Document ID: EPA-HQ-OPP-2004-0287-0013
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2004-10-14T04:00Z

PVCP­
PIPs:
The
Context
Elizabeth
A.
Milewski,
Ph.
D.

U.
S.
Environmental
Protection
Agency
Office
of
Science
Coordination
and
Policy
October
13­
15,
2004
Organization
of
Today's
Presentation:

Setting
the
Stage

Overview:
Charge
and
Context
 
Dr.
Elizabeth
Milewski,
EPA/
OPPTS

Gene
Flow
 
Dr.
Anne
Fairbrother,
EPA/
ORD

Viral
Interactions
 
Dr.
Melissa
Kramer,
EPA/
OPPTS

Other
Scientific
Considerations
 
Dr.
Elizabeth
Milewski,
EPA/
OPPTS
Charge
to
the
SAP

Provide
scientific
advice
to
assist
EPA
in
its
evaluation
of
several
technical
issues
associated
with
PVCP­
PIPs

Specifically,
respond
to
a
series
of
technical
questions
related
to
exposure
and
hazard
considerations
for
PVCP­
PIPs
What
is
a
PVCP­
PIP?

PIP
is
acronym
for
"
plant­
incorporated
protectant"

PVCP
is
acronym
for
"
plant
virus
coat
protein"
What
is
a
Plant­
Incorporated
Protectant
(
PIP)?

"
.
.
.
a
pesticidal
substance
that
is
intended
to
be
produced
and
used
in
a
living
plant,
or
in
the
produce
thereof,
and
the
genetic
material
necessary
for
production
of
such
a
pesticidal
substance.
.
.
."
A
PVCP­
PIP
is
.
.
.

.
.
.
a
PIP
created
from
the
gene,
or
a
segment
of
the
gene,
that
codes
for
a
coat
protein
of
a
virus
that
naturally
infects
crop
plants.
Benefits
of
PVCP­
PIPs
An
effective
means
of
controlling
virus
infection
has
economic
benefits
 
Higher
yield
 
Reduce
use
of
chemical
pesticides
to
control
insect
vectors
 
In
some
cases,
only
option
Technical
Questions
for
SAP

Gene
flow

Viral
interactions

Other
scientific
considerations
Gene
Flow
EPA
is
seeking
SAP
assistance:

 
To
better
understand

circumstances
in
which
the
flow
of
PVCP­
PIPs
from
transgenic
plants
to
wild
or
weedy
relatives
occurs,
and

the
potential
for
adverse
impacts
from
such
gene
flow
 
To
identify
and
evaluate
conditions
that
might
minimize
gene
flow,
should
minimization
be
seen
as
appropriate
Viral
Interactions
EPA
is
seeking
SAP
assistance
to
identify
and
evaluate:

 
Circumstances
wherein
interactions
between
introduced
virus
sequences
and
invading
viruses
might
be

more
frequent
than
expected
to
occur
in
natural
mixed
virus
interactions,
or

unlike
those
expected
to
occur
in
such
circumstances.

 
Conditions
that
might
minimize
such
occurrences,

should
minimization
be
seen
as
appropriate.
Other
Scientific
Considerations
EPA
is
seeking
SAP
assistance
in
evaluating:

 
Several
other
technical
questions
associated
with
PVCP­
PIPs

Boundaries
of
assumptions?

Additional
considerations
for
minimizing
risk?
SAP
Role

Assist
EPA
in
better
understanding:

 
Degree
of
risk
for
each
issue
 
Degree
of
certainty
of
estimates
 
Relevance
of
hypothetical
vs
data­
supported
 
Direction
science
taking
for
the
specific
issues
raised

Provide
technical
recommendations
and
advice
on
the
technical
questions
posed
How
Will
SAP
Advice
Be
Used?

Advice
will
be
used
within
the
parameters
established
by
the
statutes
under
which
EPA
operates
EPA
Legal
Context

Federal
Insecticide,
Fungicide,
and
Rodenticide
Act
(
FIFRA)

 
Protect
environment
 
Protect
human
health

Federal
Food,
Drug,
and
Cosmetic
Act
section
408
(
FFDCA)

 
Determine
safe
levels
of
pesticide
residues
in
food/
feed
PVCP­
PIPs
and
FIFRA?

FIFRA
defines
a
pesticide
as:

 
"
any
substance
or
mixture
of
substances
intended
for
preventing,
destroying,
repelling,
or
mitigating
any
pest;
.
.
."

