1!

Bioinformatik – virologiska aspekter

Magnus Lindh

Klinisk virologi

Viral genetisk variabilitet!ü Methods!!ü Phylogeny!

§  Tracing source, evolution!ü Mutationer!

§  Resistance ! ! !ü Genotyper!

§  Pathogenesis!§  Function!§  Treatment response!

Today!ü Tomas Bergström!

§  HSV, TBE!ü Peter Norberg!

§  Recombination!ü Heléne Norder!

§  Genotypes!ü Practical session!

2!

Example, HCV!ü  Molecular diagnostics:!

§  Confirmation by PCR!§  Typing prior to treatment!§  Monitoring treatment response!§  Investigating transmission routes!§  Resistance mutations (?)!

Traditional virus diagnostics!

ü Culture���

ü Antibodies ���

ü Detection ü Elektron microscopy ü Antigen detection

ü IF, EIA

CPE

PCR!•  Rapid testing (< 1 d.)!

•  CNS (HSV, VZV) ! ! !!

•  Opportunistic (CMV, EBV, HSV) ! !!

•  Gastroenteritis (calici) ! ! !!

•  Respiratory infections ! ! ! !

•  Quantification!•  HIV, CMV, EBV, hepatitis B and C!

•  Typing!–  HSV, HCV, HBV, HPV!

3!

Herpes simplexvirus (HSV) 1&2!

•  Blisters, genital, oral !

•  CNS !•  encephalitis, meningitis!

Cytomegalovirus (CMV)!§  Mononucleosis in immuncompetent!§  Ksevere in transplant recipients!§  Pneumonitis, hepatitis, febver!§  Quantification!

Epstein-Barrvirus (EBV)!•  Mononucleosis in immuncompetent!•  Lymphoma (PTLD) in immunsuppressed!

Post transplantation lymphoproliferative disease!

4!

Hepatit C virus (HCV)!•  Cirrhosis, liver transplantation !•  Genotyping prior to treatment (type 1-6)!•  Quantification during treatment!

0

1

2

3

4

5

6

7

8

9

0 20 40 60 80 100

Time

Lo

g H

CV

RN

A

Liver cirrhosis!

Hepatit B virus (HBV)!

§  Cirrhosis, HCC!§  Quantification important for prognosis! ! !<10,000 copies/mL ≈”healthy”!

! >100,000 copies/mL – often progressive!

§  Typing!!!

HIV!

§  Monitoring treatment!§  Resistance!

5!

Respiratory infections!• Rhinovirus (100 typer)!• Coronavirus (4 typer)!• Influensa A - B (- C)!• RSV !!• Parainfluensa 1 - 2 - 3!• Adenovirus (40 typer)!• Enterovirus (80 typer)!• Metapneumovirus!• Mykoplasma pneumonia!• Ch. pneumoniae !!

Svar inom 24 tim • Kan undvika onödig antibiotikabehandling • Behandling av influensa • Behandling av mykoplasmabakterie • Nöjd läkare • Nöjd patient

Multianalys på viruslab

HPV (humant papillomvirus)!•  100 subtypes!

- 30 genital subtypes!

•  13 subtyper associated with cervixcancer!

!

Metodutveckling PCR!

ü Ofta stor variabilitet i virusgenom!ü Alignment av representativa sekvenser!ü Val av targetregion!

§  Påvisning, kvantifiering: konserverad (maximal säkerhet och känslighet)!

§  Typning: variabel men konserverad!§  Fylogeni: variabel (lagom)!

6!

HCV!ü RNA-virus, 9400 nt!ü 6 genotyper, 30% diff!

Exempel, HCV!ü  9400 nt, 30% difference between genotypes!

ü  Konserverade regioner: 5’NTR, 3’NTR!ü  Variable region!

