XRB. UNIVERSIDAD DE BARCELONA

13 JULIO 2012

LUIS ENJUANES

CNB-CSIC. MADRID

MOLECULAR BASIS OF CORONAVIRUS VIRULENCE AND VACCINE DESIGN TO

PROTECT AGAINST SARS

VIRULENCE

CORONAVIRUSTGEV & SARS-CoV

PACKAGINGTRANSCRIPTION REPLICATION

VIRUS-HOST INTERACTION

CORONAVIRUS INDUCED DISEASES

MAN

ANIMALS

AS IN HUMANS &

PERITONITIS

HEPATITISNEPHRITIS

CARDIOMIOPATHY

CENTRAL NERVOUSSYSTEM

EYE

UPPER AND LOWERRESPIRATORY TRACT

IMMUNE SYSTEM CELLS

GASTROINTESTINALTRACT

RNA VIRUS GENOME SIZE IN NUCLEOTIDES

NIDO_CORONAVIRUSNIDO_TOROVIRUS

NIDO_RONIVIRIDAECLOSTEROVIRIDAE

BENYVIRUSNIDO_ARTERIVIRIDAE

SEQUIVIRIDAEPOMOVIRUS

TOGAVIRIDAECOMOVIRIDAEFLAVIVIRIDAE

FUROVIRUSTOBRAVIRUSPECLUVIRUSCHERAVIRUS

HORDEIVIRUSPOTYVIRIDAE

IFLAVIRUSDICISTROVIRIDAE

IDAEOVIRUS

MARMAVIRIDAEBROMOVIRIDAE

PICORNAVIRIDAE

ASTROVIRIDAE

CALICIVIRIDAEFLEXIVIRIDAE

TETRAVIRIDAEHEPEVIRIDAE

TYMOVIRIDAE

TOBAMOVIRUSUNCLASSIFIED

LUTEOVIRIDAENODAVIRIDAE

SOBEMOVIRUS

TOMBUSVIRIDAEUMBRAVIRUS

BARNAVIRIDAELEVIVIRIDAE

NARNAVIRIDAE

GFDVEGC-111-RTCCLCDK-11-YACWNHS-3-DYVYNPFMIDVQ-23-HVASCDAIMTR

GMDVEGA-111-KHCQ-CGT-11-YCCFKHA-3-DYVYNPYVIDIQ-23-HVASGDAIMTR

GFDVEGA-111-QKCE-CGK-11-YACFKHA-3-DYLYNPYCIDIQ-23-HVASGDAIMTR

GFDVEAT-111-QVCS-CGS-11-YACWKHC-3-DFVYNPLLVDIQ-23-HVASVDAIMTR

AIDAEFV-103- VFVGHGLNNDFK-44-HDSIEDAHTAL

GFDAEGA-111-VVCSVCTK-11-YGCWRHS-3-DYLYNPLIVDIQ-23-HVASSDAIMTR

YEAST

SARS-CoV

TGEV

IBV

HCoV-229E

MHV-A59

.. ..MUT 1 MUT 2

REP 1a REP 1b An4a 4b E M NSCPD

I

5aHEMHV

C1 D89 E91 ZN F D242 D272 Q521

nsp14

Zn F

Eckerle et al., 2007, 2010

MUTATION OF CORONAVIRUS EXON MOTIFS

c1

c1

c2

c1

c2

c2

c1

c1

c2

WT p5

p17

p1

p5

p17

p1

p5

p17

MUT 1

MUT 2

REP 1a REP 1b An4a 4b E M NSCPD

I

5aHEMHV nsp14

DISTRIBUTION OF MUTATIONS AFTER PASSAGE

Eckerle et al., 2007

CORONAVIRUS STRUCTURE AND GENE EXPRESSION

G

S

3a

E

M

N

7

3b

mRNA

-+

-+

-+

-+

-+

-+

-+

-+

AAAUUU

AAAUUU

AAAUUU

AAAUUU

AAAUUU

AAAUUU

AAAUUU

AAAUUU

+

ORF 1a ORF 1b 3a 7 UTR3b

S M NE

AAACAP

TRANSCRIPTION MECHANISM

TEMPLATE SWITCH

A HIGH FREQUENCY RECOMBINATION

ENGINEERED AN INFECTIOUS cDNA CLONE

REGULATION OF sgmRNA LEVELS

CS-L CS-B An 3’

TRS-L TRS-B

gRNA 5’ L

5’ An 3’ +

Un 5’ -3’

cTRS-B

cCS-BcL

cCS-B5’ 3’ mRNA

MULTIFACTOR REGULATION

1. TRS RNA STRUCTURE

2. TRS PRIMARY SEQUENCE

3. LONG DISTANCE INTERACTIONS

4. PROTEIN-RNA BINDING

. . . . . . . . . . . . . . . . . . . . .

