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Genotyping of acute HBV isolates from England, 1997–2001
Journal of Clinical Virology 44 (2009) 157–160
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Genotyping of acute HBV isolates from England, 1997–2001 Richard D. Sloan a,∗ , Angela L. Strang a , Mary E. Ramsay b , Chong-Gee Teo a a b
Virus Reference Division, Centre for Infections, Health Protection Agency, Colindale Avenue, London NW9 5HT, UK Communicable Disease Surveillance Centre, Centre for Infections, Health Protection Agency, Colindale Avenue, London NW9 5HT, UK
a r t i c l e
i n f o
Article history: Received 23 April 2008 Received in revised form 19 November 2008 Accepted 24 November 2008 Keywords: Hepatitis B virus Acute viral infection Viral genotype Phylogeny
a b s t r a c t Background: Increasing data shows the relevance of HBV genotypes in the outcome of infection. Most studies investigating the relationship between the genotypic characteristics of hepatitis B virus (HBV) and the clinical or epidemiological aspects of HBV infection originate from studies of patients with chronic rather than acute hepatitis B. Objectives: To study a convenience sample representing ca. 5% of reported acute hepatitis B in England between 1997 and 2001 to investigate the distribution of HBV genotypes and specific HBV variants with epidemiological risk factors, thereby providing baseline data for ongoing surveillance. Study design: From 160 serum samples, PCR was carried out to amplify the first 600 bases of the HBV S gene. Amplicons were sequenced and subjected to phylogenetic analysis and risk factor analysis. Results: Fifty-seven percent of the study samples carried HBV belonging to subtype A2, 13% to subtype D2, and the rest to genotype E (8%) and subtypes C2 and D3 (each 6%), D1 and D4 (each 3%) and B4 (1%). One particular A2 isolate was dominant, accounting for 23% of the total sample set. Drug use and homosexual transmission were equally implicated as risks within genotype A2. No mutations associated with vaccine escape or resistance to antiviral therapy were identified. Conclusion: Immigration and travel likely shape the observed genotype distribution and consequent prevalence of genotypes other than A2 or D in this population. Data suggests no genetic separation of parenteral and sexually transmitted virus. These data demonstrate the value in pursuing more extensive and recent surveillance. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) has been classified into 8 genotypes, designated A to G. These genotypes have been associated with outcomes of chronic hepatitis B such as progression to severe disease and development of liver cancer, and response to antiviral therapy based on interferons and analogues of nucleotides or nucleosides.1,2 Knowledge of the HBV genotype distribution in patients with chronic hepatitis B may aid the formulation and implementation of initiatives to lessen the morbidity and mortality burden with which it is associated. Studies in Europe enquiring into the distribution of HBV genotypes have been helpful in this regard.3–5 From a broader public health perspective, it would be important also to determine, in acutely infected people, how particular genotypes and strains of HBV are being transmitted within specific risk groups. Presented here are findings from an investigation to describe HBV genotypes, epidemiological risk factors
∗ Corresponding author at: McGill AIDS Centre, Lady Davis Institute, 3755 CôteSte-Catherine, Montréal, Québec H3T 1E2, Canada. Tel.: +1 514 340 8222; fax: +1 514 340 7537. E-mail address: [email protected] (R.D. Sloan). 1386-6532/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2008.11.010
and specific HBV variants associated with acute hepatitis B in England. 2. Materials and methods Study samples were assembled from hepatitis B surface antigen-positive sera of patients with suspected acute hepatitis B testing between January 1997 and December 2001 to the Virus Reference Division (VRD) of the Centre for Infections of the Health Protection Agency (HPA). All samples had been referred to the HPA for tertiary reference analysis. Samples were included into the study if they had been tested by the VRD to contain >200 Paul Erlich Institute International Units per ml of IgM to the anti-hepatitis B core antigen. Two hundred and twenty four samples satisfied this criterion and were thus included for analysis. Risk factors (where available), patient gender and age were also recorded from patient records provided by referring clinicians and by screening of HPA epidemiological databases. From these samples, DNA from the HBV S gene was amplified using nested PCR. Primers for first-round PCR were 5 -AGCCCTCAGGCTCAGGGCATA-3 (outer, sense) and 5 -AAACCCAGAAGACCCACA-3 (outer, antisense), and the second-round primers were 5 -TCATCCTCAGGCCATGCAGT-3 (inner, sense) and 5 -ACACACTTTCCAATCAATAG-3 (outer, sense).
