Hepatitis B virus mutation in children
http://www.100md.com
《美国医学杂志》
Department of Pediatrics, National Taiwan University Hospital, Taipei, Taiwan
Abstract
Due to the lack of proof reading activity of hepatitis B virus (HBV) polymerase, mutation/variation of the viral sequence is frequently found during long term follow-ups. In the majority of children with chronic HBV infection, wild type HBV is the dominant viral strain during the natural course of chronic HBV infection. During long-term follow-up, HBV precore mutants developed spontaneously in approximately 10 to 24% of children before HBeAg seroconversion, and in around 50% of children after HBeAg seroconversion mutants. Occasionally, children may be infected primarily by mutant strains of HBV. Approximately 36% of children with fulminant hepatitis and 30% of children with acute hepatitis B were infected by precore mutants of HBV transmitted by their mothers or blood donors. In addition, after universal HBV vaccination, HBV surface gene variants emerge or are selected under the immune pressure generated by the host or by administration of hepatitis B immune globulin and hepatitis B vaccination.[2] In HBV DNA positive children from four sequential surveys in Taiwan, the prevalence of hepatitis B surface gene a determinant mutants increased from 7.8% before the vaccination program, to 19.6%, 28.1%, and 23.1% at 5, 10, and 15 years after the program. Nucleoside analogue may also induce mutant strains, which reduces the antiviral effects. The most common example is the YMDD mutation of the HBV polymerase gene after antiviral therapy with lamivudine. It developed in 19% of the treated children. In conclusion, children may be infected primarily by mutant strains of HBV either naturally during acute HBV infection. Those infected with wild type HBV initially may develop mutant strains gradually during the course of chronic infection under the host immune pressure. Vaccine escape mutants may develop after immunoprophylaxis. In addition, antiviral therapy with nucleoside anlogues may also induce drug resistant mutant strains. Understanding the viral mutation status will help to design accurate strategies of immmunoprophylaxis and antiviral therapy against HBV infection.
Keywords: Hepatitis B virus; Mutant; Immunoprophylaxis; Antiviral therapy
Hepatitis B virus ( HBV) infection is a global health problem. HBV infection may lead to acute or fulminant hepatitis, chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Asia and Africa are hyper-endemic areas for HBV infection. In most hyperendemic areas, primary infection occurs mainly during early childhood. Perinatal transmission from mother to their infants is an important route of transmission, which leads to a very high chronicity rate.
Biology of HBV
HBV is a 3.2 Kb partially double-stranded DNA virus. It contains four partly overlapping open reading frames (ORFs): C that encodes for core protein (HBcAg) and e antigen (HBeAg); P for polymerase (reverse transcriptase) protein (POL), S for envelope proteins and X for a transcriptional trans -activator protein. The presence of antibody to HBsAg generally implies a recovery from a natural infection, or the protective effect from HBV immunization. Serum markers for chronic HBV infection includes HBsAg, HBeAg and its antibody, core antibody (anti-HBc), and HBV DNA.
In patients with chronic HBV infection, plasma HBV has a relatively short half-life of approximately 24 hours, whereas infected liver cells have a much longer and more variable half-life of 10-100 days.[1] The average total amount of plasma HBV production is 1.3 × 10 11 particles per day. The total serum virus load varies from 10 10 to 10 12 particles in different patients, with an average of 2.2 × 10 11.
Natural History of HBV Infection
HBV infection in early life may lead to high rate of chronicity with high viral replication. These long term effects may result in serious liver diseases in later life. Although most complication of HBV infection manifests in adulthood, hepatitis B related liver disease may begin during childhood. In endemic areas such as Asia and Africa, severe complications of HBV infection such as hepatoma and fulminant hepatitis, may occur during childhood and adolescents in Asia.
