1.
Genetic
stability
of
recombinant
baculoviruses
containing
foreign
genes
Jasvir
Kaur
Presented
as
final
requirement
for
the
degree
of
Master
of
Biotechnology
Oxford
Brookes
University
School
of
Life
Sciences
September
2015
2. ii
Table
of
Contents
LIST
OF
FIGURES
IV
LIST
OF
TABLES
VI
ACKNOWLEDGMENTS
VII
DECLARATION
VIII
ABSTRACT
IX
TABLE
OF
ABBREVIATIONS
XI
OVERVIEW
OF
BACULOVIRUS
1
CHAPTER
1-‐
INSIGHT
OF
BACULOVIRUS
3
1.1
HISTORY
3
1.2
CLASSIFICATION
4
1.3
BACULOVIRUS
PROTEIN
STRUCTURE
5
1.4
GENOME
OF
ACMNPV
6
1.5
BACULOVIRUS
REPLICATION
7
1.6
VIRUS
REPLICATION
IN
VIVO
10
1.7
ENTRY
INTO
THE
NUCLEI
11
1.8
EXITING
THE
CELL
NUCLEI
11
1.9
GENE
EXPRESSION
REGULATION
12
1.9.1
IMMEDIATE
EARLY
GENES
12
1.9.2
DELAYED
EARLY
GENES
13
1.9.3
LATE
AND
VERY
LATE
GENE
13
1.10
INSECT
CELL
LINES
13
1.11
TRANSFER
VECTOR
14
1.13
SELECTION
OF
POLYHEDRON-‐NEGATIVE
RECOMBINANT
BACULOVIRUSES
16
1.13.1
BEVS-‐
BACULOVIRUS
EXPRESSION
VECTOR
SYSTEM
16
1.13.2
RECOMBINANT
BACULOVIRUS
PRODUCTION
18
1.13.3
THE
BACPAK6
SYSTEM
18
1.13.4
BAC-‐TO
BAC®
19
1.13.5
FLASHBACTM
20
1.14
MULTIPLICITY
OF
INFECTION
21
1.15
SERIAL
PASSAGING
23
AIM
OF
THIS
PROJECT
25
CHAPTER
2
-‐MATERIALS
AND
METHODS
26
2.1
MATERIALS
26
2.1.1
PLASMIDS
26
2.2
PREPARATION
OF
BACTERIAL
CELLS
AND
EXTRACTION
OF
PLASMID
DNA
26
2.3
INSECT
CELLS
AND
VIRUSES
27
2.4
COTRANSFECTION
OF
INSECT
CELLS
WITH
FLASHBAC
AND
PLASMID
TRANSFER
VECTOR
DNA
27
3. iii
2.5
VIRUS
STOCK
28
2.6
TESTING
PRESENCE
OF
FOREIGN
GENE
IN
VIRUS
STOCK
28
2.7
TEMPLATE
FORMATION
WITH
THE
USE
OF
KIT
TO
TEST
THE
PRESENCE
OF
THE
GENE
IN
THE
VIRUS
29
2.8
SERIAL
PASSAGING
29
2.9
HARVESTING
OF
VIRUS
DNA
30
2.10
VIRUS
STOCK
TITRATION
30
2.10.1
PLAQUE
ASSAY
30
2.11
PURIFICATION
OF
VIRUS
DNA
31
2.12
PCR
ANALYSIS
31
2.13
AGAROSE
GEL
ELECTROPHORESIS
32
CHAPTER
3
–
RESULTS
33
3.1
VIRUSES
33
CHAPTER
4-‐
DISCUSSION
45
CONCLUSION
AND
FUTURE
DEVELOPMENTS
51
REFERENCES
53
4. iv
List
of
Figures
Figure
Figure
Caption
Page
Figure
1.1.
The
above
phylogenetic
tree
indicates
the
division
of
four
groups
in
baculovirus
family.
It
indicates
amino
acids
of
29
baculoviruses
genes
derived
from
29
sequenced
baculovirus
genomes
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
5
Figure
1.2.
Group
I
has
both
GP64
and
F
while
Group
II
only
has
F
protein
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
6
Figure1.3.
AcMNPV's
genetic
map.
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
7
Figure
1.5.
Baculovirus
structure
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
10
Figure
1.3.
A.
DNA
containing
Escherichia
coli
(E.
coli)
lacZ
inserted
at
polh
locus.
B.
Digestion
of
viral
DNA
removes
lacZ
and
partially
deletes
orf1629-‐coding
region
C.
Cotransfection
and
insertion
of
foreign
gene
into
the
virus
DNA
and
restoration
of
orf1629
and
recircularization
of
DNA
thus
permitting
replication
within
insect
cells.
D.
Plaque
assay
is
used
to
isolate
recombinant
virus
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
19
Figure
1.4.
The
flashBAC
baculovirus
expression
system
used
for
production
of
recombinant
baculoviruses.
A.)
Shows
the
deleted
genes.
B.)
Containment
of
gene,
insert,
lef2,
and
orf1629
gene
in
transfer
vector.
C.)
Recombinant
virus
with
repaired
orf1629
gene.
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
21
Figure
3.1.
A.
PCR
analysis
of
the
recombinant
genes.
B.
A
plasmid
map
of
pAcRP23.lacZ
C.
A
plasmid
map
of
pOET6NaV1.4.
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
35
Figure
3.2.
PCR
analysis
of
cell
DNA
and
plasmid
DNA.
Lane
1,
molecular
weight
markers
(New
England
Biolabs).
Lane
2,
6,
8,
are
plasmid
DNA.
Lane
2,
4,
5,
7,
are
cell
DNA.
Lane9,
10
are
control
groups
-‐-‐
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
36
Figure
3.3.
The
virus
stock
titration
done
by
plaque
assay.
A.
AcNaV1.4
virus
dilution
10-‐4.
B.
AcUK,
virus
dilution10-‐1.
C.
AcHANA
,
virus
dilution10-‐2.
D.
AcRP23.lacZ
virus
dilution10-‐5
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
37
Figure
3.4.
Genomic
DNA
isolated
from
AcMNPV
passaged
five
times
at
low
(0.1
pfu/cell,
lane
2-‐6)
and
(high
5pfu/cell,
land
7-‐11).
The
MOI
was
amplified
using
PCR
with
oligonucleotide
primers
RDP
213
and
214.
Lane
1,
molecular
weight
markers
(New
England
Biolabs).
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
38
5. v
Figure
3.5.
Genomic
DNA
isolated
from
AcRP23.lacZ
passaged
five
times
at
low
(0.1
pfu/cell,
lane
2-‐6)
and
(high
5pfu/cell,
land
7-‐11).
The
MOI
was
amplified
using
PCR
with
oligonucleotide
primers
RDP
213
and
214.