158
R.D. Sloan et al. / Journal of Clinical Virology 44 (2009) 157–160
Fig. 1. Neighbour-joining phylogenetic analysis of the 1st 600 nucleotides of HBV S gene for 160 sequences studied. Genbank accession numbers of representative reference sequences are given. Genotype and sub-genotype assignments are shown at right. Multiple identical sequences identified are collapsed to single lines, with the number of each set of identical sequences shown in parentheses. HBVPV is specifically identified and shaded grey.
R.D. Sloan et al. / Journal of Clinical Virology 44 (2009) 157–160
159
Table 1 Risk factor/epidemiological data distribution throughout genotypes. Percentages of total risk factors for specific genotypes are also indicated. IVDU = Intravenous drug user. MSM = men who have sex with men. GUM attendee = genito-urinary medicine clinic attendee. Risk Factor/epidemiological data
Genotype A
IVDU MSM Travel to/lived in endemic area Heterosexual contact Surgical GUM attendee Health care exposure Incarceration Skin piercing Transfusion
12 (92%) 13 (93%) 8 (33%) 6 (38%)
Total B
1 (4%)
1 (50%) 1 (50%) 1 (50%) 2 (67%)
EXPAND DNA Polymerase (Roche) was used in both PCRs. The final amplicon encompassed the first 600 nucleotides of the S gene. Purified PCR products were sequenced using 4 primers; 5 TCATCCTCAGGCCATGCAGT-3 , (antisense), 5 -TATCAAGGAATTCTGCCCGTTTGTCCT-3 (antisense), 5 -ACTGAGCCAAGAGAAACGGGCTGAG-3 (sense) and 5 -ACACACTTTCCAATCAATAG-3 (sense). Raw chromatograph data were analysed using the SeqMan sequence analysis software in the LASARGENE analysis package (DNAstar Inc.). Multiple alignments against GenBank sequences representing known HBV genotypes and subtypes6 were made using Clustal W, analysed with DNADIST and NEIGHBOR within PHYLIP7 and neighbour-joining dendrograms visualised using TREEVIEW.8 Sequences from study samples were checked individually for presence of mutations identified previously to be associated with antibody escape or resistance to lamivudine.9,10
3. Results One hundred and sixty of 224 (71%) serum samples were found to be HBV DNA PCR positive. The mean age of the 160 patients that were HBV DNA-positive was 36 years (range: 1 month to 87 years). 68% of the patients were male, 27% were female, and for 5% no gender data were provided. Patients were predominantly from Southeast England. Data relating to risk factors associated with hepatitis B were available for 70 (44%) patients. Risk factors reported were: travel to or having lived in endemic area (28%), heterosexual contact (19%), men who have sex with men [MSM] (17%), intravenous drug use [IVDU] (16%), surgery (5%), undefined health care exposure (5%), blood transfusion (4%), undefined sexual contact (2%), skin piercing (2%), and prison incarceration (2%). The outcome of phylogenetic analysis of HBV S gene sequences is presented in Fig. 1. HBV carried in the samples could be assigned to A1 (6%), A2 (57%), B4 (1%), C2 (6%), D1 (3%), D2 (13%), D3 (6%), D4 (3%) and E (8%). HBV belonging to genotypes F, G and H were not evident. Significantly, one particular A2 sequence comprised 23% of the total sample set. That common sequence is identical to the “prisoner variant” of HBV (HBVPV ) previously characterized11 (GenBank reference: AY374226). Upon allocation of genotype to HBV sequences an analysis of distribution of risk factors throughout genotypes was performed (Table 1). Due to sample size limitations this analysis was not extended to sub-genotypes. For HBVPV 12/36 patients had reported risk factors. Therefore HBVPV risk factors were as follows: 3/12 MSM, 5/12 IVDU, 3/12 ‘travel to/lived in endemic country’, 1/12 transfusion.