Age of infection is an important factor affecting the outcome of hepatitis B virus infection. Perinatal transmission from hepatitis B e antigen (HBeAg) positive hepatitis B surface antigen (HBsAg) carrier mothers become chronic HBsAg carriers if un-immunized. The rate of chronicity after HBV infection declined with age of infection : 25% in infected preschool children , and 3 to 10% in adolescents and young adults. Maternal HBeAg status may also affect the outcome of infection in their infants. Only less than 5 % of the infants of HBeAg seronegative mothers may lead to chronic HBV infection. Acute or fulminant hepatitis in infancy occurs mostly in infants of HBeAg negative mothers.[2]
In those with chronic HBV infection, serum HBeAg was gradually cleared and the viral replication is reduced while the child is getting older. This process of HBeAg seroconversion takes place insidiously in most individuals for a period of 2 to 7 years. Exceptionally, HBeAg/anti-HBe seroconversion may occur as early as in infancy with an unclear or brief period of HBeAg seropositive phase. However, in most occasions the annual rate of spontaneous HBeAg seroconversion is very low before 3 years of age (<2% per year), and is increased gradually to around 5% after 3 years of age.[3] This might be due to the immune tolerance to HBcAg and HBeAg in infected children. We have demonstrated that immune tolerance in perinatall transmitted children is very likely due to transplacental HBeAg, evidenced by the absence of T-cell response to hepatitis B core antigen (HBcAg) in infants and children of hepatitis B e antigen positive HBsAg carrier mothers, while the T-lymphocytes from acute hepatitis infants of hepatitis B e negative HBsAg carrier mothers response very well to HBcAg.[4] Development of liver cirrhosis or hepatocellular carcinoma is occasionally observed, but is rare during childhood. They are mostly (around 80%) anti-HBe seropositive.
Genotype and HBeAg seroconversion in children
The genotypes of HBV are defined by an intergroup divergence of more than 8% in the complete genome sequence and more than 4% at the level of the S gene.[5] Up to now, eight genotypes (A to H) have been identified. There are some differences between the genotypes of HBV in the length of the Open Reading Frames (ORFs) and the size of the protein products translated. The distribution of HBV genotypes vary in isolates from different geographic areas.[6] Genotypes B and C are more prevalent in Asia, while genotype A is more commonly found in Europe, North America, Brazil, South Africa and India. A long term (15 years) follow-up study of HBV carrier children revealed that HBV genotype B dominates in children with chronic HBV infection and HCC in Taiwan (genotype B in 73 to 86% versus genotype C in 6-27%). Genotype C carriers had a delayed HBeAg seroconversion compared with the genotype B carriers.[7]
Mutant Viruses Dveloped During the Natural Course of HBV Infection table1
Due to the lack of proof reading activity of HBV polymerase, mutation/variation of the viral sequence is frequently found during long term follow-up. The annual nucleotide substitution rate per site for HBV has been estimated to be 1.4 to 5 × 10 -5, approximately the same as retroviruses (10 -5) but 10 4 times higher than DNA viral genomes. At any time, the virus population can be composed of a number of different mutants referred to as "quasispecies". Some viral hot spots of mutation gradually develop during the natural course as a result of virus-host interaction.
Several hot spots of the hepatitis B virus (HBV) nucleotide sequences are susceptible to mutation, perhaps due to various extrinsic pressures.
Hepatitis B e antigen (HBeAg), coded by the precore and core genes, is an important infectivity marker in hepatitis B virus (HBV) infection. HBeAg has been suggested to be a target of host cytotoxic T cell, and its disappearance may suggest an immune clearance of HBV. Seroconversion from HBeAg antigenemia to anti-HBe may occur spontaneously in the natural course of chronic HBV infection. This process usually suggests that HBV breaks through the immune tolerance and enters into a low replication phase.
1. Mutants Developed During Chronic HBV Infection
(1). Preore and Core Promoter Mutants : HBV wild type is the dominant strain during the long-term course of chronic HBV infection during childhood. An important G to A mutation at nucleotide 1896 of the HBV precore gene may lead to a stop codon, which causes a failure of hepatitis e antigen production.
The proportion of the precore stop codon mutant in children with chronic HBV infection increases gradually from 10% at early HBeAg positive status to approximately 50% (39 to 52%) after HBeAg seroconversion during long term follow-up.[8] Prior to HBeAg clearance, precore stop codon mutant emerged and coexisted with the wild type in only 25% of children with chronic HBV infection. A remarkable proportion (46%) of children had no detectable precore stop codon mutant throughout the long-term follow-up course, even after HBeAg seroclearance. The HBV DNA levels were mostly low or even undetectable (10%) by nested PCR at the end of follow-up in anti-HBe seropositive children. Those in whom the precore stop codon mutant emerged before HBeAg seroconversion during follow-up tended to have higher levels of ALT, suggesting more severe hepatocellular damage
Precore nucleotide 1896 and core promoter mutations account for most hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B virus (HBV) infection in adults, yet the mutational profiles of the core promoter are less well studied in children. We have conducted an age-matched, case-control study enrolled 110 chronic HBV infected children, including 55 HBeAg seroconverters and 55 nonseroconverters. In adults with no precore gene mutation, HBV core promoter nucleotide 1762/1764 mutations is often accounted for the HBeAg seroconversion. Yet HBV core promoter nucleotide 1762/1764 mutation plays minimal role in HBeAg sero-conversion in children.[9] In contrast, HBV precore gene 1896 mutant was found in a half of childhood HBeAg seroconverters. HBeAg seroconverters had a higher frequency of HBV core promoter gene nucleotide A1775G and G1799C mutation and lower frequency of A1752G mutation. In the HBeAg seroconverter group, HBV genotype C was associated with core promoter 1762 / 1764 mutations.
