Molecular biology of VHF viruses and recent diagnostic tools

12 August 2003

Roger Hewson

Virology

World Health Organisation Collaborative Centre Virus Reference and Research

Novel and Dangerous Pathogens

HPA Centre Emergency, Preparedness& Response

Porton Down

Istanbul June 2008

Molecular biology of VHF viruses and recent diagnostic tools

Summary

•Global perspective

•Viral Haemorrhagic fever viruses

Filoviridea

Arenaviridae

Flaviridae

BunyaviridaeRVFV

CCHF : Biology / History / Genetics

•Diagnostic assays:

Utility

Real Time –PCR [context CCHF]

Field / low-tech assays

Hi –tech assays

•Conclusions

Main emerging and reMain emerging and re--emergingemergingArbovirus & VHF infections between 1990Arbovirus & VHF infections between 1990--20082008

«« DengueDengue

« West Nile

« St Louis encephalitis

«« Hantavirus (HPS)Hantavirus (HPS)

«« Dengue/DHFDengue/DHF

«« Yellow feverYellow fever

« St Louis encephalitis

«« Hantavirus (HPS)Hantavirus (HPS)

«« ArenavirusesArenaviruses

« Tick borne encephalitis« West Nile« Sinbis« Hanatvirus (HFRS)Hanatvirus (HFRS)«« CCHFCCHF

ss Yellow feverYellow fever

ss DengueDengues West Nile

s Chikungunya

ss Rift Valley feverRift Valley fever

ss CCHF CCHF Lassa fever virusLassa fever virusEbolaEbolaMarburgMarburg

u DengueDengueuJapanese encephalitis

u Murrey Valley Fever

u Ross River Virus

u Nipah Virus

u Dengue/DHFu Japanese encephalitis

u Hanatavirus (HFRS)Hanatavirus (HFRS)u Chikungunya

u Henipaviruses

u HPAI

u SARAS

Haemorrhagic fever viruses

Family Genus Virus

Arenaviridae Arenavirus Junin, MachupoLassa Sabiá, GuanaritoWhitewater Arrayo

Flaviviridae Flavivirus Dengue,Yellow fever,OHF, KFD

Bunyaviridae Nairovirus CCHFPhlebovirus RVFHantavirus HFRS, HPSOrthobunyavirus Ngari

Filoviridae Filovirus Marburg, Ebola

Filoviridae: Taxonomic classification

Marburg EbolaSudan

EbolaZaire

EbolaReston

EbolaIvory Coast

Ebola like virus

FiloviridaeFamily

Genus

Species

VP35 VP30

3’ 5’GP LNP

VP40 VP24

Non coded regions Conserved intergenic sequences

EbolaUganda

Marburg like virus

Filovirus Filovirus CharacteristicsCharacteristics

Enveloped RNA virus 80 x Enveloped RNA virus 80 x ≈≈1000 nm1000 nm

NegativeNegative--stranded genome 19 kbstranded genome 19 kb

Virion proteinsVirion proteins NP: Nucleocapsid proteinNP: Nucleocapsid protein

VP30, VP35, L: replication complexVP30, VP35, L: replication complex

VP24, VP40 VP24, VP40 -- membranemembrane--associatedassociated

GP GP →→ GP1 + GP2: surface glycoproteinsGP1 + GP2: surface glycoproteins

sGP: secreted variantsGP: secreted variant

Detail of EBOV Glycoprotein Detail of EBOV Glycoprotein

Mucin domain

•Viron attachment to receptor or lectin on cell surface•Entry by endocytosis •Fusion of viral membrane within endosome•Release of RNA genome and viral proteins into cytoplasm•High rates of replication - - pathogenic effects

Virus ReplicationVirus Replication

ArenaviridaeArenaviridae

Family contains greatest proportion of Hazard Group 4 viruses

Name derived from “arenosus” (Latin “sandy”) describing appearance of virions on examination by electron microscopy