C
6 (25%) 3 (19%)
D 1 (8%) 1 (7%) 7 (29%) 5 (31%) 3 (75%) 1 (50%) 4 (100%) 1 (50%)
E
2 (8%) 2 (13%) 1 (25%)
1 (50%) 1 (33%)
F
G
H 13 14 24 16 4 2 4 2 2 3 84
4. Discussion The A2 sub-genotype was the most dominant in serum sample set derived from cases of acute hepatitis B. Previous studies have identified A2 to be the most common HBV sub-genotype in North America and Europe.6 A study of the Scottish blood donors over a similar period found a lower prevalence (41%) of genotype A infections.4 Our previous study of 266 patients with acute hepatitis B in England and Wales sampled between 1990 and 1996 found the A2 variant HBVPV to account for 41% of acute infections.11 This study suggests that HBVPV continues to be the major HBV strain being transmitted in England. While the previous study implicated injecting drug use and prison incarceration as risk factors for the acquisition of HBVPV the presence of this variant in MSMs and a transfusion recipient suggests it has spread to the wider community. The D2 sub-genotype is second most dominant. Studies suggest that it co-circulates with A2 in North America and Europe.6 The overall prevalence of genotype D in our study (24%) was lower than has been reported from Scotland (30%).4 About a fifth of the present set of study samples were found to carry sub-genotypes A1 and subtypes of B, C and E, which are associated with infection in people indigenous to East Asia (B and C) and sub-Saharan Africa (A1 and E).6 A previous study has documented immigration as playing an important role in introducing de novo chronic hepatitis B into England and Wales.12 Data from the Netherlands suggests that immigration is responsible for the genotype distribution observed in Western European populations.13 It is probable that such carriers are reservoirs of transmission of the minority HBV types, as reflected in travel to or having lived in endemic area being reported as the risk factor in 28% of the cases. Nonetheless, the possibility cannot be excluded that acute infection by these types is due to acquisition from travel to the regions where they are prevalent. A major feature of note in our analysis is the lack of segregation between the MSM and IVDU risk factors, both found within sub-genotype A2. This finding is in contrast to data from the Netherlands13 and indicates a lack of genetic distinction between virus found in both risk groups in our study. No HBV with iatrogenic mutations could be identified. Specifically, antibody-escape mutations in the a determinant of the S gene10 and mutations in the overlapping P gene associated with resistance to antivirals14 were not observed. Such absence reflects the rarity of reservoirs carrying the mutants, their poor transmissibility, or the weakness of the selection pressure that was being exerted that would permit emergence of the mutants. During the period that study samples were collected, the U.K. was adopting a selective policy towards vaccination against hepatitis B, and
160
R.D. Sloan et al. / Journal of Clinical Virology 44 (2009) 157–160
the therapeutic use of lamivudine was not yet widespread. Other studies have used sensitive assays to identify antiviral resistanceassociated mutations of the P gene in untreated patients,15 but our current study was not focussed on identifying minority mutants. A description of the HBV genotypes and specific HBV variants associated with acute hepatitis B in England 1997–2001 is here provided. Between 1995 and 2000, an annual average of 673 cases of laboratory-confirmed acute HBV infection was reported to the HPA.10 Therefore, our study sampled a small proportion (ca. 5%) of the total number of reported acute hepatitis B cases. Nonetheless, the scale of this study is in keeping with other studies of this nature.3–5,12 A sample bias in our dataset may be possible as samples tended to be from South-Eastern England and the dataset also consisted of samples which required tertiary testing. Thus, the sample may not be fully representative of the overall molecular epidemiology of acute hepatitis B in England, limiting the potential to generalise these results. Nonetheless, our approach demonstrates the value of more extensive ongoing surveillance involving more recent cases of acute hepatitis B, with a view to monitor the changing spectrum of HBV genotypes and sub-genotypes associated with incident hepatitis B to track the emergence of iatrogenic and other unusual HBV strains. This activity requires to be further strengthened by accurate reporting and analysis of patient risk factors. Conflict of interest No conflicts of interest have been identified. Acknowledgements These studies were funded by the Health Protection Agency, England. R.D.S. was funded in part by a European Community 5th Framework grant (HepBVaR QLRT-2001-00977).