G A U

Gene 3a

TRANSCRIPTION AND LEADER-BODY CS IDENTITY

UnG A U U U G 5’ -

C U A A A CL AnC U A A A C+ 5’ 3’ +

G A U U U G

Gene 3a

TRANSCRIPTION AND LEADER-BODY CS IDENTITY

C U A A A CL AnC U A A A C+ 5’ 3’ +TRS-L

5’ -

A3

G1

G2

C4

A5

U34

U32

U30

G31

C33

C10

A9

A8

C7

C6

A24

A25

U26

A27

U28

U29

G13

C12

C16

A15

A18

A19

A14 A20

U11

U17

A23

C21

G22

WT

APICAL HEPTALOOP INCLUDING THE CS-L

MICRO STEM

INTERNAL LOOP

WILD TYPE TRS-L STRUCTURE PREDICTION

G

G

A

C

A

U

U

U

G

C

G

C

U A

C

G

A

A

C

C

AUAUU

C

G

CA

AA

A A

U

IL1

G

G

A

C

A

U

U

U

G

C

AAUAUU

ACAACC

G

C

C

G

CA

AA

A A

U

MS2WT

C

U A

G

G

G

A

C

A

U

U

U

G

C

C

A

A

C

C

AAUAUU

G C

CA

AA

A A

U

C

C G

G

G

G

A

C

A

U

U

U

G

C

C

A

A

C

C

AAUAUU

G C

CA

AA

A A

U

MS1

C

C G

U

G

A U

U A

C G

U A

U A

G

G

A

C

A

U

U

U

G

C

G C

CA

AA

A A

U

IL2

G

C

C

A

UA

G

U

C AU

G

G

A

C

A

U

U

U

G

C

AA

G

A

U

C

AACGAAA

IL3MS3

A

G

G

A

C

A

U

U

U

G

C

C

A

A

C

C

GAAAUAUU

G C

CA

AA

A

U

A

A

WT AND MUTANTS TRS-L STRUCTURES

CONCLUSION

THE EXTENT OF sg mRNA SYNTHESIS:

CORRELATES WITH TRS-L STABILITY

REQUIRES A SPECIFIC SECONDARY STRUCTURE

WITH THE AVAILABILITY OF TRS-L

TRANSCRIPTION MECHANISM

TEMPLATE SWITCH

A HIGH FREQUENCY RECOMBINATION

ENGINEERED AN INFECTIOUS cDNA CLONE

G A U

Gene 3a

TRANSCRIPTION AND LEADER-BODY CS IDENTITY

UnG A U U U G 5’ -

C U A A A CL AnC U A C A C+ 5’ 3’ +C

G

G A U U U GG

Gene 3a

TRANSCRIPTION AND LEADER-BODY CS IDENTITY

C U A A A CL AnC U A C A C+ 5’ 3’ +C

REQUIREMENT OF COMPLEMENTARITY BETWEEN CS-L AND cCS-B FOR sgmRNA SYNTHESIS

CC U A AA

GG

CC

CG

5’ 3’L-C1GL-U2GL-A3CL-A4CL-A5CL-C6G

TITER*

---

2x104

4x104

2x103

CC U A AA

GG

CC

CG

5’ 3’B-C1GB-U2GB-A3CB-A4CB-A5CB-C6G

TITER*

8x108

1x107

5x108

6x108

3x108

4x108

* pfu/ml

CS-B MUTANTS CS-L MUTANTS

5’ 3’D-C1G3’ 5’

5’ 3’D-C6G

3’ 5’

C

G

GU A AA

CA U UU

CG U A AA

GC A U UU

TITER*

---

2x105

4x105

2x103

COMPLEMENTARY MUTANTS

CC U A AA

U

UU

UU

5’ 3’L-C1U

L-A3UL-A4UL-A5UL-C6U

TITER*

2x103

5x108

3x103

5x104

2x104

1x108

WT

NON-WATSON-CRICK MUTANTS

MAIN CONCLUSION

BASE-PAIRING (ΔΔΔΔG) IS A DRIVING FORCE IN CoV TRANSCRIPTION

COMPLEMENTARY AND SYNTHESIS OF sgmRNA

2455

0

2468

4

2468

5

2471

0

2473

4

2474

6

15

25

35

45

55

65

75

PO

TE

NT

IAL

BA

SE

-PA

IRIN

G S

CO

RE

GENOME POSITION, nt

CS1CS2M1M2M3M4M7M8M9M10M5M6

CUAAACACUAAAC

ACUAAACAACUAAAC

GAACUAAACCGAACUAAAC

CGAACUAAACGAAACGAACUAAACGA

CUAAACGAAACUAAACGAACUAAACGACUAAACG

CGAACUAAACGAAA5’ 3’

