Lamivudine resistance in Hepatitis B Virus: A Brief Summary
Hepatitis B has been commonly known as a virally spread disease infecting millions worldwide1. The total population chronically infected is in the ranges of 400 million and the disease itself is responsible for over 1 million annual fatalities1, 2. The manifestation of this disease is characterised by its primary attack site - the Liver; more specifically the cells of the Liver known as Hepatocytes. Infections caused by the Hepatitis B virus (HBV) can lead to two possible outcomes, an acute or a chronic infection3. The acute form is characterised by the variability in symptoms after infection. The dormancy period for an acute infection can vary by anywhere between 6 weeks to several months3. Common symptoms include fever, anorexia, nausea, malaise, vomiting, jaundice, dark urine, clay-coloured or pale stools, and abdominal pain4. Occasionally some cases also present symptoms such as skin rashes, painful joints, and arthritis4. The chronic infection is characterised by a symptom-less infection period of over 6 months4. Individuals with a chronic infection may eventually develop symptoms similar to that of an acute infection, but in addition to that they have a significantly higher risk of developing chronic liver diseases, including cirrhosis of the liver and primary hepatocellular carcinoma5, 6. The development of acute vs. chronic infection is highly age-dependant4. Theoretically, all infections start as acute infections but depending on the age of the individual, they either develop into chronic infections or are dealt with and eradicated from the body4. For instance, the risk of developing a chronic infection when the individual is exposed in prenatal age is almost 90% while the same risk for an individual infected as an adult is 5%4.
The virus that causes Hepatitis B (HBV) is part of a family of viruses known as Hepadnaviridae7, 8. This family of viruses is known to selectively infect Hepatocytes, thus the name Hepa-dna-viridae. The Hepadnaviridae family is further divided into two sub-categories: the Orthohepadnavirus that infects mammals (HBV) and the Avihepadnavirus that infects ducks and geese (DHBV)7, 8. Structurally, a HBV particle consists of multiple units8, 9, 10, 11. The outer protein consists of lipids and the surface antigen (HbsAg). There is an inner icosahedral nucleocapsid core consists of the core antigen (HBcAg) and the pre-core antigen (HBeAG). Inside the nucleocapsid is a partially double stranded DNA (dsDNA) and a viral DNA polymerase. The extent of the double stranded nature of the viral genome is relatively variable10. It has been concluded that the partial double helix consists of a full length negative DNA strand and a partial length positive DNA strand10. The negative strand is approximately 3.2kb in length and the positive strand is approximately 2.3kb. The variability of the length of the positive strand is found to be extensive but it has been shown that a minimum of 650-700 nucleotides are always part of the single stranded region10.
The transmission mechanism of HBV has been found to be similar to retroviruses12. Transmission can occur by direct exposure to infected blood or other bodily fluids. It can also occur via sexual contact, needle-sharing and prenatally from mother-to-child13. Due to the relatively high stability of the virus, transmission is also known to occur from contaminated surfaces and environments13. The main attach site of HBV particles are the Hepatocytes (liver cells)14. This tissue specific behaviour of HBV suggests the presence of specific surface receptors on Hepatocytes that dispose them as attack sites14; however, researchers have been able to infect other endocrine cells (glandular cells) with DHBV14.
The virus enters the cells via endocytosis8. During the entry, the outer protein shell is uncoated and left outside the cell and only the nucleocapsid enters the host cell15. This characteristic has been well used as a detection mechanism because immediately following infection, the blood is found to have surface antigen (HbsAg) activity. Following that is the entry of the viral genome into the nucleus. Studies done on nucleocapsid activity in the nucleus suggest that the viral nucleocapsid is arrested outside the nucleus and only the viral partial dsDNA enters the nucleus15. The next step is the formation of a fully double stranded DNA from the original partially double stranded DNA16. This process utilizes the DNA polymerase of the virus and converts the partial dsDNA to a 3.2kb long covalently closed circular DNA (cccDNA)16, this becomes the primary transcript from which all the essential mRNA are transcribed. Despite the technological advancements since the documentation of this virus, the early events of the viral life cycle, including entry, uncoating, and delivery of the viral genome into the cell nucleus, are not well understood8.