References 1. Schaefer S. Hepatitis B virus: significance of genotypes. J Viral Hepat 2005;12:111–24. 2. Palumbo E. Hepatitis B genotypes and response to antiviral therapy: a review. Am J Ther 2007;4:306–9. 3. Bielawski KP, Dybikowska A, Lisowska-Charmuszko U, Stalke P, Gregorowicz K, Trocha H, et al. Distribution of HBV genotypes and mutants among hepatitis B infected patients from northern Poland. Int J Mol Med 2004;2: 301–4. 4. Davidson F, Lycett C, Sablon E, Petrik J, Dow BC. Hepatitis B virus genotypes and precore mutations in Scottish blood donors. Vox Sang 2005;88:87–92. 5. Echevarría JM, Avellón A, Magnius LO. Molecular epidemiology of hepatitis B virus in Spain: identification of viral genotypes and prediction of antigenic subtypes by limited sequencing. J Med Virol 2005;76:176–84. 6. Norder H, Couroucé AM, Coursaget P, Echevarria JM, Lee SD, Mushahwar IK, et al. Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, sub-genotypes, and HBsAg subtypes. Intervirology 2004;47:289–309. 7. Felsenstein J. PHYLIP—phylogeny inference package (version 3.2). Cladistics 1989;5:164–6. 8. Page RDM. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 1996;12:357–8. 9. Hepatitis B Virus Drug Resistance Working GroupLok AS, Zoulim F, Locarnini S, Bartholomeusz A, Ghany MG, et al. Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management. Hepatology 2007;46:254–65. 10. Ngui SL, Hallet R, Teo CG. Natural and iatrogenic variation in hepatitis B virus. Rev Med Virol 1999;9:183–209. 11. Hallett RL, Ngui SL, Meigh RE, Mutton KJ, Boxall EH, Teo CG. Widespread dissemination in England of a stable and persistent hepatitis B virus isolate. Clin Infect Dis 2004;39:945–52. 12. Hahne S, Ramsay M, Balogun K, Edmunds WJ, Mortimer P. Incidence and routes of transmission of hepatitis B virus in England and Wales, 1995–2000: implications for immunisation policy. J Clin Virol 2004;29:211–20. 13. van Steenbergen JE, Niesters HG, Op de Coul EL, van Doornum GJ, Osterhaus AD, Leentvaar-Kuijpers A, et al. Molecular epidemiology of hepatitis B virus in Amsterdam 1992–1997. J Med Virol 2002;66:159–65. 14. Sloan RD, Ijaz S, Moore PL, Harrison TJ, Teo CG, Tedder RS. Antiviral resistance mutations potentiate hepatitis B virus immune evasion through disruption of its surface antigen a determinant epitopes. Antivir Ther 2008;13:439– 47. 15. Punia P, Cane P, Teo CG, Saunders N. Quantitation of hepatitis B lamivudine resistant mutants by real-time amplification refractory mutation system PCR. J Hepatol 2004;40:986–92.
