Coronavirus Pandemic Potential and Research Response Mark R. Denison, M.D. Departments of...

Coronavirus Pandemic Potential and Coronavirus Pandemic Potential and Research ResponseResearch Response

Mark R. Denison, M.D.Mark R. Denison, M.D.

Departments of Pediatrics, Microbiology & ImmunologyDepartments of Pediatrics, Microbiology & Immunology

The Elizabeth B. Lamb Center for Pediatric ResearchThe Elizabeth B. Lamb Center for Pediatric Research

Vanderbilt University Medical CenterVanderbilt University Medical Center

• Vanderbilt University Vanderbilt University Department of Pediatrics, Department of Pediatrics, Department of Microbiology & Department of Microbiology & ImmunologyImmunology

• Elizabeth B. Lamb Center for Elizabeth B. Lamb Center for Pediatric Research Pediatric Research

• NIH R01 AI26603 -15 NIH R01 AI26603 -15 R01 AI50083 -R01 AI50083 - 01 (S1)01 (S1)

Disclosures: Disclosures: nonenone

Denison LabDenison Lab

• Xiaotao LuXiaotao Lu• Erik PrenticeErik Prentice• Rachel GrahamRachel Graham

UNC Chapel HillUNC Chapel Hill

• Ralph BaricRalph Baric

• Boyd YountBoyd Yount

• Amy SimsAmy Sims

Course of the PandemicCourse of the Pandemic

• February 2003February 2003

““Dad why don’t you work on an ‘important’ virus”Dad why don’t you work on an ‘important’ virus”

• April 2003April 2003

““Daddy, I think you are getting kind of ‘full of yourself’”Daddy, I think you are getting kind of ‘full of yourself’”

• August 2003August 2003

““Dad, why haven’t you found a cure for SARS yet?”Dad, why haven’t you found a cure for SARS yet?”

ObjectivesObjectives

• Describe the role of basic research in SARS Describe the role of basic research in SARS

• Describe coronavirus life cycle and replicationDescribe coronavirus life cycle and replication

• Discuss genetic systems coronavirus researchDiscuss genetic systems coronavirus research

• Summarize research goals Summarize research goals

SARS, Public Health, and ResearchSARS, Public Health, and Research

SARS - CoV SARS - CoV

Pandemic PotentialPandemic Potential

High mortalityHigh mortality

Worldwide spreadWorldwide spread

Naive populationNaive population


Pandemic potentialPandemic potential



Immune naive populationImmune naive population


Pandemic PotentialPandemic Potential



Naive populationNaive population

Public Health ResponsePublic Health Response



Pandemic potentialPandemic potential



Immune naive populationImmune naive population

No DiseaseNo Disease

New models for detection and New models for detection and intervention of emerging infectionsintervention of emerging infections

Coronavirus replication, disease, Coronavirus replication, disease, and preventionand prevention

Antivirals and vaccines for Antivirals and vaccines for coronaviruses coronaviruses


• Escape / mutationEscape / mutation

• PersistencePersistence

• Animal reservoirAnimal reservoir










• Continued human diseaseContinued human disease

• Re-emergence of epizootic, Re-emergence of epizootic, epidemic or pandemic diseaseepidemic or pandemic disease

• New SARS-CoV disease New SARS-CoV disease









• Continued human diseaseContinued human disease


• New SARS-CoV disease New SARS-CoV disease

• Need for research in biology, Need for research in biology, pathogenesis, therapeutics and pathogenesis, therapeutics and vaccinesvaccines







• Civet, Raccoon Dog, other?Civet, Raccoon Dog, other?


• Singapore - September 2003Singapore - September 2003


• ASx infection animals / humans ASx infection animals / humans

• Need for research in biology, Need for research in biology, pathogenesis, therapeutics and pathogenesis, therapeutics and vaccinesvaccines





Big QuestionsBig Questions• Is SARS-CoV still present in human populations? Is SARS-CoV still present in human populations?