506

220 mRNA CS2

Mock CS1 CS2wt 1 62 3 4 597 8 10

MUTANTS

mRNA-3a.2

mRNA 3a.2

mRNA CS2

5’ TRS MUTANTS

- ΔΔ ΔΔG

, kca

l/mo

l

2

6

10

14

0 50 100 150 200 250 300

M4

M1

M3

M2

CS-S2

mRNA, u.r.

COMPLEMENTARITY BETWEEN NASCENT RNA AND TRS-LSEQUENCE AND mRNA LEVELS

mRNA LEVELS AND BASE-PAIRING SCORE

10

30

50

70

90

110

S 3a E M N 7

mRNA

BASE-PAIRING

RE

LA

TIV

E U

NIT

S

mRNA

REGULATION OF sg mRNA-N LEVELS

CS-N An 3’

TRS-L TRS-N

gRNA 5’ L CS-L dE pE

Un 5’ -

3’

cTRS-N

cCS-N

cL

5’

An 3’ +

7nt460nt

ATTACATAT

TAATGTATA

N-GENE

COMPLEMENTARITY AND ENHANCER ACTIVITY

50

100

150

200

10 20150 5 25∆∆∆∆G, Kcal/mol

sgm

RN

A-N

, RE

LA

TIV

E U

NIT

S

REQUIREMENT OF pE AND dE FLANKING SEQUENCES

TRS-N

dE-113-158

dE-173-20

E2-TRS-N

pE-20

dE-45-158

pEdEMUTANT

sgmRNA-N, %

AD 0 20010050 150

OPTIMIZED TRM

ORF 1aORF 1bORF 1a ORF 1b AAA7 AAAN 7

dE pE CS

20 7 620173 699

250 ntOriginal TRM 642 nt ����

ENHANCEMENT MECHANISM

CS-NpE

dE

3’

5’

5’

AD

ENHANCEMENT MECHANISM

CS-N3’

5’

5’

AD

REQUIREMENT OF LONG DISTANCE INTERACTION

REP-1

sgmRNA-N/gRNA

0

AD-TRS-N

TRMopt-N

TRS-N

5 10 15

TRMpEdE CS-N

REP-1

TRS-N

AD-TRS-N

NAD

TRMopt-NTRMopt

MUTANT

AD

ENHANCEMENT MECHANISM

CS-NpE

dE

3’

5’

5’

AD

80% ENHANCEMENT

ACTIVE DOMAIN REGIONS REQUIREMENT

A

B

CΔΔΔΔA ΔΔΔΔB ΔΔΔΔC

0

5

10

15

20

25

+ -

sgm

RN

A-N

, RE

LA

TIV

E U

NIT

S

ACTIVE DOMAIN REGION C REQUIREMENT

ΔΔΔΔA-C’ΔΔΔΔA

B

C

0

5

10

15

20

25

ΔΔΔΔA-C’ΔΔΔΔA

sgm

RN

A-N

, RE

LA

TIV

E U

NIT

S

ΔΔΔΔ[A-B]

B

∆∆∆∆A-B*2r ∆∆∆∆A-B*1r ∆∆∆∆A-B*1r4 ∆∆∆∆A-B*1r3∆∆∆∆A

ACTIVE DOMAIN REGION B REQUIREMENT

0

5

10

15

20

25

∆∆∆∆A ∆∆∆∆A-B*2r ∆∆∆∆A-B*1r (-)

sgm

RN

A-N

, RE

LA

TIV

E U

NIT

S

∆∆∆∆A-B*2r4 ∆∆∆∆A-B*1r3

nt 218-229

INTRAGENOMIC RNA-RNA INTERACTIONS INVOLVING B RNA MOTIF

∆∆∆∆G = -12.7 ∆∆∆∆G = -12.9

cB-M

nt 477-489

cB-M

nt 26408 - 26421

B-M

nt 26408 - 26421

B-M

TGEV REPLICONS WITH MUTATED B AND cB RNA MOTIFS

wt/∆∆∆∆B

wt/B

5’ 3’