Lane
1,
molecular
weight
markers
(New
England
Biolabs).
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
38
Figure
3.6.
Genomic
DNA
isolated
from
AcUK
passaged
five
times
at
low
(0.1
pfu/cell,
lane
2-‐6)
and
(high
5pfu/cell,
land
7-‐11).
The
MOI
was
amplified
using
PCR
with
oligonucleotide
primers
RDP
213
and
214.
Lane
1,
molecular
weight
markers
(New
England
Biolabs).
A.
Ethidium
bromide
nucleic
acid
gel
stain
used.
B.
SYBR
Gold
nucleic
acid
gel
stain
used.
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
39
Figure
3.7.
Genomic
DNA
isolated
from
AcRP23.lacZ
passaged
five
times
at
low
(0.1
pfu/cell,
lane
2-‐6)
and
(high
5pfu/cell,
land
7-‐11).
The
MOI
was
amplified
using
PCR
with
oligonucleotide
primers
RDP
213
and
214.
Lane
1,
molecular
weight
markers
(New
England
Biolabs).
A.
Ethidium
bromide
nucleic
acid
gel
stain
used.
B.
SYBR
Gold
nucleic
acid
gel
stain
used.
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
40
Figure
3.8.
Genomic
DNA
isolated
from
AcNaV1.4
passaged
five
times
at
low
(0.1
pfu/cell,
lane
2-‐6)
and
(high
5pfu/cell,
land
7-‐11).
The
MOI
was
amplified
using
PCR
with
oligonucleotide
primers
RDP
213
and
743.
Lane
1,
molecular
weight
markers
(New
England
Biolabs).
A.
Ethidium
bromide
nucleic
acid
gel
stain
used.
B.
SYBR
Gold
nucleic
acid
gel
stain
used.
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
42
Figure
3.
9.
Genomic
DNA
isolated
from
AcHANA
passaged
five
times
at
low
(0.1
pfu/cell,
lane
2-‐6)
and
(high
5pfu/cell,
land
7-‐11).
The
MOI
was
amplified
using
PCR
with
oligonucleotide
primers
RDP
213
and
214.
Lane
1,
molecular
weight
markers
(New
England
Biolabs).
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
43
Figure
3.10.
PCR
analysis
of
virus
DNA
provided
by
Robert
Possee
-‐-‐-‐-‐-‐-‐-‐-‐
43
6. vi
List
of
Tables
Table
Table
Caption
Page
Table
2.1
Plasmid
Transfer
Vectors
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
27
Table
2.2:
Viruses
used
in
this
experiment
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
28
Table
3.1.
DNA
concentration
of
virus
cells
using
Nanovue
Spectrophotometer
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
36
Table
3.2.
Titration
using
plaque
assay
of
each
virus.
-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐-‐
37
7. vii
Acknowledgments
I
would
like
to
acknowledge
and
thank
the
following
individuals
who
helped
me
perform
the
work
presented
in
this
thesis.
Prof.
Possee
who
was
always
available
to
help
me
and
guided
me
throughout
the
research.
I
thank
him
for
giving
me
the
opportunity
to
work
in
his
lab.
He
has
invested
significant
amounts
of
time
and
been
a
great
mentor
to
me.
My
parent
for
their
support
and
their
encouragement
in
everything
I
did.
This
master’s
research
thesis
was
not
possible
without
my
parent’s
support
and
love.
8. viii
Declaration
I,
Jasvir
Kaur
declare
that
this
thesis
and
the
work
present
in
it
are
my
own
and
have
been
generated
by
me
as
the
result
of
my
own
original
research.
Wherever
contributions
of
others
are
involved,
it
is
made
clear
with
due
reference
to
literature.
The
work
was
performed
under
the
guidance
of
Prof.
Robert
Possee,
at
Oxford
Brookes
University.
9. ix
Abstract
Baculoviruses
are
mostly
found
in
order
Lepidoptera
and
are
found
among
600
different
insect
species.
Baculoviruses
were
originally
used
in
production
of
insecticides,
but
now
they
are
widely
used
for
the
production
of
many
recombinant
proteins.
The
baculovirus
expression
system
was
developed
decades
ago
and
has
improved
in
expression
of
foreign
genes
in
insect
cell
lines.
Cotransfection
is
commonly
used
to
generate
recombinant
viruses.
The
Sf9
cells
were
co-‐transfected
with
the
use
of
flashBAC
DNA
and
plasmid
transfer
vectors
(pACRP23.lacZ,
pAcUK,
pOET6.NaV1.4,
and
pAcHANA).
The
populations
of
the
viruses
were
monitored
up
to
five
passages
at
both
low
(0.1)
and
high
(5)
moi.
The
viruses
were
passaged
in
cell
culture
at
5ml,
from
which
the
virus
particles
were
purified
from
and
DNA
analysis
was
performed.
The
data
collected
in
this
study
reveals
that
two
control
viruses
AcMNPV,
AcRP23.lacZ
and
AcHANA
generated
in
this
study
were
all
genetically
stable
during
passage.
However,
AcUK,
which
contains
the
mammalian
gene
urokinase,
AcRP23.lacZ
(generated
in
this
study),
containing
bacterial
beta-‐glacatosidase
gene
and
AcNav1.4,
which
contains
mammalian
Nav1.4
gene
were
found
to
be
genetically
unstable.
Thus,
nonspecific
bands
were
found
in
the
viruses
AcUK
and
AcRP23.lacZ.
The
results
with
the
newly
generated
AcRP23.lacZ
were
surprising
since
the
control
virus
with
the
same
gene
appeared
to
be
stable
after
passage
at
both
low
and
high
moi’s.
This
may
be
due
to
defective
interfering
particles
(DIPs)
or
few
polyhedra
(FP)25K
mutation.
DIP
occurs
over
time
during
viral
infection
and
FP25K
mutation
takes
place,
if
small
amount
of
cells
have
10. x
polyhedron
and
ODV,
which
can
cause
nonspecific
bands
to
occur.
The
verification
of
plasmid
cell
DNA
in
the
cotransfected
cells
all
had
high
molecular
weight
bands.
The
PCR
analysis
of
the
plasmid
DNA
showed
no
such
results
among
AcUK,
AcRP23.lacZ,
and
AcNaV1.4.