• Is there risk of human animal transmission? Is there risk of human animal transmission?

• Will SARS-CoV adapt to better survive in human Will SARS-CoV adapt to better survive in human populations?populations?

• Will SARS reemerge as a “seasonal disease”Will SARS reemerge as a “seasonal disease”

• Can vaccines and therapeutics for SARS be Can vaccines and therapeutics for SARS be developed, tested and used?developed, tested and used?


• Describe the role of basic research in SARS Describe the role of basic research in SARS

• Describe coronavirus life cycle and replicationDescribe coronavirus life cycle and replication

• Discuss genetic systems coronavirus researchDiscuss genetic systems coronavirus research


100 nm100 nm

SS

EE

MM RNA + NRNA + N

SARS Coronavirus SARS Coronavirus (SARS-CoV)(SARS-CoV)

CoronaviridaeCoronaviridaeGroup 1Group 1

TGEVTGEV

HCoV229EHCoV229E

Group 2Group 2MHVMHV

BCoVBCoV

HCoV-OC43HCoV-OC43

Group 3Group 3

IBVIBV

Nidovirales OrderNidovirales Order

SARS-CoVSARS-CoV

Snijder et al 2003 J. Mol. Biol. (2003) 331, 991–1004Snijder et al 2003 J. Mol. Biol. (2003) 331, 991–1004

Coronavirus Genome Organization Coronavirus Genome Organization

MHV Genome + RNA 32 kbMHV Genome + RNA 32 kb

genes 2-7genes 2-7Replicase gene 22 kbReplicase gene 22 kbLeaderLeader

AANN

SS EE MM NNORF 1aORF 1aORF 1bORF 1b

SARS Genome + RNA 30 kbSARS Genome + RNA 30 kb

genes 2-9genes 2-9Replicase gene 20 kbReplicase gene 20 kbLeaderLeader

AANN

SS EE MM NNORF 1aORF 1aORF 1bORF 1b

Coronavirus Replicase Coronavirus Replicase

p213p213 polpol helhelp70 p70 3CLpro3CLprop20p20

PLP2PLP2SARSSARS

10001000 20002000 30003000 1400014000 50005000 60006000 70007000aaaa

PLP1PLP1

p210p210 polpol hel hel p65p65 3CLpro3CLprop28p28

PLP-2PLP-2MHVMHV

Snijder et al 2003

doi:10.1016/S0022-2836(03)00865-9 J. Mol. Biol. (2003) 331, 991–1004

nucleusmaturationmaturationassemblyassembly

attachmentattachment

entry

releasereleaseReplication ComplexesTranslationProcessingRNA synthesis

Coronavirus Life CycleCoronavirus Life Cycle



entry





entry



MHV-A59 infected DBT cellsMHV-A59 infected DBT cells

DIC/HelDIC/Hel

Coronavirus genome and replicationCoronavirus genome and replication

replicase polyproteinreplicase polyprotein

polpol

Genome + RNA 32 kbGenome + RNA 32 kb

SS EE MM NN

genes 2-7genes 2-7gene 1 (replicase)gene 1 (replicase)R CR C

R CR C

LeaderLeader

775'5'GenomeGenomeRNARNA ((++))

3'3'22 33 44 55 66

AAnn

Coronavirus Transcription and ReplicationCoronavirus Transcription and Replication

Replicase geneReplicase gene genes 2-7genes 2-7

775'5'

3'3'

GenomeGenomeRNARNA ((++))

((--))

3'3'22 33 44 55 66

IntermediateIntermediateRNARNA

AAnn

5'5'

UUnn



5'5' 3'3'

AAnn


775'5'

3'3'


3'3'22 33 44 55 66

AAnn

AAnn

5'5'



775'5'

3'3'


3'3'22 33 44 55 66

AAnn

AAnn

5'5'



775'5'

3'3'