5’ 3’B dE pE N

pE N

5’ 3’

5’ 3’B*

*

5’ 3’B**

cB-477*/B*-14 5’ 3’B**

pE N

dE pE N

dE pE N

dE pE N

cB-218*/∆∆∆∆B

cB-218*/B

5’ 3’

5’ 3’B*

cB-218*/B* 5’ 3’B**

pE N

dE pE N

dE pE N

*

cB-218

cB-477

cB-477*/∆∆∆∆B

cB-477*/B

cB-477*/B*

dE

dE

dE

5’ 3’

218-229 477-486 26,212-26,221

TRANSCRIPTION AND COMPLEMENTARY LEVELS

0

5

10

15

20

25

30

35

40

45

wt/ ∆∆ ∆∆

B

wt/B

cB-2

18*/ ∆∆ ∆∆

B

cB-2

18*/B

cB-2

18*/B

*

sgm

RN

A N

, RE

LA

TIV

E U

NIT

S

cB-4

77*/B

*-14

cB-4

77*/ ∆∆ ∆∆

B

cB47

7*/B

cB-4

77*/B

*

5’ 3’B dE pE N

218 477 26,212

EVOLUTION OF VIRUSES WITH MUTATED B AND cB RNA MOTIFS

RELEVANCE OF THE INTERACTION BETWEEN B AND cB RNA MOTIFS ON sgmRNA AND VIRAL TITERS

MECHANISM OF DISCONTINUOUS TRANSCRIPTION

5’(+) 3’(+)CS-NpEdE

AD

B-McB-MTRS-L

pE

dE

AD

5’(+)

CS-N3’(+)

CS-LB-M

cB-M

5’(-)cCS-NdE

AD

5’(+)

CS-N3’(+)

CS-L

B-McB-M

pE

TRS-L STABILITY AND SECONDARY STRUCTURE

COMPLEMENTARITY BETWEEN NASCENT MINUS RNAs AND TRS-L

CoV TRANSCRIPTION IS REGULATEDAT THREE LEVELS

LONG DISTANCE RNA-RNA INTERACTIONS

ORF 1a ORF 1b

N

3a 3b

N A

N 7MS Ewt-TGEV

TRS-3a

dE pE CS

TRMopt-3a

CS

AD

dE pE

sgmRNA-3a/gRNA

TRMopt-19-3a

0 2 4 6

TRMopt*-3a

TRM ACTIVITY WITHIN INFECTIOUS VIRUSES

GEOGRAPHICAL AREA ORIGIN OF SARS

SEVERE ACUTE RESPIRATORY SYNDROME

Neumonia High fever

Lymphopenia Death

SPREADING OF SARS

…JULY 5th, 03 END EPIDEMY BY WHO

Beijin Apr F 26y, BSL-4 Beijin Apr M 44y, BSL-4

NOV 02 JAN 03 FEB 03 DEC03 to JAN04FOSHAN GUANGZHOU HONG KONG

MOST RECENT COMMON ANCESTOR

FIRSTMAJOROUTBREAKHOSPITAL

Dr. ANephro-logist

HOTEL M

VIETNAMCANADASINGAPOREUSATWO ADDDITIONALSSE IN HK (>100 CONTACTS)

FOUR NON-LABORATORYISOLATED CASES

FOUR LABORATORY CASES

RACOON

CIVET CATFERRETS

GENUS αααα

GENUS ϒϒϒϒGENUS ββββ

CORONAVIRUS EVOLUTION

ENGINEERING OF A SARS-CoV INFECTIOUS cDNA CLONE AS A BAC

EM

NREP 1a REP 1b SL

An3a

3b

6

7a

7b

8a8b 9b

CMV

T by C10338

T by A11163

pBAC-SARS-CoV-URB*

BASIS OF SARS-CoV VIRULENCE

Structural: E

Accessory: 6, 7a, 7b, 8a, 8b, 9b

Combinations: E, 6, 7a, 7b, 8a, 8b, 9b

GENEDELETED

8a

L REP 1a REP 1b S 3a3b

EM

67a

7b8b

N9b

An

BSL-3 CNB. CSIC. MADRID

GROWTH KINETICS OF SARS-CoV DELETION MUTANTS

CaCo-2

GENES: E, 6, 7a, 7b, 8a, 8b, 9b ARE NON ESSENTIAL

TIME POST-INFECTION, HOUR

VERO E6

0 24 48 72

101

103

105

107

VIR

US

TIT

ER

, p

fu/m

l

ΔΔΔΔE,6-9b

0 24 48 72

wt

ΔΔΔΔ6-9b

ΔΔΔΔE

MORPHOGENESIS OF SARS-CoV IN ERGIC

SARS-CoVSARS-CoV-∆∆∆∆ [6-9b]