11. xi
Table
of
Abbreviations
AcMNPV:
Autographa
californica
nucleopolyhedrovirus
BAC:
bacterial
artificial
chromosome
BEVs:
baculovirus
expression
vectors
bp:
base
pairs
BV: budded virions
DNA:
deoxyribonucleic
acid
DI:
defective
interfering
dpi:
days
post
infection
FCS:
foetal
calf
serum
Fp25k:
Autographa
californica
multiple
nucleopolyhedrovirus
with
the
fp25k
gene
removed
FP:
few
polyhedra
Gp64:
globular
protein
64
GV:
granulovirus
H:
hours
hpi:
hours
post
infection
LB:
luria
broth
MOI:
multiplicity
of
infection
NPV:
nucleopolyhedrovirus
N-‐terminal:
amino-‐termina
OBs:
occlusion
bodies
ODV:
occlusion-‐derived
virions
ORF:
open
reading
frame
P10:
baculovirus
10
kDa
protein
PBS:
phosphate
buffered
saline
PCR:
polymerase
chain
reaction
PE:
polyhedral
envelope
PFU:
plaque
forming
units
pi:
post-‐infection
Polh:
polyhedrin
rpm:
rotations
per
minute
12. xii
Sf:
Spodoptera
frugiperda
T.
ni:
Trichoplusi
ni
TBS:
tris
buffered
saline
13. 1
Overview
of
Baculoviruses
Baculoviruses
are
commonly
found
in
nature.
They
are
large
DNA
viruses
isolated
from
arthropods
found
on
land
and
in
aquatic
environments.
They
mainly
tend
to
infect
butterflies,
moths,
sawflies,
wasps,
flies
and
beetles.
However,
most
baculoviruses
have
been
isolated
from
species
in
the
order
Lepidoptera.
They
are
believed
to
have
coevolved
with
their
insect
hosts
over
millions
of
years.
Baculoviruses
have
the
ability
to
survive
easily
in
soil
or
in
clefts
of
plants
for
long
periods
(decades)
since
they
have
the
capability
of
surviving
outside
their
host
within
highly
resistant
protein
occlusion
bodies.
The
recent
literature
claims
that
baculovirus
diseases
are
found
in
more
than
600
different
insect
species.
New
information
is
also
emerging
about
the
genetic
diversity
of
baculoviruses.
The
gene
organization
of
NPVs
is
conserved,
but
the
existence
or
truancy
of
auxiliary
genes
can
result
in
variation
among
the
viruses.
Baculoviruses
were
originally
studied
for
their
use
as
biological
insecticides.
Each
baculovirus
isolate
only
infects
particular
species
and
will
not
harm
non-‐
invertebrate
hosts.
Hence
they
are
regarded
as
very
safe
viruses.
This
was
a
major
advantage
in
their
second
main
use.
Currently
they
are
also
widely
employed
as
gene
expression
vectors
to
make
recombinant
proteins
and
as
vectors
for
mammalian
cell
transduction,
where
genes
are
introduced
under
control
of
promoters
active
in
the
target
cells.
One
of
the
most
widely
studied
members
of
the
family
Baculoviridae
is
Autographa
californica
multiple
nucleopolyhedrovirus
(AcMNPV),
as
it
infects
around
30
species
belonging
to
the
lepidoptera.
Most
studies
of
virus
structure
have
been
based
on
this
isolate.
Baculovirus
nucleocapsids
are
rod-‐shaped
and
contain
double
stranded
circular
genome
DNA.
Some
of
the
features
of
baculoviruses
include
two
types
of
virions:
occlusion-‐derived
virions
(ODV)
with
occlusion
bodies
–responsible
for
horizontal
transmission
between
insects
and
budded
virions
(BV)-‐
known
to
spread
virus
from
cell
to
cell
within
a
host
insect.
The
baculoviruses
are
capable
of
14. 2
expressing
foreign
genes
in
baculovirus
expression
vector
system
(BEVS),
as
they
are
nuclear-‐replicating
DNA
viruses
that
encode
DNA
directed
RNA
polymerase,
which
is
used
to
transcribe
late
and
very
late
genes
(Clem
&
Passarelli,
2013).
The
BEVS
has
been
used
to
express
genes
from
plants,
fungi,
bacteria,
and
viruses
in
insect
cells.
One
of
the
benefits
of
baculoviruses
is
that
they
have
large
genomes,
which
allows
easy
insertion
of
the
foreign
genes.
The
basis
of
the
system
is
the
removal
of
the
occlusion
body
protein
gene
(polyhedron)
and
its
replacement
with
a
foreign
coding
region.
An
important
aspect
of
the
BEVs
is
the
method
used
to
insert
genes
into
the
recombinant
virus.
flashBACTM
produced
by
Oxford
Expression
technologies
has
reduced
the
time
to
produce
recombinant
viruses
to
a
one
step
process
(Possee
et
al.,
2008).
However,
it
is
also
important
that
we
have
a
better
understanding
of
the
stability
of
these
viruses
as
they
are
passaged
in
insect
cells
to
generate
infectious
stocks
used
for
subsequent
recombinant
protein
production.
15. 3
Chapter
1-‐
Insight
of
Baculoviruses
1.1
History
Baculoviruses
do
not
cause
any
infection
in
humans,
mammals
and
researchers
have
known
of
their
existence
for
several
hundred
years.
The
earliest
information
on
baculoviruses
can
be
traced
in
ancient
Chinese
literature,
which
describes
the
culture
of
silkworms.
Marco
Vida
of
Cremona,
an
Italian
bishop
of
16th
century,
provided
the
first
explanation
of
baculovirus
disease.
He
described
the
disease
of
silk
worms
in
a
poem
called,
“De
Bombyce”.
In
the
poem
he
described
the
symptoms
of
the
infection.
Baculoviruses
were
not
observed
directly
until
19th
century.
Polyhedral
crystals
were
observed
by
microscopy
and
associated
with
disease
in
insects.
During
research
carried
out
in
the
20th
century,
it
was
found
that
the
virus
particles
are
embedded
in
the
polyhedral
crystals.
In
addition
it
was
found
that
baculoviruses
played
a
major
role
in
regulating
insect
populations.
The
granuloviruses
(GVs)
were
also
identified.
In
comparison
to
nucleopolyhedrovirus
(NPV),
the
GVs
are
found
to
be
small
and
granular.
They
contain
only
one
virion
per
occlusion
body.
During
the
same
period,
baculoviruses
were
observed
and
were
affecting
the
insect
pest.
Introducing
baculoviruses
in
North
America
effectively
controlled
sawfly.
From
the
early
1950’s
to
1975,
the
development
of
baculovirus
as
control
agents
of
insect
pests
was
on
the
rise.
From
1970
to
1985,
various
pathological
and
genetic
understandings
of
baculoviruses
were
developed.
Discovering
the
fact
that
there
are
two
different
forms
of
baculoviruses,
a
budded
virus
(BV)
and
occluded
virus
(OV)
form
was
important
in
realizing
the
behavior
of
the
virus.
The
behavior
helped
in
cell
cultures
and
in
insect
host
pathology.
OV
are
more
infectious
in
the
mid-‐gut
of
an
insect,
while
BV
spreads
the
infections
in
cell
cultures
or
in
other
tissues.
