baric-sars safety symposium2

8/2/2019 Baric-sars Safety Symposium2

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SARS-CoV: Reverse Genetics and Pathogenesis

Boyd Yount, Kris Curtis, Amy Sims

100 nmKindly providedby the CDC


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Coronaviridae

-Group 1TGEV

HCV229E

FIPHuCV-NL

-Group 2

MHVBCV

HCV-OC43

SARS-CoV

-Group 3

IBV

NIDOVIRALES ORDER

Molecular Clones available for TGEV,

HCV229E, MHV, SARS-CoV and IBV


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SARS-CoV

CASE MORTALITY RATES Overall Mortality Rate: 10-12%; ~20% require intensive care and/or

mechanical ventilation 50% Age 65 or older

Future of the SARS Outbreak?

Three laboratory acquired infections in 2003-2004 (Taiwan, Singapore and China Four SARS-CoV infections reported in China in Dec 2003-04; source: likely

zoonosis transmission

~1-2% of serum samples collected in Hong Kong and in Guangdong province in2001-2002 were seropositive to SARS-CoV

SARS-CoV likely introduced multiple times into human population in Southeast A


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OBJECTIVES Coronavirus Life Cycle and Replication

Coronavirus Molecular Genetics Techniques Targeted Recombination

Coronavirus reverse genetics

SARS CoV Reverse Genetics Pathogenesis, replication and vaccine development

Replication Competent Propagation defective viruses

Safety Measures Facilities

Personnel

Community

Summary


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SARS-CoV Schematic

Nucleocapsid protein (N)

Spike

Protein (S)

Envelopeprotein (E)

RNA

Membrane

glycoprotein (M)

S glycoprotein

interacts with humACE2 molecule foentry into cells inculture; Majordeterminant of hosrange; neutralizingepitopes

E and M driveassembly of matuparticles into anintermediatecompartment betwgolgi and rER

Readily inactivate

65o

C, 70-100%alcohol, or phenolbased disinfectant

ss, linear, positive polarity RNA genome of ~29 Kb in length


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0 10 2025

30 K b

Leader

Leader

T R S

O R F 1 a

O R F 1 b

2

3a

b

4

5

6

7 a

b

8 a

b

9a

9b

nsp1 2 3 4 5 6-11

PLP1 / PLP2

3CLpro

12 13 14 15 16

2 - O- MTXenduU

ExoN

Hel1R d R p

T m 1 T m 2

SA R S G roup S pec ific G enes

O RF 3ab , O RF 6 , O RF 7ab

O R F 8 a /b a n d O R F 9 b

Trans cr ip t ion R eg ulatory Se que nce (TR S)

C S - A C G A A C ORF8a/b 29 bp insertion in CiviCoV produces a single ORF;


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0 10 2025

30 K b

Leader

T R S

O R F 1 a

O R F 1 b

2

3a

b

4

5

6

7 a

b

8 a

b

9a

9b

S G lycoprote in

O R F 3 a - b

E pro te in (OR F4)

M g lycopro te in (ORF 5)

O R F 6

O R F 7 a - b

O R F 8 a - b

ORF9a-b )

nucleocapsid

SA R S G roup Sp ec ific G enes

O R F 3 a b , O R F 6 , O R F 7 a b

O R F 8 a /b a n d O R F 9 b

m R N A 2

m R N A 9

Trans cr ip t ion R egu latory Se que nce (TR S)

C S - A C G A A C ORF8a/b 29 bp insertion in CiviCoV produces a single ORF;


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Replicase

Subgenomic ORFs

5'- 3'

5'

5'

S

ACGAAC ACGAAC ACGAAC

Transcription Attenuation

of incompletet negative strands

Leader RNA


Sawicki and Sawicki 1990, Sethna and Brian 1989-90;

Schaad et al., 1993; van Marle G. et al., 1999, Baric et al., 2

(+)

(-)

(-)


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Replicase

Subgenomic ORFs

5'- 3'

5'

5'

5'

S




Leader RNA




(+)

(-)

(-)


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Replicase

Subgenomic ORFs

5'- 3'

5'

5'

5'

5'3'

3'5'

S


S Protein

Subgenomic minus

Subgenomic plus



Leader RNA

Anti-leader

RNA




Derived from

Body CS

(+)

(-)

(-)

(+)

(-)


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Coronavirus RNA Recombination

(Positive or Negative RNA Synthesis- ~20%)

Replicase

Replicase Subgenomic ORFs

Subgenomic ORFs

A

Full-length

minus

strands

Recombinant

Genomes 5' 3'

5'

5'

3'

