hepatitis b virus genetic diversity in spain. · 2007. 10. 18. · test 1 test1new 145a (1993-01)...

Hepatitis B Virus Geneticdiversity in Spain.

Hepatitis Reference Laboratory

National Centre of Microbiology

Ana Avellón & José Manuel Echevarría

http://images.google.es/imgres?imgurl=http://protist.i.hosei.ac.jp/virus/virusalbum/virusalbum/enlargement/B/HBV-2.JPG&imgrefurl=http://protist.i.hosei.ac.jp/virus/virusalbum/virusalbum/enlargement/B/HBV-2B&h=176&w=200&sz=11&tbnid=Vj1-y0LXXPcJ:&tbnh=87&tbnw=99&start=82&prev=/images%3Fq%3Dhbv%26start%3D80%26hl%3Des%26lr%3D%26rls%3DGGLD,GGLD:2004-47,GGLD:es%26sa%3DN

VHB, Hepadnaviridae

nveloped -ddDNA, circularised 3200nt


VHB, Genome

Pre S1, Pre S2, S

XP

C

4 ORF 7 proteins

HBxAgViral replicationtransactivator

HBcAgCoreProtein

HBeAgDNA-polymerase-RT

HBsAg, gp36 and gp42Neutralization, Join to the receptor


VHB, Genome

Pre S1, Pre S2, S

XP

C

GATTG CTG TLeu LeuMetVal

GA

TGT TGC TGTCys Cys Cys

HBsAg Polymerase

VHB, Genome

Pre S1, Pre S2, S

XP

C

4 ORF 7 proteins

HBxAgViral replicationtransactivator

HBcAgCoreProtein

HBeAgDNA-polymerase-RT

HBsAg, gp36 and gp42Neutralization, Join to the receptor


VHB, Genome

4 ORF 7 proteins

Pre S1, Pre S2, S

XP

C

•Genotypes•Antigenic subtypes•Variants

PolimeraseHBsAgCoreX Gene

High geneticvariability

(10%)


VHB, Genotypes & antigenic subtypes115

116

118

120

122

124

126

129

130

133

134

144145

112

212

121

135

146

139

137

140141

142

147148

149

151

152

153

155156

157

158

159

160

161

162

163

143

113

114

117

119

123

125

127

128

131

132

136

138

150

154

164

165

166

167

168

169

HBsAga

y/dw/r

PRATFPRayr

PKGS/TF/YL/IRayw4

PKGTYTRayw3

PKGTYPRayw2

PKATFPRayw1

PRVTFPKadrq-

PRATFPKadr

QKGSFLKadw4q-

PKGTYTKadw3

PKATFPKadw2

178160159140134127122Subtype

PRATFPRayr

PKGS/TF/YL/IRayw4

PKGTYTRayw3

PKGTYPRayw2

PKATFPRayw1

PRVTFPKadrq-

PRATFPKadr

QKGSFLKadw4q-

PKGTYTKadw3

PKATFPKadw2

178160159140134127122Subtype

Genotypes

Antigenic subtypes


VHB, Genotypes & antigenic subtypes A adw2ayw1B

adw2adw3ayw1

C

adrq-adrq+adw2adw3 ayrayw3

Dadw3ayw2ayw3ayw4

E ayw4

Fadw2adw4ayw4

G

H adw4

adw2

A adw2ayw1

Badw2adw3ayw1

C

adrq-adrq+adw2adw3 ayrayw3

Dadw3ayw2ayw3ayw4

E ayw4

Fadw2adw4ayw4

G

H adw4

adw2

B/adw2, ayw1C/adw, adr, ayrB/adw2, ayw1C/adw, adr, ayrE/ayw4E/ayw4A/adw2; D/ayw2-3

A/ayw1; D/ayw2 F/adw4; HF/adw4; H D/ayw3 C/adrq-, ayw3


VHB, Variants

Pre S1, Pre S2, S

XP

C

HBxAgX gene variants:Anomalous replicationand expression ofantigens. Inefficientimmune stimulation.

HBeAgPre-core defectivesvariants : Truncatedproteins due to codon

stops. Anomalousexpression of HBeAg.

Bad prognosis

DNA-polymerase-RTAntiviral resistance

Immunotherapy resistance

Vaccine failures

HBsAg detection failures

HBsAg, gp36 , gp42148

147149

146 150136

115

116

118

120

122

124

126

129

130

133

134

145 151137135 144

112

212

121

139

140141

142

152138 143

153

132 154

155131 156113

157114169

158

128 168 159117

127 167 160

119166 161

125165 162

163123 164


Methods

Pre S1, Pre S2, S

XP

C

(León et al 2004)Nested PCR

303ntDirect sequencing

•Genotypes•Antigenic subtypes•Variants

PolymeraseHBsAg

http://tppmsgs/msgs20.htm

HBV GENOTYPES & ANTIGENIC SUBTYPES IN

SPAIN

0

C

H F

E

B

G

A1

A2

D

Gibbon Echevarria et al 2006


HBV GENOTYPES & ANTIGENIC SUBTYPES IN

SPAIN

Gibbon Echevarria et al 2006

Genotype Predicted HBsAg subtype A B C D E F Total adw2 57 2 59 adw3 2 2

adw4q+ 4a 4 adw4q- 5 5

adr 3 3 adrq- 1 1 ayw1 5b 1 6 ayw2 81c 81 ayw3 77d 77 ayw4 13 9 22 ayw? 3 3 ayr 1 2 1 4