The
most
studied
member
of
the
Baculoviridae
family
is
Autographa
californica
nucleopolyhedrovirus
(AcMNPV).
It
became
popular
for
study
because
it
was
easy
to
grow
in
cell
cultures.
In
1979,
National
Institutes
of
16. 4
Health
allowed
cloning
of
baculovirus
in
Escherichia
coli.
The
cloning
was
used
to
analyze
particular
genes
and
their
functions.
The
usefulness
of
baculovirology
led
to
major
success
in
human
gene
therapy,
molecular
biology
and
genetics
with
cell
culturing.
It
also
led
to
mass
production
of
pesticides
for
the
benefit
in
the
field
of
agriculture.
1.2
Classification
The
Baculoviridae
viruses
are
rod-‐shaped
with
circular
double
stranded
DNA
genome
of
88-‐180
kbp,
consisting
of
up
to
180
genes,
in
a
rod-‐
shaped
virus
particle
with
the
size
of
200-‐400nm
in
length
and
36nm
wide
(Cheng
et
al.,
2013).
These
viruses
are
categorized
as
arthropod-‐specific
viruses.
(Shi
et
al.,
2015).
There
are
two
virion
pheotypes
found
in
baculoviruses
occlusion-‐derived
virion
(ODV)
and
budded
virions
(BV).
Budded
virion
(BV)-‐
spreads
the
infecton
from
tissue
to
tissue
and
occlusion
derived
virion
(ODV),
which
is
consumed
via
oral
route
and
spreads
infection
between
individual
insects.
In
the
two
virions,
the
nucleocapsid
protein
and
genetic
material
are
identical
to
each
other.
Both
of
the
virions
are
formed
in
the
virogenic
stroma
(VS)
(Shi
et
al.,
2015).
ODV
are
occluded
in
a
crystalline
protein
matrix
to
form
granules
or
polyhedra.
The
viruses
that
form
ganules
are
known
as
granuloviruses
and
the
viruses
that
form
polyhedra
are
known
as
nucleopolyhedroviruses
(NPVs).
Granuloviruses
produce
small
occlusion
bodies
(OBs)
ranging
between
the
size
of
(0.13-‐
0.50μm),
containing
one
to
two
encapsulated
virions
in
a
protein
known
as
granulin(Jehle
et
al.,
2006).
Nucleopolyhedroviruses
produce
occlusion
bodies
ranging
from
(0.15
to
3μm)
and
they
contain
many
ODVs
(Jehle
et
al.,
2006).
NPVs
have
been
commonly
found
among
insect
orders
Lepidoptera,
Diptera
and
Hymenoptera,
while
GVs
have
been
only
found
among
Lepidoptera.
For
a
long
time,
baculoviruses
have
been
classified
into
two
groups
granuloviruses
(GVs)
and
nucleopolyhedroviruses
(NPVs)
(Kelly
et
al.,
2008).
The
Baculoviridae
family
is
sectioned
into
four
generas.
The
Alphabaculoviruses
are
lepidoperan-‐specific
nuclopolyhedroviruses
with
either
single
or
multipe
nuclocapsids,
which
produces
both
BV
and
ODV
(Jehle
et
al.,
2006).
17. 5
The
Betabaculoviruses
are
composed
of
lepidoteran
specific
genus
Granulovirus
and
also
produces
both
BV
and
ODV.
Deltabaculoviruses
and
Gammabculoviruses
are
made
up
of
NPVs
that
only
infect
dipteran
and
hymenopteran
(Jehle
et
al.,
2006).
The
phylogentic
tree
shown
below
in
Figure
1.1
shows
the
division
of
Baculoviridae
family.
Figure
1.5.
The
above
phylogenetic
tree
indicates
the
division
of
four
groups
in
baculovirus
family.
It
indicates
amino
acids
of
29
baculoviruses
genes
derived
from
29
sequenced
baculovirus
genomes
(Jehle
et
al.,
2006).
1.3
Baculovirus
protein
structure
The
baculoviruses
have
the
potential
to
encode
more
than
150
proteins
due
to
their
large
genomes
(Ahrens
et
al.,
1997).
The
viral
particles
consist
of
nucleocapsids
of
which
the
DNA
is
associated
with
the
p6.9
protein,
which
is
a
54
amino
acid
protein.
In
additon,
vp39
has
also
been
recognized,
which
is
about
39
kDa
and
forms
most
of
the
capsid
structure.
The
occlusion
bodies,
budded
virus
(BV)
and
occlusion
derived
virus
(ODV)
envelopes
and
proteins
are
building
blocks
of
nucleocapsids
(Rohrmann,
2013).
One
of
the
best
characterised
baculovirus
protein
is
group
I
NPV
gp64,
this
protein
is
18. 6
important
for
BV
infectivity
but
genome
sequence
analysis
indicateded
that
many
baculoviruses
lack
homologs
of
the
gp64
gene(Rohrmann,
2013).
It
was
later
discovered
that
those
that
lack
gp64
possess
a
different
protein,
called
F
(fusion)
protein
(e.g.
LD130
from
Lymantria
dispar
MNPV)
Granuloviruses
and
group
II
NPVs
do
not
have
GP64,
therefore
the
F
protein
is
used
as
an
alternative
(Rohrmann,
2013).
It
is
assumed
that
group
I
viruses
use
GP64
to
enter
BV
into
cells,
while
the
baculoviruses
that
lack
gp64
homolog
use
F
protein
as
their
envelope
protein
(Figure
1.2)
(Rohrmann,
2013).
Figure
1.6.
Group
I
has
both
GP64
and
F
while
Group
II
only
has
F
protein
(Rohrmann,
2013).
1.4
Genome
of
AcMNPV
The
AcMNPV
was
first
described
in
the
early
1970s
and
a
decade
later
genetic
research
was
done.
The
genetic
research
was
influenced
by
virus
replication
in
cells
from
Spodoptera
frugiperda
and
Trichoplusia
ni
(Rohrmann,
2013).
These
discoveries
lead
to
the
development
of
a
bacmid
system,
which
produced
recombinant
virus
with
transposition
plasmids
in
the
AcMNPV
genome
in
artificial
bacterial
chromosome.
The
artificial
bacterial
chromosome
replicated
the
entire
AcMNPV
genome
in
bacteria
(Rohrmann,
2013).
This
technology
allowed
specific
deletion
gene
knockout
19. 7
in
bacteria,
which
could
later
be
investigated
with
the
use
of
transfection
in
insect
cells
(Rohrmann,
2013).
AcMNPV
is
a
vector
that
is
used
frequently
for
recombinant
protein
production
in
baculovirus
expression
system
(Harrison,
2009).
AcMNPV
infections
have
been
reported
in
43
lepidopteron
species
belonging
to
11
different
families
caused
by
AcMNPV.