SARS-CoV-∆∆∆∆ESARS-CoV-∆∆∆∆ [E,6-9b]

ACTIVITY WHEEL USED TO QUANTIFY CLINICAL ILLNESS

DAY

INFECTION

0

200

400

600

800

1000

1200

-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4

RE

VO

LU

TIO

NS

/ hr

MOCK

∆∆∆∆E

wt

SARS-CoV-∆∆∆∆E-INFECTED HAMSTER WHEEL ACTIVITY

2 4 6 8

25

50

75

100

SU

RV

IVA

L, %

TIME POSTINOCULATION, days

0

ΔΔΔΔE

ΔΔΔΔE,6-9b

ΔΔΔΔ6-9b

ATTENUATION OF SARS-CoV DELETION MUTANTSIN TRANSGENIC MICE EXPRESSING hACE-2

2 4 6 870

80

90

110

ST

AR

TT

ING

WE

IGH

T, %

100

wt

ΔΔΔΔE

ΔΔΔΔE,6-9b

ΔΔΔΔ6-9b

TIME POSTINOCULATION, days

12000 pfu intranasally

CLINICAL DISEASE LETHALITY

SUMMARY

SARS-CoV-∆∆∆∆E IS ATTENUATED

E GENE CONDITIONS TISSUE SPECIFIC TROPISM

GENE E IS A VIRULENCE FACTOR

MECHANISM OF E PROTEIN VIRULENCE

E PROTEIN AND HOST GENE EXPRESSION

±±±±E

8a

L REP 1a REP 1b S 3a3b

EM

67a

7b8b

N9b

An

10 12 144 6 8210 12 144 6 82 10 12 144 6 82

10

12

14

4

6

8

2

10

12

14

4

6

8

2

MA104

65 hpi

wt vs mock ΔΔΔΔE vs mock ΔΔΔΔE vs wt

LOG CONTROL

LO

G E

XP

ER

IME

NT

Vero E6

15 hpi

DIFFERENTIAL GENE EXPRESSION IN ∆∆∆∆E AND WT INFECTED CELLSDIFFERENTIAL GENE EXPRESSION IN ΔΔΔΔE AND WT

INFECTED CELLS

GENES DIFFERENTIALLY EXPRESSED IN SARS-CoV-ΔΔΔΔE VS FULL-LENGTH VIRUS INFECTED CELLS

INFLAMMATION

APOPTOSIS

SIGNAL TRANSDUCTION

IMMUNOREGULATION

DSCR1 + 5.9 --DUSP1 + 4.0 --DUSP10 -- + 2.7YWHAG + 2.5 --WASL + 2.5 --MAP2K3 + 2.1 --FOLR1 -- +15.6PDE4B -- + 2.5K-RAS -- - 2.6

DEDD2 + 2.6 + 5.0CLU + 2.1 + 2.0PHLDA1 + 3.2 --STK17B -- + 2.2

NFKBIZ + 2.9 --ADAMTS1 + 2.1 --CCL2 -- - 3.2CXCL2 -- - 2.2

GENE VERO MA-104

FOLD CHANGE

FKBP4 + 4.7 + 5.5RIPK2 + 2.3 --ZC3H12A -- + 2.9

GENE VERO MA-104

FOLD CHANGE

STRESS RESPONSE

Hsp10 HspE1 + 3.3 --Hsp20 B5 CRYAB -- + 3.2Hsp27 B1 +19.0 --Hsp40 DNAJB1 +14.8 +25.9Hsp40 DNAJA1 + 8.7 + 7.2Hsp40 DNAJB6 + 3.9 + 2.1Hsp40 DNAJA4 -- + 2.6Hsp40 DNAJB4 -- + 4.1Hsp47 SERPINH1 + 6.7 + 2.5Hsp60 D1 + 4.1 + 4.7Hsp70 A1A +22.3 +29.3Hsp70 A1B +18.8 --Hsp70 A8 + 5.8 + 4.9Hsp70 A4L -- + 2.3Hsp90 AA1 +15.1 + 6.5Hsp90 AB1 + 6.1 + 2.7Hsp105/110 +35.3 +13.4UBB + 3.9 + 4.4UBC + 2.3 + 2.7BAG3 + 7.2 + 3.9CCT4 + 3.3 --CCT3 + 3.3 --AHSA1 + 4.7 --CRYAB -- + 3.2TCP1 -- + 2.3STIP1 -- + 2.1