3'

Koetzner et al., 1992 J. Virol. 66, 1841-1848; Fu and Baric, 1993 Virology


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Coronavirus RNA Recombination

(Positive or Negative RNA Synthesis- ~20%)

Replicase

Replicase Subgenomic ORFs

Subgenomic ORFs

A

B

Subgenomic minus strand

Full-length

minus

strands

Recombinant

Genomes 5' 3'

5'

5'

3'

3'

5'3'

Koetzner et al., 1992 J. Virol. 66, 1841-1848; Fu and Baric, 1993 Virology


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Coronavirus Genetics

Targeted RNA Recombination

Kuo et al., 2000 J. Virol. 74, 1393-1406


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MHV MHVFIPV

1a 2a HE S E M N fMHV

1a pMH5

MHV MHV

HE S E M N1a

Infect AKD

with fMHV

Electroporate pMH54

transcripts into AKDAKD Feline

Cell

DBT

Cell

Select for growth

on Murine cells

Targeted RNA Recombination

RNA Recombination

fMHV

pMH54

MHV-A59parent

encodingSARS 3 endregulatory

sequences

SARS 3-end


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SARS Targeted Recombination

P L P L 3cPro

5 '

O R F 1 aO R F 1 b p 3 0

E M NH E

M H V -A 5 9 /S A R S S 3 ' E n d

M H V

Leade r

R N A

p 12

S A R S S

P L 3cPro

5 '

O R F 1 aO R F 1 b

m S A R S S C h im e raM H VLeade rR N A

P L P L 3cPro

5 '

O R F 1 aO R F 1 b p 3 0

E M NH E

M H V

Leade r

R N A

p 12

3 '

= S A R S S e q u e n ce

3a

4

5

6

7 a

b

8a

b

9a

9b

M H V S

Goebel et al., 2004 J. Virology

7846-7851


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SARS Targeted Recombination

P L P L 3cPro

5 '

O R F 1 aO R F 1 b p 3 0

E M NH E

M H V -A 5 9 /S A R S S 3 ' E n d

M H V

Leade r

R N A

p 12

S A R S S

P L 3cPro

5 '

O R F 1 aO R F 1 b

m S A R S S C h im e raM H VLeade rR N A

P L P L 3cPro

5 '

O R F 1 aO R F 1 b p 3 0

E M NH E

M H V

Leade r

R N A

p 12

3 '

= S A R S S e q u e n ce

3a

4

5

6

7 a

b

8a

b

9a

9b

M H V S

Concerns: Humanized coronavirus encoding SARS S glycoprotein/SARS genetic backbone

targeted to murine cells and rodents

Weakly pathogenic coronaviruses encoding SARS sequences or virulence allele

Recombinant SARS-CoV encoding genes from other viruses or cells

Goebel et al., 2004 J. Virology

7846-7851


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Coronavirus Infectious Clone

Large Size of the Viral GenomeStable Cloning Vectors

Regions of Chromosomal Toxicity

Synthesizing Infectious Transcripts

Ease of Manipulationthe availability of rare cutting restriction sites for reverse genetic applications

Solutions: Clone genome into BAC vectors, Vaccinia Vectors, oruse Systematic Assembly of component clones

HCV 229E IBV*


18/40Thiel V et al., 2001. J. Gen. Virol 82, 1273-1281

HCV 229E, IBV*

TGEV*

Almazan, F. et al., 2000. PNAS USA 97, 5516-55

DNA launch using host RNApolymerase

**Full length genomes in self-replicating vectors (vaccinia or BAC),easily renewable resource


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Bgl I 5.GCCNNNNNGGC.3

3.CGGN

NNNNCCG.5

Systematic Assembly of Adjoining cDNA Clones(MHV, TGEV, SARS-CoV)

Class IIS Restriction Endonucleases unique interconnecting junction

Recursive Assembly Strategy: Bgl1 = 264 fragments; Sfi1 (8 cutter with 3

nucleotide variable overhang) = 264 fragments of ~64Kb.