(2) Core Gene Mutants : Because hepatitis B core antigen is the target for the cytotoxic T lymphocyte mediated lysis of HBV infected hepatocytes, it is also susceptible to mutation. Mutations of the core gene may change the conformation of the core protein and allow HBV to escape immune surveillance via loss or change of immunodominant epitopes. Alternatively, they could be virulent strains that induce persistent host immune attacks and lead to severe liver disease. Previous studies of HBV core gene mutations in adults showed that the core gene was highly conserved during the immune tolerance phase of chronic HBV infection. As patients passed through the immune tolerance phase, an increasing number of mutations develops in the core gene, where is rich in B and T cell epitopes.
We have investigated the changes and implication of HBV core gene sequence in children with chronic HBV infection and /or hepatocellular carcinoma. Four serial serum samples before and after hepatitis B e antigen seroconversion from 31 children with chronic HBV infection, and one serum sample taken from 12 children with hepatocellular carcinoma were studied. The accumulation of mutations of HBV core gene is more frequently seen in children with hepatocellular carcinoma and the pattern of mutation is different from those in chronic HBV infected children.[10] Mutations accumulated as chronic infection persisted and most frequently occurred at core gene codon 21, 147 , and 65 (16-29%) in the chronic HBV infected children. Core gene mutation sites in children with hepatocellular carcinoma were identified at core codons 74, 87, and 159. Children with hepatocellular carcinoma had more mutations in the core gene than those with chronic HBV infection.
We also have screened out HBV core gene deletion mutants in 18 (4.9%) among the long-term followed 365 children with chronic HBV infection for more than 10 years. Most patients with core gene deletion mutants heralded HBeAg sero-conversion. Those deletion mutants disappeared after HBeAg seroconversion. Core gene deletion mutants could appear as early as during 5 years of age. The duration of their appearance was 0.5 to 5 years. Horizontal rather than perinatal transmission of HBV was a favorable factor for those mutants to develop. The emergence of the mutants was likely the result of host-viral interaction and mostly signified HBeAg sero-conversion within 1 year.[11]
2. Primary Infection By Mutants During Acute or Fulminant Hepatitis table1
Our previous studies revealed that approximately 36% of children with fulminant hepatitis and 30% of children with acute hepatitis B were infected by precore mutants of HBV. Those mutants were transmitted mainly by their HBeAg negative mothers or blood donors.[12]
Infection By HBV Mutants After Mass Immunization Program table1
While the available treatment of hepatitis B is not satisfactory, prevention is the best way toward its control. Universal immunization program of HBV with combination of hepatitis B immunoglobulin at birth and hepatitis B vaccines during early infancy, has been proved to be effective in reducing hepatitis B carrier rate to approximately one-tenth of the prevalence before the vaccination program in endemic areas.[13] The incidence of hepatocellular carcinoma in children has also been reduced significantly.[14],[15] Recently, we have also demonstrated the reduction of the incidence of fulminant hepatitis B in children.[16]
The viral sequences can be changed after active or passive immunization. HBV surface gene variants emerge or are selected under the immune pressure generated by the host or by HBIG and hepatitis B vaccination. In HBV DNA positive children from four sequential surveys in Taiwan, the prevalence of hepatitis B surface gene a determinant mutants increased from 7.8% before the launch of the vaccination program, to 19.6% at 5 years after, 28.1% at 10 years after, and 23.1% at 15 years after the program.[17]
Serum hepatitis B virus (HBV) DNA from infants with fulminant hepatitis B, acute self-limited hepatitis B, and infants with chronic HBV infection were studied for the sequences of the region of HBV genome encoding the major antigenic epitopes of hepatitis B surface antigen (HBsAg). All infants were born to carrier mothers and administered immunoprophylaxis from birth. Serum HBV DNA from carrier children born to carrier mothers who did not receive immunoprophylaxis and had comparable length of infection were studied as controls. In the majority of the mothers of the infants, corresponding mutations in HBsAg were not detected in serum. The surface gene mutants detected in the carrier infants were not found in their mothers' serum after cloning and sequencing of 10 DNA clones from each maternal sample. None of the control patients had detectable surface gene mutants.