3'3'22 33 44 55 66

AAnn

UnUn

5'5'



775'5'

3'3'


3'3'22 33 44 55 66

AAnn

UUnn

5'5'



AAnn

((--) strand ) strand subgenomic subgenomic RNARNA

((++) strand ) strand Subgenomic mSubgenomic mRNARNA

Spike (S)Spike (S)


77RNARNA

5'5'

3'3'

GenomicGenomicRNARNA

LeaderLeaderRNARNA

((++))

((++))

((--))

3'3'

Spike (S)Spike (S)

MM

NN

EE

NSNS

NSNS

Gene 1Gene 1

ReplicaseReplicase

ProteinsProteins

1122 33 44 55 66

Genes 2-7Genes 2-7

RNARNA

33

44

55

66

77

22

5'5' 3'3'

AAnn

AAnn

AAnn

AAnn

AAnn

AAnn

AAnn

AAnn

5'5'

UUnn

SubgenomicSubgenomicRNAsRNAs

(( +/-+/- ))

GenomicGenomicRNARNA ((++)) ReplicaseReplicase



polpol


SS EE MM NN


R CR C

LeaderLeader



polpol


SS EE MM NN


R CR C

LeaderLeader



polpol


SS EE MM NN


R CR C

LeaderLeader

Proteinase inhibitors result in shutoff of viral Proteinase inhibitors result in shutoff of viral RNA synthesis at any time during MHV infectionRNA synthesis at any time during MHV infection

[[33 H] c

pm

H] c

pm

101033

101044

101055

Time (hours p.I)Time (hours p.I)11 22 33 44 55 66 77 88 99 1010

E64-dE64-d

Kim et al 1995Kim et al 1995



entry





entry





entry



Coronavirus Replication and Coronavirus Replication and Targets for InhibitionTargets for Inhibition

• S protein, receptorS protein, receptor

• Fusion, uncoatingFusion, uncoating

• Replicase polyprotein expression and processingReplicase polyprotein expression and processing

• Virus assembly and releaseVirus assembly and release

• Novel functions (polymerase, helicase, methyltransferase, Novel functions (polymerase, helicase, methyltransferase, exonuclease, CoMt)exonuclease, CoMt)

• Cellular functions used by virusCellular functions used by virus

– CholesterolCholesterol

– Membrane TraffickingMembrane Trafficking

– AutophagyAutophagy


• Describe the role of basic research in Describe the role of basic research in SARS SARS

• Describe coronavirus life cycle and Describe coronavirus life cycle and replicationreplication

• Discuss genetic systems coronavirus Discuss genetic systems coronavirus researchresearch


• High theoretical mutation rate: 10High theoretical mutation rate: 1044 per template per template per replication (3 changes per genome)per replication (3 changes per genome)

• RNA-RNA homologous recombination (20%)RNA-RNA homologous recombination (20%)

• Result:Result: rapid adaptation, recovery from rapid adaptation, recovery from deleterious mutations, mechanisms to acquire deleterious mutations, mechanisms to acquire and regain virulence.and regain virulence.

• Genome tolerates deletions, mutations, Genome tolerates deletions, mutations, substitutionssubstitutions

Coronavirus Replication and Molecular Biology: Coronavirus Replication and Molecular Biology:

General ThemesGeneral Themes

Coronavirus Diseases: General ThemesCoronavirus Diseases: General Themes

• Repeat infectionsRepeat infections

• Persistent infectionsPersistent infections

• Disease enhancement in vaccinated animalsDisease enhancement in vaccinated animals

• Trans-species transmissionTrans-species transmission

• Changes in virus transmission, tropism and Changes in virus transmission, tropism and disease - disease - **New Coronavirus Diseases**New Coronavirus Diseases

Coronavirus Vaccines: General Themes Coronavirus Vaccines: General Themes

• Live attenuated, inactivated virus, protein, heterologous Live attenuated, inactivated virus, protein, heterologous virus, DNA have been usedvirus, DNA have been used

