is high. Undetected and low levels ranging about 300 particles or copies in a milliliter shows inactive infection (Iannacone, Sitia, and Ruggeri, 2007). Hepatitis B Virus (HBV) DNA quantification is necessary for the confirmation of HBV infection. The quantification in serum of people with HBV infection earlier identified as hepatitis B surface antigen-positive (Tong, Li, and Wands, 1999). It is also important for monitoring the progression of chronic HBV infection or reaction to anti-HBV therapy. Presence of HBV DNA in blood is reliable for indicating active HBV replication. With HBV DNA, the infection is detected within 30 days after invasion by the virus. The levels often reach peak during hepatitis before gradually decreasing and eventually disappearing after the infection resolves spontaneously (Iannacone, Sitia, and Ruggeri, 2007). Testing for HBV DNA in case of acute hepatitis is a useful adjunction in the diagnosis of acute infection. This is often applicable with viral hepatitis with ambivalent HBsAg. HBV DNA is easily detected in 21 days before HBsAg appears in the serum. Remarkably, HBV DNA levels are important for determining whether chronic HBV infection is active or inactive (Dane DS, 2005). Active HBV infections expose patients to greater risk of other liver diseases compared to inactive infections. HBV RNA Background HBV replicates itself in a very curious way. As much as it is a DNA virus, HBV uses RNA pro-viral intermediary that should be copied to DNA. The copying process is not a usual function of uninfected cell. It is found in retroviruses that have RNA genome and a DNA intermediary often incorporated in chromosomes of the host cells. For the sake of copying RNA to DNA, HBV and retroviruses have varying encoded DNA polymerase also called transcriptase (Iannacone, Sitia, and Ruggeri, 2007). Once the HBV attaches to a cell surface receptor, the viral membrane combines with the cell membrane then releases the core to the cytoplasm. Core proteins then dissociate from the partly double stranded DNA. Polymerase completes the DNA making it completely double-stranded. The double stranded DNA now enters the cell nucleus with ends litigated to a virus of the circular episome form (Dane DS, 2005). The viral DNA links with host cell nucleus and is then transcribed by the cellular RNA polymerase II to mRNAs. Contrary to the case of retroviruses, the DNA type of HBV is not blended into cellular DNA. It instead exists as an autonomous episome. The HBV buds via the endoplasmic reticulum or Golgi body membranes of host cells where it acquires HBsAg (Alter and MJ, 2003). When utilized to transcribe RNA to DNA, viral DNA polymerase acts as a reverse transcriptase similar to that of retroviruses. Studies have shown that retroviral reverse and HBV DNA polymerase are very similar that they may have common ancestors (Lurman, 2005; Wittwer, 2007). Virus particles containing DNA or RNA at various replication stages are found in bloodstreams at times. The impression is that the replication of nucleic acid is not strictly controlled with death of cells (Tong, Li, and Wands, 1999). Hepatitis C virus (HCV) DNA Background Approximately 80% of hepatitis C virus (HCV) infections eventually become chronic infections with lifelong viremia. There are about 200 million chronic HCV infections in the world with about 4 million of these in United States (Williams, 2014). Although HCV is not resistive to immunization, treatment of the state is not yet explored satisfactorily. The virus exists in various genotypes in various quasispecies. Therefore, it is advisable to formulate a strategy to induce a strong immune reaction to various variable and conserved regions of HCV. Immunization against the virus involves generating cell-mediated immunity for purposes of mediating the killing and downgrading of infected cells (Lurman, 2005). DNA based immunization is highly recommended and preferred way of generating cell-mediated immunity. Studies have revealed numerous methods for enhancing DNA based immune response. Available approaches used today include co-admission of cytokine to express plasmids, administration of immune-stimulatory DNA sequence, salmonella vectors, and targeting DNA to dendritic cells (Hunt, 2013). Studies on DNA based immunization against HCV have revealed that using recombinant poxvirus months after immunization with DNA produced strong booster effect (Alter and MJ, 2003). This is in comparison to when DNA is used as a booster. Immune responses triggered by HCV DNA canary pox virus boost are robust and appear early