Enveloped virus, round or pleomorphic, 50-300 nm in diameter

Single-stranded genome divided into 2 RNA segments: S (3.4kb) & L (7.1kb)

Whitewater Arroyo

New World

Arenavirus Arenavirus Phylogenetic Phylogenetic TreeTree

Lassa Josiah

Lassa LPLassa GA391

Mobala

Ippy

Mopeia AN21366Mopeia AN20410

LCM WELCM Armstrong

Machupo

JunínTacaribe

Amapari

Guanarito Sabiá

Latino Oliveros

Flexal Paraná

Pichinde

Tamiami

Pirital

Old World

A

C

B

50 steps

Species causing

VHF

Virus ReplicationVirus Replication

Jiro YASUDA

GPNP VP35 VP30 VP24 L (Polymerase)VP40

PTAPPEY

L-domain

Virus assembly / BuddingVirus assembly / Budding

PTAPPPY Z ProteinS

L

Lassa virus

Ebola / Marburgvirus

L (Late) –Budding domains

Host Proteins: Tsg101, Nedd4

Viral budding using -Vesicular budding pathway

Receptor

ubiquitin

Endocytosis

Early Endosomes

Late Endosomes（MVB9Õ

Lysosomes

Exosomes

Endosomemembrane

Cytoplasmicmembrane

MVB sorting

Virus budding

Ubiquitin ligase（Nedd4 etc.�å

Ubiquitin

Normal Cargo Proteins

Eap45Eap30

Eap20

ESCRT-II

Chmpproteins

ESCRT-III

Vps4ATP

ADP

DUB?

MVB

Virion

ESCRT-I

Vps28

Vps37C

Viral Gag/Matrix proteins

Eap45Eap30

Eap20

ESCRT-II

Chmp

ESCRT-III

Vps4ATP

ADP

DUB?AIP1/ALIX

Tsg101

ESCRT-I

Vps28Vps37?

Hrs PT/SAP

Tsg101

PT/SAP YPxL

Multi-vesicular body (MVB) sorting & Virus buddingMulti-vesicular body (MVB) sorting & Virus budding

proteins

Ebola, Marburg and Lassa viruses utilize cellular MVB sorting pathway (like retoviruses)

Flaviviridae Haemorrhagic Fever Viruses

v Dengue

vYellow fever

•Estimated 200,000 case annually(South America and Africa)

•Estimated 30,000 deathsVaccine

Aedes sp

S. America Africa

[ X Asia ]

v Dengue

vYellow fever

WHO estimates half population of Tropics at risk (>2.3 Billion)

DHF/ DSS emerged as most important arboviral disease of humans

Dengue

Haemorrhagic fever viruses

Dengue Viruses Flaviviridae

Transmitted by mosquitoesTransmitted by mosquitoes

Human DEN infections can lead to: Human DEN infections can lead to: ••Dengue fever Dengue fever

••Fever, Headache, Muscle / joint painFever, Headache, Muscle / joint painNausea/vomitingNausea/vomiting

••Dengue haemorrhagic fever (DHF)Dengue haemorrhagic fever (DHF)••Fever, Haemorrhage, Low platelet countFever, Haemorrhage, Low platelet count

Leaky capillariesLeaky capillaries

••Dengue shock syndrome (DSSDengue shock syndrome (DSS))••Features of DHF & circulatory failureFeatures of DHF & circulatory failure

E

Four serotypes [DEN - 1, 2, 3, 4]•Ades aegypti

•Each serotype provides specific lifetime immunityEach serotype provides specific lifetime immunity••No cross protectionNo cross protection••Genetic variation within serotypesGenetic variation within serotypes

SS RNA Genome

Bunyaviridae

BunyaviridaeBunyaviridae

•Segmented Genome [Small Medium Large] •Single Stranded -ve sense RNA •Lipid enveloped viruses•Arthropod borne [x Hanta] •Some viruses cause disease in humans

