MOLECULAR BASIS OF CORONAVIRUS VIRULENCE AND VACCINE DESIGN TO PROTECT AGAINST SARS - XRB Annual Meeting 2012

1. MOLECULAR BASIS OF CORONAVIRUS
VIRULENCE AND VACCINE DESIGN TO
PROTECT AGAINST SARS
XRB. UNIVERSIDAD DE BARCELONA
13 JULIO 2012
LUIS ENJUANES
CNB-CSIC. MADRID

2. CORONAVIRUS
TGEV & SARS-CoV
REPLICATION TRANSCRIPTION PACKAGING VIRULENCE
VIRUS-HOST INTERACTION

3. CORONAVIRUS INDUCED DISEASES
MAN
CENTRAL NERVOUS
SYSTEM
ANIMALS
EYE
UPPER AND LOWER AS IN HUMANS &
RESPIRATORY TRACT PERITONITIS
IMMUNE SYSTEM CELLS
HEPATITIS
NEPHRITIS
GASTROINTESTINAL CARDIOMIOPATHY
TRACT

4. RNA VIRUS GENOME SIZE IN NUCLEOTIDES
NIDO_CORONAVIRUS
NIDO_TOROVIRUS
NIDO_RONIVIRIDAE
CLOSTEROVIRIDAE
BENYVIRUS
NIDO_ARTERIVIRIDAE
SEQUIVIRIDAE
POMOVIRUS
TOGAVIRIDAE
COMOVIRIDAE
FLAVIVIRIDAE
FUROVIRUS
TOBRAVIRUS
PECLUVIRUS
CHERAVIRUS
HORDEIVIRUS
POTYVIRIDAE
IFLAVIRUS
DICISTROVIRIDAE
MARMAVIRIDAE
BROMOVIRIDAE
IDAEOVIRUS
PICORNAVIRIDAE
CALICIVIRIDAE
FLEXIVIRIDAE
TETRAVIRIDAE
HEPEVIRIDAE
ASTROVIRIDAE
TOBAMOVIRUS
UNCLASSIFIED
TYMOVIRIDAE
LUTEOVIRIDAE
NODAVIRIDAE
SOBEMOVIRUS
UMBRAVIRUS
TOMBUSVIRIDAE
BARNAVIRIDAE
LEVIVIRIDAE
NARNAVIRIDAE

5. MUTATION OF CORONAVIRUS EXON MOTIFS
MHV REP 1a REP 1b nsp14 CPD HE S 4a 4b 5a E M N An
I
C1 D89 E91 ZN F D242 D272 Q521
YEAST
MUT 1
..
AIDAEFV-103-
Zn F
.MUT 2.
VFVGHGLNNDFK-44-HDSIEDAHTAL
MHV-A59 GFDAEGA-111-VVCSVCTK-11-YGCWRHS-3-DYLYNPLIVDIQ-23-HVASSDAIMTR
SARS-CoV GFDVEGC-111-RTCCLCDK-11-YACWNHS-3-DYVYNPFMIDVQ-23-HVASCDAIMTR
HCoV-229E GMDVEGA-111-KHCQ-CGT-11-YCCFKHA-3-DYVYNPYVIDIQ-23-HVASGDAIMTR
TGEV GFDVEGA-111-QKCE-CGK-11-YACFKHA-3-DYLYNPYCIDIQ-23-HVASGDAIMTR
IBV GFDVEAT-111-QVCS-CGS-11-YACWKHC-3-DFVYNPLLVDIQ-23-HVASVDAIMTR
Eckerle et al., 2007, 2010

6. DISTRIBUTION OF MUTATIONS AFTER PASSAGE
MHV REP 1a REP 1b nsp14 CPD HE S 4a 4b 5a E M N An
I
WT p5 c1
c2
p17 c1
MUT 1 p1
c1
p5
c2
p17 c1
MUT 2 p1
c1
p5
c2
p17 c2
Eckerle et al., 2007

7. CORONAVIRUS STRUCTURE AND GENE
EXPRESSION
3b
ORF 1a ORF 1b S 3a E M N 7 UTR
+ CAP AAA
mRNA
+ AAA
- UUU
G
+ AAA
- UUU S
+ AAA
- UUU 3a
+ AAA
- UUU 3b
+ AAA
- UUU E
+ AAA
- UUU M
+ AAA
- UUU N
+ AAA
- UUU 7

