HBV genotypes E and A deletions and recombination: two sides of the same

coin?

Penelope Garmiri1, Andre Loua2, Daniel Candotti3

and Jean-Pierre Allain1

1 Dept of Haematology, University of Cambridge2National Blood Transfusion Centre, Conakry, Guinea

3NHSBT, Cambridge

• HBV genotype E became prevalent in West Africa over the past 300 years

• HBsAg carriage ranging between 12% and 25% is the highest in the world

• In Guinea, the prevalence of HBsAg+ in 1st time blood donors is 15%

• Very high HBV exposure in population (75%)

HBV prevalence in Guinea, West Africa

Phylogenetic analysis of full genome sequences

from Guinea

• 81 full genome sequences

• Genotype A3 : 1 seq

• Genotype E : 77 seq

• Seq in blue: Recombinants

0.1

AY090454 Nicaragua HAY090457 Nicaragua H

X69798 Brazil FX75658 France F

X75663 Colombia FAB036910 Venezuela F

AB036920 Venezuela FM54923 Indonesia B

D23678 Japan BD50522 Japan B

D50519 Japan CM38636 Korea C

D23680 Japan CAB194949 Cameroon A3

GU1616*GU1607*

GH2537 Ghana*AM180623 Mali A4

GU1021^AY934764 Gambia A3

AB194952 Cameroon A3AB194951 Cameroon A3

AM180624 Cameroon A3AB194950 Cameroon A3

AM184126 Gabon A3AM184125 Gabon A3

AY934763 Gambia A3AF297624 South Africa A2

AF143302 A2DQ788725 Germany A2

AB116078 USA A2EF208113 Germany A2DQ020002 Congo A1

AY934772 Uganda A1AY934773 Tanzania A1AM494718 Cent African Republ A1

AY934766 Somalia A1AY934765 Somalia A1AB246336 South Africa A1

AY934771 Somalia A1AY903452 South Africa A1AY934767 Somalia A1

AY934768 Somalia A1AY934769 Somalia A1AY934770 Somalia A1

AB056515 USA GAB056513 USA G

AB064311 USA GGH16 Ghana*

AB033558 Japan D5AB048703 Australia D4

AY233291 S. Africa D3X80925 Greece D2

Z35716 Poland DAB104710 Egypt D1

X02496 Latvia DGU754*

GU763*AB091256 Cote Ivoire E

AB106564 Ghana E

X75657 France E

X75664 Senegal E

AB091255 Cote Ivoire E

77 Guinea E^

100

98

100

98

100

100

100

84

100

100

100

100

100

100

E

A3

A2

A1

7 Guinean strains with deletions in the core region

A

3222 bpEcoRI

Pre-core Core

1818 2454

Wild type

1 1979 2241262 bp

2 2314200749 bp 147 bp

4 2010 2235

78 bp37 bp

HyperL

adder

II

HyperL

adder

II

GU

489

GU

732

GU

1086

GU

1405

GU

1365

GU

1410

GU

1520

+ c

ontr

ol

- contr

ol

2000180016001400

1200

1000

800

700

600

500

400

300

Size (bp)

943862747681

Size (bp)

B

Recombinant strains

• Indication

– Differential clustering of sequences during assembling

– Unusual clustering of samples during phylogenetic

analysis

• Confirmation

– NCBI/ Genotyping function

– SIMPLOT software

Guinean recombinant strains

• GU1616, GU754, GU763: full genome

• GU1125, GU1127, GU1607: partial sequences

• GH16, GH2537: full genome from Ghana

• Further confirmation

- Cloning

Evidence of recombination between HBV

genotype E and A and E and D in

strains originating from Guinea and

Ghana

Red: genotype A

Blue: genotype E

Orange: genotype D

Schematic representation of Guinean and Ghanaian HBV A/E and E/D recombinant strains

GU754

GU763

GU1616

E EA

A E A

E EA1287 1896

23941542

1896 2419

GU1125 A E A24191896

11931143

GU1127

GH2537

GH16

A E A

D E D E D

A E A

2419

2419

1896

1019 17662120

1651 2406 30812823

GU1607 A E A24231906

0 500 1000 1500 2000 2500 3000

EcoR I

Pol Pol

X

pre-core core pre-S1pre-S2 S

880 1044

Schematic representation of clone diversity from a single plasma sample from Ghana (GH99)

¥ 3 A

¥ 2 E

¥ 1 E EA402 778

¥ 1 A E753

1270 bp

¥ 1 A E1018362

E

pre-S1

Clones

2,12, 22

4, 20

24

3

1

SIMPLOT analysis of GU1127 recombinant clones

SIMPLOT analysis of GU1607 recombinant clones

SIMPLOT analysis of GU1616 recombinant clones

Overview of the recombination and deletion points in West African strains

Deletions and recombinations occur in similar core regions suggesting preferential DNA re-arrangement in this area of

HBV genome

0 500 1000 1500 2000 2500 3000

EcoRI

RECs

DELs

Pol Pol

X

pre-core core pre-S1pre-S2 S

Viral Load distribution of HBsAg+ Guinean samples

• The ratio E/A3 is 77/1 but 7% of donors carry recombinant forms (RFs)

• Recombination points are identical in multiple strains

- Recombination occurs in the same positions of the HBV genome

and/or

- RFs are infectious (CRFs)

It most likely happened earlier when the balance of E and A genotypes in the population was different

• Viral Load of Rec samples is similarly distributed to the WT samples

Origin & epidemiology of Rec HBV in Guinea

Summary

• Genotype E is most prevalent in Guinea (93.9%)

• Core deletions in Guinea strains (8.5%); evidence of multiple populations

• Recombination events between genotypes E and A are frequent in Guinea (7.1%)

• Several E/A infectious recombinant forms might be circulating in the region

• Deletions and recombinations occur in the core region suggesting preferential DNA rearrangement in this area of HBV genome

Acknowledgements

• The staff of National Blood Transfusion Centre, Conakry for collecting and testing the donor samples

• Dr I.H.A. Barnes for determining the HBV BCP/PC sequences of the strains included in this study

• Dr B. Meldal for her input in the phylogenetic analysis

• Funding from National Health Service Blood and Transplant (NHSBT)