Contents lists available at ScienceDirect
Journal of Clinical Virology journal homepage: www.elsevier.com/locate/jcv
Short communication
Genotyping of acute HBV isolates from England, 1997–2001 Richard D. Sloan a,∗ , Angela L. Strang a , Mary E. Ramsay b , Chong-Gee Teo a a b
Virus Reference Division, Centre for Infections, Health Protection Agency, Colindale Avenue, London NW9 5HT, UK Communicable Disease Surveillance Centre, Centre for Infections, Health Protection Agency, Colindale Avenue, London NW9 5HT, UK
a r t i c l e
i n f o
Article history: Received 23 April 2008 Received in revised form 19 November 2008 Accepted 24 November 2008 Keywords: Hepatitis B virus Acute viral infection Viral genotype Phylogeny
a b s t r a c t Background: Increasing data shows the relevance of HBV genotypes in the outcome of infection. Most studies investigating the relationship between the genotypic characteristics of hepatitis B virus (HBV) and the clinical or epidemiological aspects of HBV infection originate from studies of patients with chronic rather than acute hepatitis B. Objectives: To study a convenience sample representing ca. 5% of reported acute hepatitis B in England between 1997 and 2001 to investigate the distribution of HBV genotypes and specific HBV variants with epidemiological risk factors, thereby providing baseline data for ongoing surveillance. Study design: From 160 serum samples, PCR was carried out to amplify the first 600 bases of the HBV S gene. Amplicons were sequenced and subjected to phylogenetic analysis and risk factor analysis. Results: Fifty-seven percent of the study samples carried HBV belonging to subtype A2, 13% to subtype D2, and the rest to genotype E (8%) and subtypes C2 and D3 (each 6%), D1 and D4 (each 3%) and B4 (1%). One particular A2 isolate was dominant, accounting for 23% of the total sample set. Drug use and homosexual transmission were equally implicated as risks within genotype A2. No mutations associated with vaccine escape or resistance to antiviral therapy were identified. Conclusion: Immigration and travel likely shape the observed genotype distribution and consequent prevalence of genotypes other than A2 or D in this population. Data suggests no genetic separation of parenteral and sexually transmitted virus. These data demonstrate the value in pursuing more extensive and recent surveillance. © 2008 Elsevier B.V. All rights reserved.
1. Introduction Hepatitis B virus (HBV) has been classified into 8 genotypes, designated A to G. These genotypes have been associated with outcomes of chronic hepatitis B such as progression to severe disease and development of liver cancer, and response to antiviral therapy based on interferons and analogues of nucleotides or nucleosides.1,2 Knowledge of the HBV genotype distribution in patients with chronic hepatitis B may aid the formulation and implementation of initiatives to lessen the morbidity and mortality burden with which it is associated. Studies in Europe enquiring into the distribution of HBV genotypes have been helpful in this regard.3–5 From a broader public health perspective, it would be important also to determine, in acutely infected people, how particular genotypes and strains of HBV are being transmitted within specific risk groups. Presented here are findings from an investigation to describe HBV genotypes, epidemiological risk factors
∗ Corresponding author at: McGill AIDS Centre, Lady Davis Institute, 3755 CôteSte-Catherine, Montréal, Québec H3T 1E2, Canada. Tel.: +1 514 340 8222; fax: +1 514 340 7537. E-mail address: [email protected] (R.D. Sloan). 1386-6532/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2008.11.010
and specific HBV variants associated with acute hepatitis B in England. 2. Materials and methods Study samples were assembled from hepatitis B surface antigen-positive sera of patients with suspected acute hepatitis B testing between January 1997 and December 2001 to the Virus Reference Division (VRD) of the Centre for Infections of the Health Protection Agency (HPA). All samples had been referred to the HPA for tertiary reference analysis. Samples were included into the study if they had been tested by the VRD to contain >200 Paul Erlich Institute International Units per ml of IgM to the anti-hepatitis B core antigen. Two hundred and twenty four samples satisfied this criterion and were thus included for analysis. Risk factors (where available), patient gender and age were also recorded from patient records provided by referring clinicians and by screening of HPA epidemiological databases. From these samples, DNA from the HBV S gene was amplified using nested PCR. Primers for first-round PCR were 5 -AGCCCTCAGGCTCAGGGCATA-3 (outer, sense) and 5 -AAACCCAGAAGACCCACA-3 (outer, antisense), and the second-round primers were 5 -TCATCCTCAGGCCATGCAGT-3 (inner, sense) and 5 -ACACACTTTCCAATCAATAG-3 (outer, sense).