• ““Classical” live-attenuated vaccines have been most Classical” live-attenuated vaccines have been most licensed and employedlicensed and employed

• Challenges for vaccines : poor protection, enhanced Challenges for vaccines : poor protection, enhanced disease, altered disease, reversion to virulence, disease, altered disease, reversion to virulence, recombinationrecombination

• Every coronavirus has required different approachesEvery coronavirus has required different approaches

• **Multiple pathways for SARS vaccine development**Multiple pathways for SARS vaccine development

Coronavirus Reverse Genetics Coronavirus Reverse Genetics

• Infectious clones : MHV, IBV, TGEV, HCoV-229EInfectious clones : MHV, IBV, TGEV, HCoV-229E

– In vitro assembly, Vaccinia recombinants, BACIn vitro assembly, Vaccinia recombinants, BAC

• Applicable to entire genomeApplicable to entire genome

• Example of mutations yielding viable virusesExample of mutations yielding viable viruses

– Gene deletions, gene duplicationsGene deletions, gene duplications

– Gene substitution (GFP)Gene substitution (GFP)

– Gene order rearrangement Gene order rearrangement

– Replicase polyprotein cleavage site deletionReplicase polyprotein cleavage site deletion

In vitro ligationIn vitro ligation

RT / PCR RT / PCR cDNA clonescDNA clones

Genome +strand RNA (32 kb)Genome +strand RNA (32 kb)

Transcribe Transcribe + strand RNA+ strand RNA

Transfect cellsTransfect cells

nucleusnucleus

Yount, Denison, Weiss and Baric 2002Yount, Denison, Weiss and Baric 2002






nucleusnucleus







nucleusnucleus


Mutagenesis of MHV genome Mutagenesis of MHV genome

Replicase gene (22kb)5’

3’

replicase polyprotein

K G Y R G V K P

CS1 P5 P4 P3 P2 P1 P1’ P2’ P3’

K G Y R G V K PK G Y R G V K P

CS1CS1 P5 P4 P3 P2 P1 P1’ P2’ P3’P5 P4 P3 P2 P1 P1’ P2’ P3’

Mut 3 Mut 3 HH NO YESNO YES

Mut 4 Mut 4 AA NO YESNO YES

Mut 5 Mut 5 HH VV NO YESNO YES

Mut 8 Mut 8 AA YES YESYES YES

Mut 9 Mut 9 HH YES YESYES YES

Cleavage VirusCleavage VirusIn VitroIn Vitro

Mutation of CS1Mutation of CS1

MHV replicase cleavage site mutations block MHV replicase cleavage site mutations block cleavage during infection cleavage during infection

wtwt icwticwt

p93p93

p65p65

p28p28

mut8 mut9 mut3 mut4 mut5mut8 mut9 mut3 mut4 mut5

p28p28 p65p65

p93p93

CS1CS1

CleavingCleavingNon-Non-cleavingcleaving

Cleavage site mutants are viable Cleavage site mutants are viable

WildtypeWildtypeVirusVirus

““Assembled”Assembled”WildtypeWildtype

CleavingCleavingMutantMutant

Non-CleavingNon-CleavingMutantMutant

Time (hours post infection)

0

1

2

3

4

5

6

7

8

0 5 10 15 20 25 30

wticwtmut9

mut3mut4mut5

Viru

s tit

er (

Log

PF

U/m

l)Noncleaving mutants grow to lower peak titersNoncleaving mutants grow to lower peak titers

Cleaving

Non-Cleaving

Application of Genetics to Application of Genetics to SARS-CoV Research SARS-CoV Research

• Functions of replicase, structural and “accessory” Functions of replicase, structural and “accessory” genesgenes

• Pathogenic determinantsPathogenic determinants

• Recapitulation of animal virusesRecapitulation of animal viruses

• Deteminants of trans-species adaptationDeteminants of trans-species adaptation

• Capacity for recombination, reversion, and Capacity for recombination, reversion, and resistanceresistance