S

LMS

L

Ortho Bunyavirus Phlebovirus Hantavirus Nairovirus TospovirusGenus

48

172 (+3)

Rodents

Mosquito

BUN

9

37 (+16)

Ruminants

Mosquito

RVF

22

49

Rodents

Nil

HPS / HFRS

7

39

Ruminants

Tick

CCHF

8

8 (+6)

Plants

Thrips

TSWV

Species

Subtypes

Host / Res

Vector

Virus / Disease

Genome Organisation & Replication

Orthobunyavirus:

~960 nts3’ 5’

NP

NSs

Gn Gc

5’3’~4460nts

5’3’

L

~6880 nts

S Segment M Segment

~1970 nts3’ 5’

NP Gn Gc5’3’

~3940nts5’3’

L

~6530 nts

Hantavirus:

Nairovirus:

~1710 nts3’ 5’

NP

5’3’~4900nts

5’3’

L

~12225 nts

Phlebovirus:5’3’

LGn Gc

5’3’~3230nts ~6400 nts~1720 nts

3’ 5’

NP

NSs

Tospovirus:5’3’

L

~8897 nts~2900 nts3’ 5’

NP

NSs

Gc Gn 5’3’

~4800nts

NSm

L Segment

NSm

NSm

Gc Gn NSm

Rift Valley Fever VirusPhlebovirus

A viral zoonosis (affectingprimarily domestic livestock).Can be passed to humanscausing fever.

Segmented genome

(i) Mild illness with fever, headache, myalgia and liver abnormalities.

(ii) But some times haemorrhagic fever syndrome, meningoencephalitis (inflammation of the brain),

Human RVF infections can lead to:Human RVF infections can lead to:

Segmented genome is ambisense ~6400 nts

RVF risk map 98-99

Rift Valley Fever Virus

Vector •Mosquito vectors

•Rain fall

Predictive mapping

•Past epidemiological data

Good general prediction e.g. Somalia 2007

Saudi Arabia 443 cases

85 deaths

Yemen653 cases80 deaths

Saudi-Yemeni Boarder 2006

2006-7

Kenya 684cases/ 155 deaths Somalia, 114 cases/ 51 deathsTanzania 264 cases/ 109 deaths

But

Spread to Saudi Arabia

Rift Valley Fever Virus2006-7

443 cases / 85 deaths

Predictive outbreaks in Africa

Genus Nairovirus: CCHF

S

LL- Large segment

M- Medium segment

S- Small segment

M - Glycoprotein spikes L - RNA dependent RNA Polymerase

S - Nuclear capsid

M - Glycoprotein spikes L - RNA dependent RNA Polymerase

S - Nuclear capsid

Transmission EM X20,000–ve staining

Transmission EM X20,000–ve staining

Gc 5’3’

~4900nts

Gn NSm

Pre Gn Pre Gc

Mucin - GP38 Gn Gc

L- Protein

NP + Genome

Virus assembly

Nairovirus: Glycoprotein processingNairovirus: Glycoprotein processing

Maturation and budding into the Golgi

Golgi membranes fuse with plasma membrane releasing particles

HPA

HPA

CCHF Virus Morphogenesis and budding in Golgi cisternaeCCHF Virus Morphogenesis and budding in Golgi cisternae

•1944 Viral origin - passage through human volunteers

CCHF –Historical Perspective (i)

First descriptions of CCHF type disease

•1136 Tajikistan (Zayn al-Din Sayyed)

•1941 Crimea first medical description

Isolation / registration - Yale 1968 (Crimean HF)

M.P Chumakov et al.,

First descriptions of Congo fever

•Stanleyville March 1956: 13 year old presented with fever / bruising

•Isolated / adapted to mice / maintained by passage - Unidentified

CCHF –Historical Perspective (ii)