PVCP
gene
sequences
can
confer
resistance
to
the
virus
from
which
it
was
derived,
and
often
to
related
viruses,
to
the
recipient
plant.
U.
S.
Biotech
Regulation:

Coordinated
Framework
Three
U.
S.
regulatory
agencies
play
complementary
roles
 
EPA
 
regulation
of
pesticidal
substances
 
FDA
­
food
safety,
except
for
pesticides
 
USDA
 
plant
pest
risks
History
of
PVCP­
PIPs
at
EPA

1994:
Proposals
to
exempt
PVCP­
PIPs
 
All
 
Some
depending
on
potential
for
weediness

1997:
Supplemental
request
for
comment
on
proposal
to
exempt
PVCP­
PIPs

2001:
Supplemental
request
for
comment
on
proposal
to
exempt
PVCP­
PIPs
Wide
Range
of
Comments

Examples
of
support
for
exemption
proposal
 
Wild
species
.
.
.
are
generally
already
resistant
or
exhibit
a
high
degree
of
tolerance
to
infection.
(
29/
300369)

 
Since
viral
coat
proteins
do
not
act
in
toxic
manner,
all
viral
coat
proteins
should
be
exempt.
(
69/
300369)

Examples
opposing
exemption
proposal
 
The
sexual
transfer
of
engineered
virus­
resistance
would
readily
confer
an
advantage
to
weedy
populations.

(
29/
300371)

 
[
G]
enetically
engineered
virus­
resistant
crops
present
serious
ecological
risks:
i)
new
viral
strains
may
emerge
through
recombination
and
transcapsidation
.
.
.
.

(
05/
300370)
2000
NRC
Report

"
Genetically
Modified
Pest­
Protected
Plants:

Science
and
Regulation"

 
National
Research
Council
of
the
National
Academy
of
Sciences

Raised
a
number
of
questions
still
being
discussed
today,
e.
g.,

 
About
the
potential
for
gene
flow
from
transgenics
to
weedy
relatives
 
Suggestions
that
transgenics
could
be
constructed
with
mitigating
controls
to
reduce
potential
for
viral
interactions
Relevance
For
the
SAP

Long
history
involving
complicated
issues
 
but
this
meeting
narrowly
focused

Provide
scientific
advice
to
assist
EPA
in
its
evaluation
of
several
technical
issues
associated
with
PVCP­
PIPs

Focus
on
series
of
specific
questions
posed
by
EPA
Gene
Flow
in
Viral
Coat
Protein
Transgenic
Plants
Anne
Fairbrother,
D.
V.
M.,
Ph.
D.

U.
S.
Environmental
Protection
Agency
Office
of
Research
and
Development
October
13­
15,
2004
Gene
Flow

Gene
flow
(
for
the
purposes
of
this
discussion)
refers
to
movement
of
genes
(
including
transgenes)
from
crops
to
weeds,
wild
relatives
or
other
crops

Introgression
is
when
the
gene
becomes
fixed
in
the
recipient
population
Gene
Flow
Reviews
General

Ellstrand,
2003

GM
Science
Review
Panel
2003

NRC
2000

Snow
2002

Stewart
2003
PVCP­
PIPs

Bartsch
et
al.
1996

Bartsch
1997

Bartsch
et
al.
2001

Fuchs
et
al.
2004

Ilardi
&
Barba
2001

Power
2002

Spencer
&
Snow
2001

Tepfer
2002
Gene
Flow

Gene
flow
will
occur
every
year

Introgression
may
happen
quickly
or
take
multiple
years
and
introductions,
depending
upon:

 
Rates
of
pollen
and
seed
dispersal
 
Population
size
of
donor
and
recipient
plants
 
Selection
pressures
 
Reproductive
times
Gene
Flow
Concerns
as
discussed
in
the
literature

Potential
effects
on
recipient
plants
 
Increase
or
decrease
fitness

Potential
effects
on
plant
community
 
Changed
competitive
advantage
 
Extinction
risks

Potential
effects
on
genetic
diversity
 
"
genetic
swamping"

Potential
indirect
effects
Gene
Flow
Lessons
Learned

Transgenes
act
like
conventional
genes
for
dispersal
and
introgression
rates

Gene
flow
can
be
wide­
spread
and
happen
regularly

Increases,
decreases,
or
no
changes
in
fitness
can
result
from
gene
flow
Snow,
A.
June
02
Nature
Biotech
20:
542

Sterile
F1s
can
propagate
widely
via
asexual
reproduction

Genes
not
on
the
chromosomes
can
be
transferred

Fitness
changes
occur:

 
only
after
release
from
strong
limiting
factors
Snow,
A.
June
02
Nature
Biotech
20:
542
Gene
Flow
Lessons
Learned
Ecological
Fitness

Ecological
fitness
is
the
relative
ability
to
contribute
offspring
to
the
next
generation
 
May
be
measured
by
the
finite
rate
of
increase
of
a
population
or
number
of
offspring
from
an
individual

Fitness
strategies
in
plants
include:

 
Increased
number
or
size
of
seed
produced
 
Faster
rate
of
maturity
 
Greater
resistance
to
stress
(
drought,
temperature
extremes,
disease,
parasites,
soil
properties,
etc.)