Studying the viral genome in further detail; it consists of four genes and the genome codes for four different mRNA fragments17, 18, 19. The four genes are named as P, S, C and X and the four different lengths of mRNA produced are 3.5kb, 2.4kb, 2.1kb and 0.7kb. It is noteworthy that the four genes and the four mRNAs do not correlate. The P gene codes for the viral DNA polymerase and the partial dsDNA8, 20. The S gene codes for the viral envelop and the surface antigen (HbsAg). The C gene codes for the core (HBcAg) and the pre-core (HBeAG) antigens. The function of the X gene has not been well documented but it has been suggested that it codes for two regulatory proteins required for HBV replication21. Moreover, studies have also concluded that the X gene may also be a co-factor in the development of hepatocellular carcinoma due to the over expression of this gene in mice and their subsequent higher rate of developing hepatocellular carcinoma22.
In terms of mRNA, the longest mRNA (3.5kb) is translated into forming the constituents of the nucleocapsid. This includes the core and pre-core antigens, the dsDNA and the viral DNA polymerase18, 19, 20. To make the partial dsDNA, the mRNA obtained from gene P is first reversed transcribed to from the negative DNA strand. The DNA polymerase then partially base pairs the negative strand with a positive strand leaving a part of the negative strand unpaired. The mechanism by which the polymerase arrests the double-stranding of the genome is yet to be described. The 2.4kb and 2.1kb mRNA fragments code the surface antigen and associated proteins8f. The combination of the two mRNA fragments allows for the formation of three different sized (large, medium and small) envelope proteins for the outer protein coat2. The smallest 0.7kb mRNA codes for the two protein encoded by gene X21.
The virus relies on the host cells machinery for the translation and synthesis of its proteins - another aspect by which HBVs are similar to retroviruses12. Once all the required components are translated and combined, new virions are formed which either exit the host cell in infect other uninfected cells or re-infect the same host cell and produce more copies of the virus24. Due to the relatively long life cycles of Hepatocytes and very infrequent cell divisions, the virus can easily replicate and store itself in the cells for long periods of time. In addition, the direct contact of Hepatocytes with blood and other Hepatocytes means that the virus can easily infect the entire Liver in a short amount of time making it relatively lethal and difficult to treat8.
Furthermore, 8 different genotypes of Hepatitis B variants have been identified and labelled for A to H25, 26. These genotypes are identified on the basis of their geographical distributions27. Although all the genotypes have been documented, genotypes A, B and C are usually found in majority25, 26.
A major part of the treatment for Hepatitis B involves use of anti-viral drug therapy. Multiple drugs have been qualified as therapy options; one such drug is Lamivudine27. Lamivudine is an orally taken anti-retro viral drug that has been widely used as a treatment for chronic Hepatitis B27. Due to the extensive use of this drug in various anti-viral therapies, HBV is now known to develop resistance against Lamivudine after a set period of time28, 29, 30. Studies have shown that resistance to Lamivudine develops in 53% to 76% of patients after 3 years of treatment28. Another independent study showed that the rate oflamivudineresistance particles in the individual increasedfrom 6% at 12 months to 51% at 48 months30.
Several studies directed towards developing a theory explaining the cause of resistance in HBV against Lamivudine have concluded a similar result - mutation28, 29, 30. The viral genome is known to have a catalytic motif for the restriction transcriptase enzyme. This catalytic motif is known as YMDD and is located in domain C of the polymerase molecule28. Mutations directed towards this domain provide the primary cause of resistance against Lamivudine28. The mutations are base-substitution mutations that result in the translation of an incorrect amino acid due to the change in codons. Various mutations have been recorded in Lamivudine resistant mutants. The most common mutation documented is the M204V mutation29. The M204V refers to the presence of a Valine residue instead of a Methionine at amino acid position 20428. Other mutations which have a similar effect as the M204V are M204I and M204S - presence of Isoleucine and Serine (respectively) instead of Methionine at amino acid position 20428. These mutations, although do provide resistance against Lamivudine, result in the decreased replication efficiency. To compensate for this loss in efficiency, these Lamivudine resistant mutations are frequently found co-existing with mutation such as L180M (Methionine substituted for Leucine) and V173L (Leucine substituted for Valine) that improve the replication efficiency of the virus28. Studies have shown that mutants with both L180M and M204V were fully resistant to Lamivudine but mutants with only L180M were only predisposed to developing a fully Lamivudine resistance29.
The mechanisms underlying the development of resistance via the observed mutations have not yet been fully understood. It is proposed that steric hindrance plays an important role in the development of resistance31. Steric hindrance is defined as the inhibition of the action of a substance due to the occupancy of the reactive center/attack site by another substance. Studies suggest that hindrance between the substituted amino acid side-chain and Lamivudine may be the cause of resistance31. More specifically, the conflict between the methyl groups of Valine of Isoleucine with the sulphur atom in the oxathiolane ring present in Lamivudine might inhibit the action of Lamivudine31. Moreover, with regard to the L180M mutation, the increase in replication efficiency may be related to the “rearrangement of the deoxynucleoside triphosphate-binding pocket residues31” caused by the mutation.