•Sent to EAVRI / Entebbe 1957

Entebbe

Kisangani

East Africa Virus Research Institute

Registered - Arbovirus catalogue Yale 1961 - Congo Virus

Dir: Sandy Haddow: John Woodall, David Simpson:

Simpson et al., 1967

New virus (1956)

Woodall et al., 1962

MP Chumakov: 1968 Isolation of Crimean HF virus

1969 - Identical to Congo

1973 –CCHF virus

Registration at Yale

David Simpson: Director Virology Porton Down // 1960 - 75David Simpson: Director Virology Porton Down // 1960 - 75

Global distribution of CCHF

•Name synonymous with CCHF virus

•Amassed wide range of different strains

•Often called upon by WHO to assistin VHF outbreaks

•Part of Special Pathogens Reference collection - WHO CC

S

L M

S

L

Implications for diagnostic assays / vaccines

Evidence of reassortment (& recombination)

•Evolutionary opportunities

Mapping strains to geographic regions

Genome studies (in relation to developing better diagnostic assay)

Genome studies (in relation to developing better diagnostic assay)

Genetic Variation and Evolution•General

•Specific to CCHF

Elements of stability AND flexibility

0.1

M86624 Hazara

U04958 Greece

U84636 South Africa

U88416 Uganda

U84635 South Africa

AF404507 Kosovo

AF428144 Kosovo

AF428145 Kosovo

AF449482 Albania

AF432118 South Russia



AF481802 Russia Stvropol


U88412 Russia Astrakhan


AY062026 South Russia

AY062027 South Russia


AY0455062 Russia Volograd




U88410 Nigeria

U15093 Burkina Faso

U15092 Central African Republic

U88415 South Africa

U84638 South Africa

U84637 South Africa

U84639 South Africa

U15089 Mauretania

U15091 Senegal

U15090 Senegal

U75668 United Arab Emirates

S82581 United Arab Emirates




AF527810 Pakistan

U88414 Pakistan

U75677 Pakistan


AJ538198 Pakistan

U15024 Madagascar

AJ538196 Iraq


AF358784 China XinJiang



AJ010649 China XinJiang

AY029157 China XinJiang

AJ010648 China XinJiang

AF481799 Uzbekistan


AF362746 Kazakhstan

AF362743 Kazakhstan

AF481805 Tadjikistan

AY223475 Uzbekistan

U88413 China XinJiang

M86625 China

U88411 Senegal

U15021 Senegal

U15023 Mauretania

U15022 Iran

Africa 1

Russia

Asia 2

Asia 1

Greece

Africa 3

Africa 2

97

53

92

88

69

84

76

Variation within CCHF virus S segment

•7 Distinct lineages of S segment related to geographical area of virus isolation•Evidence of long range connections over wide areas (Trade in livestock, bird migration)

*

Phylogenetic trees and grouping patterns of full length CCHF RNA segments

0.1

AP92 Greece

Hazara

Europe 2

DAK 8194 Senegal

Semunya Uganda *

Drosdov Russia

HU29223 Russia

TI10145 Uzbekistan

66019 China XinJiang

HY13 China XinJiang


7803 China XinJiang

C68031 China


8402 China XinJiang

Hodzha Uzbekistan *


7001 China XinJiangBaghdad-12 Iraq*

Matin Pakistan

JD206 Pakistan

SR3 Pakistan*

IbAr10200 Nigeria*

SPU4/81 S. Africa*

Europe 1

Africa 2

Asia 1

Africa 1

Asia 2

Africa 3

S Segments

Distinct geographical patterns formed by S and L segments are not maintained by M segments