12
out
of
the
13
most
important
conventionally
bred
world
food
crops
have
demonstrated
gene
flow
to
wild
relatives
 
7
have
introgressed
(
Ellstrand
et
al.,
1999)

 
Also
numerous
of
the
less
important
crops
(
Snow
and
Morand­
Pama,
1997)

Gene
Flow
Lessons
Learned

Tens
of
thousands
of
potential
natural
hybridizations
among
plants
 
165
confirmed
(
65
sufficiently
documented)

Can
be
confused
with
evolutionary
convergence
and
lineage
sorting
 
New
molecular
markers
(
e.
g.,
DNA
polymorphisms)
increase
ability
to
determine
degree
&
type
of
relatedness
Stewart,
2003
Nat.
Rev.
Gen.
4:
306
Gene
Flow
Lessons
Learned
Gene
Flow
Lessons
Learned
Example
of
natural
introgression
Iris
fulva
2n
=
42
Salt
marshes
Male
parent
Iris
hexagona
2n
=
44
Freshwater
swamps
Female
parent
Stewart,
2003
Nat.
Rev.
Gen.
4:
306
I.
fulva­
like:
intermediate
or
higher
fitness
I.
hexagona­
like:
intermediate
or
same
fitness
Iris
nelsonii
:
Fixed
derivative
of
the
hybridization
of
I.
fulva
X
I.
hexagona
X
I.
brevicaulis
(
rDNA,
allozymes,
cpDNA)
Gene
Flow
Lessons
Learned
Example
of
natural
introgression
(
I.
fulva
X
I.
hexagona
X
I.
brevicaulis)
Stewart,
2003
Nat.
Rev.
Gen.
4:
306

Local
geographical
formation
of
hybrid
swarms

Gene
flow
beyond
the
range
of
original
hybridization
zone

Formation
of
a
new
stabilized
taxon

Introgression
of
transgenes
from
GM
crops
to
wild
plant
populations
 
More
difficult
than
from
wild
plants
to
crops
 
Linkage
to
domestication
alleles
imposes
a
barrier
 
Domestication
genes
reduce
ecological
fitness
Stewart,
2003
Nat.
Rev.
Gen.
4:
306
Gene
Flow
Lessons
Learned

With
rare
exceptions
transgenic
traits
in
plants
are
almost
all
dominant
traits
 
Transgenic
hybrids
will
always
express
the
trait
 
Will
be
immediately
subject
to
selection
pressures
Reviewed
by:

Ellstrand,
N.
2003
Dangerous
Liaisons?

Gene
Flow
Lessons
Learned

Traits
that
distinguish
cultivated
plants
are
usually
recessive
alleles
at
individual
loci
with
major
fitness
effect
 
sometimes
modified
by
a
few
extra
loci
of
minor
fitness
effect
Reviewed
by:

Ellstrand,
N.
2003
Dangerous
Liaisons?

Gene
Flow
Lessons
Learned

Mathematical
models
argue
that
even
very
low
transmission
rates
of
transgenes
to
wild
populations
can
eventually
result
in
fixation
of
the
gene
What
is
the
ecological
significance
of
low
levels
of
gene
flow?
Haygood
et
al.,
2003
Proc.
Res.
Soc.
Lond.

Gene
Flow
Lessons
Learned

Rates
of
mating
of
crops
with
wild
relatives
are
no
different
for
transgenic
crops
than
for
conventional
crops
 
Exceptions
are
those
crops
engineered
to
reduce
fertility
Reviewed
by:

Ellstrand,
N.
2003
Dangerous
Liaisons?

Gene
Flow
Lessons
Learned

Potential
risk
of
crop
to
wild
introgression
of
transgenes
categorized
by:

 
Co­
location
with
wild
relatives
 
Evidence
for
crop­
to­
wild
gene
introgression
 
Genetic
differentiation
between
crop
and
wild
relative(
s)
Stewart,
2003
Nat.
Rev.
Gen.
4:
306
Gene
Flow
Lessons
Learned
Gene
Flow
Concerns
for
PVCP­
PIP
Crops

Studies
have
shown
significant
negative
impacts
of
virus
infection
on
growth,

survivorship
and
reproduction
of
plants.

Examples
include:

 
Brassica
spp.

 
Wild
squash
 
Purslane
 
Chickweed
 
Wild
oats
Reviewed
by:

Tepfer,
M.
2002
Ann.
Rev.
Phytophysiol.
40:
467­
91

Viruses
are
controlling
factors
for
some
plant
populations

Therefore,
concerned
with
increased
weediness
&
competitive
advantage
of
plants
with
virus
resistance
genes
Reviewed
by:

Tepfer,
M.
2002
Ann.
Rev.
Phytophysiol.
40:
467­
91
Gene
Flow
Concerns
for
PVCP­
PIP
Crops
Barley
Yellow
Dwarf
Virus
(
BYDV)

A
luteovirus

Significant
amount
of
crop
damage

Conventional
breeding
has
not
developed
resistance
or
tolerance

Some
BYDV
strains
move
(
aphid
transmitted),

into
wild
hosts
(
wild
oats
and
squirreltail
grass)

which
have
no
natural
resistance
&
show
signs
of
infection
Power,
2002
Ch
5
in
Letourneau
&
Barrows
Barley
Yellow
Dwarf
Virus
(
BYDV)
 
Wild
Oats

Wild
oats
are
agronomic
weeds
in
cultivated
cereal
crops.