158
R.D. Sloan et al. / Journal of Clinical Virology 44 (2009) 157–160
Fig. 1. Neighbour-joining phylogenetic analysis of the 1st 600 nucleotides of HBV S gene for 160 sequences studied. Genbank accession numbers of representative reference sequences are given. Genotype and sub-genotype assignments are shown at right. Multiple identical sequences identified are collapsed to single lines, with the number of each set of identical sequences shown in parentheses. HBVPV is specifically identified and shaded grey.
R.D. Sloan et al. / Journal of Clinical Virology 44 (2009) 157–160
159
Table 1 Risk factor/epidemiological data distribution throughout genotypes. Percentages of total risk factors for specific genotypes are also indicated. IVDU = Intravenous drug user. MSM = men who have sex with men. GUM attendee = genito-urinary medicine clinic attendee. Risk Factor/epidemiological data
Genotype A
IVDU MSM Travel to/lived in endemic area Heterosexual contact Surgical GUM attendee Health care exposure Incarceration Skin piercing Transfusion
12 (92%) 13 (93%) 8 (33%) 6 (38%)
Total B
1 (4%)
1 (50%) 1 (50%) 1 (50%) 2 (67%)
EXPAND DNA Polymerase (Roche) was used in both PCRs. The final amplicon encompassed the first 600 nucleotides of the S gene. Purified PCR products were sequenced using 4 primers; 5 TCATCCTCAGGCCATGCAGT-3 , (antisense), 5 -TATCAAGGAATTCTGCCCGTTTGTCCT-3 (antisense), 5 -ACTGAGCCAAGAGAAACGGGCTGAG-3 (sense) and 5 -ACACACTTTCCAATCAATAG-3 (sense). Raw chromatograph data were analysed using the SeqMan sequence analysis software in the LASARGENE analysis package (DNAstar Inc.). Multiple alignments against GenBank sequences representing known HBV genotypes and subtypes6 were made using Clustal W, analysed with DNADIST and NEIGHBOR within PHYLIP7 and neighbour-joining dendrograms visualised using TREEVIEW.8 Sequences from study samples were checked individually for presence of mutations identified previously to be associated with antibody escape or resistance to lamivudine.9,10
3. Results One hundred and sixty of 224 (71%) serum samples were found to be HBV DNA PCR positive. The mean age of the 160 patients that were HBV DNA-positive was 36 years (range: 1 month to 87 years). 68% of the patients were male, 27% were female, and for 5% no gender data were provided. Patients were predominantly from Southeast England. Data relating to risk factors associated with hepatitis B were available for 70 (44%) patients. Risk factors reported were: travel to or having lived in endemic area (28%), heterosexual contact (19%), men who have sex with men [MSM] (17%), intravenous drug use [IVDU] (16%), surgery (5%), undefined health care exposure (5%), blood transfusion (4%), undefined sexual contact (2%), skin piercing (2%), and prison incarceration (2%). The outcome of phylogenetic analysis of HBV S gene sequences is presented in Fig. 1. HBV carried in the samples could be assigned to A1 (6%), A2 (57%), B4 (1%), C2 (6%), D1 (3%), D2 (13%), D3 (6%), D4 (3%) and E (8%). HBV belonging to genotypes F, G and H were not evident. Significantly, one particular A2 sequence comprised 23% of the total sample set. That common sequence is identical to the “prisoner variant” of HBV (HBVPV ) previously characterized11 (GenBank reference: AY374226). Upon allocation of genotype to HBV sequences an analysis of distribution of risk factors throughout genotypes was performed (Table 1). Due to sample size limitations this analysis was not extended to sub-genotypes. For HBVPV 12/36 patients had reported risk factors. Therefore HBVPV risk factors were as follows: 3/12 MSM, 5/12 IVDU, 3/12 ‘travel to/lived in endemic country’, 1/12 transfusion.