a-w? 1 1 2

0

C

H F

E

B

G

A1

A2

D

0

C

H F

E

B

G

A1

A2

D


05

101520253035404550556065707580859095

100105

Pol

469>

S11

3

Pol

471>

S11

5

Pol

473>

S11

7

Pol

475>

S11

9

Pol

477>

S12

1

Pol

479>

S12

3

Pol

481>

S12

5

Pol

483>

S12

7

Pol

485>

S12

9

Pol

487>

S13

1

Pol

489>

S13

3

Pol

491>

S13

5

Pol

493>

S13

7

Pol

495>

S13

9

Pol

497>

S14

1

Pol

499>

S14

3

Pol

501>

S14

5

Pol

503>

S14

7

Pol

505>

S14

9

Pol

507>

S15

1

Pol

509>

S15

3

Pol

511>

S15

5

Pol

513>

S15

7

Pol

515>

S15

9

Pol

517>

S16

1

Pol

519>

S16

3

Pol

521>

S16

5

Pol

523>

S16

7

Pol

525>

S16

9

Pol

527>

S17

1

Pol

529>

S17

3

Pol

531>

S17

5

Pol

533>

S17

7

Pol

535>

S17

9

Pol

537>

S18

1

Pol

539>

S18

3

Pol

541>

S18

5

Pol

543>

S18

7

Pol

545>

S18

9

Pol

547>

S19

1

Pol

549>

S19

3

Pol

551>

S19

5

Pol

553>

S19

7

Pol

555>

S19

9

Pol

557>

S20

1

Pol

559>

S20

3

Pol

561>

S20

5

Pol

563>

S20

7

2004 2001_2002

HBV, Variants: POLYMERASE. 2001-2002 vs 2004

Polymerase/RT (domains B y C)

YMDD

521POL(173RT)

528POL(180RT)

B C

B C


HBV, Variants: POLYMERASE

0.9%

3.9%

0%

0%

3.9%

No

No

2%/3.3%

2%

0%


HBV, Variants: HBsAg

Insertions & deletions

115

116

118

120

122

124

126

129

130

133

134

144145

112

212

121

135

146

139

137

140141

142

147148

149

151

152

153

155156

157

158

159

160

161

162

163

143

113

114

117

119

123

125

127

128

131

132

136

138

150

154

164

165

166

167

168

169

(2)(2)

172

182

196

199

154


HBV, Variants: HBsAg. 2001-2002 vs 2004

Point mutations

111 153120

127

135 142

EESS

TT126

IISSAA NN

QQHH 129RR

MM133 LL

KKEE

141

DDAA144

GG145

AA RR

SS

TTNN116

151

Vaccine escape

6.6 % 2001-2002 2004

2.3% to 0%



9.2 %

111 153EE

TT

QQHH

TTSS

MMII

KK SS

DDAA

GGAA RR

NN SS

II

GGSS

NN

TTNNRRYY

TT

AANN

GGRR

RR YY SSSSTTSS

SSRR

TTAA RR

LL

FF YY

127

135 142

120151

Point mutationsImmunotheraphy

resistance

2001-2002 2004

6.5 to 0%



Point mutations

111 153TT TT TT

KKTT

QQ

TT

MM

FF SS

DD

GG

TT

SSAA SS SS

QQ

NNAA

NN

II

TT

NNLL

SS YY

LL

LL

GG

KK

FFRR

HH

120127

135 142

151

HBsAg detection failures

12.5 % 2001-2002 2004

10.4 to 0%


HBV, Variants: HBsAg detection

Pointmutationsassociatedwith HBsAgdetectionfailures(n=34)

2001-2002

Avellon et al 2006


HBV, Variants: HBsAg 2001-2002

ABC

DFE

Avellon et al 2006


HBV, Variants: HBsAg detection

120

126

144145

142133

134

more frequent

143

129

more detectionfailuresPoint

mutationsassociatedwith HBsAgdetectionfailures


HBV, Variants: HBsAg

Only a few amino acid changes have a relevant effecton the HBsAg detection.

Multiple amino acid changes have low reactivity in allthe studied HBsAg tests.


VHB, Variantes estables: AgHBs detection

Only a few amino acid changes have a relevant effecton the HBsAg detection.

Multiple amino acid changes have low reactivity in allthe studied HBsAg tests.

Commonly samples with virus with the same amino acid substitution have different behaviour in the samediagnostic test.

Low viral load is an added factor.

Mutant and wild population rate is also relevant.


Test 1 Test1new

Positive Positive

Positive

D144Awt

144A+ 133I (3039)DNA 6000pg/ml

LowPositive144A+ 120Q+115N+131K (601)

DNA 143pg/ml

Test 1 Test1new

145A (1993-01)DNA2300pg/ml

145A stop 196 (1641_02)DNA 0.001pg/ml

145A (475-04)DNA 0.001pg/ml

145A + 136Y (936-04)DNA (n.s.)

wt

Positive Positive

G145APositive Positive

Negative LowPositive

LowPositiveNegative

CONCLUSIONS

Genetic diversity of HBV was observed in Spain througha variety of genotypes, antigenic subtypes and stablevariants, with some geographic and year of studydifferences.

Frequency of amino acid substitutions in polymeraseassociated to antiviral resistance in non selected chroniccarriers seem to be low.

Frequency of amino acid substitutions in HBsAg is high, but its effect on the diagnostic tests seem to be minimaland associate to specific substitutions with added factorsas viral load, and mixed wild/mutated populations.


Hepatitis B Virus Geneticdiversity in Spain.

Hepatitis Reference Laboratory

National Centre of Microbiology

Ana Avellón & José Manuel Echevarría

Thanks to:

María Cabrerizo

Beatriz Tendero

Mayte Arias

Rocío Villalba

Inés PareraThank you for your attention

hepatitis b virus genetic diversity in spain. · 2007. 10. 18. · test 1 test1new 145a (1993-01)...

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