The
first
fully
sequenced
AcMNPV
was
the
C6
clone
(Ayreus
et
al.,1994).
The
genome
of
AcMNPV
contains
a
133.9
kbp,
circular
double
stranded
DNA
genome
in
rod-‐
shaped
virion
(Lee,
et
al.,
2015).
AcMNPV
is
also
made
up
of
about
154
ORFs,
which
have
little
redundant
sequence
between
them
(Dickison
et
al.,
2012).
AcMNPV
lead
to
the
development
of
the
original
baculovirus-‐insect
cell
expression
system.
Figure1.3.
AcMNPV's
genetic
map.
Adapted
from
(Maciag,
Olszowka,
&Klein,2014)
1.5
Baculovirus
replication
The
replication
cycle
consists
of
two
type
of
virions,
one
known
as
occluded
and
the
other
as
budded.
The
occluded
is
responsible
for
infection
and
stability
in
insects
midgut
cells.
Whereas
the
budded
virions
are
only
responsible
in
spreading
the
infection
from
cell
to
cell.
The
replication
starts
when
the
occlusion
body
is
absorbed
by
the
insect.
The
viral
particles
are
20. 8
then
released
inside
insects
midgut
due
to
high
pH
(Figure
1.4).
The
viral
particles
(ODV)
after
being
released,
infect
three
different
cell
types,
such
as
regenerative(R
),
columnar
epithelium
(CE),
and
goblet
cells
(G).
(McCarthy
&
Theilmann,
2008).
Figure
1.4.
Baculovirus
infection
in
insect
larvae
(O’Reilly
et
al.,
1994)
The
replication
system
for
OB
and
NPVs
differ,
first
it
produces
budded
virus
and
then
it
goes
onto
developing
occluded
form.
At
around
12
hour
post-‐
infection
(h
p.i),
nucleocapsids
buds
through
plasma
membrane,
further
enveloping
the
BV
(Figure
1.4).
After
20
h
p.
i.,
ODVs
tend
to
form
nucleocapsids
and
then
they
are
transported
between
nuclear
membrane
and
VS.
Hence,
it
forms
occlusion
bodies
or
polyhedra
(Shi
et
al.,
2015).
The
genetic
expression
is
also
divided
into
three
phases:
early,
late
and
very
late.
In
the
late
phase
of
the
infection,
production
of
budded
virus
starts
and
the
production
of
polyhedron
begin
in
the
very
late
phase
(Kelly
et
al.,
2008).
The
NPV
virus
are
found
in
the
matrix
of
polyhedra
and
they
can
remain
stable
in
the
environment
(Lynn,
2003).
When
the
larvae
eats
the
occlusion
bodies,
the
ODVs
are
then
released
in
the
midgut
alkaline
environment.
This
phenomenon
leads
to
infection
in
the
epithelium(Cheng
et
al.,
2013,
Lynn,
2003).
The
ODV
then
attaches
to
the
microvilli
of
the
midgut
leading
to
an
infection.
The
infection
spreads
with
budded
virus
(BV)
from
midgut
to
other
tissues
(Figure
1.5)(Lynn,
2003).
Occluded
virus
is
responsible
for
causing
the
primary
infection
and
only
infects
insect-‐to-‐insect
transmission
of
the
virus.
The
BVs
bud
out
from
infected
cells
acquiring
their
envelopes
21. 9
from
the
plasma
membranes,
thereafter
it
continues
to
cause
infection
to
other
tissues
(Cheng
et
al.,
2013).
The
ODVs
are
found
occluded
in
protein-‐
polyhedra,
which
consists
of
29
kDa
polyhedrin
protein
(Cheng
et
al.,
2013).
The
ODV
obtain
their
envelope
from
the
protein
FP25K
from
inside
nuclear
membrane
(INM)
(Cheng
et
al.,
2013).
The
FP25K
is
found
in
the
cytoplasm
and
nuclei
of
AcMNPV-‐
infected
cells
(Harrison
&
Summers,
1995a).
Polyhedrin
and
granulin
are
very
similar
to
each
other
and
are
major
components
of
occlusion
bodies.
Polyhedrins
are
one
of
the
abundant
proteins
found
in
the
infected
baculovirus
cells.
They
tend
to
form
crystalline
cubic
lattice
that
is
enclosed
by
virions
(Rohrmann,
2013).
The
polyhedron
envelope
(PE)
or
calyx
is
what
surrounds
polyhedra
(Rohrmann,
2013).
The
function
of
PE
is
to
increase
the
stability
and
seal
the
surface.
When
polyhedra
are
given
alkaline
treatment,
the
crystalline
lattice
dissolves
but
polyhedron
envelope
does
not
and
that
is
where
all
the
virions
are
trapped
(Rohrmann,
2013).
Those
viruses
that
do
not
sysnthesize
occluded
form
are
known
as
“nonoccluded”
baculoviruses.
The
PE
protein
is
linked
to
p10
fibrillar
and
it
is
required
for
assembly
of
the
polyhedron
envelope
(Rohrmann,
2013).
If
p10
is
deleted
it
causes
reduction
in
lysising
of
cells
and
increase
in
virus.
The
deletion
of
p10
gene
makes
a
positive
impact
on
recombinant
protein
production
(Hitchman
et
al.,
2011).
The
homologs
of
p10
are
found
in
all
group
I
and
II
NPVs
and
also
among
some
GVs.
Although
the
exact
funtion
of
p10
(10kDA)
is
still
unknown
in
previous
studies
it
was
concluded
that
knock-‐out
of
p10
function
by
lacZ
had
no
impact
on
polyhedral
envelope
(Hitchman
et
al.,
2011).
Although
it
is
not
proven
it
is
thought
that
P10
may
help
polyhedra
bundle
separate
into
individual
particles
to
spread
in
the
environment
and
thus
aid
horizontal
transmission.
However,
OB
bundles
spread
easily
if
p10
orf
is
deleted
(Hitchman
et
al.,
2011).
22. 10
Figure
1.5.
Baculovirus
structure
(Maciag,
Olszowka,
&
Klein,
2014)
1.6
Virus
replication
in
vivo
Infection
in
vivo
occurs
when
the
alkali-‐soluble
occlusion
body
is
ingested
by
a
host
and
dissolves
in
the
high
pH
of
the
insect’s
midgut,
thus
releasing
virions.
In
the
first
stage
of
the
infection
BV
are
produced.
This
further
spreads
the
infection
from
cell
to
cell
in
the
insect.
The
nucleocapsids
are
then
transported
from
nucleus
to
the
cytoplasm.
From
cytoplasm,
the
nucleocapsids
obtain
an
envelope
when
they
bud
through
the
plasma
membrane.
The
membrane
has
been
modified
by
the
glycoprotein
known
as
gp64.
