Scientific and technical aspects of

research on Influenza, SARS and MERS

Kanta Subbarao
Laboratory of Infectious Diseases, NIAID, NIH
NAS GoF symposium
December 15, 2014

Topics

The types of Gain of Function (GoF) research

What impact does virological research typically have on

the viruses being studied, in terms of gain or loss of
function?

What do we know or not know about flu, SARS, and

MERS and can GoF research help fill the knowledge
gaps?

Where does virological research cross the line into GoF

research as defined by the U.S. government?

Questions virologists ask

Why/how does the virus infect and kill mammals?

Do antiviral drugs work and how does the virus become resistant?
Do current or novel vaccines provide protection and can the virus
escape?
How does the virus spread within animals?
How does the virus spread from animals to humans and from
humans to humans?
Could the virus cause a pandemic?
What is the likelihood of (re)emergence?
Adapted from Paul Duprex; image Russell Kightley Science Photo Library

Reverse Genetics
Generating viruses de novo
cDNA copy of genome

RNA
(+)

introduce into cells

Adapted from Paul Duprex

recombinant
virus

New Technologies in Virology Research

Reverse genetics: flu, SARS and MERS

Deep sequencing: viral quasispecies at different sites

can be characterized

Virus-host interactions: siRNA screens

Human MAbs generated from phage display libraries or

plasmablasts or by immortalizing B cells

Modifying the host genome: CRISPR/Cas9

Applications of Gain of Function (GoF) Research

Biology of viruses

Identification of novel viral gene products

Identification of receptor(s) used

Virus Ecology

Characterizing viruses from animal hosts

Identification of host range determinants

Viral Pathogenesis

Identification of virulence determinants

Airborne spread

Virus-host interactions including innate and adaptive immunity

Applications of Gain of Function (GoF) Research-2

Development of animal models

Adaptation to specific animal species to induce clinical signs of

disease e.g. SARS MA15 and MA20

Antiviral drugs

Mechanisms of antiviral drug resistance

Evaluation of the fitness cost of antiviral resistance mutations

Immunoprophylaxis/immunotherapy

Identification of epitope(s) targeted by Abs, especially broadly

neutralizing MAbs

Evaluation of the virulence and fitness of MAb resistance

mutants

Applications of Gain of Function (GoF) research-3

Vaccine development

Molecular characterization of antigenic variants

Generation of vaccine seed viruses with appropriate properties

including enhanced yield and immunogenicity while preserving
antigenicity; example H1N1pdm vaccine

Elucidating the biological basis for adverse outcomes

associated with vaccine candidates (e.g. SARS vaccines)

Determining the molecular basis for attenuation of vaccine

viruses

Determining the stability of live attenuated vaccine candidates

How do influenza virologists identify viral determinants of

virulence?
Virus A
Lethal for
mice

Generate single gene reassortants to

decide which gene(s) to compare

Compare viral gene sequences

Virus B
Not lethal
for mice

Determine role of specific amino acids

by introducing single or combinations
of mutations by site directed
mutagenesis

Test virulence of mutant viruses in

mice

PB2x
PB1
PA
HA
NP
NA
M
NS

x
PB2
PB1
PA
HA
NP
NA
M
NS

PB2
PB1
PA
HAx
NP
NA
M
NS

Transmissibility
Indonesia/2005 H5N1 virus: Ron Fouchier (2012)
10 times

T156A
N158D
Q222L
G224S
Q226L
N224K

H103Y

T318I

infection in the respiratory

tract

Is the virus transmissible? Yes

mutations in 7 segments, 2 new changes

(red) in HA

Is it more virulent than the original virus? No

Adapted from Paul Duprex

How and why do virologists study evolution of

viruses under immune or antiviral pressure?
How?
Generate a panel of escape mutants that replicate in the
presence of MAbs or antiviral drugs
Virus