8. TRANSCRIPTION MECHANISM
TEMPLATE SWITCH
A HIGH FREQUENCY RECOMBINATION
ENGINEERED AN INFECTIOUS cDNA CLONE

9. REGULATION OF sgmRNA LEVELS
TRS-L TRS-B
gRNA 5’ L CS-L CS-B An 3’
5’ An 3’ + MULTIFACTOR REGULATION
1. TRS RNA STRUCTURE
3’ cL cCS-B Un 5’ -
2. TRS PRIMARY SEQUENCE
cTRS-B
3. LONG DISTANCE INTERACTIONS
4. PROTEIN-RNA BINDING
5’ cCS-B 3’ mRNA
.....................

10. TRANSCRIPTION AND LEADER-BODY CS IDENTITY
Gene 3a
+ 5’ L CUAAAC CUAAAC An 3’ +
GAUUUG Un 5’ -

11. TRANSCRIPTION AND LEADER-BODY CS IDENTITY
TRS-L Gene 3a
+ 5’ L CUAAAC CUAAAC An 3’ +
GAUUUG 5’ -

12. WILD TYPE TRS-L STRUCTURE PREDICTION
U
C16 17 A18 APICAL HEPTALOOP
A15 A19
INCLUDING THE CS-L
A14 A20
G13 C21
C12 G22
MICRO STEM
U11 A23
C10 A24
A9 A25
U26
A8 INTERNAL LOOP
A27
C7
U28
C6 U29
A5 U30
C4 G31
A3 U32
G2 C33
G1 U34
WT

13. WT AND MUTANTS TRS-L STRUCTURES
U A U U A
U A U A U A C C A C
C C C A A A A
A A U A A A A A A A
C A A A A A A
A A A A A A A
A G C C G C
G C G C G C G
A A A G G
A C G
C G G C C G C G A A
A
U A A U U A
U A C G C G C A
C A A A C A A C A U A
C G A
A A C A A A U A U
A A A U A
U A U U A
A A U A C A U A U
A A A A U
C A C
U C U C U C U C G C G
U
C U C U C C U C G
U A U C G
A U A U A U
A U C G A U A U
C G C G C G U
C G A U C G
A U A U C G
A U A U A U
G C A U
G C G C G C
G C G U G C
G U G U G C
G U G U G U
G U
WT MS2 IL1 MS1 MS3 IL3 IL2

14. CONCLUSION
THE EXTENT OF sg mRNA SYNTHESIS:
CORRELATES WITH TRS-L STABILITY
WITH THE AVAILABILITY OF TRS-L
REQUIRES A SPECIFIC SECONDARY
STRUCTURE

15. TRANSCRIPTION MECHANISM
TEMPLATE SWITCH
A HIGH FREQUENCY RECOMBINATION
ENGINEERED AN INFECTIOUS cDNA CLONE

16. TRANSCRIPTION AND LEADER-BODY CS IDENTITY
Gene 3a
+ 5’ L CUAAAC CUACAC An 3’ +
G A UG U G
U Un 5’ -

17. TRANSCRIPTION AND LEADER-BODY CS IDENTITY
Gene 3a
+ 5’ L CUAAAC CUACAC An 3’ +
G AUG U G
U

18. REQUIREMENT OF COMPLEMENTARITY BETWEEN
CS-L AND cCS-B FOR sgmRNA SYNTHESIS
CS-B MUTANTS CS-L MUTANTS
TITER* TITER*
5’ C U A A A C 3’ 5’ C U A A A C 3’
B-C1G G 8x108 L-C1G G -
B-U2G G 1x107 L-U2G G -
B-A3C C 5x108 L-A3C C -
B-A4C C 6x108 L-A4C C 2x104
B-A5C C 3x108 L-A5C C 4x104
B-C6G G 4x108 L-C6G G 2x103
NON-WATSON-CRICK MUTANTS COMPLEMENTARY MUTANTS
TITER* TITER*
5’ C U A A A C 3’ 5’ G U A A A C 3’
L-C1U U 2x103
D-C1G -
3’ C A U U U G 5’
WT 5x108 -
L-A3U U 3x103 -
5x104 2x105
L-A4U U
L-A5U U 2x104 5’ C U A A A G 3’ 4x105
D-C6G
L-C6U U 1x108 3’ G A U U U C 5’ 2x10
3
* pfu/ml