once immunization is done. It is possible to detect HCV antigens in the cytoplasm of infected hepatocytes that have antibodies. This is particularly in the case of frozen tissues that have immunohistochemistry. It is then possible to detect HCV RNA in fixed liver tissues through situ-hybridization. The test is considerably more sensitive compared to immunohistochemistry (Iannacone, Sitia, and Ruggeri, 2007). HCV RNA Background HCV RNA tests are crucial tools that help to confirm the diagnosis and treatment of HCV infections. Hepatitis C RNA test may be qualitative or quantitative. Qualitative tests reveal the absence or presence of HCV RNA. Results are reported as positive or negative implying detected or not detected respectively. This information and hence the test is useful for patients with positive RIBA test to verify active infection. Quantitative test uses PCR to measure HCV RNA copies also called viral load in patient’s blood (Lurman, 2005). HCV RNA is the genetic content or material for HCV. Doctors measure the viral load before, during and after treatment for progress mastery. A decrease less than “2 log” may imply that one is not responding appropriately to the therapy (Campbell, Keene, and Brackney, 2008). Qualitative HCV RNA tests help to detect hepatitis C virus in patient’s blood with high precision. It is among the sensitive tests used today. Qualitative tests, further, are used to evaluation the effects antiviral therapy. Long-term treatment is preferred where HCV RNA is undetected after completing the course of treatment and after testing the same six months later (Williams, 2014). This test is also commendable for confirming HCV diagnosis when patients have very low viral loads. Examples of low viral loads include cases of immunocompetent/immunosuppressed persons and patients with high aminotransferases (Afonina and Ankoudinova, 2008). Indeterminate Hepatitis C viral antibody tests help to differentiate between current and past infections. The tests, therefore, help to detect acute infections before sero conversions as well as when aminotransferase levels are slightly elevated or even normal (Hunt, 2013; Timofeeva and Skrypina, 2007). Measuring DNA DNA is measured and expressed in internationally agreed upon units “copies per milliliter abbreviated as IU/mL. Since there are very many copies of DNA in a drop of blood, labs use mathematical expressions to illustrate viral load. Expressions such as 5 log or 105 stand for 100,000 copies/ml. Every log fall or rise is equal to ten-fold decrease or increase respectively. The change from 100 to 1000 is a 1-log increment while a change from 100 to 10000 is expressed as a 2-log increase. Doctors monitor DNA levels keenly whenever treating patients. A 1 or 2-log decrease implies that an antiviral is effectively working (Guangyu and Wei, 2013). The reverse implies that the administered antiviral has ceased working and viral resistance developed. Undetectable viral load implies that there is less than 300 copies of DNA in a milliliter hence laboratory equipment cannot identify them. Moderate levels of DNA start at 10,000 copies while high levels may exceed 100,000 copies in a milliliter (Sallie, 2012). Experimental Virus Model: HCV Permissive Model In the pursuit to develop hepatitis C virus (HCV) permissive model, I shall show that HCV JFH-1 sub-genomic replicas and full-length genomes can replicate in cells of mice. The model will show that species block at HCV RNA replication level can possibly be overcome although additional blocks in post-replication stages of progeny virus production process may emerge (Catherine, 2010). In terms of virus entry, the model will determine species-specific determinants for the virus entry into mice cells to pose as human occluding and human CD81 (Williams, 2014; Campbell, Keene, and Brackney, 2008). Expression of these human receptors in fibroblast cell lines often result GCVpp passiveness. Remarkably, though, infectious HCVcc entry may not be easily demonstrated implying that more entry factors are necessary for the entry of HCVcc entry into cells of mice. This is from the view that earlier attempts and advances to develop an HVC permissive mouse model have not satisfactorily achieved this goal (Catherine, 2010). Measurement of RNA in Virus The existence of full-length accompaniments of viral genomic RNA is a characteristic of RNA virus replication in infected cells. Mechanisms of determining and measuring particular strands of viral RNA in not only infected cells but also in infected tissues are crucial in studying