• Basis for stable live-attenuated virus vaccinesBasis for stable live-attenuated virus vaccines

Genetics Research Needs Genetics Research Needs

• Develop reverse genetic systemsDevelop reverse genetic systems

• Viruses from animals, humans, lab-adapted, and Viruses from animals, humans, lab-adapted, and passagedpassaged

• Rapid sequencing capacity for “natural”, cloned, Rapid sequencing capacity for “natural”, cloned, recovered, and modified viruses.recovered, and modified viruses.

Research NeedsResearch Needs

• GeneticsGenetics

• Animal models Animal models

• Protein structure and functionProtein structure and function

• Emergence / persistenceEmergence / persistence

• Immune response / protectionImmune response / protection

• Vaccines and AntiviralsVaccines and Antivirals

• Animal ModelsAnimal Models

– SARS-CoV animal reservoirSARS-CoV animal reservoir

– SARS-CoV animal modelsSARS-CoV animal models

• Primate and small animalPrimate and small animal

• Replication, pathogenesis, disease, persistenceReplication, pathogenesis, disease, persistence

• Adaptation of SARS-CoV in different speciesAdaptation of SARS-CoV in different species

• Mutant virus studiesMutant virus studies

– Natural and Trans-species Coronavirus ModelsNatural and Trans-species Coronavirus Models

• PRCoV (pigs), BCoV (cattle), MHV (mice)PRCoV (pigs), BCoV (cattle), MHV (mice)

• Pathogenesis, persistence, immune response, protection, reinfectionPathogenesis, persistence, immune response, protection, reinfection

Research Needs: Preparing for SARSResearch Needs: Preparing for SARS

Big QuestionsBig Questions• Is SARS-CoV still present in human populations? Is SARS-CoV still present in human populations?

• Is there risk of human animal transmission? Is there risk of human animal transmission?

• Will SARS-CoV adapt to better survive in human Will SARS-CoV adapt to better survive in human populations?populations?

• Will SARS reemerge as a “seasonal disease”Will SARS reemerge as a “seasonal disease”

• Can vaccines and therapeutics for SARS be Can vaccines and therapeutics for SARS be developed, tested and used?developed, tested and used?

Assumptions, Hypotheses, Myths and RumorsAssumptions, Hypotheses, Myths and Rumors

• SARS is a respiratory diseaseSARS is a respiratory disease

• Global public health response was responsible for control / elimination Global public health response was responsible for control / elimination of SARSof SARS

• SARS is an acute disease SARS is an acute disease

• If SARS-CoV reemerges it will be a “seasonal” diseaseIf SARS-CoV reemerges it will be a “seasonal” disease

• SARS-CoV will adapt and become less virulentSARS-CoV will adapt and become less virulent

• SARS-CoV will adapt and become more virulentSARS-CoV will adapt and become more virulent

• The barriers for trans-species movement of coronaviruses are highThe barriers for trans-species movement of coronaviruses are high

• Immune response equals recovery and protectionImmune response equals recovery and protection

• Response of animals correlates with protection in humansResponse of animals correlates with protection in humans

SARS Clinical DiseaseSARS Clinical Disease

• Prolonged incubation period with late Prolonged incubation period with late development of progressive respiratory diseasedevelopment of progressive respiratory disease

• Lack of upper respiratory prodromeLack of upper respiratory prodrome

• Evidence for systemic infection - multisystem Evidence for systemic infection - multisystem disease, and lab findingsdisease, and lab findings

• Disease in children clinically milderDisease in children clinically milder

• Development of antibody responses 10-20 daysDevelopment of antibody responses 10-20 days

Models for SARS PathogenesisModels for SARS Pathogenesis

SARS as a measles wannabe:SARS as a measles wannabe:

• Respiratory transmission Respiratory transmission

• Systemic infectionSystemic infection

• Severe progressive pulmonary disease in naïve adultsSevere progressive pulmonary disease in naïve adults

• Less severe in childhood primary infectionLess severe in childhood primary infection

• Direct epithelial destruction + immune diseaseDirect epithelial destruction + immune disease

• Multi-system diseaseMulti-system disease

• Concerns about late immune disease, virus mutationsConcerns about late immune disease, virus mutations

• Enhanced disease after killed vaccine?Enhanced disease after killed vaccine?