C
6 (25%) 3 (19%)
D 1 (8%) 1 (7%) 7 (29%) 5 (31%) 3 (75%) 1 (50%) 4 (100%) 1 (50%)
E
2 (8%) 2 (13%) 1 (25%)
1 (50%) 1 (33%)
F
G
H 13 14 24 16 4 2 4 2 2 3 84
4. Discussion The A2 sub-genotype was the most dominant in serum sample set derived from cases of acute hepatitis B. Previous studies have identified A2 to be the most common HBV sub-genotype in North America and Europe.6 A study of the Scottish blood donors over a similar period found a lower prevalence (41%) of genotype A infections.4 Our previous study of 266 patients with acute hepatitis B in England and Wales sampled between 1990 and 1996 found the A2 variant HBVPV to account for 41% of acute infections.11 This study suggests that HBVPV continues to be the major HBV strain being transmitted in England. While the previous study implicated injecting drug use and prison incarceration as risk factors for the acquisition of HBVPV the presence of this variant in MSMs and a transfusion recipient suggests it has spread to the wider community. The D2 sub-genotype is second most dominant. Studies suggest that it co-circulates with A2 in North America and Europe.6 The overall prevalence of genotype D in our study (24%) was lower than has been reported from Scotland (30%).4 About a fifth of the present set of study samples were found to carry sub-genotypes A1 and subtypes of B, C and E, which are associated with infection in people indigenous to East Asia (B and C) and sub-Saharan Africa (A1 and E).6 A previous study has documented immigration as playing an important role in introducing de novo chronic hepatitis B into England and Wales.12 Data from the Netherlands suggests that immigration is responsible for the genotype distribution observed in Western European populations.13 It is probable that such carriers are reservoirs of transmission of the minority HBV types, as reflected in travel to or having lived in endemic area being reported as the risk factor in 28% of the cases. Nonetheless, the possibility cannot be excluded that acute infection by these types is due to acquisition from travel to the regions where they are prevalent. A major feature of note in our analysis is the lack of segregation between the MSM and IVDU risk factors, both found within sub-genotype A2. This finding is in contrast to data from the Netherlands13 and indicates a lack of genetic distinction between virus found in both risk groups in our study. No HBV with iatrogenic mutations could be identified. Specifically, antibody-escape mutations in the a determinant of the S gene10 and mutations in the overlapping P gene associated with resistance to antivirals14 were not observed. Such absence reflects the rarity of reservoirs carrying the mutants, their poor transmissibility, or the weakness of the selection pressure that was being exerted that would permit emergence of the mutants. During the period that study samples were collected, the U.K. was adopting a selective policy towards vaccination against hepatitis B, and
160
R.D. Sloan et al. / Journal of Clinical Virology 44 (2009) 157–160
the therapeutic use of lamivudine was not yet widespread. Other studies have used sensitive assays to identify antiviral resistanceassociated mutations of the P gene in untreated patients,15 but our current study was not focussed on identifying minority mutants. A description of the HBV genotypes and specific HBV variants associated with acute hepatitis B in England 1997–2001 is here provided. Between 1995 and 2000, an annual average of 673 cases of laboratory-confirmed acute HBV infection was reported to the HPA.10 Therefore, our study sampled a small proportion (ca. 5%) of the total number of reported acute hepatitis B cases. Nonetheless, the scale of this study is in keeping with other studies of this nature.3–5,12 A sample bias in our dataset may be possible as samples tended to be from South-Eastern England and the dataset also consisted of samples which required tertiary testing. Thus, the sample may not be fully representative of the overall molecular epidemiology of acute hepatitis B in England, limiting the potential to generalise these results. Nonetheless, our approach demonstrates the value of more extensive ongoing surveillance involving more recent cases of acute hepatitis B, with a view to monitor the changing spectrum of HBV genotypes and sub-genotypes associated with incident hepatitis B to track the emergence of iatrogenic and other unusual HBV strains. This activity requires to be further strengthened by accurate reporting and analysis of patient risk factors. Conflict of interest No conflicts of interest have been identified. Acknowledgements These studies were funded by the Health Protection Agency, England. R.D.S. was funded in part by a European Community 5th Framework grant (HepBVaR QLRT-2001-00977).