Introduced
wild
oats
already
out
compete
native
grasses
in
CA.

Cultivated
oats
hybridize
readily
with
wild
oats
(
4.8%)

Fitness
(
growth
and
reproduction)
could
be
enhanced
with
a
PVCP­
PIP
against
BYDV
Power,
2002
Ch
5
in
Letourneau
&
Barrows
Barley
Yellow
Dwarf
Virus
(
BYDV)
Power,
2002
Ch
5
in
Letourneau
&
Barrows

Release
from
BYDV
infection
could
increase
competitive
advantage
 
Increased
weediness
(
oats,
barley,
wheat)

 
Increased
invasion
of
grasslands

Might
occur
only
in
the
absence
of
other
mitigating
environmental
factors.
Beet
Necrotic
Yellow
Vein
Virus
(
BNYVV)

Sea
beet
(
Beta
vulgaris
L.
subsp.

maritima)

 
Progenitor
of
cultivated
beet
(
subsp.

vulgaris)

 
Susceptible
to
BNYVV

BNYVV
absent
in
brackish
environment
where
sea
beets
grow
 
Fungal
vector
does
not
tolerate
salty
soils
Reviewed
by:

Tepfer,
M.
2002
Ann.
Rev.
Phytophysiol.
40:
467­
91
Beet
Necrotic
Yellow
Vein
Virus
(
BNYVV)

Receipt
of
transgene
that
confers
resistance
to
BNYVV
by
the
sea
beet
 
No
selective
advantage
or
disadvantage
 
Lack
of
vector
for
transmitting
virus
Reviewed
by:

Tepfer,
M.
2002
Ann.
Rev.
Phytophysiol.
40:
467­
91

Sexual
compatibility

Grown
in
the
same
vicinity

Overlapping
flowering
times

F1
hybrids
must
persist
for
>
1
generation

F1
must
be
fertile
and
backcross
Features
that
Increase
the
Likelihood
of
Gene
Flow
Features
that
Increase
the
Likelihood
of
Introgression

Dominance

Selective
advantage

Absence
of
association
with
deleterious
crop
alleles
or
traits

Location
on
a
shared
genome

Location
on
a
homologous
chromosome

Location
on
non­
rearranged
chromosomes
Approaches
to
decrease
likelihood
of
gene
flow
and
introgression

Placement
on
non
transferred
chromosome

Linkage
to
deleterious
crop
alleles
or
traits

Insertion
into
maternally­
transmitted
organelle
DNA
(
e.
g.,
chloroplasts)

Induced
sterility
(
e.
g.,
decrease
pollen
formation,

seed
development
or
germination)

Deployment
in
areas
where
crops
have
no
known
wild
relatives
Power,
2002
Ch
5
in
Letourneau
&
Barrows
Conclusions

Gene
flow
and
gene
introgression
can
occur
between
crops
and
wild
and
weedy
relatives
 
Likelihood
and
consequences
vary
depending
upon
crop
species,
recipient
species,
and
genes
transferred

Questions
remain
about
how
to
characterize
potential
for
risks
of
crops
with
PVCP­
PIPs
Viral
Interactions
in
Viral
Coat
Protein
Transgenic
Plants
Melissa
G.
Kramer,
Ph.
D.

U.
S.
Environmental
Protection
Agency
Office
of
Science
Coordination
and
Policy
October
13­
15,
2004
Virus
Infection

Virus
enters
plant
through
mechanical
breach
of
cell
wall

Virus
sheds
protein
coat
and
replicates

Movement
proteins
modify
plasmodesmata
and
allow
the
virus
to
cross
cell
walls
and
spread
throughout
the
plant

Virus
available
for
transmission
to
new
plant
Transgenic
Virus
Resistance

Many
types
of
transgenes
have
been
used
experimentally
to
confer
resistance
 
Plant
viral
coat
proteins
(
PVCPs)

 
Viral
replicase
 
Movement
proteins
 
Nuclear
inclusion
genes
 
Nonviral
sequences

PVCPs
are
the
most
common

Report
of
first
PVCP­
transgenic
plant
published
1986
Plant
Viral
Coat
Proteins
(
PVCPs)