19. MAIN CONCLUSION
Δ
BASE-PAIRING (ΔG) IS A DRIVING FORCE
IN CoV TRANSCRIPTION

20. COMPLEMENTARY AND SYNTHESIS OF sgmRNA
mRNA CS2
5’ CGAACUAAACGAAA 3’
75
POTENTIAL BASE-PAIRING SCORE
CS1 ACUAAAC
CS2 CUAAAC 65
M1 ACUAAAC
M2 AACUAAAC 55
M3 GAACUAAAC mRNA 3a.2
M4 CGAACUAAAC 45
M7 CUAAACG
M8 CUAAACGA 35
M9 CUAAACGAA
M10 CUAAACGAAA 25
M5 CGAACUAAACGA
M6 CGAACUAAACGAAA 15
24550
24684
24685
24710
24734
24746
GENOME POSITION, nt
MUTANTS
Mock CS1 CS2wt 1 2 3 4 7 8 9 10 5 6
506
mRNA-3a.2
220
mRNA CS2

21. COMPLEMENTARITY BETWEEN NASCENT RNA AND TRS-L
SEQUENCE AND mRNA LEVELS
14
M4
10 M3
-ΔG, kcal/mol
M2
M1
6
CS-S2
5’ TRS MUTANTS
Δ
2
0 50 100 150 200 250 300
mRNA, u.r.

22. mRNA LEVELS AND BASE-PAIRING SCORE
110
90 BASE-PAIRING
RELATIVE UNITS
70
50
30
mRNA
10
S 3a E M N 7
mRNA

23. REGULATION OF sg mRNA-N LEVELS
TRS-L TRS-N
N-GENE
gRNA 5’ L CS-L dE pE CS-N An 3’
460nt 7nt
5’
3’ cL TAATGTATA
ATTACATAT An 3’ +
cCS-N Un 5’ -
cTRS-N

24. COMPLEMENTARITY AND ENHANCER ACTIVITY
sgmRNA-N, RELATIVE UNITS
200
150
100
50
0 5 10 15 20 25
∆G, Kcal/mol

25. REQUIREMENT OF pE AND dE FLANKING
SEQUENCES
sgmRNA-N, %
MUTANT
AD dE pE 0 50 100 150 200
E2-TRS-N
TRS-N
dE-113-158
dE-45-158
dE-173-20
pE-20

26. OPTIMIZED TRM
dE pE CS
ORF 1a 77
ORF 1a ORF 1b
ORF 1b N AAA
173 9 20 20 9 7 6 6
Original TRM 642 nt 250 nt

27. ENHANCEMENT MECHANISM
pE CS-N
3’
dE 5’
AD
5’

28. ENHANCEMENT MECHANISM
CS-N
3’
5’
AD
5’

29. REQUIREMENT OF LONG DISTANCE INTERACTION
TRM
MUTANT AD dE pE CS-N N
REP-1
TRS-N
AD
AD-TRS-N
TRMopt
TRMopt-N
0 5 10 15
REP-1
TRS-N
AD-TRS-N
TRMopt-N
sgmRNA-N/gRNA

30. ENHANCEMENT MECHANISM
pE CS-N
3’
dE 5’
AD
5’
80% ENHANCEMENT

31. ACTIVE DOMAIN REGIONS REQUIREMENT
A 25
sgmRNA-N, RELATIVE UNITS
20
B 15
10
5
C
0 +
- ΔA ΔB ΔC

32. ACTIVE DOMAIN REGION C REQUIREMENT
ΔA ΔA-C’
25
sgmRNA-N, RELATIVE UNITS
B
20
15
10
C 5
0
ΔA ΔA-C’ Δ[A-B]

33. ACTIVE DOMAIN REGION B REQUIREMENT
∆A ∆A-B*2r ∆A-B*1r ∆A-B*1r4 ∆A-B*1r3
B
25
sgmRNA-N, RELATIVE UNITS
20
15
10
5
0
∆A ∆A-B*2r ∆A-B*1r ∆A-B*2r4 ∆A-B*1r3 (-)

34. INTRAGENOMIC RNA-RNA INTERACTIONS
INVOLVING B RNA MOTIF
cB-M B-M cB-M B-M
nt 218-229 nt 26408 - 26421 nt 477-489 nt 26408 - 26421
∆G = -12.9 ∆G = -12.7