0.1

Hazara

30908 S Russia

K229-243 S. Russia

Baghdad Iraq

Matin Pakistan

Semunya Uganda

Hodzha Uzbekistan

TADJ/HU8966 Uzbekistan

IbAr10200 Nigeria

SPU128/81/7 S. Africa

IbAn10248 Nigeria

SPU4/81 S. Africa

Europe 1

Asia 1

Africa 2

Asia 2

Africa 3

L Segments

SR3 Pakistan

VLG- TI29414

Kosovo/9553/2001

SR3

SPU4/81 *

IbAr10200 *

SPU128-81*

U2-2-002--U-6415

Baghdad *

75024

7803

Hodzha

Tadj-HU8966

66019

Matin

HY13

8402

8816

M4

M2

M1

SEMUNYA *

CONGO

UG3010 *

79121

7001

M5

M3

Hazara

M Segments

0.1

Discrepancies between the phylogenetic grouping of M segments can be explained by either recombination or reassortment

Q. Do phylogenies generated from these windows show different topologies?

Different regions of sequence alignment from complete segments were used to build phylogenetic trees

Test these hypotheses :

W1 W2 W3 W4 W5 W6 Wn

Full length window

0.1

Hazara

SEM

Matin

Baghdad

Tadj

30908

10200

0.1

Hazara

SEM

Matin

Baghdad

Tadj

30908

10200

0.1

Hazara

SEM

Matin

Baghdad

Tadj

30908

10200

Examples of trees generated from windows

A. No evidence of phylogenetic incongruence

Phylogenetic trees are congruent

Therefore discrepancies in M segment are due to reassortment

0.1

VLG- TI29414

Kosovo/9553/2001

SR3

SPU4/81

IbAr10200

SPU128-84

U2-2-002--U-6415

Baghdad

75024

7803

Hodzha

Tadj-HU8966

66019

Matin

HY13

8402

8816

SEMUNYA

CONGO 3010

UG3010

79121

7001

M4

M2

M1

M5

M3

Hazara

M Segments

0.1

AP92 Greece

Hazara

Europe 2

DAK 8194 Senegal

Semunya Uganda

Drosdov Russia

HU29223 Russia

TI10145 Uzbekistan


HY13 China XinJiang


7803 China XinJiang

C68031 China


8402 China XinJiang

Hodzha Uzbekistan


7001 China XinJiangBaghdad-12 Iraq

Matin Pakistan

JD206 Pakistan

SR3 Pakistan

IbAr10200 Nigeria

SPU4/81 S. Africa

Europe 1

Africa 2

Asia 1

Africa 1

Asia 2

Africa 3

S Segments

Best example of reassortment is provided by Matin strain

Likely that other strains have arisen through segment reassortment

0.1

Hazara

30908 S Russia

K229-243 S. Russia

Baghdad Iraq

Matin Pakistan

Semunya Uganda

Hodzha Uzbekistan

TADJ/HU8966 Uzbekistan

IbAr10200 Nigeria

SPU128/81/7 S. Africa

IbAn10248 Nigeria

SPU4/81 S. Africa

Europe 1

Asia 1

Africa 2

Asia 2

Africa 3

L Segments

SR3 Pakistan

Phylogenetic analysis of S, L and M segments shows segment reassortment has occurred in some viruses

S

L

L

S

MS

L

M

S

LS

LS

M

•Co-replication of two strains in the same cell / organism. •Ticks are suitable hosts to support reassortment. •Reassortment events involving strains separated by vast geography suggest co-replication events are common

•Global reservoir of CCHF virus with local sub-reservoirssupporting high levels of virus circulation.

•This permits frequent co-infection, in which migratory birds play a role in virus dispersion.

+

=S

LS

M

Conclusions:

Segment reassortment in CCHF viruses leads to new viruses and new CCHF disease

SL

S

L

M

SL

S

L

M

N

L

L

S

S

M

M

Budding reassortant viruses

Newly reassortent virus may be brought into

contact with a new host

Adaptation in new host and new CCHF virus type

Exchange of M segments between different viruses can influence host range

Envelope glycoproteins influence

cellular tropism

… .. commonly associated with altered pathogenicity

Global and dynamic reservoir

Genetic variation in CCHF underlines importance of global surveillance

Diagnostics / potential vaccines should be based on strains isolated globally

Multiple strains of CCHF circulating in Kosovo

“… ..alternative clinical presentation” •Prof Salih AhmetiUH - Prishtina

Recent Diagnostic tools

•General overview / time line

•Utility

•Real Time –PCR [context CCHF]