HBV surface gene variants emerge or are selected under the immune pressure generated by the host or by administration of hepatitis B immune globulin and hepatitis B vaccination. An surface gene mutant (residue 129, Gln to Arg) found in one mother-infant pair suggested a direct maternal-infant transmission, resulting in immunoprophylaxis failure. None of the family members of children infected with Arg-145 variant had the same variant infection, implying this variant's low transmissability.[18],[19]
Viral Mutation after Antiviral Therapy table1
During antiviral therapy, mutation of HBV genome may develop to escape the drug effect. This event occurs mainly during therapy using nucleoside analogue, and not seen in interferon therapy. In an multicentered clinical trail in children, 191 children were randomly assigned to receive lamivudine and 97 to receive placebo. The rate of virologic response at week 52 was higher among children who received lamivudine than among those who received placebo (23% vs 13%). Lamivudine therapy was associated with higher rates of HBeAg seroconversion, normalization of alanine aminotransferase levels, and suppression of HBV DNA.[20] At week 52 after therapy, change of HBV polymerase gene codon 552, i.e. YMDD mutation (methionine to valine or isoleucine), was detected in 19% of patients in the lamivudine group, while none was found in the placebo group. The serum HBV DNA level tended to be higher at base line in the patients in whom the YMDD variant was present at 52 weeks. Demographic characteristics, aminotransferase levels, and histologic activities were similar between patients with YMDD-variant HBV DNA and patients with wild type sequence. In another study of lamivudine therapy in children of HBV carrier mothers, YMDD mutants developed in 34% of the lamivudine treated children after one year therapy.[21]
Conclusions
Children may be infected by mutant strains of HBV naturally during acute or fulminant hepatitis B, or infected by wild type HBV initially and develop mutant strains gradually during the course of chronic infection under the immune pressure of the host. In addition, infection with vaccine escape mutant strains may occur after immunoprophylaxis. Antiviral therapy using nucleoside analogues may also induce drug resistant mutant strains. Understanding the viral mutation status will help to design accurate strategies of immmunoprophylaxis and antiviral therapy against HBV infection.
References
1. Nowak MA, Bonhoeffer S, Hill AM, Boehme R, Thomas HC, McDade H. Viral dynamics in hepatitis B virus infection. Proc Natl Acad Sci USA 1996; 93 : 4398- 4402.
2. Chang MH, Lee CY, Chen DS, Hsu HC, and Lai MY : Fulminant Hepatitis in children in Taiwan : The Important Role of Hepatitis B Virus. J Pediatr 1987; 111 : 34-39.
3. Chang MH, Hsu HY, Hsu HC, Ni YH, Chen JS, Chen DS. The significance of spontaneous HBeAg seroconversion in childhood : with special emphasis on the clearance of HBeAg before three years of age. Hepatology 1995; 22 : 1387-1392.
4. Hsu HY, Chang MH, Hsieh KH et al. Cellular immune response to hepatitis B core antigen in maternal-infant transmission of hepatitis B virus. Hepatology 1992; 15: 770-776.
5. Kramvis A, Kew M, Francois G. Hepatitis B virus genotypes. Vaccine 2003; 23 : 2409-2423.
6. Liu CJ, Kao JH, Chen DS. Therapeutic implications of hepatitis B virus genotypes. Liver International 2005; 25: 1097-1107.
7. Ni YH, Chang MH, Wang KJ et al. Clinical relevance of hepatitis B virus genotype in children with chronic infection and hepatocellular carcinoma. Gastroenterology 2004; 127 : 1733-1738.
8. Chnag MH, Hsu HY, Ni YH et al. Precore stop codon mutant in chronic hepatitis B virus infection in children : Its relation to hepatitis B e seroconversion and natural hepatitis B surface antigen. J Heptology 1998; 28 : 915-922.
9. Ni YH, Chang MH, Hus HY, Tsuei DJ. Longitudinal study on mutation profiles of core promoter and precore regions of the hepatitis B virus genome in children. Pediatr Research 2004; 56: 396-399.
10. Ni YH, Chang MH, Hsu HY, Tsuei DJ. Different hepatitis B virus core gene mutations in children with chronic infection and hepatocellular carcinoma. GUT 2003; 52 : 122-125.