*allows for directional assembly of recombinant or synthetic DNAs into full

length, million base pair microbial genomes

-synthetic cDNAs of ~5-8 Kb in length are readily available commercially

Yount et al., J.Virol 74, 10600-10611 (2000)

GCCNNNNNGGC B l1 Sit


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SARS ASSEMBLY STRATEGY

GCCNNNNNGGC- Bgl1 Site

Systematic assem

of contiguous cDNclones

64 possible ends;most ends are notcompatible with another ends

Assemble 264

fragments using a

recursive assemblstrategy

Yount et al., 2003. PNAS USA 100, 12995-13000


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A B C D E F

Purify plasmid DNA containing SARS CoV fragments

Digest with Bgl2 restriction endonuclease and purify

Ligate fragments

Transfect Vero E6 cells

Transcribe genome length RNA

Finite source of non-replicating full length

cDNA that is consumedin the reaction

N transcripts (N protein)boots infectivity by 10->1000 fold (enhances

transcription)

Set of

Contiguous ~Kb pieces


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SARS Reverse GeneticsBuilt full length constructs and

performed in vitro transcriptionwith T7 RNA polymerase

Electroporated cells, performedFA at about 24 hrs post-transfection

+/- N transcripts

Virus yields determined byplaque assay


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icSARS-CoV Pathogenesis

2 NHPs (Cynomolgus macaques) where infected by bronchialinstillation and intranasal routes with icSARS-CoV (3x106 PFU/ml); 2

NHPs inoculated with wildtype SARS Urbani

NHPs where monitored daily for clinical signs of disease

Even days chest radiographic films were taken (AP and Lateral)

Even days nostrils, throat, blood, urine, and feces were assayed forviral load by TaqMan RT-PCR

J.Paragas et al., unpublished


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Wildtype and Recombinant icSARS-CoV

NHP Results

Recombinant icSARS-CoV and wildtype are comparable

No clinical signs of disease

No CBC or chemistry changes

No temperature changes No reduction in oxygen saturation

Viral load is pending RADIOGRAPHIC EVIDENCE OF MULTI-FOCAL LOWER

LOBE PNEUMONIA in icSARS-CoV INFECTED ANIMALS

Jason Paragas et al, unpublishe


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Day 0 Day 8

Right lower lobe pneumonia on X-Ray


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Summary SARS-CoV Pathogenesis

icSARS-CoV produces radiographic pneumonia; reminiscent of clinicacourse of SARS in children less than 13 years of age.

Neutralization titers of 1:640 and 1:1280 against SARS-CoV

Animals are protected from wildtype challenge

Results clearly not as dramatic as reported by other groups in theliterature (Fouchier RAM et al., Nature 2003).

icSARS-CoV and wildtype virus pathogenesis are comparable inmacaques

icSARS-CoV and wildtype virus replicate to similar titers in the mouse


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Recombinant SARS-CoV

Leader

Leader

TRS

ORF1a

ORF1b

2

3a

b

4

5

6

7a

b

8a

b

9a

9b

icSARS-WT

Leader

LeaderTRS

ORF1a

ORF1b

2

3a

b

4

5

6

8a

b

9a

9b

icSARS X4-GFP

GFP

X4

Leader

Leader

TRS

ORF1a

ORF1b

2

3a

b

4

5

68a

b

9a

9b

icSARS X4

Accessory ORF function in replication and pathogenesis?


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Indicator Virus to Screen Compounds

with anti SARS- CoV Activity

Platform for rapid screening of compounds with anti-SARS activity


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Are ORF3a and ORF6 accessory proteins essential?

Why? Deletion of group specific genes attenuates all othercoronaviruses tested but little effect on in vitro replication=candidateattenuated viruses and identification of virulence alleles


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SARS-CoV Recombinant Virus GrowthPanel C: Recombinant Virus Growth in MA104

Cells

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

2 6 12 22 32

Hrs Postinfection

Virus

Titer(Log

10

PFU/m

l)

Urbani

icSARS

icSARS X1

icSARS X3

icSARS X4

GFP

icSARS X4

Luciferase

Panel A: Recombinant Virus Growth in Vero

Cells

1.00E+03

1.00E+04

1.00E+05

1.00E+06

1.00E+07

1.00E+08

2 6 12 22 32

Hrs Postinfection

V

irusTiter(Log

10

PFU/m

l)

Urbani

icSARS

icSARS X1

icSARS X3

icSARS X4

GFP

icSARS X4

Luciferase


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Attenuated SARS-CoV or Safer Laboratory Strains

Leader

Leader

TRS

ORF1a

ORF1b

2

b

4

5 7a

b

8a

b

9a

9b

ExoNCS1 and/or CS2 Cleavage

Site 2 aa deletions

3a

ORF6-X3 Deletion

Combinations: Viable

X1, X2, X3 Yes

CS1, ExoN Y-H, X3 Yes CS2, ExoN Y-H, X3 Yes

Live viruscandidatesseed stockfor KilledVaccines

Attenuating mutations in MHVwhose sequence targets arealso conserved in SARS-CoV

8.0 x 106

5.0 x 106

7.5 x 106


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Replication Competent Propagation Deficient Coronaviruse