Escape
Mutants

CellmonolayerX2h
+Antibody/Antiviral
+Antibody/Antiviral
4days37C

30min37C

OR

Experimental
animalmodel

Recover
virus

Virus

+Antibody/Antiviral

Sequenceto
identify
Escape
Mutants

How and why do virologists study evolution of

viruses under immune or antiviral pressure?
Sequence their genomes (or target gene segments)
Introduce each substitution individually and in
combinations into a reverse-genetics derived virus
Examine the fitness and resistance phenotypes in vitro or
in vivo
Why?
To understand the biology of the virus
To map epitopes of antibodies
To develop robust counter measures
(antiviral drugs and vaccines)

Coronavirus targets for vaccines and

therapeutics
CoV genome ~30kb +sense RNA
5

nsp3

nsp3
PL-protease

nsp5

nsp5
protease

Proteases

nsp12

nsp12
RdRp

nsp14 nsp16

nsp16
nsp14
Exonuclease 2O MT-ase

Polymerase
Proofreading

EM

Spike

Entry
Immune
Host Range
Evasion
Immunity

defined by reverse genetics and adaptive experimental evolution

An3

Topics

The types of Gain of Function (GoF) research

What impact does virological research typically have on

the viruses being studied, in terms of gain or loss of
function?

What do we know or not know about flu, SARS, and

MERS and can GoF research help fill the knowledge
gaps?

Where does virological research cross the line into GoF

research as defined by the U.S. government?

Gain of Function
The term gain of function is a vague and unsatisfactory term
for microbiologists
One suggested alternative is aTRIP: an acronym for an
experiment that uses one or more of the DURC agents and
produces, aims to produce, or can be reasonably
anticipated to alter Transmission, Range and resistance,
Infectivity/immunity or Pathogenesis
Another suggestion is Gain of Virulence or Transmissibility

Duprex and Casadevall mBio in press; Mark Denison

The impact of virological methods on gain

of function in virology
Research
maneuver
Passage in
cell
culture/eggs

Comment/
Example

GoF?

Other
consequences:
LoF?

aTRIP/G
OVT?

Yes: usually
desired

Possible for other

cells

no

The impact of virological methods on gain of

function in viruses
Research
maneuver

Comment/
Example

Passage in
cell
culture/eggs
Serial
passage in
experimental
animals

SARS CoV
MA15

GoF?

Other
consequences:
LoF?

aTRIP/GO
VT?

Yes: usually
desired

Possible for
other cells

no

Possible:
often desired

Possible for
other species

RP/V

The impact of virological methods on gain of

function in viruses
Research
maneuver

Comment/
Example

Passage in
cell
culture/eggs

GoF?

Other
consequences:
LoF?

aTRIP/GO
VT?

Yes: usually
desired

Possible for other no

cells

Serial
passage in
experimental
animals

SARS CoV
MA15

Possible:
often desired

Possible for other RP/V

species

Genetic
reassortment
of influenza
viruses

Influenza
vaccines GoF
yield, LoF
virulence

Yes; often
desired

Possible: often
desired

no

The impact of virological methods on gain of

function in viruses
Research
maneuver

Comment/
Example

GoF?

Antiviral or
MAb resistant
mutants

Valuable
Yes: usually
information on desired
suitable
antiviral
strategies

LoF?

aTRIP/GO
VT?

Possible
reduction in
virulence and
fitness

no

The impact of virological methods on gain of

function in viruses
Research
maneuver

Comment/
Example

GoF?

Antiviral or
MAb resistant
mutants

Valuable
Yes: usually
information on desired
suitable
antiviral
strategies

Site directed
mutagenesis

Used to
conclusively
prove the
molecular
basis of a
phenotype

Possible

LoF?

aTRIP/GO
VT?

Possible
reduction in
virulence and
fitness

no

Possible

No or
TRIP/VT

Topics

The types of Gain of Function (GoF) research

What impact does virological research typically have on

the viruses being studied, in terms of gain or loss of
function?

What do we know or not know about flu, SARS, and

MERS and can GoF research help fill the knowledge
gaps?

Where does virological research cross the line into GoF

research as defined by the U.S. government?

What do we know about influenza, SARS and

MERS coronaviruses?
Biology of the virus
Ecology; reservoir
Several host range and virulence
determinants
Innate and adaptive immunity
Antiviral drugs and Vaccines for influenza but
not for coronaviruses

What do we not know about influenza viruses?

Why are some viruses more virulent than others?

Why do some viruses spread efficiently while others dont?