11. Ni YH, Chang MH, Hsu HY, Chen HL. Long term follow-up study of core gene deletion mutants in children with chronic hepatitis B virus infection. Hepatology 2000; 32 : 124-128.
12. Hsu HY, Chang MH, Lee CY et al. Precore mutant of hepatitis B virus in childhood fulminant hepatitis B : an infrequent association. J Infect Dis 1995; 171: 776-781.
13. NI YH, Chang MH, Huang LM et al. Hepatitis B virus infection in children and adolescents in a hyperendemic area : 15 years after mass hepatitis B vaccination. Ann Intern Med 2001; 135 : 796-768.
14. Chang MH, Chen CJ, Lai MS et al. Universal hepatitis B vaccination in Taiwan and the incidence of hepatocellular carcinoma in children. N Engl J Med 1997; 336 : 1855-1859.
15. Chang MH, Shau WY, Chen CJ et al. Hepatitis B vaccination and hepatocellular carcinoma rates in boys and girls. JAMA 2000; 284: 3040-3042.
16. Chen HL, Chang CJ, Kong MS et al. Pediatric fulminant hepatic failure in endemic areas of hepatitis B infection: 15 years after universal hepatitis B vaccination. Hepatology 2004; 39 : 58-63.
17. Hsu HY, Chang MH, Ni YH, Chen HL. Survey of hepatitis B surface variant infection in children 15 years after a nationwide vaccination program in Taiwan. GUT 2004; 53: 1499-1503.
18. Hsu HY, Chang MH, Ni YH, Lin HH, Wang SM, Chen DS. Surface gene mutants of hepatitis B virus in infants who develop acute or chronic infections despite immunoprophylaxis. Hepatology 1997; 26 : 786-791.
19. Hsu HY, Chang MH, Liaw SH, NI YH, Chen HL. Changes of hepatitis B surface antigen variants in carrier children before and after universal vaccination in Taiwan. Hepatology 1999; 30; 1312-1317.
20. Jonas MM, Mizerski J, Badia IB et al. Clinical trial of lamivudine in children with chronic hepatitis B. N Engl J Med 2002; 346 : 1706-1713.
21. Ni YH, Hunag FC, Kong MS et al. Lamivudine treatment in maternally transmitted chronic hepatitis B virus infection patients. Pediatrics International 2005; 47: 372-377.(Chang Mei-Hwei)
Abstract
Due to the lack of proof reading activity of hepatitis B virus (HBV) polymerase, mutation/variation of the viral sequence is frequently found during long term follow-ups. In the majority of children with chronic HBV infection, wild type HBV is the dominant viral strain during the natural course of chronic HBV infection. During long-term follow-up, HBV precore mutants developed spontaneously in approximately 10 to 24% of children before HBeAg seroconversion, and in around 50% of children after HBeAg seroconversion mutants. Occasionally, children may be infected primarily by mutant strains of HBV. Approximately 36% of children with fulminant hepatitis and 30% of children with acute hepatitis B were infected by precore mutants of HBV transmitted by their mothers or blood donors. In addition, after universal HBV vaccination, HBV surface gene variants emerge or are selected under the immune pressure generated by the host or by administration of hepatitis B immune globulin and hepatitis B vaccination.[2] In HBV DNA positive children from four sequential surveys in Taiwan, the prevalence of hepatitis B surface gene a determinant mutants increased from 7.8% before the vaccination program, to 19.6%, 28.1%, and 23.1% at 5, 10, and 15 years after the program. Nucleoside analogue may also induce mutant strains, which reduces the antiviral effects. The most common example is the YMDD mutation of the HBV polymerase gene after antiviral therapy with lamivudine. It developed in 19% of the treated children. In conclusion, children may be infected primarily by mutant strains of HBV either naturally during acute HBV infection. Those infected with wild type HBV initially may develop mutant strains gradually during the course of chronic infection under the host immune pressure. Vaccine escape mutants may develop after immunoprophylaxis. In addition, antiviral therapy with nucleoside anlogues may also induce drug resistant mutant strains. Understanding the viral mutation status will help to design accurate strategies of immmunoprophylaxis and antiviral therapy against HBV infection.
Keywords: Hepatitis B virus; Mutant; Immunoprophylaxis; Antiviral therapy
Hepatitis B virus ( HBV) infection is a global health problem. HBV infection may lead to acute or fulminant hepatitis, chronic hepatitis, liver cirrhosis and hepatocellular carcinoma. Asia and Africa are hyper-endemic areas for HBV infection. In most hyperendemic areas, primary infection occurs mainly during early childhood. Perinatal transmission from mother to their infants is an important route of transmission, which leads to a very high chronicity rate.
