Viral load distribution, BCP/PC mutations and HBV genotypes
If Barnes, Daniel Candotti, Diane Doucet and Jean-Pierre AllainDivision of Transfusion Medicine
University of Cambridge, UK
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
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
Origin of samples studied and HBV genotypes
A2
F
D
E
A1 A2/C
B/C
A2/A1
Whole genome
3 Kb
BCP/PC 0.3 Kb
Methods: amplification of HBV genome
Pre-S-S 1.2 Kb
Q-PCR 0.1 Kb
Strategy for HBV strain analysis
Plasma HBsAg+ve
0.5 ml extraction
Quantification
BCP/PCwhole genomeSuccessful Unsuccessful
Sequence
Ultracentrifugation
BCP/PCwhole genomeBCP/PC 0.3 Kb
whole genome 3 Kb
Successful
Unsuccessful
Pre-S-S 1.2 kb
Ghana E
GuineaE
TunisiaD
TurkeyD
PolandA2/D
101
102
103
104
105
106
107
108
109
Limit of detection
Viral load distribution
Distribution of viral load in genotype E with an assay sensitivity (10 IU/ml)
<10 101 102 103 104 105 106 107 108 109
40
30
20
10
0
Per
cent
age
of to
tal
Pool of 10
Ghana
Guinea
Viral load IU/ml
Distribution of viral load in genotype D with an assay sensitivity (10 IU/ml)
40
30
20
10
0
Per
cent
age
of to
tal
Pool of 10
Tunisia
Turkey
<10 101 102 103 104 105 106 107 108 109
Viral load IU/ml
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
Viral load compared to percentage of samples negative by Q-PCR
Basal Core Promoter Pre-core Region Core RegionUpper
RegulatoryRegion
1742 18491814 1901
Core Promoter and Pre-core mutations occurring prior to HBe conversion
Deletions
A1762T G1764A Mutated ATG G1896A
GENOME POSITIONbp
1785
Pre Core mRNA
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
Summary of the BCP/PC mutations
in Guinea (E) and Tunisia (D)
HBV DNA load Vs G1896A mutation
Mann Whitney test P value <0.0001
wt G1896A10
100
1000
10000
100000
1000000
1.0x1007
1.0x1008
1.0x1009
Mann Whitney test P value 0.2072
TunisiaD
GuineaE
wt G1896A1
10
100
1000
10000
100000
1000000
1.0x1007
1.0x1008
1.0x1009
Mann Whitney test
P value 0.0069
Mann Whitney test
P value 0.0841
HBV DNA load Vs 1762/64 mutations
wt 1762/641
10
100
1000
10000
100000
1000000
1.0x1007
1.0x1008
1.0x1009
wt 1762/6410
100
1000
10000
100000
1000000
1.0x1007
1.0x1008
1.0x1009
TunisiaD
GuineaE
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
Acknowledgements Division of Transfusion Medicine, University of Cambridge, UK
Dr. Daniel CandottiDr. Diane DoucetProf. 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