RNA viruses. Strand-specific quantitative PCR (ssqPCR) essays are now highly depended upon for this purpose (Vargas, 2008). The precision of these essays is dependent on an assumption that the measured cDNA in the quantitative PCR (qPCR) stage accurately shows the quantity of the specific viral RNA strand in the RT reaction. This assumption is tested by developing multiple ssqPCR assays for positive stranded RNA virus o’nyong-nyong abbreviated (ONNV) (Alter and MJ, 2003; Wang and Sung, 2002). The various parameters of ONNV-specific assays are then compared based on prevalent ssqPCR assay designs in literature. References Afonina, I, and Ankoudinova M 2008, Primers with 5′ flaps improve real-time PCR, Biotechniques , 772-774. Agada, G O 2009, Hepatitis C Prevention, Treatment and Care, Guidelines for Australian Custodial Settings, Evidence base for the guidelines, viewed from Australian Government, DEpartment of Health, March 1, 2014, Alter, and MJ 2003, Epidemiology and prevention of hepatitis B, Seminars in liver disease , 39-46. Anwar, A and August, J. 2006, A stem-loop-mediated reverse transcription real-time PCR for the selective detection and quantification of the replicative strand of an RNA virus, Anal Biochem , 120–128. Campbell, C, Keene, K, and Brackney, D 2008, Aedes aegypti uses RNA interference in defense against Sindbis virus infection, BMC Microbiol , 47. Catherine, K 2010, June 21, Hepatitis B Virus HBV) DNA Detection and Quantification by Real-Time PCR, Serum: Mayo Clinic and Medical Laboratorie, viewed March 1, 2014, Dane DS 2005, Virus-like particles in serum of patients with Australia-antigen-associated hepatitis, Journal of Hepatology , 610-114. Echeonwu, G 2012, November 14, HEPATITIS/HIV PROJECT BACKGROUND: Treatment Action Group, viewed March 1, 2014, Guangyu, X, and Wei C 2013, An Analysis of the Molecular Evolution of Hepatitis B Viral Genotypes A/B/D Using a Bayesian Evolutionary Method, Virology Journal , 256. Gunji, T, Kato, N, and Hijikata, M 2004, Specific detection of positive and negative stranded hepatitis C viral RNA using chemical RNA modification Arch Virol 134 , 293–302. Hedson, C 2009, April 3, Hepatitis B Information for the Public: Center for Disease Control and Prevention, viewed March 1, 2014, Hunt, R 2013, January 19, Virology, Hepatitis Viruses: Microbiology and Immunology, viewed March 1, 2014, Iannacone, M, Sitia, G, and Ruggeri, Z M 2007, HBV pathogenesis in animal models, Recent advances on the role of platelets, Journal of Hepatology , 719–726. Lurman, A 2005, Eine icterus epidemic, Berl Klin Woschenschr , 22-23. Mayer, T K 2011, August 23, Hepatitis B Assays in Serum, Plasma and Whole Blood on Filter Paper, Medscape Multispeciality, viewed March 1, 2014, Nwankiti, O. O, and Ndako, J. A. 2010, March 17, Hepatitis C Virus infection in apparentenly healthy individuals with family history of diabetes in Vom, Plateau State Nigeria. viewed February 1, 2014, < http,//www.virologyj.com/content/6/1/110> Nwosuh, CI, and Nwosuh, CI 2011, February 23, Background and Rationale for Hepatitis C. Department of Health, New York State, Viewed March 1, 2014, Rumenapf, T, and Strauss, E 2004, Regulation of Semliki Forest virus RNA replication, a model for the control of alphavirus pathogenesis in invertebrate hosts, Virology 323 , 153–163. Sallie, R 2012, February 13, Selective detection of hepatitis B virus RNA by PCR. Retrieved March 1, 2014, from Genome Research, genome.cshlp.org Sanchez, S 2007, Hepatitis B virus taxonomy and hepatitis B virus genotypes, World Journal of Gastroenterology , 1421. Timofeeva, A, and Skrypina, N 2007, Background activity of reverse transcriptases, Biotechniques , 22–24. Tong, S L, and Wands, JR 1999, Carboxypeptidase D is an avian hepatitis B virus receptor, Journal of Virology , 96-102. Vargas, R L 2008, November 12, Background and Rationale for Hepatitis B. Retrieved March 1, 2014, from New York State Department of Health, http,//www.health.ny.gov/diseases/communicable/hepatitis/strategic/background_rationale_hepatitis_b.htm Wang, W, and Sung, T 2002, Detection of dengue virus replication in peripheral blood mononuclear cells from dengue virus type 2-infected patients by a reverse transcription-real-time PCR assay, J Clin Microbiol 40 , 243-245. Williams, SA 2014, February 28, Hepatitis B, Health Guide. Retrieved March 1, 2014, from The New York Times, http,//www.nytimes.com/health/guides/disease/hepatitis-b/background.html Wittwer, C 2007, Continuous fluorescence monitoring of rapid cycle DNA amplification, Biotechniques , 134–138. Read More