At
the
later
stage
of
the
infection,
the
virions
stay
within
the
nuclei
of
the
cell
where
they
become
occluded.
Theses
occluded
virions
are
then
released
from
decaying
insect
and
contaminate
foliage.
Thereafter
other
insects
consume
the
foliage.
The
total
infection
time
takes
around
5-‐7
days.
During
the
infection
stage,
the
host
goes
through
various
phases.
In
the
first
phase
the
skin
tends
to
swell
and
a
change
is
observed
in
the
luster
of
the
skin.
In
the
second
stage,
the
muscular
tissues
dissolve
and
thereafter
the
larva
becomes
a
pouch
of
fluid.
In
the
final
stage,
the
larva
bursts
and
releases
polyhedral
particles.
The
two
viral
structures
are
different
in
their
composition,
although
their
nucleocapsids
are
similar.
The
differences
among
these
viruses
exist
due
to
their
different
functionality.
For
example
the
BV
tends
to
infect
insect
tissues,
while
the
envelope
of
ODV
is
responsible
for
midgut
infection.
It
also
plays
an
important
role
while
interacting
with
polyhedron
structure.
The
23. 11
ODV
occluded
form
is
considered
to
be
stable
in
harsh
environment
of
the
insect
gut.
Thus,
it
spreads
the
infection
from
one
insect
to
another
over
the
period
of
one
year.
1.7
Entry
into
the
nuclei
After
the
virus
enters
the
cell,
the
NPV
nucleocapsids
are
taken
to
the
nuclear
membrane,
where
actin
polymerization
takes
place
(Rohrmann,
2013).
Many
studies
suggest
that
nucleocapsids
are
carried
via
nuclear
pore.
Even
the
observation
of
empty
nucleocapsids
found
in
the
nuclei
of
cells
support
this
evidence
(Rohrmann,
2013).
Finding
nuclocapsids
in
nuclei,
leads
to
the
suggestion
that
they
do
not
need
cell
dividing
or
neccessity
of
nuclear
membrane
breakdown
to
migrate
into
nuclei
(Rohrmann,
2013;
Van
Loo
et
al.,
2001).
Studies
have
found
that
some
baculoviruses
inject
their
DNA
through
nuclear
pores
with
the
use
of
fluorescent
tag
attached
to
vp39
capsid
protein
and
also
to
WT
vp39
protein
(Rohrmann,
2013;
Ohkawa
et
al.,
2010).
The
fluorescence
was
observed
inside
nuclei
and
also
found
that
nucleocapsids
restrict
to
the
nuclear
pore.
Thus,
this
concludes
that
nucleocapsids
are
transported
through
nuclear
pore
complex
(Rohrmann,
2013).
The
nuclear
pore
have
been
characterised
to
have
channels
of
38-‐78
nm.
The
virion
are
measured
to
be
around
30-‐60
nm
in
diameter.
Hence,
these
measurements
allow
them
to
move
through
the
pores
(Rohrmann,
2013).
1.8
Exiting
the
cell
nuclei
The
virions
have
the
ability
to
enter
and
exit
with
the
use
of
nuclear
pores,
with
nucleocapsids
injected
into
Xenopus
oocytes.
In
a
previous
study,
it
was
found
that
treatment
by
colchicine
tends
to
obstruct
the
formation
of
the
microtubule.
Further,
it
leads
to
the
reduction
in
production
in
BV.
This
process
indicates
that
the
structure
might
play
a
role
in
virion
movement
out
of
the
cell
(Rohrmann,
2013).
Kinesin
is
one
of
the
major
protein
that
transports
material
to
the
cell
periphery.
The
BV
has
been
found
to
interact
24. 12
with
kinesin,
which
transports
them
from
microtubules
to
the
cell
membrane
(Au,
2012;
Danguah
et
al.,
2012;
Rohrmann,
2013).
Nucleocapsids
obtain
their
envelope,
which
consist
of
one
viral
protein
GP16.
It
happens
when
the
nucleocapsids
replicate
in
the
nucleus
they
are
then
released
from
midgut
cells
by
budding
via
the
nuclear
membrane.(Pearson
et
al.,
2001).
The
envelope
then
gets
lost
on
its
way
to
the
cytoplasm
and
parts
of
the
envelope
protein
assembly
to
the
plasma
membrane.
This
contains
both
GP64
(Ac128)
and
the
F
protein
(Ac23)
for
the
group
I
NPVs
(Blissard
&
Rohrmann,
1989).
The
GP64
protein
modifies
the
host
membrane,
this
mechanism
then
allows
the
occurrence
of
virus
budding
and
secondary
infection
to
take
place,
which
infects
midgut
cells
(Pearson
et
al.,
2001;
Blissard
&
Rohrmann,
1989;
Rohrmann,
2013).
If
the
AcMNPV
is
used
to
infect,
it
tends
to
go
toward
basal
and
lateral
regions
of
the
cells
allowing
the
infection
to
target
tissues
rather
than
the
gut
lumen
(Keddie
et
al.,
1989;
Rohrmann,
2013).
1.9
Gene
expression
regulation
Baculoviruses
gene
expression
is
split
into
three
different
stages,
each
one
depends
on
the
prior
stage:
early,
late
and
very
late.
The
viral
RNA
polymerase
II
transcribes
the
early
genes
while
RNA
polymerase
transcribes
late
and
very
late
genes
(Clem
&
Passarelli,
2013).
1.9.1
Immediate
early
genes
The
immediate
early
genes
are
the
ones
that
can
be
transcirbed
even
when
the
protein
synthesis
inhibitors
are
present,
for
example
cycloheximide
(King
&
Possee,
1992).
This
stage
has
been
further
studied
and
has
been
known
that
copies
of
these
genes
are
transcriptionally
active
when
inserted
into
plasmids.
It
should
be
considered
that
genes
should
be
active
even
after
transfection
into
uninfected
insect
cells.
25. 13
1.9.2
Delayed
early
genes
In
the
second
phase
of
gene
expression,
the
use
of
cycloheximide
and
other
inhibitors
is
important
in
defining
this
stage
(King
&
Possee,
1992).
When
cells
are
treated
with
such
inhibitors
and
returned
to
normal
condition,
a
pattern
of
protein
expression
is
observed
(King
&
Possee,
1992).
Some
proteins
express
instantly
when
the
inhibitors
were
removed
while
the
others
are
expressed
after
a
delay.
Thus,
it
defines
the
division
between
immediate
and
delayed
genes
(King
&
Possee,
1992).
1.9.3
Late
and
very
late
gene
The
third
phase,
in
which
the
virus
genes
are
expressed
in
infected
cells
with
replication
of
virus
DNA
(King
&
Possee,
1992).
If
the
virus
DNA
replication
is
inhibited
by
aphidicolin
then
the
late
genes
are
not
transcribed.