Due to the emergence of lamivudine resistant HBV after treatment by lamivudine drug therapy (from an otherwise lamivudine resistant-free HBV), there needs to be a simplified laboratory test that could be used to indicate presence of resistant strains in an individual. One such test is the INNO- Line probe assay (LiPA) for HBV32. This test is very sensitive in providing information about the types of mutated variants present in an individual. The test works on the basis of reverse hybridization principle32. The test is done on a membrane-based strip32. The strip is lined with immobilized specific short probes (approximately 20 bases in length) that are characteristic to a particular mutation in the viral genome. The probes are immobilized in parallel lines on the strip32. To test for the presence of mutations, the suspected viral genome is denatured and allowed to hybridise against the strip. The presence of any mutations will develop as bands in an assigned area on the strip. The patterns of hybridisation are then observed on the developed strip and compared over time to observe emergence of lamivudine-resistant strains32.
In conclusion, the scientific literature discussing Lamivudine resistance is enormous. Multiple studies have been conducted and have shown similar results as the ones discussed in this paper. However, there still are various mechanisms associated with HBV replication and Lamivudine resistance that are yet unclear and need to by fully observed and explained. Moreover, even though the INNO- Line probe assay for HBV is a fairly sensitive test to determine presence of resistant viral strains and provides results with very little error, it is still incapable of detecting novel mutations which arise spontaneously and can result in fatal circumstances if not detected32.
1. Aggarwal, R., & Ranjan, P. (2004). Preventing and treating hepatitis b infection.British Medical Journal, 329, 1080-1086.
2. Chisari, F.V. (2000). Viruses, immunity, and cancer: lessons from hepatitis b.American Journal of Pathology, 156(4), 1117-1132.
3. Mahoney, F.J. (1999). Update on diagnosis, management, and prevention of hepatitis b virus infection.Clinical Microbiology Reviews,12(2), 351-366.
4. McMahon, B. J., W. L. M. Alward, D. B. Hall, W. L. Heyward, T. R. Bender, D. P. Francis, & J. E. Maynard. (1985). Acute hepatitis B virus infection: relation of age to the clinical expression of disease and subsequent development of the carrier state. Journal of Infectious Diseases, 151, 599-603.
5. Beasley, R. P. (1988). Hepatitis B virus: The major etiology of hepatocellular carcinoma. Cancer, 61, 1942-1956.
6. Liaw, Y.F., D.I. Tai, C.M. Chu, & T.J. Chen. 1988. The development of cirrhosis in patients with chronic type B hepatitis: a prospective study. Hepatology, 8, 493-496.
7. Mason, W. S., G. Seal, & J. Summers. (1980). Virus of Pekin ducks with structural and biological relatedness to human hepatitis B virus. Journal of Virology, 36, 829-836.
8. Seeger, C., & Mason, W.S. (2000). Hepatitis b virus biology.Microbiology and Molecular Biology Reviews, 64(1), 51-68.
9. Birnbaum, F., & Nassal, M. (1990). Hepatitis b virus nucleocapsid assembly: primary structure requirements in the core protein.Journal of Virology, 64(7), 3319-3330.
10. Delius , H., Gough , N.M., Cameron, C.H., & Murray, K. (1983). Structure of the hepatitis b virus genome. Journal of Virology,47(2), 337-343.
11. Satoh, O., Imai, H., Yoneyama, T., Miyamura, T., Utsumi, H., Inoue, K., & Umeda, M. (2000). MembraneStructureof theHepatitisBVirus Surface Antigen Particle. Journal of Biochemistry, 127(4), 543-550.
12. Miller, R.H., & Robinson, W.S. (1986). Common evolutionary origin of hepatitis B virus and retroviruses.Evolution,83, 2531-2535.
13. Bond, W.W., Petersen, N.J., & Favero, M.S. (1977).Viral hepatitis B: aspects of environmental control. Health Laboratory Science, 14, 235-52.
14. Halpern, M. S., Egan, J., Mason, W.S., & England J.M. (1984). Viral antigen in endocrine cells of the pancreatic islets and adrenal cortex of Pekin ducks infected with duck hepatitis B virus. Virus Research, 1, 213-223.