Encapsidate
viral
nucleic
acid

Important
in
every
stage
of
infection
 
Replication
 
Movement
throughout
an
infected
plant
 
Transport
from
plant
to
plant
Proposed
Mechanisms
of
CP­
mediated
resistance

Protein
mediated
 
level
of
protection
correlated
with
level
of
mRNA
and
protein
accumulation
 
Transgenic
CPs
block
uncoating
of
virions
upon
entry
into
cell

Nucleic
acid
mediated
 
no
correlation
between
mRNA
and
level
of
protection
 
Post­
translational
transgene
silencing
suppresses
expression
of
the
transgene
and
accumulating
viral
RNA
that
shares
sequence
homology
with
the
transgene
Mixed
Virus
Infections

Viral
genomes
from
different
strains/
species
simultaneously
infect
the
same
plant

Can
be
extremely
common

In
rare
cases,
have
been
implicated
in
adverse
agricultural
or
environmental
events
 
e.
g.,
Ugandan
cassava
mosaic
disease

In
PVCP­
transgenic
plants,
every
infection
is
a
mixed
infection
with
respect
to
the
PVCP
gene
Critical
Question
Are
the
risks
associated
with
virus
interactions
in
PVCP­
transgenic
plants
greater
in
degree
or
different
in
kind
than
in
natural
mixed
infections?
Issues
to
Consider

For
(
1)
recombination,
(
2)
heterologous
encapsidation,
and
(
3)
synergy
 
Occurrence
under
natural
conditions
 
Potential
to
occur
in
VCP­
transgenic
plants
 
Ways
to
reduce
the
frequency
if
warranted

Field
evaluations
 
ecological
significance

Is
the
frequency
of
virus
interactions
in
PVCPtransgenic
plants
different
than
in
natural
mixed
infections?

Is
the
nature
of
virus
interactions
in
PVCP­
transgenic
plants
different
than
in
natural
mixed
infections?
Recombination

Recombination:
segments
from
different
parental
molecules
form
chimeric
molecules

Mechanism
in
RNA
viruses:
template
switching
of
the
viral
replicase
during
replication
Recombination
Under
Natural
Conditions

Rarely
leads
to
new,
viable
viruses

Still
a
significant
role
in
virus
evolution

More
likely
among
closely
related
viruses

Both
virus­
virus
recombination
and
virus­
host
recombination
Recombination
in
Transgenic
Plants
with
Virus
Transgenes

Nucleic
acids
of
viruses
that
infect
the
host
available
for
recombination
with
host
transgenes

Lab
experiments
show
that
such
recombination
can
occur
 
High
selection
pressure
 
Ecological
significance
unclear
Reducing
Frequency
of
Recombination

Remove
3'
untranslated
region

Exclude
replicase
recognition
sites
or
other
hotspots

Reduce
extent
of
shared
sequence
similarity

Use
smallest
viral
fragment
possible

Insert
GC­
rich
sequences
downstream
of
AU­
rich
sequences
Heterologous
Encapsidation
Coat
protein
subunits
of
one
virus
surround
the
nucleic
acid
of
a
different
virus
Heterologous
Encapsidation
Under
Natural
Conditions

Can
affect
virus­
vector
interactions

Regular
occurrence
among
some
plant
viruses
 
May
be
required
for
transmission
 
Natural
part
of
virus
epidemiology

More
likely
among
closely
related
viruses
Limited
Environmental
Concern
due
to
Heterologous
Encapsidation

Vector
specificity
often
only
partially
determined
by
coat
protein

Vectors
may
carry
a
heterologously
encapsidated
virus
only
to
plants
it
already
infects

If
virus
replicates
in
novel
host,
it
is
encapsidated
in
its
own
coat
protein

However 

 
With
high
frequency
of
heterologous
encapsidation,

secondary
transmission
among
new
host
plants
may
not
be
needed
for
impact
 
Virus
may
rapidly
evolve
in
a
new
host
 
Virus
may
be
exposed
to
new
vectors
once
in
novel
host
Heterologous
Encapsidation
in
Transgenic
Plants
with
Viral
Transgenes
Protein
(
when
produced)
from
PVCP
transgenes
can
encapsidate
even
unrelated
infecting
viruses
Reducing
the
Impact
of
Heterologous
Encapsidation

Certain
regions
affect
aphid
transmission
specificity
 
Readthrough
domain
of
the
CP
 
Major
capsid
protein
 
Loop
structure
of
the
CP
 
DAG
3­
amino
acid
sequence

PVCP
gene
modifications
can
reduce
frequency
of
heterologous
encapsidation/
vector
transmission
 
Mutations
in
assembly
motif
of
CP
 
DAG
triplet
deleted
 
First
420
nucleotides
deleted
Synergy
Disease
severity
of
two
viruses
together
is
greater
than
expected
based
on
severity
of
each
alone
Synergy
Under
Natural
Conditions

Many
known
viral
synergisms

More
common
among
some
viruses
than
others

Coat
protein
less
likely
to
be
responsible
than
other
regions
Synergy
in
Transgenic
Plants
with
Viral
Transgenes