1a.HCV-1_complete_M623211a.HCV-H_complete_M674631b.AB016785_complete_AB0167851b.Con1_complete_AJ2387991c.AY051292_complete_AY0512921c.HC-G9_complete_D148532a.G2AK1_complete_AF1690032a.JFH-1_complete_AB0476392b.HC-J8_complete_D109882b.JPUT971017_complete_AB0309072c.BEBE1_complete_D504092k.VAT96_complete_AB0316633a.CB_complete_AF0468663a.NZL1_complete_D177633b.HCV-Tr_complete_D493743b.JP1996056672-A/1_complete_E103k.JK049_complete_D638214a.ED43_complete_Y116045a.EUH1480_complete_Y131845a.SA13_complete_AF0644906a.EUHK2_complete_Y120836b.TH580_complete_D842626d.VN235_complete_D842636g.JK046_complete_D638226h.VN004_complete_D842656k.VN405_complete_D84264

2280 2300 2320 2340 2360 2380 2400 2420 2440 2460 2480 2500 2520 2540 2560 2580 2600 2620 2640 2660 2680 2700

HCV, diagnostic target!ü 5’UTR!

1a.HCV-1_complete_M623211a.HCV-H_complete_M674631b.AB016785_complete_AB0167851b.Con1_complete_AJ2387991c.AY051292_complete_AY0512921c.HC-G9_complete_D148532a.G2AK1_complete_AF1690032a.JFH-1_complete_AB0476392b.HC-J8_complete_D109882b.JPUT971017_complete_AB0309072c.BEBE1_complete_D504092k.VAT96_complete_AB0316633a.CB_complete_AF0468663a.NZL1_complete_D177633b.HCV-Tr_complete_D493743b.JP1996056672-A/1_complete_E103k.JK049_complete_D638214a.ED43_complete_Y116045a.EUH1480_complete_Y131845a.SA13_complete_AF0644906a.EUHK2_complete_Y120836b.TH580_complete_D842626d.VN235_complete_D842636g.JK046_complete_D638226h.VN004_complete_D842656k.VN405_complete_D84264Gt1 F-probe-RLIPA

0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420

7!

Herpes simplexvirus!ü HSV1, HSV2!ü DNA-virus, 150,000 nt!

AF133601AF133600AF133599AF133598AF133597AF133596AF133595AF133594AF133593AF133592AF133591AF133589CMV_Zibin_78197-78218

1510 1520 1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650 1660 1670 1680 1690 1700 1710 1720 1730 1740 1750 1760 1770

U12174 HSV-2AF097023 HSV-1Namvar Tqm _HSV1Namvar Tqm _HSV2

1530 1540 1550 1560 1570 1580 1590 1600 1610 1620 1630 1640 1650 1660 1670

Herpesgruppens virus!ü Low degree of variability!ü Long sequences required for phylogeny!ü Rekombination common!

AF133601AF133600AF133599AF133598AF133597AF133596AF133595AF133594AF133593AF133592AF133591AF133589CMV_Zibin_78197-78218

3280 3290 3300 3310 3320 3330 3340 3350 3360 3370 3380 3390 3400 3410 3420 3430 3440 3450 3460 3470 3480 3490 3500 3510 3520 3530 3540

Phylogeni!ü Type of algorithm!

§  Distance matrix, parsimony, ML!ü Substitution model (JK, Kimura, HKY, TN)!

§  C<-->T more common than C<-->A and C<-->G!ü Tree!

§  Roted, unrooted!§  Distance!

ü Bootstrapping!ü Recombination!

8!

Molecular epidemiology!ü Source of outbreak!

!ü Transmission chain!

§  Nosocomial!§  Sexual!

00 7400

99 7684

99 11398

99 8747

X76918

D14305

98 12331Genotype 1

68

100

93

100

64

4785

12

17

//

0.05

Genotype 3

100

Patient 1, April 5, 2000

Patient 2, June 10, 2000

Patient 2, Nov 3, 2000

Patient 3, Nov 17, 2000

Patient 1, Sept 20, 2000

Patient 1, June 13, 2000

Selection of resistance!