The
virus
genes
expressed
in
this
stage
are
those
that
are
encoding
structural
elements
of
the
virus
particles
(basic
protein,
capsid
protein,
and
the
virus
membrane
glycoprotein
(gp67))
(King
&
Possee,
1992).
The
transcription
of
these
genes
begins
with
RNA
polymerase,
which
is
α-‐
amanitin
resistant.
It
is
prompted
in
the
late
infection
of
the
cells.
The
very
late
genes
are
transcribed
when
the
virus
is
producing
occlusion
bodies
in
the
nucleus
of
an
infected
cell
from
15
h
p.i.
onwards.
This
stage
includes
polyhedron
protein,
which
helps
in
forming
the
matrix
of
the
occlusion
body
and
p10
protein.
Further,
it
plays
the
role
in
the
production
of
polyhedra.
The
p10
forms
crystalline
matrix
in
the
nucleus
of
the
infected
cells
is
also
associated
with
formation
of
polyhedra.
1.10
Insect
Cell
lines
The
cell
lines
that
are
used
in
baculovirus-‐based
expression
studies
are
usually
Sf21,
Sf9,
and
Trichoplusia
ni
(Tn).
The
two
cell
lines
(Sf21
and
Sf9)
were
obtained
from
ovarian
cells
of
Spodoptera
frugiperda
–(fall
army
worm)
(Haines
et
al.,
2006).
The
Trichoplusia
ni
(Tn)
were
obtained
from
26. 14
adult
tissues
of
cabbage
loopers
(Hink,
1970).
T.ni
is
a
very
important
pest
in
agriculture
and
highly
similar
to
AcMNPV.
The
Sf-‐9
cells
were
cloned
from
Sf-‐21
cells
to
provide
the
maximum-‐production
of
Beta-‐galactosidase
during
the
process
of
baculovirus
expression.
The
cells
Sf21
or
Sf9
are
used
for
co-‐
transection,
amplification
of
virus
and
in
plaque
assays
(Haines
et
al.,
2006).
The
insect
cells
are
easier
to
maintain
when
compared
to
mammalian
cell.
They
can
be
grown
in
either
a
shake
or
stirred
flask,
or
if
monolayer
needs
to
be
maintained
T-‐flasks
or
culture
dishes
can
be
used
easily
(Haines
et
al.,
2006).
These
cell
culture
lines
do
not
require
a
CO2
incubator,
as
they
can
be
easily
grown
at
25OC
to
30OC
temperature
(Haines
et
al.,
2006).
1.11
Transfer
Vector
There
are
many
transfer
plasmids
available
which
contain
different
sequences
for
various
purposes
(Hitchman
et
al.,
2011).
One
of
the
main
purposes
of
transfer
vector
is
to
allow
the
insertion
of
a
foreign
gene
of
a
specific
promoter
gene.
The
transfer
vectors
are
composed
of
polyhedrin
or
p10
very
late
promoters.
This
then
produces
high
concentration
virus
within
the
infected
cells
(Possee
&
King,
2007).
After
the
insertion
into
the
virus
genome,
the
two
genes
have
well
characterized
promoters
and
results
in
high-‐level
transcription
or
recombinant
sequence
(Possee
&
King,
2007).
One
of
the
benefits
of
using
transfer
vector
is
that
it
is
easy
to
operate
in
vitro.
It
is
also
simple
to
find
the
primary
genetic
structure
of
recombinant
components
(Possee
&
King,
2007).
A
transfer
vector
is
combined
with
virus
DNA
and
then
used
to
co-‐transfect
insect
cells
to
develop
an
infection.
The
infected
cells
with
a
virus,
face
the
occurrence
of
recombination
in
between
homologous
sequences
in
the
plasmid
transfer
vector
and
virus
genome
(Possee
&
King,
2007).
The
native
virus
gene
is
usually
discarded
in
the
event
of
double
cross-‐over
and
substituted
with
a
foreign
coding
region
(Possee
&
King,
2007).
27. 15
1.12
Glycosylation
The
N-‐linked
glycosylation
takes
place
in
endoplasmic
reticulum
of
both
insect
and
mamalian
cells.
In
insect
and
mammalian
cells,
N-‐linked
glycosylation
can
be
repressed
if
cells
are
treated
with
tunicamycin
(King
&
Possee,
1992).
Eukaryotic
proteins
are
modified
by
adding
covalent
to
carbohydrate
side
chains.
There
are
three
glycosylation
pathways
in
eukaryotes:
N-‐glycosylation,
O-‐glycosylation,
and
O-‐linked
N-‐
actylglucosamine
(O-‐GlcNAc).
Insect
cells
consists
of
all
these
three
pathways
but
do
not
match
up
with
eukaryotes
(Jarvis,
1997).
Insect
cells
contain
low
levels
of
fucose,
galactose
and
sialic
acid
transferases.
Thus,
insect
cells
lack
the
ability
undertake
oligosaccharide
(King
&
Possee,
1992).
The
recombinant
proteins
tend
to
be
sensitive
to
endo
H,
endo
F
and
N-‐
glycanase,
which
is
responsible
in
removal
of
immature
oligosaccharides
(King
&
Possee,
1992).
Abnormal
glycosylation
can
result
in
disease
and
such
developmental
deficiency,
neuro
disorders
or
it
can
lead
to
serious
tumors.
Therefore,
it
is
important
to
have
functional
glycoprotein
for
analyses
and
medical
treatment
for
many
symptoms
(Morokuma,
et
al.,
2015).
As
we
know
N-‐linked
glycan
increases
the
protein
stability
and
also
regulate
cell-‐cell
and
protein-‐protein
interactions
(Morokuma,
et
al.,
2015).
Thus,
it
is
necessary
to
have
complex
linkage
of
glycoprotein
for
proper
function
and
medical
treatment
of
many
diseases.
Up
until
today,
mammalian
expression
system
is
used
to
produce
glycoproteins
even
when
it
carries
many
disadvantages.
Those
disadvantages
could
be
such
as
high
cost
and
low
productivity
(Morokuma,
et
al.,
2015).
The
proteins
retrieved
from
insect
cells
are
paucimannosidic-‐type
N-‐glycan
therefore,
it
removes
the
terminal
GlcNAc
of
hybrid
structures
(Morokuma,
et
al.,
2015).
28. 16
1.13
Selection
of
polyhedron-‐negative
recombinant
baculoviruses
1.13.1
BEVS-‐
Baculovirus
Expression
Vector
System
The
BEVS
consists
of
three
important
parts,
a
transfer
plasmid
with
gene
of
interest
that
needs
to
be
transferred
into
the
virus
genome,
a
baculovirus
vector
(AcMNPV)
and
insect
cell
line
(Sf9
or
Sf21)
(Hitchman
et
al.,
2011).
