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