Biology of HBV
HBV is a 3.2 Kb partially double-stranded DNA virus. It contains four partly overlapping open reading frames (ORFs): C that encodes for core protein (HBcAg) and e antigen (HBeAg); P for polymerase (reverse transcriptase) protein (POL), S for envelope proteins and X for a transcriptional trans -activator protein. The presence of antibody to HBsAg generally implies a recovery from a natural infection, or the protective effect from HBV immunization. Serum markers for chronic HBV infection includes HBsAg, HBeAg and its antibody, core antibody (anti-HBc), and HBV DNA.
In patients with chronic HBV infection, plasma HBV has a relatively short half-life of approximately 24 hours, whereas infected liver cells have a much longer and more variable half-life of 10-100 days.[1] The average total amount of plasma HBV production is 1.3 × 10 11 particles per day. The total serum virus load varies from 10 10 to 10 12 particles in different patients, with an average of 2.2 × 10 11.
Natural History of HBV Infection
HBV infection in early life may lead to high rate of chronicity with high viral replication. These long term effects may result in serious liver diseases in later life. Although most complication of HBV infection manifests in adulthood, hepatitis B related liver disease may begin during childhood. In endemic areas such as Asia and Africa, severe complications of HBV infection such as hepatoma and fulminant hepatitis, may occur during childhood and adolescents in Asia.
Age of infection is an important factor affecting the outcome of hepatitis B virus infection. Perinatal transmission from hepatitis B e antigen (HBeAg) positive hepatitis B surface antigen (HBsAg) carrier mothers become chronic HBsAg carriers if un-immunized. The rate of chronicity after HBV infection declined with age of infection : 25% in infected preschool children , and 3 to 10% in adolescents and young adults. Maternal HBeAg status may also affect the outcome of infection in their infants. Only less than 5 % of the infants of HBeAg seronegative mothers may lead to chronic HBV infection. Acute or fulminant hepatitis in infancy occurs mostly in infants of HBeAg negative mothers.[2]
In those with chronic HBV infection, serum HBeAg was gradually cleared and the viral replication is reduced while the child is getting older. This process of HBeAg seroconversion takes place insidiously in most individuals for a period of 2 to 7 years. Exceptionally, HBeAg/anti-HBe seroconversion may occur as early as in infancy with an unclear or brief period of HBeAg seropositive phase. However, in most occasions the annual rate of spontaneous HBeAg seroconversion is very low before 3 years of age (<2% per year), and is increased gradually to around 5% after 3 years of age.[3] This might be due to the immune tolerance to HBcAg and HBeAg in infected children. We have demonstrated that immune tolerance in perinatall transmitted children is very likely due to transplacental HBeAg, evidenced by the absence of T-cell response to hepatitis B core antigen (HBcAg) in infants and children of hepatitis B e antigen positive HBsAg carrier mothers, while the T-lymphocytes from acute hepatitis infants of hepatitis B e negative HBsAg carrier mothers response very well to HBcAg.[4] Development of liver cirrhosis or hepatocellular carcinoma is occasionally observed, but is rare during childhood. They are mostly (around 80%) anti-HBe seropositive.
Genotype and HBeAg seroconversion in children
The genotypes of HBV are defined by an intergroup divergence of more than 8% in the complete genome sequence and more than 4% at the level of the S gene.[5] Up to now, eight genotypes (A to H) have been identified. There are some differences between the genotypes of HBV in the length of the Open Reading Frames (ORFs) and the size of the protein products translated. The distribution of HBV genotypes vary in isolates from different geographic areas.[6] Genotypes B and C are more prevalent in Asia, while genotype A is more commonly found in Europe, North America, Brazil, South Africa and India. A long term (15 years) follow-up study of HBV carrier children revealed that HBV genotype B dominates in children with chronic HBV infection and HCC in Taiwan (genotype B in 73 to 86% versus genotype C in 6-27%). Genotype C carriers had a delayed HBeAg seroconversion compared with the genotype B carriers.[7]
Mutant Viruses Dveloped During the Natural Course of HBV Infection table1
Due to the lack of proof reading activity of HBV polymerase, mutation/variation of the viral sequence is frequently found during long term follow-up. The annual nucleotide substitution rate per site for HBV has been estimated to be 1.4 to 5 × 10 -5, approximately the same as retroviruses (10 -5) but 10 4 times higher than DNA viral genomes. At any time, the virus population can be composed of a number of different mutants referred to as "quasispecies". Some viral hot spots of mutation gradually develop during the natural course as a result of virus-host interaction.