Agroeconomic
concern

Evaluated
before
deployment

Farmers
would
quickly
abandon
products
with
synergistic
infections
Reducing
the
Frequency
of
Synergy

Constructs
can
be
engineered
to
reduce
likelihood
 
Avoid
particular
transgenes
 
Use
defective
copies
of
genes

Stacking
multiple
resistances
in
the
same
plant
Field
Evaluations
of
Viral
Interactions
in
Transgenic
Plants

Most
experiments
have
been
done
in
the
lab

Field
evaluations
are
critical
for
assessing
impacts
and
likelihood
of
events

Limited
number
of
published
evaluations
provide
no
evidence
of
adverse
effects
Thomas,
et
al.
1998

25,000
potato
plants
442
lines
transformed
with
16
PLRV
CP
constructs

Exposed
to
field
infection
over
6
years

No
new
viruses
or
viruses
with
altered
transmission
or
disease
characteristics
detected
Molecular
Breeding
4:
407­
417
Fuchs
et
al.
1998

Transgenic
melon
and
squash
containing
CP
from
aphid
transmissible
strain
of
CMV

Plants
infected
with
aphid
non­
transmissible
strain
of
CMV

Found
no
aphid­
vectored
spread
of
nontransmissible
strain
Transgenic
Research
7:
449­
462
Fuchs
et
al.
1999

Transgenic
squash
containing
CP
from
aphid
transmissible
strain
of
WMV

Plants
infected
with
aphid
non­
transmissible
strain
of
ZYMV

No
ZYMV
transmission
in
nontransgenic
fields

ZYMV
transmitted
to
77/
3700
(
2%)
of
plants
in
transgenic
fields
(
likely
due
to
heterologous
encapsidation),
but
no
epidemic
developed
Transgenic
Research
8:
429­
439
Lin
et
al.
2003

Estimated
biological
and
genetic
diversity
of
CMV
isolates
before
and
after
development
of
transgenic
squash
containing
CP
from
CMV,
ZYMV,
and
WMV

Most
CMV
isolates
showed
no
significant
sequence
changes
after
infecting
transgenic
squash

One
isolate
did
differ,
but
not
due
to
recombination
or
selection
Journal
of
General
Virology
84:
249­
258
Vigne
et
al.
2004

Transgenic
grapevines
containing
CP
of
GFLV

Nontransgenic
scions
grafted
onto
transgenic
and
nontransgenic
rootstocks;
exposed
over
3
years
to
GFLV
infection

Transgenic
grapevines
did
not
assist
emergence
of
viable
GFLV
recombinants
or
affect
molecular
diversity
of
indigenous
population
Transgenic
Research
13:
165­
179
Does
the
Frequency
of
Interactions
Change
in
PVCP­
Transgenic
Plants?

Difficult
to
measure
directly
due
to
rarity
of
events

Some
factors
suggest
a
decrease
in
frequency
in
transgenic
systems
 
Lower
concentration
of
cellular
RNA
transcripts
from
transgene
than
infecting
virus
 
Concentration
of
infecting
virus
reduced

Some
factors
suggest
a
possible
increase
in
frequency
in
transgenic
systems
 
Constitutive
promoters
 
Natural
temporal/
spatial
expression
patterns
obscured
Could
PVCP­
Transgenic
Plants
Lead
to
Novel
Viral
Interactions?

Transgenic
multi­
resistance

Heterologous
resistance

Use
of
exotic
strain's
CP

Expression
in
new
cells/
tissues

Altered
CP
genes
Summary:
Overarching
Issues
for
Panel
to
Consider

Are
viral
interactions
in
PVCP­
transgenic
plants
an
environmental
concern
above
and
beyond
what
occurs
naturally?

 
Potential
for
increased
frequency
of
interactions?

 
Are
novel
interactions
likely
to
occur
and
have
any
adverse
environmental
impacts?

Of
what
value
are
mechanisms
to
reduce
likelihood
of
some
interactions?
Other
Scientific
Considerations
Elizabeth
A.
Milewski,
Ph.
D.

U.
S.
Environmental
Protection
Agency
Office
of
Science
Coordination
and
Policy
October
13­
15,
2004
Other
Scientific
Considerations

Boundaries
of
assumptions?

Additional
considerations
for
minimizing
risk?
Boundaries
of
Assumptions?

EPA
is
seeking
SAP
assistance
in
examining
how
far
a
PVCP­
PIP
can
be
modified
while
still
supporting
assumptions
of
 
Dietary
safety
for
humans
 
No
new
effects
on
nontarget
species
 
No
potential
for
novel
viral
interactions
Dietary
Safety

Assumption
is
that
humans
have
consumed
viral
coat
proteins
for
generations
as
part
of
food
supply

To
what
degree
and
in
what
ways
might
a
PVCP
gene
be
modified
and
the
PVCP­
PIP
still
present
no
new
human
dietary
exposures?

