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Viral load distribution, BCP/PC mutations and HBV genotypes

Viral load distribution, BCP/PC mutations and HBV genotypes If Barnes, Daniel Candotti, Diane Doucet and Jean-Pierre Allain Division of Transfusion Medicine University of Cambridge, UK. Introduction. HBV viral load is dependent on the virus and / or the host

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Viral load distribution, BCP/PC mutations and HBV genotypes

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  1. Viral load distribution, BCP/PC mutations and HBV genotypes If Barnes, Daniel Candotti, Diane Doucet and Jean-Pierre Allain Division of Transfusion Medicine University of Cambridge, UK

  2. Introduction • HBV viral load is dependent on the virus and / or the host • Differences in viral load may be related to specific mutations within the HBV genome • Viral load differences within a genotype may reflect differences in host factors

  3. Aim • Provide information on HBV DNA load between different blood donor populations and relationship to genotype / host • Determine the world-wide genotype distribution in HBsAg +ve blood donors • Obtain and analyse full genome sequences

  4. Origin of samples studied and HBV genotypes A2/A1 A2 D B/C E F A1 A2/C

  5. Methods: amplification of HBV genome Q-PCR 0.1 Kb Pre-S-S 1.2 Kb Whole genome 3 Kb BCP/PC 0.3 Kb

  6. Strategy for HBV strain analysis Plasma HBsAg+ve 0.5 ml extraction Quantification BCP/PC whole genome Successful Unsuccessful Ultracentrifugation Sequence BCP/PC whole genome Successful BCP/PC 0.3 Kb whole genome 3 Kb Unsuccessful Pre-S-S 1.2 kb

  7. Viral load distribution 10 9 10 8 10 7 10 6 10 5 10 4 10 3 10 2 10 1 Limit of detection Ghana E Guinea E Tunisia D Turkey D Poland A2/D

  8. Distribution of viral load in genotype E with an assay sensitivity (10 IU/ml) Pool of 10 40 30 20 10 0 Ghana Guinea Percentage of total <10 101 102 103 104 105 106 107 108 109 Viral load IU/ml

  9. Distribution of viral load in genotype D with an assay sensitivity (10 IU/ml) Pool of 10 40 30 20 10 0 Tunisia Turkey Percentage of total <10 101 102 103 104 105 106 107 108 109 Viral load IU/ml

  10. Viral load compared to percentage of samples negative by Q-PCR Country Dominant Median % HBsAg +ve Pool of 10 genotype viral load & DNA -ve (10 IU/ml) (IU/ml) (10 IU/ml) Ghana E 200 4.4 43.9 Guinea E 36,600 0.4 10 Tunisia D 94 20.5 50.1 Turkey D 1,195 11.6 24.6 Poland A2/D 4,880 5.6 11.3

  11. Core Promoter and Pre-core mutations occurring prior to HBe conversion Pre Core mRNA Upper Regulatory Region Basal Core Promoter Pre-core Region Core Region 1785 1814 1742 1849 1901 GENOME POSITION bp G1896A A1762T G1764A Mutated ATG Deletions

  12. Summary of the BCP/PC mutations in Guinea (E) and Tunisia (D) Mutation GUINEA % TUNISIA % P Value (n=241) (n=173) (Chi-squared) A1762T/R/W/Y/C/G 12.48 24.86 0.0011 G1764A/R/T/K/C 11.61 45.67 0.0004 G1896A/R 33.61 75.72 <0.0001 1762/1764 double mutations 7.47 21.38 <0.0001 1762/1764/1896 triple mutations 6 16.18 0.0011 Pre-core start codon mutations 0.83 13.87 <0.0001

  13. HBV DNA load Vs G1896A mutation Guinea E Mann Whitney test P value <0.0001 Tunisia D Mann Whitney test P value 0.2072

  14. HBV DNA load Vs 1762/64 mutations Guinea E Mann Whitney test P value 0.0069 Tunisia D Mann Whitney test P value 0.0841

  15. Summary • Significant differences in viral load distribution were observed between genotypes and between populations of the same genotype • Significant differences in the frequency of several BCP/PC key mutations between different genotypes • In genotype E, but not in genotype D, 1762/64 double mutation and 1896 stop codon were significantly correlated with lower viral load

  16. Acknowledgements • Division of Transfusion Medicine, University of Cambridge, UK • Dr. Daniel Candotti • Dr. Diane Doucet • Prof. Jean-Pierre Allain • Dr. Shirley Owusu-Ofori, Transfusion Medicine Unit, Department of Medicine, Komfo Anokye Teaching Hospital, Ghana • Dr. Andre Loua, National Transfusion Centre, Conakry, Guinea • Prof. Kamel Boukef, National Transfusion Centre, Tunis, Tunisia • Prof. Onder Arslan, Department of Haematology, Ankara University, Turkey • Prof. Ewa Brojer, Institute of Haematology and Transfusion Medicine, Warsaw, Poland

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