Several hot spots of the hepatitis B virus (HBV) nucleotide sequences are susceptible to mutation, perhaps due to various extrinsic pressures.
Hepatitis B e antigen (HBeAg), coded by the precore and core genes, is an important infectivity marker in hepatitis B virus (HBV) infection. HBeAg has been suggested to be a target of host cytotoxic T cell, and its disappearance may suggest an immune clearance of HBV. Seroconversion from HBeAg antigenemia to anti-HBe may occur spontaneously in the natural course of chronic HBV infection. This process usually suggests that HBV breaks through the immune tolerance and enters into a low replication phase.
1. Mutants Developed During Chronic HBV Infection
(1). Preore and Core Promoter Mutants : HBV wild type is the dominant strain during the long-term course of chronic HBV infection during childhood. An important G to A mutation at nucleotide 1896 of the HBV precore gene may lead to a stop codon, which causes a failure of hepatitis e antigen production.
The proportion of the precore stop codon mutant in children with chronic HBV infection increases gradually from 10% at early HBeAg positive status to approximately 50% (39 to 52%) after HBeAg seroconversion during long term follow-up.[8] Prior to HBeAg clearance, precore stop codon mutant emerged and coexisted with the wild type in only 25% of children with chronic HBV infection. A remarkable proportion (46%) of children had no detectable precore stop codon mutant throughout the long-term follow-up course, even after HBeAg seroclearance. The HBV DNA levels were mostly low or even undetectable (10%) by nested PCR at the end of follow-up in anti-HBe seropositive children. Those in whom the precore stop codon mutant emerged before HBeAg seroconversion during follow-up tended to have higher levels of ALT, suggesting more severe hepatocellular damage
Precore nucleotide 1896 and core promoter mutations account for most hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B virus (HBV) infection in adults, yet the mutational profiles of the core promoter are less well studied in children. We have conducted an age-matched, case-control study enrolled 110 chronic HBV infected children, including 55 HBeAg seroconverters and 55 nonseroconverters. In adults with no precore gene mutation, HBV core promoter nucleotide 1762/1764 mutations is often accounted for the HBeAg seroconversion. Yet HBV core promoter nucleotide 1762/1764 mutation plays minimal role in HBeAg sero-conversion in children.[9] In contrast, HBV precore gene 1896 mutant was found in a half of childhood HBeAg seroconverters. HBeAg seroconverters had a higher frequency of HBV core promoter gene nucleotide A1775G and G1799C mutation and lower frequency of A1752G mutation. In the HBeAg seroconverter group, HBV genotype C was associated with core promoter 1762 / 1764 mutations.
(2) Core Gene Mutants : Because hepatitis B core antigen is the target for the cytotoxic T lymphocyte mediated lysis of HBV infected hepatocytes, it is also susceptible to mutation. Mutations of the core gene may change the conformation of the core protein and allow HBV to escape immune surveillance via loss or change of immunodominant epitopes. Alternatively, they could be virulent strains that induce persistent host immune attacks and lead to severe liver disease. Previous studies of HBV core gene mutations in adults showed that the core gene was highly conserved during the immune tolerance phase of chronic HBV infection. As patients passed through the immune tolerance phase, an increasing number of mutations develops in the core gene, where is rich in B and T cell epitopes.
We have investigated the changes and implication of HBV core gene sequence in children with chronic HBV infection and /or hepatocellular carcinoma. Four serial serum samples before and after hepatitis B e antigen seroconversion from 31 children with chronic HBV infection, and one serum sample taken from 12 children with hepatocellular carcinoma were studied. The accumulation of mutations of HBV core gene is more frequently seen in children with hepatocellular carcinoma and the pattern of mutation is different from those in chronic HBV infected children.[10] Mutations accumulated as chronic infection persisted and most frequently occurred at core gene codon 21, 147 , and 65 (16-29%) in the chronic HBV infected children. Core gene mutation sites in children with hepatocellular carcinoma were identified at core codons 74, 87, and 159. Children with hepatocellular carcinoma had more mutations in the core gene than those with chronic HBV infection.