35. TGEV REPLICONS WITH MUTATED B AND cB RNA
MOTIFS
218-229 477-486 26,212-26,221
5’ 3’
B dE pE N
wt/B 5’ 3’
dE pE N
∆
wt/∆B 5’ 3’
cB-218 dE pE N
* B
cB-218*/B 5’ 3’
dE pE N
∆
cB-218*/∆B 5’ * 3’
* B* dE pE N
cB-218*/B* 5’ 3’
cB-477
B dE pE N
cB-477*/B 5’ * 3’
dE pE N
∆
cB-477*/∆B 5’ * 3’
B* dE pE N
cB-477*/B* 5’ * 3’
B* dE pE N
cB-477*/B*-14 5’ * 3’

36. sgmRNA N, RELATIVE UNITS
0
5
10
15
20
25
30
35
40
45
5’
w
t/B
w
t/∆
B
218
477
cB
-2
18
cB * /B
-2
18
*/∆
cB B
-2
18
* /B
*
cB
47
7*
cB /B
B
-4
26,212
77
*/∆
cB B
dE
-4
cB 77
-4 */ B
pE
77 *
*/ B
N
*-
14
3’
TRANSCRIPTION AND COMPLEMENTARY LEVELS

37. EVOLUTION OF VIRUSES WITH MUTATED B AND cB RNA
MOTIFS

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

39. MECHANISM OF DISCONTINUOUS TRANSCRIPTION
AD
cB-M
B-M
TRS-L
dE pE CS-N
5’(+) 3’(+)
pE CS-N
3’(+)
5’(-)
dE cCS-N
5’(+)
CS-L
B-M
cB-M
AD
cB-M
CS-L
5’(+)

40. CoV TRANSCRIPTION IS REGULATED
AT THREE LEVELS
TRS-L STABILITY AND SECONDARY STRUCTURE
COMPLEMENTARITY BETWEEN NASCENT
MINUS RNAs AND TRS-L
LONG DISTANCE RNA-RNA INTERACTIONS

41. TRM ACTIVITY WITHIN INFECTIOUS
VIRUSES
ORF 1a ORF 1b S 3a 3b E M N 7
wt-TGEV N N A
dE pE
0 2 4 6
CS
TRS-3a
AD dE pE CS
TRMopt-3a
TRMopt*-3a
TRMopt-19-3a
sgmRNA-3a/gRNA

42. GEOGRAPHICAL AREA ORIGIN OF SARS

43. SEVERE ACUTE RESPIRATORY SYNDROME
Neumonia High fever
Lymphopenia Death

45. SPREADING OF SARS
…JULY 5th, 03 END EPIDEMY BY WHO
NOV 02 JAN 03 FEB 03 DEC03 to JAN04
FOSHAN GUANGZHOU HONG KONG
FOUR LABORATORY CASES
Dr. A VIETNAM
CANADA FOUR NON-LABORATORY
MOST FIRST Nephro- ISOLATED CASES
SINGAPORE
RECENT MAJOR logist USA
COMMON OUTBREAK TWO ADDDITIONAL
ANCESTOR HOSPITAL HOTEL M SSE IN HK Beijin Apr F 26y, BSL-4
(>100 CONTACTS) Beijin Apr M 44y, BSL-4

46. RACOON
CIVET CAT
FERRETS

47. CORONAVIRUS EVOLUTION
GENUS α
GENUS β GENUS ϒ

48. ENGINEERING OF A SARS-CoV INFECTIOUS
cDNA CLONE AS A BAC
T by C T by A
10338 11163
8a
L 3b M 7a 8b 9b
CMV REP 1a REP 1b S 3a E 6 7b N
An
pBAC-SARS-CoV-URB*

49. BASIS OF SARS-CoV VIRULENCE
8a
3b M 7a 8b 9b
L REP 1a REP 1b S 3a E 6 7b N
An
DELETED GENE
Structural: E
Accessory: 6, 7a, 7b, 8a, 8b, 9b
Combinations: E, 6, 7a, 7b, 8a, 8b, 9b

50. BSL-3 CNB. CSIC. MADRID

51. GROWTH KINETICS OF SARS-CoV DELETION
MUTANTS
VERO E6 CaCo-2
107
wt
VIRUS TITER, pfu/ml
Δ6-9b
105
ΔE
103 ΔE,6-9b
101
0 24 48 72 0 24 48 72
TIME POST-INFECTION, HOUR
GENES: E, 6, 7a, 7b, 8a, 8b, 9b ARE NON ESSENTIAL