References 1. Schaefer S. Hepatitis B virus: significance of genotypes. J Viral Hepat 2005;12:111–24. 2. Palumbo E. Hepatitis B genotypes and response to antiviral therapy: a review. Am J Ther 2007;4:306–9. 3. Bielawski KP, Dybikowska A, Lisowska-Charmuszko U, Stalke P, Gregorowicz K, Trocha H, et al. Distribution of HBV genotypes and mutants among hepatitis B infected patients from northern Poland. Int J Mol Med 2004;2: 301–4. 4. Davidson F, Lycett C, Sablon E, Petrik J, Dow BC. Hepatitis B virus genotypes and precore mutations in Scottish blood donors. Vox Sang 2005;88:87–92. 5. Echevarría JM, Avellón A, Magnius LO. Molecular epidemiology of hepatitis B virus in Spain: identification of viral genotypes and prediction of antigenic subtypes by limited sequencing. J Med Virol 2005;76:176–84. 6. Norder H, Couroucé AM, Coursaget P, Echevarria JM, Lee SD, Mushahwar IK, et al. Genetic diversity of hepatitis B virus strains derived worldwide: genotypes, sub-genotypes, and HBsAg subtypes. Intervirology 2004;47:289–309. 7. Felsenstein J. PHYLIP—phylogeny inference package (version 3.2). Cladistics 1989;5:164–6. 8. Page RDM. TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 1996;12:357–8. 9. Hepatitis B Virus Drug Resistance Working GroupLok AS, Zoulim F, Locarnini S, Bartholomeusz A, Ghany MG, et al. Antiviral drug-resistant HBV: standardization of nomenclature and assays and recommendations for management. Hepatology 2007;46:254–65. 10. Ngui SL, Hallet R, Teo CG. Natural and iatrogenic variation in hepatitis B virus. Rev Med Virol 1999;9:183–209. 11. Hallett RL, Ngui SL, Meigh RE, Mutton KJ, Boxall EH, Teo CG. Widespread dissemination in England of a stable and persistent hepatitis B virus isolate. Clin Infect Dis 2004;39:945–52. 12. Hahne S, Ramsay M, Balogun K, Edmunds WJ, Mortimer P. Incidence and routes of transmission of hepatitis B virus in England and Wales, 1995–2000: implications for immunisation policy. J Clin Virol 2004;29:211–20. 13. van Steenbergen JE, Niesters HG, Op de Coul EL, van Doornum GJ, Osterhaus AD, Leentvaar-Kuijpers A, et al. Molecular epidemiology of hepatitis B virus in Amsterdam 1992–1997. J Med Virol 2002;66:159–65. 14. Sloan RD, Ijaz S, Moore PL, Harrison TJ, Teo CG, Tedder RS. Antiviral resistance mutations potentiate hepatitis B virus immune evasion through disruption of its surface antigen a determinant epitopes. Antivir Ther 2008;13:439– 47. 15. Punia P, Cane P, Teo CG, Saunders N. Quantitation of hepatitis B lamivudine resistant mutants by real-time amplification refractory mutation system PCR. J Hepatol 2004;40:986–92.