Critical for understanding seasonal influenza and for assessing

pandemic potential of novel viruses

What drives the evolution of antigenic change and antiviral

resistance?

Critical for vaccines and antiviral drugs

Are there viral targets that will not escape under immune pressure?

Critical for the development of universal vaccines

What do we not know about SARS and MERS-CoV?

Will SARS or a SARS-like CoV re-emerge from bats or other animal

hosts?

What are the critical host range and virulence determinants of

MERS-CoV?

Can we develop a candidate vaccine that is safe, immunogenic and

efficacious?

Can MAbs be used safely for prevention and treatment?

Can we identify antiviral drugs that are safe and effective?

Critical because of the ongoing outbreak of MERS and in preparing for

possible re-emergence of SARS-CoV

What safety and oversight measures are in place?

HPAI and
H7N9
influenza

Measures

MERS

SARS

Non-HPAI
influenza

BSL3 with
respirators

Medical
surveillance
and support

Vaccines and
antivirals

N/A

N/A

Select agent
regulations

DURC
questionnaire

Where does virological research cross the

line into GoF research as defined by the
U.S. government?
The line:
all influenza viruses, SARS-CoV, MERS CoV
Experiments that are reasonably anticipated to
increase
pathogenicity
or
transmissibility
in any mammalian species

Where does virological research cross the line into

GoF research as defined by the U.S.
government?

Adaptation of MERS CoV to animal models

Elucidating the molecular determinants of transmissibility by the

airborne route (influenza)

Elucidating the biological basis for adverse outcomes associated

with candidate SARS vaccines

Conclusive experiments to demonstrate the biological significance of

novel gene products

genetic differences between isolates from animals and/or humans for

newly emerged viruses e.g. H7N9, H5N8, H5N2, H10N7 and H10N8
influenza and MERS CoV

Virulence determinants of newly emerged viruses e.g. H7N9, H5N8,

H5N2, H10N7 and H10N8 influenza and MERS CoV

Molecular basis for resistance to antiviral drugs and MAbs

Viral evolution under immune pressure

Viral evolution in the presence of antiviral drugs

The perspective of many influenza and

coronavirologists
Concerns about aerosol-transmissible highly pathogenic
avian influenza viruses have expanded to include

MERS-CoV and SARS-CoV

Research in rodent models

All influenza viruses including laboratory strains that are

avirulent for humans

An open scientific discussion is needed about the rationale and

justification for doing so.

Risk assessment should be based on

biosafety measures and PPE that are in use
numbers that represent actual experience and
laboratory exposures and accidents with SARS,
MERS and highly pathogenic avian influenza viruses

Priorities

Replace GOF with microbiological terms that precisely and

appropriately define the experiments of concern

Lift the restrictions on MERS for 3 critical reasons:

there is an urgent public health need for medical countermeasures with

cases and deaths reported every week

there is no evidence of transmission in any animal model of MERS or

SARS

no laboratory acquired infections in the US in over 10 years of work on

SARS and MERS

Lift the restrictions on rodent models because adaptation to rodents

does not enhance virulence for humans

Focus only on influenza viruses that are of concern

Lift the restrictions on characterizing escape from antibodies,

antivirals and vaccines

Relevant outbreaks in 2014

H5N8 H10N7

MERS CoV

H5N2
H7N9
H10N8

Approachestovirologyresearch
gainoffunc onpandemicthreat
universalgeneticapproach

ferretsareausefulmodelforhumaninfluenza
convergenceofresults

NAbescapemutantsariseinpatients
howmuchselection?

riskmitigationisavailable
engineeredstrains
vaccinationoflabpersonnel

GainofFunctionResearchandVirology
Usesbasicmethodscommontoallareasofmicrobiology
[e.g.passageindifferentspecies,reassortment,adaptation,selection]

Goodreasonsforitandoftennoalternatives
[e.g.drugresistancemechanisms]

Notaquestionofwhether,buthow
Thoroughriskassessmentneeded
Emphasisontransmissibilityandvirulence

Currentruleiscounterproductive
[e.g.MERSCoV smallanimalmodelforvaccine/drugevaluation;
notallfluarealike]