15. Guidotti, L.G., Martinez, V., Loh, Y.T., Rogler, C.E., & Chisari, F.V.(1994). Hepatitis B virus nucleocapsid particles do not cross the hepatocyte nuclear membrane in transgenic mice. Journal of Virology, 68, 5469-5475.
16. Summers, J., Mason, W.S. (1982). Replication of the genome of a hepatitis B-like virus by reverse transcription of an RNA intermediate. Cell, 29, 403-415
17. Lau, J. Y. N., and T. L. Wright. (1993). Molecular virology and pathogenesis of hepatitis B. Lancet, 342, 1335-1340.
18. Gish, R.G., & Gadano, A.C. (2006). Chronic hepatitis B: current epidemiology in the Americas and implications for management. Journal of Viral Hepatitis, 13, 787-798.
19. Wei, Y., & Tiollais, P.K. (1999). Molecular biology of hepatitis B virus. Clinics in Liver Disease, 3, 189-219
20. Hunt, C.M., McGill, J.M., Allen, M.I., & Condreay, L.D. (2000). Clinical relevance of hepatitis B viral mutations. Hepatology, 3, 1037-1044.
21. Balsano, C., Billet, O., Bennoun, M., Cavard, C., Zider, A., Grimber, G., Natoli, G., Briand, P., & Levrero, M. (1994). Hepatitis B virus X gene product acts as a transactivator in vivo. Journal of Hepatology, 21, 103-109
22. Kim, C.M., Koike, K., Saito, I., Miyamura, T., & Jay, G. (1991). HBx gene of hepatitis B virus induces liver cancer in transgenic mice. Nature, 351, 317-320
23. Ganem, D., & Varmus, H.E. (1987). The molecular biology of the hepatitis B viruses. Annual Review of Biochemistry, 56, 651-693
24. Elgouhari, H.M., Tamimi, T.I.A, & Carey, W.D. (2008). Hepatitis b virus infection: understanding its epidemiology, course, and diagnosis.Cleveland Clinic Journal of Medicine,75(12), 881-889.
25. Norder, H., Courouce, A.M., Coursaget, P., et al. (2004). Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, subgenotypes, and HBsAg subtypes. Intervirology, 47, 289-309.
26. Chu, C.J., Keeffe, E.B., Han, S.H., et al. (2003) Hepatitis B virus genotypes in the United States: results of a nationwide study. Gastroenterology, 125, 444-451.
27. Bottecchia, M., Souto, F.J.D, Ó, K.M.R, Amendola, M., Brandão, C.E., Niel, C., & Gomes, S.A. (2008). Hepatitis b virus genotypes and resistance mutations in patients under long term lamivudine therapy: characterization of genotype g in Brazil.BMC Microbiology,8, 11.
28. Pallier, C., Castera, L., Soulier, A., Hezode, Nordmann, P., Dhumeaux, D., C., & Pawlotsky, J.M. (2006). Dynamics of hepatitis b virus resistance to lamivudine.Journal of Virology,80(2), 643-653.
29. Alvarado-Esquivel, C., Carrera-Gracia, M.A., Conde-Gonzalez, C.J., Juarez-Figueroa, Ruiz-Maya, l., Aguilar-Benavides, S., Torres-Valenzuela, A., L., & Sablon, E. (2006). Genotypic resistance to lamivudine among hepatitis b virus isolates in Mexico.Journal of Antimicrobial Chemotherapy,57, 221-223.
30. Thompson, A.J., Ayres, A., Yuen, J.,Bartholomeusz, A., Bowden, D.S., Iser, D.M., Chen, R.Y.,Demediuk, B., Shaw, G., Bell, S.J., Watson, K.J., Locarnini, S.A., & Desmond, P.V. (2007). Lamivudineresistanceinpatients with chronichepatitisB. Journal of Gastroenterology and Hepatology, 22(7), 1078-1085.
31. Das, K., Xiong, X., Yang, H., Westland, C.E., Gibbs, C.S., Sarafianos, S.G., & Arnold, E. (2001). Molecular modeling and biochemical characterization reveal the mechanism of Hepatitis B virus polymerase resistance to lamivudine (3TC) and emtricitabine (FTC).Journal of Virology,75(10), 4771-4779.
32. Osiowy, C., & Giles, E. (2003). Evaluation of the INNO-LiPA HBV genotyping assay for determination of Hepatitis B virus genotype.Journal of Clinical Microbiology,41(12), 5473-5477.