ü Genetic barrier!§  One or several mutations required!

ü Fitness!ü Compensatory mutations !!

!

Mutationer!ü Rates!

§  DNA-virus !10−6 to 10−8 per bas per generation!§  RNA-virus !10−3 to 10−5 per bas per generation!

ü Spread, competition!ü Quasispecies!

9!

Mutation rates in HBV!Mother and children (adult)

Early ≈0 Late ≈1-2%

Hepatit B-virus!ü Hominider!

§  schimpans, gorilla, orangutang, gibbon !

ü Sydamerikansk ullapa!

ü Murmeldjur!ü  Jordekorre!ü Pekinganka!ü Snögås!ü Häger!

Hepatit B-virus!ü  3200 nt!ü  4 ORF, 7 proteiner!ü Replikerar via RNA!ü Episomalt DNA i

nukleus!ü  8 genotyper !!

§  När separerade de!§  3000 år sedan?!§  5 milj år sedan?!

10!

HBV-genotyper!ü 8% nt-differens mellan genotyper!

F_X75663

H AY090454

B6 AB287321 Greenland

B6 DQ463787 Canada

B6 AB287316 Alaska

B1 D50521 Japan

B1 AB073853 Japan

B5 AB219427_Phi l ippines

B5 AB219426 Philippines

B3 AB033554 Indonesia

B3 D00331 Indonesia

B7 EF473972 Indonesia

B7 EF473971 Indonesia

B2 AB073825 China

B2_AF121243 Vietnam

B2 AF121251 Vietnam

B4 AY033073 Vietnam

B4 AB100695 Vietnam

B4 AB115551 Cambodja

C4 AB048705 Australia

C4 AB048704 Australia

C5 AF241411 Vietnam

C7 AP011099 Indonesia

C5 EU410081_Phi l ippines

C6 EU410079 Philippines

C3 X75656 Polynesia

C3 X75665 New Caledonia

C1 {CCT} AF068756 Thailand

C1 {CCC} AF223957 Vietnam

C1 {TCC} DQ089789 China {Hongkong}

C2 X04615 Japan

C2 AY247032 Korea

A2 Z35717_Poland

A3 ik3346_Gambia

A1 27178_Somalia

A1 AY373429

D4 AB048701 Australia

D3 X85254 Italy

D2 AB205127 Russia

D1 AF121241 Turkey

D1 AF280817 China

G AB064310

0.02

A1 27178_SomaliaA1 AY373429A2 Z35717_PolandA3 ik3346_GambiaB1 D50521 JapanB1 AB073853 JapanB2 AB073825 ChinaB2_AF121243 VietnamB2 AF121251 VietnamB3 AB033554 IndonesiaB3 D00331 IndonesiaB4 AB100695 VietnamB4 AB115551 CambodjaB4 AY033073 VietnamB5 AB219427_PhilippinesB5 AB219426 PhilippinesB6 DQ463787 CanadaB6 AB287316 AlaskaB6 AB287321 GreenlandB7 EF473972 IndonesiaB7 EF473971 IndonesiaC1 {CCC} AF223957 VietnamC1 {TCC} DQ089789 China {Hongkong}C1 {CCT} AF068756 ThailandC2 X04615 JapanC2 AY247032 KoreaC3 X75656 PolynesiaC3 X75665 New CaledoniaC4 AB048704 AustraliaC5 AF241411 VietnamC5 EU410081_PhilippinesC6 EU410079 PhilippinesC7 AP011099 IndonesiaD1 AF121241 TurkeyD1 AF280817 ChinaD2 AB205127 RussiaD3 X85254 ItalyD4 AB048701 AustraliaF_X75663G AB064310H AY090454

1520 1540 1560 1580 1600 1620 1640 1660 1680 1700 1720 1740 1760 1780 1800 1820 1840 1860

HBV-genotypning!ü  RFLP !