We also have screened out HBV core gene deletion mutants in 18 (4.9%) among the long-term followed 365 children with chronic HBV infection for more than 10 years. Most patients with core gene deletion mutants heralded HBeAg sero-conversion. Those deletion mutants disappeared after HBeAg seroconversion. Core gene deletion mutants could appear as early as during 5 years of age. The duration of their appearance was 0.5 to 5 years. Horizontal rather than perinatal transmission of HBV was a favorable factor for those mutants to develop. The emergence of the mutants was likely the result of host-viral interaction and mostly signified HBeAg sero-conversion within 1 year.[11]
2. Primary Infection By Mutants During Acute or Fulminant Hepatitis table1
Our previous studies revealed that approximately 36% of children with fulminant hepatitis and 30% of children with acute hepatitis B were infected by precore mutants of HBV. Those mutants were transmitted mainly by their HBeAg negative mothers or blood donors.[12]
Infection By HBV Mutants After Mass Immunization Program table1
While the available treatment of hepatitis B is not satisfactory, prevention is the best way toward its control. Universal immunization program of HBV with combination of hepatitis B immunoglobulin at birth and hepatitis B vaccines during early infancy, has been proved to be effective in reducing hepatitis B carrier rate to approximately one-tenth of the prevalence before the vaccination program in endemic areas.[13] The incidence of hepatocellular carcinoma in children has also been reduced significantly.[14],[15] Recently, we have also demonstrated the reduction of the incidence of fulminant hepatitis B in children.[16]
The viral sequences can be changed after active or passive immunization. HBV surface gene variants emerge or are selected under the immune pressure generated by the host or by HBIG and hepatitis B vaccination. In HBV DNA positive children from four sequential surveys in Taiwan, the prevalence of hepatitis B surface gene a determinant mutants increased from 7.8% before the launch of the vaccination program, to 19.6% at 5 years after, 28.1% at 10 years after, and 23.1% at 15 years after the program.[17]
Serum hepatitis B virus (HBV) DNA from infants with fulminant hepatitis B, acute self-limited hepatitis B, and infants with chronic HBV infection were studied for the sequences of the region of HBV genome encoding the major antigenic epitopes of hepatitis B surface antigen (HBsAg). All infants were born to carrier mothers and administered immunoprophylaxis from birth. Serum HBV DNA from carrier children born to carrier mothers who did not receive immunoprophylaxis and had comparable length of infection were studied as controls. In the majority of the mothers of the infants, corresponding mutations in HBsAg were not detected in serum. The surface gene mutants detected in the carrier infants were not found in their mothers' serum after cloning and sequencing of 10 DNA clones from each maternal sample. None of the control patients had detectable surface gene mutants.
HBV surface gene variants emerge or are selected under the immune pressure generated by the host or by administration of hepatitis B immune globulin and hepatitis B vaccination. An surface gene mutant (residue 129, Gln to Arg) found in one mother-infant pair suggested a direct maternal-infant transmission, resulting in immunoprophylaxis failure. None of the family members of children infected with Arg-145 variant had the same variant infection, implying this variant's low transmissability.[18],[19]
Viral Mutation after Antiviral Therapy table1
During antiviral therapy, mutation of HBV genome may develop to escape the drug effect. This event occurs mainly during therapy using nucleoside analogue, and not seen in interferon therapy. In an multicentered clinical trail in children, 191 children were randomly assigned to receive lamivudine and 97 to receive placebo. The rate of virologic response at week 52 was higher among children who received lamivudine than among those who received placebo (23% vs 13%). Lamivudine therapy was associated with higher rates of HBeAg seroconversion, normalization of alanine aminotransferase levels, and suppression of HBV DNA.[20] At week 52 after therapy, change of HBV polymerase gene codon 552, i.e. YMDD mutation (methionine to valine or isoleucine), was detected in 19% of patients in the lamivudine group, while none was found in the placebo group. The serum HBV DNA level tended to be higher at base line in the patients in whom the YMDD variant was present at 52 weeks. Demographic characteristics, aminotransferase levels, and histologic activities were similar between patients with YMDD-variant HBV DNA and patients with wild type sequence. In another study of lamivudine therapy in children of HBV carrier mothers, YMDD mutants developed in 34% of the lamivudine treated children after one year therapy.[21]
Conclusions
Children may be infected by mutant strains of HBV naturally during acute or fulminant hepatitis B, or infected by wild type HBV initially and develop mutant strains gradually during the course of chronic infection under the immune pressure of the host. In addition, infection with vaccine escape mutant strains may occur after immunoprophylaxis. Antiviral therapy using nucleoside analogues may also induce drug resistant mutant strains. Understanding the viral mutation status will help to design accurate strategies of immmunoprophylaxis and antiviral therapy against HBV infection.
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