•Field / low-tech assays

•Hi –tech assays

1985 1990 1995 2000--

PCR Amplification

1st PCR kits (HLA)

Semi-automated systems

Automated extraction systems

1st DNA PCR kits for infectious disease Real Time

Technologies: ‘water bath to chip’

New agents

Lab Based technologies

Nt / Prtoein CHIPELISA Lateral Flow dev

Diagnostic assays for VHF’sDiagnostic assays for VHF’s

5 10

Fever

Incubation Disease 15

- Viraemia- IgM Antibodies- Neutralising IgG Antibodies

Days

Convalescence

Re:CCHF

What to measure

5 10

Fever

Incubation Disease 15

Days

Convalescence

RT-PCR

IgM

IgGAvailable assays

Utility of diagnostic tests for CCHF

Block assay

Indirect Immunofluorescence

Cells infected and fixed in high containment

IgG / IgM ELISA

Diagnostic assays for VHF’sDiagnostic assays for VHF’sDiagnostic assays for VHF’sDiagnostic assays for VHF’s

Real Time / RT-PCR’s

Conventional RT-PCR

[~ 2-5 hrs]

[~ 2-3 hrs]

[~ 2-3 hrs] [~ 40 –60 mins]

Real Time / RT-PCR’s

[~ 40 –60 mins]

Diagnostic assays for Real time [CCHF] Diagnostic assays for Real time [CCHF] Diagnostic assays for Real time [CCHF] Diagnostic assays for Real time [CCHF]

Rapid, Sensitive, Reliable --- Useful

Design of good assay dependent on:

•Good sequence information

•Ideally non variable region

•Global distribution of strains

e.g. Nucleotide sequence alignment of CCHF virus S segmentse.g. Nucleotide sequence alignment of CCHF virus S segments

250 500 750 1000 1250 1500

ORF Region

CCHF v S segment (cRNA)

Sequence data not always complete

56 1504

More sequence entriesMake highlighted regionmost represented

CCHF shows high variabilityCCHF shows high variability

Focus on most represented (and conserved) area :

Use several probes Use several probes

F primer R primer

ATCTACATGCACCCTGCTGTGTTGACAFAM- -TAMRA

ATTTACATGCACCCTGCCGTCTTACAFAM- -TAMRA

AGCTTCTTCCCCCACTTCATTGGAGTFAM- -TAMRA

Broad range probe

5’3’CAAGGGGTACCAAGAAAATGAAGAAGGC GCCACAGGGATTGTTCCAAAGCAGAC

EU Assay: EID July 2007R Wölfel, J Paweska, N Petersen, A Grobbelaar, P Leman R Hewson, M-C Georges, A Papa, S Günther, C DrostenBundeswehr Institute of Microbiology, Munich,Germany; Bernhard Nocht Institute for TropicalMedicine, Hamburg, Germany; National Institutefor Communicable Diseases, Sandringham,South Africa; Health Protection Agency, PortonDown, Salisbury, United Kingdom; InstitutePasteur, Lyon, France; Aristotle University ofThessaloniki, Thessaloniki, Greece

Tested against global strain representation

Variability question:

CCHF shows high variability 0.1

IbAr10200

SPU415-85SPU4/81

SPU128/81/7

Bul/HU517

STV/HU29223DROSDOV

ROS/TI28044

DAK8194

AP92

Semunya

3010

TI1014566019

881668402

HY13

68031

75024

7803

Hodzha

791217001

Baghdad

Matin

JD206

SR3

Europe 1

Asia 1

Africa 2

Asia 2

Africa 3

Europe 2

Africa 1

97

99

98

98

100

82

98

Un-rooted phylogentic tree of available CCHF S Segments

Importance of strain representation Importance of strain representation

Missed in original assay

Hybridisation to complementary target resulting in hyper-stabilisation.