Can
the
SAP
provide
a
succinct
statement
describing
the
boundary?
No
New
Effects
on
Nontargets

Assumption
is
that
species
that
interact
with
nontransgenic
comparator
plants
have
been
exposed
to
viral
coat
proteins
for
generations

To
what
degree
and
in
what
ways
might
a
PVCP
gene
be
modified
and
the
PVCP­
PIP
still
present
no
new
effects
on
nontarget
species?

Can
the
SAP
provide
a
succinct
statement
describing
the
boundary?
No
Potential
for
Novel
Viral
Interactions

To
what
degree
and
in
what
ways
might
a
PVCP
gene
be
modified
before
it
becomes
a
concern
that
novel
viral
interactions
could
occur
because
the
gene
could
be
significantly
different
from
any
existing
in
nature?

Can
the
SAP
provide
a
succinct
statement
describing
the
boundary?
Additional
Considerations
for
Minimizing
Risk?

Are
there
any
additional
considerations
related
to
the
PVCP­
PIP
construct
that
should
be
considered
when
attempting
to
identify
risk?

Are
there
any
scientific
considerations
beyond
gene
flow,
recombination,
and
heterologous
encapsidation
as
posed
in
EPA's
questions?
Charge
to
the
SAP

Provide
scientific
advice
to
assist
EPA
in
its
evaluation
of
several
technical
issues
associated
with
PVCP­
PIPs

Specifically,
respond
to
a
series
of
technical
questions
related
to
exposure
and
hazard
considerations
for
PVCP­
PIPs
 
Gene
flow
 
Viral
interactions
 
Other
scientific
considerations
Q1
What
scientific
evidence
supports
or
refutes
the
idea
that
plant
viruses
have
significant
effects
on
reproduction,
survival,
and
growth
of
plant
populations
in
natural
settings?
Is
there
scientific
evidence
that
plant
populations
freed
from
viral
pressure
could
have
increased
competitive
ability
leading
to
changes
in
plant
population
dynamics?
Q2
Please
comment
on
the
validity
of
the
Agency
list
of
crops
that
have
no
wild
or
weedy
relatives
in
the
United
States
with
which
they
can
produce
viable
hybrids
in
nature
(
i.
e.,
tomato,
potato,
soybean,
and
corn).
Q3
Please
identify
other
crops
that
have
no
wild
or
weedy
relatives
in
the
United
States
with
which
they
can
produce
viable
hybrids
in
nature,
e.
g.,
papaya,
peanut,
and/
or
chickpea.
Q4
What
laboratory
techniques
used
to
achieve
genetic
exchange
between
species
(
e.
g.,

embryo
rescue,
use
of
intermediate
bridging
crosses,
protoplast
fusion)
are
not
indicative
of
possible
genetic
exchange
between
these
species
in
the
field?
Conversely,
what
techniques,
if
any,
used
in
laboratory
or
greenhouse
experiments
provide
the
most
reliable
indication
of
ability
to
hybridize
in
the
field?
Q5
Given
that
current
bioconfinement
techniques
are
not
100%
effective,
what
would
the
environmental
implications
be
of
extremely
low
transfer
rates
of
virus­
resistance
genes
over
time?
Q6
Please
comment
on
the
prevalence
of
tolerance
and/
or
resistance
to
viruses
in
wild
relatives
of
crops.
Q7
Please
specify
techniques
that
do
or
do
not
provide
measures
of
tolerance
and/
or
resistance
that
are
relevant
to
field
conditions.
Q8
How
do
environmental
or
other
factors
(
e.
g.,

temporal
variations)
affect
tolerance
and/
or
resistance?
Given
the
expected
variability,

what
measures
of
tolerance
and/
or
resistance
would
be
reliable?
Q9
What
would
be
the
ecological
significance
if
a
plant
population
acquired
a
small
increase
in
viral
tolerance
and/
or
resistance
above
a
naturally­
occurring
level?
Q10
Please
comment
on
how
necessary
and/
or
sufficient
these
conditions
are
to
minimize
the
potential
for
the
PVCP­
PIP
to
harm
the
environment
through
gene
flow
from
the
plant
containing
the
PVCP­
PIP
to
wild
or
weedy
relatives.
Would
any
other
conditions
work
as
well
or
better?
Q10
Conditions

The
plant
into
which
the
PVCP­
PIP
has
been
inserted
has
no
wild
or
weedy
relatives
in
the
United
States
with
which
it
can
produce
viable
hybrids
in
nature,
e.
g.,
corn,
tomato,
potato,
or
soybean;
or

Genetic
exchange
between
the
plant
into
which
the
PVCP­
PIP
has
been
inserted
and
any
existing
wild
or
weedy
relatives
is
substantially
reduced
by
modifying
the
plant
with
a
scientifically
documented
method
(
e.
g.,
through
male
sterility);
or