52. MORPHOGENESIS OF SARS-CoV IN ERGIC
SARS-CoV SARS-CoV-∆E
∆
SARS-CoV-∆ [6-9b]
∆ SARS-CoV-∆ [E,6-9b]
∆

53. ACTIVITY WHEEL USED TO QUANTIFY
CLINICAL ILLNESS

54. ∆
SARS-CoV-∆E-INFECTED HAMSTER WHEEL
ACTIVITY
INFECTION
1200
∆E
1000
REVOLUTIONS/ hr
800
MOCK
600
wt
400
200
0
-7 -6 -5 -4 -3 -2 -1 0 1 2 3 4
DAY

55. ATTENUATION OF SARS-CoV DELETION MUTANTS
IN TRANSGENIC MICE EXPRESSING hACE-2
12000 pfu intranasally
CLINICAL DISEASE LETHALITY
110 ΔE 100
STARTTING WEIGHT, %
ΔE,6-9b
SURVIVAL, %
ΔE
100 75
90 ΔE,6-9b 50
80 wt Δ6-9b 25
0 Δ6-9b
70
2 4 6 8 2 4 6 8
TIME POSTINOCULATION, days
TIME POSTINOCULATION, days

56. SUMMARY
E GENE CONDITIONS TISSUE SPECIFIC TROPISM
∆
SARS-CoV-∆E IS ATTENUATED
GENE E IS A VIRULENCE FACTOR

57. MECHANISM OF E PROTEIN VIRULENCE
E PROTEIN AND HOST GENE EXPRESSION
8a
3b M 7a 8b 9b
L REP 1a REP 1b S 3a E 6 7b N
An
±E

58. DIFFERENTIAL GENE EXPRESSION IN ∆E
DIFFERENTIAL GENE EXPRESSION IN ΔE AND WT
AND WT INFECTED CELLS
INFECTED CELLS
wt vs mock ΔE vs mock ΔE vs wt
14
12
10
Vero E6
8
6 15 hpi
LOG EXPERIMENT
4
2
14
12
10
MA104
8
6 65 hpi
4
2
2 4 6 8 10 12 14 2 4 6 8 10 12 14 2 4 6 8 10 12 14
LOG CONTROL

59. Δ
GENES DIFFERENTIALLY EXPRESSED IN SARS-CoV-ΔE
VS FULL-LENGTH VIRUS INFECTED CELLS
FOLD CHANGE FOLD CHANGE
GENE VERO MA-104 GENE VERO MA-104
STRESS RESPONSE IMMUNOREGULATION
Hsp10 HspE1 + 3.3 -- FKBP4 + 4.7 + 5.5
Hsp20 B5 CRYAB -- + 3.2 RIPK2 + 2.3 --
Hsp27 B1 +19.0 -- ZC3H12A -- + 2.9
Hsp40 DNAJB1 +14.8 +25.9
Hsp40 DNAJA1 + 8.7 + 7.2
INFLAMMATION
Hsp40 DNAJB6 + 3.9 + 2.1
Hsp40 DNAJA4 -- + 2.6 NFKBIZ + 2.9 --
Hsp40 DNAJB4 -- + 4.1 ADAMTS1 + 2.1 --
Hsp47 SERPINH1 + 6.7 + 2.5 CCL2 -- - 3.2
Hsp60 D1 + 4.1 + 4.7 CXCL2 -- - 2.2
Hsp70 A1A +22.3 +29.3
APOPTOSIS
Hsp70 A1B +18.8 --
DEDD2 + 2.6 + 5.0
Hsp70 A8 + 5.8 + 4.9
CLU + 2.1 + 2.0
Hsp70 A4L -- + 2.3
PHLDA1 + 3.2 --
Hsp90 AA1 +15.1 + 6.5
STK17B -- + 2.2
Hsp90 AB1 + 6.1 + 2.7
Hsp105/110 +35.3 +13.4 SIGNAL TRANSDUCTION
UBB + 3.9 + 4.4
DSCR1 + 5.9 --
UBC + 2.3 + 2.7
DUSP1 + 4.0 --
BAG3 + 7.2 + 3.9
DUSP10 -- + 2.7
CCT4 + 3.3 --
YWHAG + 2.5 --
CCT3 + 3.3 --
WASL + 2.5 --
AHSA1 + 4.7 --
MAP2K3 + 2.1 --
CRYAB -- + 3.2
FOLR1 -- +15.6
TCP1 -- + 2.3
PDE4B -- + 2.5
STIP1 -- + 2.1
K-RAS -- - 2.6