Use TaqMan Minor Grove Binder (MGB) ProbesUse TaqMan Minor Grove Binder (MGB) Probes

Allows use of shorter probe sequences

Regular probe cleavage (5’ nuclease PCR assay) Real time PCR - TaqMan assay

MGB –dihydrocyclopyrroloindole tripeptide (DPI3)Conjugated to 3’ end of probe

Real Time assays enable measurement of viral load:

•Assessing patients infectivity

•Clinical outcome

Roche LightCycler II / HPA RT assay

Kazakh Centre for Quarantine Zoonotic Disease //

HPA Collaboration : Kazakhstan [CCHF / HFRS]HPA Collaboration : Kazakhstan [CCHF / HFRS]

Pakistan, Georgia, Tajikistan, Kyrgyzstan : [Arboviruses and VHF’s]

Rapid Field / Laboratory tests Rapid Field / Laboratory tests

Mobile Light Cycler / Smart Cyclers

High technology

Ease of use??

CCHF2006 1.5 MicroarrayDr Roman Wolfel

•PCR (thermal cycler)

•Hybridization (37◦C)

•Wash

•Develop

•Analysis Hybridization pattern enables strain identification

RNA extraction

30 min

RT-PCR130 min

Sample

Results

5 min

Detection

45 minChip

CCHFCCHF20062006 1.51.5

4 hours

More to come : Lassa / Dengue

LD - Microarray

RT- Loop Mediated isothermal amplification RT- Loop Mediated isothermal amplification

•Rapid / Specific / Simple

•Requires limited hard ware

- Water bath

EBOV RNA

101 102 103 WSN -VE

RT- PCR•4 Primers •AMB-RT•Bst DNA Polymerase•63°C / 60 min RT / Pol•80°C / 2 min Terminate

Other Assay Systems.. Other Assay Systems..

Microarrays

Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry and Proteomics[MALDI / TOF]

Real time sequencing and analysis pyrosequencing

HIGH THROUGHPUT CAPABILITIES[Still dependent on high quality sequence data to start with]

HIGH THROUGHPUT CAPABILITIES[Still dependent on high quality sequence data to start with]

•Nucleotide / Protein

•Accurate / Rapid / Sensitive analysis of nucleic acids

•Rapid sequence analysis using

Recombinant proteins for ELISA systems

Most appropriate expression methodologies

•What proteins to be expressed •Vector system•Cell culture system •Purification

•Hazard-free recombinant protein production

•Purification via 6xHis-tag

•Retains antigenicity …

Rec EBOV NP / Baculovirus

NP

WB: anti EBOV serumProtein stain

12 24 48 72 EB 12 24 48 EB

Strain collection(s) - built up over years very important- based on new / circulating strains

•Design rapid diagnostic tools

•Understanding evolutionary opportunities of virus variation

•Important implications for testing assays / potential vaccines

•Evidence of new viruses ?

ConclusionsConclusions

•Shearing new sequences and strains is important

•Information feeds broader range high throughput assays

•Standardise diagnostic assays / +ve controls

Acknowledgements Acknowledgements

Galina Karganova

Anatoly Gmyl

Svetlana Smirnova

Larissa Gmyl

Chumakov Inst

Bushra Jamil

Rumina Hasan

AKU Hospital

John Chamberlain

Howard Tolley

Nicola Cook

Porton Down

Valerie Mioulet

Graham Lloyd

Hannah Love

Barry Atkinson

Groups of:

Jiro Yasuda

Tsugunori Notomi

Stuart Nichol

Robert Doms

Roman Wolfel

Christian Drosten

Janusz Paweska

Anna Papa

Stephan Gunther

Salih Ahlmeti

TEŞEKKÜRLER

Documents

Molecular biology of VHF viruses and recent diagnostic tools