Targets for Immunity Targets for Immunity

Spike- SSpike- S

– FunctionsFunctions

• Receptor BindingReceptor Binding

• FusionFusion

• Host rangeHost range

• TropismTropism

– ImmunityImmunity

• AntibodiesAntibodies

• Cell mediated immunityCell mediated immunity

• NeutralizationNeutralization

• Fusion inhibitionFusion inhibition

Nucleocapsid- NNucleocapsid- N– FunctionsFunctions

• EncapsidationEncapsidation

• ? Replication? Replication

• ? Nuclear interactions? Nuclear interactions


• Cell mediatedCell mediated

• Antibodies- non Antibodies- non neutralizingneutralizing

E

S

M

Targets for Immunity Targets for Immunity

Membrane - MMembrane - M


• Virion formationVirion formation

• Incorporation of RNAIncorporation of RNA


• CMI - not protectiveCMI - not protective

• Antibodies - non Antibodies - non neutralizingneutralizing

Small Membrane- ESmall Membrane- E


• Virion formation / Virion formation / maturationmaturation


• ??

E

S

M

Targets for Immunity Targets for Immunity ReplicaseReplicase

SS EE MM NNReplicaseReplicase

• Functions: polymerase, helicase, proteinase, transcription Functions: polymerase, helicase, proteinase, transcription factors, membrane interactions, intracellular movement, ?factors, membrane interactions, intracellular movement, ?exonuclease, exonuclease,

• Immunity: unknownImmunity: unknown

Non-structural proteinsNon-structural proteins

• Functions: ? Pathogenesis, host-specific functions, IFN-Functions: ? Pathogenesis, host-specific functions, IFN-cytokine induction ?cytokine induction ?


Targets for Immunity Targets for Immunity ReplicaseReplicase

SS EE MM NNReplicaseReplicase

• Functions: polymerase, helicase, proteinase, transcription Functions: polymerase, helicase, proteinase, transcription factors, membrane interactions, intracellular movement, ?factors, membrane interactions, intracellular movement, ?exonuclease, exonuclease,


Non-structural proteinsNon-structural proteins

• Functions: ? Pathogenesis, host-specific functions, IFN-Functions: ? Pathogenesis, host-specific functions, IFN-cytokine induction ?cytokine induction ?


Possible Vaccine Strategies for SARS Possible Vaccine Strategies for SARS

• Inactivated whole virusInactivated whole virus

• ProteinProtein

– Spike (S), nucleocapsid (N), membrane (M), others Spike (S), nucleocapsid (N), membrane (M), others

– Protein, DNA, viral (adenovirus, VEE, paramyxovirus)Protein, DNA, viral (adenovirus, VEE, paramyxovirus)

• Live-attenuatedLive-attenuated

– Passage, Passage,

– Chemical,UV, cold adaptationChemical,UV, cold adaptation

– EngineeredEngineered

• Targeted recombinationTargeted recombination

• Infectious cloneInfectious clone

Snijder et al 2003

doi:10.1016/S0022-2836(03)00865-9 J. Mol. Biol. (2003) 331, 991–1004

Targeted RecombinationTargeted Recombination

NNMHV - SMHV - S

ReplicaseReplicase FIPV - SFIPV - S NN

ReplicaseReplicase MHV - SMHV - S NN

GrowthGrowth

feline cellsfeline cells

murine cellsmurine cells

recombination vectorrecombination vector

• Takes advantage of high rate of homologous recombination:Takes advantage of high rate of homologous recombination:

• Powerful selection for mutations- growth on specific cellsPowerful selection for mutations- growth on specific cells

• Applicable to 3’ 10 kb of genomeApplicable to 3’ 10 kb of genome

• Includes all stuctural and “non-replicase” nonstructural genesIncludes all stuctural and “non-replicase” nonstructural genes

• Has been well utilized for studies of S, M, N and E genesHas been well utilized for studies of S, M, N and E genes

22 kb22 kb 10 kb10 kb

5’5’ 3’3’MM

MM

MM

EE

EE

EE

Why Should We Pursue SARS Public Why Should We Pursue SARS Public Health Policies and Vaccines Health Policies and Vaccines

ProposedProposed SARS SARS

Non-human reservoirNon-human reservoir Civet, Raccoon Dog, otherCivet, Raccoon Dog, other

Naive populationNaive population 6 billion 6 billion

Variable disease penetrationVariable disease penetration Asymptomatic? - deathAsymptomatic? - death

Nonspecific diseaseNonspecific disease Flu-like, gastrointestinal Flu-like, gastrointestinal

Multiple modes of transmissionMultiple modes of transmission Respiratory, fomites, fecal?Respiratory, fomites, fecal?

Spread by world travelSpread by world travel DocumentedDocumented

Genetic change and adaptationGenetic change and adaptation Mutation, deletion, Mutation, deletion, recombinationrecombination

Vaccines for SARS Vaccines for SARS • The genome organization, proteins, and replication strategy The genome organization, proteins, and replication strategy

likely are similar to other coronaviruses, especially group 2.likely are similar to other coronaviruses, especially group 2.

• Determinants of pathogenesis, immune response and Determinants of pathogenesis, immune response and protection have yet to be determinedprotection have yet to be determined

• Animal models for SARS, both primate and small animal, Animal models for SARS, both primate and small animal, will be critical to development of vaccineswill be critical to development of vaccines

• No single vaccine approach has been highly effective in No single vaccine approach has been highly effective in animal coronavirus diseasesanimal coronavirus diseases

• Inactivated virus, recombinant protein, vectored expression, Inactivated virus, recombinant protein, vectored expression, DNA, heterologous virus, and live-attenuated virus DNA, heterologous virus, and live-attenuated virus approaches all need to be pursued for SARS vaccines approaches all need to be pursued for SARS vaccines

SARS and MHV Replicase Genes: Introduction of SARS and MHV Replicase Genes: Introduction of multiple attenuating and stabilizing mutationsmultiple attenuating and stabilizing mutations

p213p213 polpol helhelp70 p70 3CLpro3CLprop20p20

PLP2PLP2

p210p210 polpol hel hel p65p65 3CLpro3CLprop28p28

PLP-2PLP-2

SARSSARS

MHVMHV

Genes 2-9Genes 2-9S, E,M, NS, E,M, N

Genes 2-7 Genes 2-7

S, E, M, NS, E, M, N

Replicase GeneReplicase Gene

PLP1PLP1

Live-attenuated vaccines for SARS-CoV Live-attenuated vaccines for SARS-CoV

Potential AdvantagesPotential Advantages

• Impaired replication, normal immune determinants and presentationImpaired replication, normal immune determinants and presentation

• Base for live vaccine for emerging variants, serotypesBase for live vaccine for emerging variants, serotypes

• Limited immune selectionLimited immune selection

• Structural limitations to reversionStructural limitations to reversion

• Reverse genetics to introduce multiple attenuating and stabilizing Reverse genetics to introduce multiple attenuating and stabilizing mutationsmutations

Potential DisadvantagesPotential Disadvantages

• Pathogenesis, diseasePathogenesis, disease

• Recombination, reversion Recombination, reversion

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Coronavirus Pandemic Potential and Research Response Mark R. Denison, M.D. Departments of...

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