60. UNFOLDED PROTEIN RESPONSE
RE CYTOPLASM NUCLEUS
ACTIVATION
IN E VS WT-
Δ
GRP78 GRP78
PERK eIF2 TRANSLATIONAL INFECTED CELLS
BLOCK
PERK eIF2
P
ATF4
TRANSLATION
ATTENUATION -
CHOP
GRP78
GENE
GRP78
CLEAVED
GRP78
CORRECT
ATF6
ATF6
EXPRESSION
PROTEIN
FOLDING
-
CHOP
GRP78
GRP78 GRP78
XBP1
IRE1 mRNA
IRE1 sXBP1
sXBP1
PROTEIN
DEGRADATION +
p58IPK
mRNA

61. EFFECT OF E PROTEIN ON THE STRESS PRODUCED
Δ
BY SARS-CoV-ΔE INFECTION
E PROTEIN PROVIDED IN TRANS
12 HSP A1A HSP90 AA1 HSP H1 SERPIN H1 HSP E1 18S
E+
FOLD CHANGE, relative units
10 E-
8
6
4
2

62. SARS-CoV E PROTEIN AND STRESS PRODUCED
BY RSV INFECTION
E PROTEIN PROVIDED IN TRANS
350 7
HSP90 AA1 UBB HSP H1 SERPIN H1 HSP E1 18S
300 6
E+
FOLD CHANGE, relative units
250 5
E-
200
4
150
3
100
2
50
1
2 24 48 2 24 48 2 24 48 2 24 48 2 24 48 2 24 48
TIME POST-INFECTION, hours

63. CONCLUSION
E PROTEIN REDUCED CELLULAR STRESS
INDUCED BY:
SARS-CoV INFECTION
RSV INFECTION
THAPSIGARGIN - Ca++
TUNICAMYCIN - GLYCOSYLATION

64. Δ
SARS-CoV-ΔE INFECTION AND INFLAMMATION
STRESS INFLAMMATORY
RESPONSE RESPONSE
+E
-E

65. MOUSE ADAPTED SARS-CoV
SARS-CoV-MA15
High titers in lungs
Viremia, extrapulmonary spread
Neutrophilia
Pathological changes in lungs
Death
K. SUBBARAO AND R. BARIC LABS

66. CONSTRUCTION OF A MOUSE ADAPTED SARS-CoV
8a
3b M 7a 8b 9b
L REP 1a REP 1b S 3a E 6 7b N
An
nsp5 nsp9
10384 12814
nsp13 S (RBD) M
16117 22797 26428
nsp5
10793
ENGINEERED MUTANTS wt
Δ[6-9b]
ΔE
Δ[E,6-9b]

67. CONCLUSION
Δ
SARS-CoV-MA15-ΔE IS ATTENUATED AND
FULLY
PROTECTS BOTH YOUNG AND OLD MICE
THEREFORE IT IS A PROMISING VACCINE
Δ
CANDIDATE, BETTER THAN SARS-CoV-ΔE

68. CNB. CSIC. MADRID COLLABORATORS
REPLICATION PROTEOMICS
FERNANDO ALMAZAN SILVIA JUAREZ. CNB-CSIC
AITOR NOGALES ALEXANDER AKOULICHEV. OXFORD
SILVIA MARQUEZ
GENOMICS
TRANSCRIPTION AND ASSEMBLY IRENE LOPEZ-VIDRIERO
ISABEL SOLA MARTA GODOY
PEDRO A. MATEOS JUAN CARLOS OLIVEROS
SONIA ZUÑIGA
MARTINA BECARES
ANIMAL MODELS
KANTA SUBBARAO. NIH
VIRUS-HOST INTERACTION: SARS
STANLEY PERLMAN. UNIV. IOWA
MARTA L. DeDIEGO
JOSE LUIS NIETO
JOSE MANUEL JIMENEZ ELECTRON MICROSCOPY
JOSE ANGEL REGLA MARIA TERESA REJAS. CBM, CSIC
RAUL FERNANDEZ CRISTINA PATIÑO. CNB. CSIC
CARLOS M. SANCHEZ
SARHAY ROS
LUIS ENJUANES MARGARITA GONZALEZ