It
has
been
empirically
demonstrated
that
all
existing
wild
or
weedy
relatives
in
the
United
States
with
which
the
plant
can
produce
a
viable
hybrid
are
tolerant
or
resistant
to
the
virus
from
which
the
coat
protein
is
derived.
Q11
To
what
extent
are
novel
viral
interactions
(
e.
g.,

recombination,
heterologous
encapsidation)

involving
a
viral
transgene
an
environmental
concern?
Q12
What
conclusions
can
be
drawn
as
to
whether
the
likelihood
of
recombination
and/
or
heterologous
encapsidation
would
be
increased
or
decreased
in
a
transgenic
plant
compared
to
its
non­
bioengineered
counterpart?
Q13
How
effective
is
deleting
the
3'
untranslated
region
of
the
PVCP
gene
as
a
method
for
reducing
the
frequency
of
recombination
in
the
region
of
the
PVCP
gene?
Is
this
method
universally
applicable
to
all
potential
PVCP­
PIP
constructs?
Would
any
other
methods
work
as
well
or
better?
Which
methods
are
sufficiently
effective
and
reproducible
such
that
actual
measurement
of
rates
to
verify
rate
reduction
would
be
unnecessary?
Q14
Are
any
methods
for
inhibiting
heterologous
encapsidation
or
transmission
by
insect
vectors
universally
applicable
to
all
PVCPPIPs
Which
methods
are
sufficiently
effective
and
reproducible
such
that
actual
measurement
of
rates
to
verify
rate
reduction
would
be
unnecessary?
Q15
How
technically
feasible
would
it
be
to
measure
rates
of
recombination,
heterologous
encapsidation,
and
vector
transmission
in
PVCP­
PIP
transgenic
plants
in
order
to
show
that
rates
are
reduced?
Q16
Please
comment
on
how
necessary
and/
or
sufficient
each
of
these
conditions
is
to
minimize
the
potential
for
novel
viral
interactions.
Please
address
specifically
what
combination
would
be
most
effective
or
what
conditions
could
be
modified,

added,
or
deleted
to
ensure
that
potential
consequences
of
novel
viral
interactions
in
PVCP­
PIP
transgenic
plants
are
minimized.
Q16
Conditions

The
genetic
material
of
the
PVCP­
PIP
is
translated
and/
or
transcribed
in
the
same
cells,
tissues,
and
developmental
stages
naturally
infected
by
every
virus
from
which
any
segment
of
a
coat
protein
gene
used
in
the
PVCP­
PIP
was
derived.

The
genetic
material
of
the
PVCP­
PIP
contains
coat
protein
genes
or
segments
of
coat
protein
genes
from
viruses
established
throughout
the
regions
where
the
crop
is
planted
in
the
United
States
and
that
naturally
infect
the
crop
into
which
the
genes
have
been
inserted.

The
PVCP­
PIP
has
been
modified
by
a
method
scientifically
documented
to
minimize
recombination,
(
e.
g.,
deletion
of
the
3'

untranslated
region
of
the
coat
protein
gene).

The
PVCP­
PIP
has
been
modified
by
a
method
scientifically
documented
to
minimize
heterologous
encapsidation
or
vector
transmission,
or
there
is
minimal
potential
for
heterologous
encapsidation
because
no
protein
from
the
introduced
PVCP­
PIP
is
produced
in
the
transgenic
plant
or
this
virus
does
not
participate
in
heterologous
encapsidation
in
nature.
Q17
To
what
degree
and
in
what
ways
might
a
PVCP
gene
be
modified
(
e.
g.,
through
truncations,
deletions,
insertions,
or
point
mutations)
while
still
retaining
scientific
support
for
the
idea
that
humans
have
consumed
the
products
of
such
genes
for
generations
and
that
such
products
therefore
present
no
new
dietary
exposures?
Q18
What
are
the
potential
adverse
effects,
if
any,

of
such
modifications
on
nontarget
species
(
e.
g.,
wildlife
and
insects
that
consume
the
PVCP­
PIP)?
Q19
To
what
degree
and
in
what
ways
might
a
PVCP
gene
be
modified
(
e.
g.,
through
truncations,
deletions,
insertions,
or
point
mutations)
before
it
would
be
a
concern
that
novel
viral
interactions
due
to
the
modifications
could
occur
because
the
PVCP
gene
would
be
significantly
different
from
any
existing
in
nature?
Q20
Would
any
additional
requirements
related
to
PVCP­
PIP
identity
and
composition
(
e.
g.,

demonstration
that
the
transgene
has
been
stably
inserted)
be
needed
for
significant
reduction
of
risks
associated
with
PVCPPIPs
Q21
Are
there
any
considerations
beyond
gene
flow,
recombination,
and
heterologous
encapsidation
as
posed
in
the
preceding
questions
that
the
Agency
should
consider
in
evaluating
the
risk
potential
of
PVCPPIPs
(
e.
g.,
synergy)?