Leader

Leader

TRS

ORF1a

ORF1b

2

3a

b

6

7a

b

8a

b

9a

9b

icSARS-CoV lacking Eand M ORFs

S Gene

N

S and N Protein

Progeny genomes

No

105-107 viral replicon particles (VRPs) Curtis K et al., 2002; Ortego J. et al., 2


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Leader

Leader

TRS

ORF1a

ORF1b

2

3ab

6

7a

b

8a

b

9a

9b


S Gene

N

CMV SARS E pA

CMV SARS M pA

E and M glycoprotein

provided in trans

S and N Protein

Helper RNAs

Progeny genomes

105-107 viral replicon particles (VRPs) Curtis K et al., 2002; Ortego J. et al., 2


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Leader

Leader

TRS

ORF1a

ORF1b

2

3ab

6

7a

b

8a

b

9a

9b


S Gene

N

CMV SARS E pA

CMV SARS M pA

E and M glycoprotein

provided in trans

S and N Protein

Helper RNAs

Progeny genomes

105-107 viral replicon particles (VRPs)

E is absolutely essential for

TGEV VRP formation, but notMHV

What is essential for SARS

particle production?Most would likely agree that SE, M deficient replicationcompetent viruses would not

produce progeny virions

Very Safe

Recombination repair?

Helper RNA or exogenouscoronavirus

2/3 genome length is replicasin coronaviruses

Curtis K et al., 2002; Ortego J. et al., 2


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Safety Precautions

Facility Personnel

Community

Developed in collaboration with UNCEnvironmental Health Department (EHS),

University Employee Occupational Health Center(UEOHC), Local and State Public Health Offices


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Enhanced Features

in the UNC SARS-CoV BSL3 Facility

Shower in and out facilities, dual anteroom access

HEPA filter exhaust

Redundant exhaust fans

Increased Security Card key access, alarm system to Public Health/Campus Police, Lab

controlled combination lock

Techniplast SealsafeTM Hepa filtered animal housing


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Personnel Precautions

Observed in UNC SARS-CoV Facility

Training Program University safety training programs (BSL2 and BSL3 training, Laboratory Safety, chemical,

radiation and Occupational health, Blood borne pathogen, etc.), Proficiency withnumerous virological assays/mildly pathogenic coronaviruses, Proficiency with PAPR andprotective gear, Partnered training in BSL3 (~6 months)

PAPRs with Hepa filters and hoods worn at all times

Yearly Flu vaccines

Archived serum samples

Protective disposable clothing: Disposable coveralls

Shoe covers

Disposable wrap around gown

Double gloves


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Worker/Community Protective Measures

Cease all SARS-CoV work 14 days prior to any travel by masstransit

High Risk Exposure Event (aerosol generating activity with failure ofadequate work practices and/or PPE, needle stick or animal bite)

Low Risk Exposure (e.g. no aerosol generating activity at time offailure of work practices and/or PPE):

No Recognized Exposure Involving Breach of PPE or Inadequate WorkPractices, but Employee Develops Respiratory Symptoms with Fever:

Response: Various times of self quarantine at home. Worker has mask andthermometer and is in daily contact with supervisor, EHS and physicians/staff at


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Recombinant Coronaviruses Stability in nature?

Delete ORFs, rearrange ORFs alter replicase protein processing and

expression, replication competent propagation defective (engineer to be fairlysafe)

Recombination with existing circulating strains?

Recombination repair of defective allele Distribution of foreign sequences or virulence alleles into related

coronaviruses

Recombination/reversion proof coronaviruses?

Pathogenesis studies and identification of virulence alleles

Platform for development of safe laboratory strains or attenuatedviruses for live or killed vaccine applications, drug testing

Recombinant coronavirus vaccines for humans and animals


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SARS CoV-Related Research

NIH AI23946, AI059136, AI061819

Baric Laboratory (UNC) Boyd Yount

Kris Curtis

Will McRoy

Amy Sims

Damon Deming Eric Donaldson

Tim Sheehan

UNC UEOHC Drs. Brian Boehlecke and David

Weber

UNC EHS Department

Ray Hackney Deb Howard

USAMRIID Group Lisa Hensley

Elizabeth Fritz

Thomas Geisbert

Peter Jahrling

Jason Paragas, PhD James Lawler, MD, LCDR,USN

Aura Garrison

Mallory Tate, DVM, MAJ, USA

John Huggins, PhD Tim Endy, MD, COL, USA

Denison Laboratory (Vanderbilt) Mark Denison

Erik Prentice

baric-sars safety symposium2

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