UNFOLDED PROTEIN RESPONSE

RE

PERK

GR

P78

eIF2 TRANSLATIONALBLOCK

ATF6CLEAVED

XBP1mRNA

sXBP1mRNA

sXBP1

CYTOPLASM NUCLEUS

ACTIVATION IN ΔΔΔΔE VS WT-

INFECTED CELLS

GENEEXPRESSION

TRANSLATION ATTENUATIONCHOP

CORRECT PROTEINFOLDINGCHOP

PROTEIN DEGRADATIONp58IPK

-

+

GRP78

GRP78

GR

P78

GR

P78

GR

P78

GR

P78

GR

P78

PERK

eIF2

ATF6

PATF4

IRE1

IRE1

-

EFFECT OF E PROTEIN ON THE STRESS PRODUCED BY SARS-CoV-ΔΔΔΔE INFECTION

FO

LD

CH

AN

GE

, rel

ativ

e u

nit

s

2

4

6

8

10

12

E+

E-

SERPIN H1HSP90 AA1 HSP H1 HSP E1HSP A1A 18S

E PROTEIN PROVIDED IN TRANS

SARS-CoV E PROTEIN AND STRESS PRODUCEDBY RSV INFECTION

TIME POST-INFECTION, hours

FO

LD

CH

AN

GE

, rel

ativ

e u

nit

s

1

2

3

4

5

6

7

50

100

150

200

250

300

350

2 24 48 2 24 48

HSP90 AA1 UBB

E+

E-

2 24 48 2 24 48

HSP H1 SERPIN H1

2 24 48

HSP E1 18S

2 24 48

E PROTEIN PROVIDED IN TRANS

CONCLUSION

E PROTEIN REDUCED CELLULAR STRESSINDUCED BY:

SARS-CoV INFECTION

THAPSIGARGIN - Ca++

TUNICAMYCIN - GLYCOSYLATION

RSV INFECTION

SARS-CoV-ΔΔΔΔE INFECTION AND INFLAMMATION

INFLAMMATORYRESPONSE

-E

+E

STRESS RESPONSE

MOUSE ADAPTED SARS-CoV

High titers in lungs

Viremia, extrapulmonary spread

Neutrophilia

Pathological changes in lungs

Death

K. SUBBARAO AND R. BARIC LABS

SARS-CoV-MA15

CONSTRUCTION OF A MOUSE ADAPTED SARS-CoV

nsp510384

nsp510793

nsp912814

nsp1316117

S (RBD)22797

M26428

ENGINEERED MUTANTS wt

ΔΔΔΔEΔΔΔΔ[E,6-9b]

ΔΔΔΔ[6-9b]

8a

L REP 1a REP 1b S 3a3b

EM

67a

7b8b

N9b

An

CONCLUSION

SARS-CoV-MA15-ΔΔΔΔE IS ATTENUATED AND FULLYPROTECTS BOTH YOUNG AND OLD MICE

THEREFORE IT IS A PROMISING VACCINECANDIDATE, BETTER THAN SARS-CoV-ΔΔΔΔE

REPLICATIONFERNANDO ALMAZANAITOR NOGALESSILVIA MARQUEZ

TRANSCRIPTION AND ASSEMBLYISABEL SOLAPEDRO A. MATEOSSONIA ZUÑIGAMARTINA BECARES

VIRUS-HOST INTERACTION: SARSMARTA L. DeDIEGOJOSE LUIS NIETOJOSE MANUEL JIMENEZJOSE ANGEL REGLARAUL FERNANDEZ

PROTEOMICSSILVIA JUAREZ. CNB-CSICALEXANDER AKOULICHEV. OXFORD

CNB. CSIC. MADRID COLLABORATORS

LUIS ENJUANES

ANIMAL MODELSKANTA SUBBARAO. NIHSTANLEY PERLMAN. UNIV. IOWA

ELECTRON MICROSCOPYMARIA TERESA REJAS. CBM, CSICCRISTINA PATIÑO. CNB. CSIC

CARLOS M. SANCHEZSARHAY ROSMARGARITA GONZALEZ

GENOMICSIRENE LOPEZ-VIDRIEROMARTA GODOYJUAN CARLOS OLIVEROS