It
usually
begins
with
cloning
of
a
preferred
gene
into
a
transfer
plasmid
between
sequences
that
flank
the
polyhedrin
(polh)
locus
in
the
genome
of
the
virus
(Hitchman
et
al.,
2011).
The
baculovirus
expression
vector
system
(BEVS)-‐
is
a
very
important
system.
It
has
high
expression
level
and
has
the
capability
of
insertion
of
large
DNA
fragments,
glycosylation
modification,
acylation,
phosphorylation
and
amidation
(Shu-‐ffen
et
al.,
2012).
A
BEV
is
a
double-‐stranded
circular
DNA
genome,
that
has
been
genetically
modified
to
contain
a
foreign
gene
of
interest.
They
are
also
capable
of
transferring
foreign
genes
into
the
eukaryotic
host
cells.
Since
baculovirus
has
many
interesting
features,
the
BEVS
is
used
not
only
for
protein
production
of
interest
but
also
for
gene
therapy
and
surface
display
(Shu-‐ffen
et
al.,
2012).
Over
the
past
three
decades,
baculovirus
has
been
efficiently
used
in
pharmaceutical
and
vaccine
production
(Shu-‐ffen
et
al.,
2012).
The
BEV
system
has
been
intensively
used
in
basic
research
for
the
production
of
many
different
recombinant
proteins.
The
BEV
system
has
the
capability
to
produce
a
large
quantity
of
foreign
proteins.
The
BEV
system
can
make
perfect
foreign
proteins
as
it
has
the
ability
to
process
eukaryotic
protein
products.
The
BEV
system
does
have
fewer
limitations
such
as
protein
to
protein
variation
in
production.
Also,
the
glycoproteins
are
produced
in
low
levels,
this
can
further
lead
to
unsuitable
clinical
uses
due
to
the
difference
in
the
N-‐linked
glycan
structure
among
insects
and
mammals.
(Morokuma,
et
al.,
2015).
This
can
also
lead
to
a
low
production
of
recombinant
proteins
such
as
membrane-‐bound
and
secreted
glycoproteins
(Jarvis,
1997).
Another
limitation
that
BEV
has
is
when
the
insect
cell
protein
processing
pathway
are
not
identical
to
eukaryotes.
Thus,
it
leads
to
differentiation
in
29. 17
protein
production
compared
to
native
proteins
due
to
modifications
in
covalent
bonds
(Jarvis,
1997).
One
of
the
properties
that
lead
to
the
production
of
baculoviruses
expression
vectors
is
their
ability
to
produce
a
larger
quantity
of
polyhedrin
during
infection.
The
production
of
the
BEV
can
be
done
by
replacing
a
polh
gene
in
the
nucleotide
sequence
of
wild-‐type
viral
genome
with
a
particular
foreign
gene
of
interest.
This
then
allows
the
wild
type
to
be
distinguished
due
to
its
phenotype
of
inability
to
produce
polyhedra.
It
also
leads
to
isolation
and
expression
of
human
β-‐interferon
or
even
Escherichia
coli
β-‐
galactosidase
in
insect
cell
cultures
(Jarvis,
1997).
This
method
was
the
original
way
of
producing
recombinant
baculoviruses,
but
it
was
quite
difficult
for
non-‐virologists.
But
the
new
methods
have
improved
such
difficulties
with
systems
such
as
Bac-‐toBac,
BacPAK6,
and
flashBAC.
The
Bac-‐to-‐Bac
technology
depends
on
production
of
recombinant
baculovirus
via
site-‐specific
transposition
in
E.coli.
The
recombinant
virus
DNA
is
then
purified
and
used
to
transfect
insect
cells
to
recover
infectious
virus
particles.
The
BacPAK6
method
consists
of
AcMNPV
with
lacZ
insert
in
place
of
the
polyhedrin
gene,
which
can
be
linearized
prior
to
cotranfection
of
insect
cells
with
the
transfer
vector.
Recombinant
viruses
can
then
be
recovered
at
high
frequency
(>95%).
The
flashBAC
system
is
similar
to
BacPAK6,
but
it
doesn’t
require
linearization
to
use
and
it
is
also
fast
and
easy.
These
systems
allow
production
of
larger
quantities
of
expression
(Possee
&
King,
2007).
One
of
the
most
important
purposes
of
baculovirus
systems
is
its
uses
to
produce
proteins
in
insect
cells.
One
of
the
earliest
BEVS
had
been
based
on
polyhedra-‐negative
viruses,
their
polyhedrin
coding
region
was
replaced
with
a
foreign
gene
inorder
to
produce
recombinant
baculovirus
(Hitchman
et
al.,
2012).
The
expression
of
the
foreign
was
influenced
by
the
polyhedrin
promoter
(Hitchman
et
al.,
2012).
Transfer
vectors
are
used
to
insert
foreign
gene
as
the
baculovirus
genome
is
too
big
to
insert
the
foreign
gene
directly.
The
recombinant
virus
genome
results
when
a
foreign
gene
is
inserted
into
the
virus
genome
(Pennock
et
al,
1984).
This
produces
both
original
parental
30. 18
viruses
and
recombinat,
thus
plaque-‐purification
is
necessary
to
separate
the
two
but
this
process
can
be
time-‐consuming
(Kitts
et
al.,
1990).
1.13.2
Recombinant
Baculovirus
Production
There
are
several
different
techniques
that
had
been
used
to
produce
recombinant
baculoviruses
consisting
the
gene(s)
of
interest.
Some
of
these
techniques
aim
to
produce
a
pure
recombined
virus
with
little
to
none
recombinant
virus
as
possible.
These
techniques
use
transfer
vector
with
the
gene
of
interest
with
control
of
many
promoters
in
baculovirus
genome.
The
transfer
plasmid
consist
of
gene
of
interest
downstream
of
promoter
sequence
and
is
flanked
with
complementary
sequence
to
baculovirus
genome,
which
helps
recombination
of
the
promoter
gene
DNA
into
unessential
region
of
the
baculovirus
(polyhedrin)
gene.
One
of
the
methods
includes
the
use
of
bacteria
by
inserting
the
gene
of
interest
into
bacmid
consisting
the
baculovirus
genome,
and
using
marker
gene
to
produce
recombinant
baculovirus
(Lee
et
al.,
1993).
Another
method
uses
in-‐vitro
site
specific
transposition
to
insert
a
foreign
gene
and
eliminates
non-‐
recombined
baculovirus
by
adding
negative
selection
marker
(Zhao
et
al,
2003).
1.13.3
The
BacPAK6
system
As
the
original
method
of
purification
of
the
recombinant
virus
was
time
consuming,
it
was
then
further
improved
with
linearized
baculovirus
genomes
at
insert
site,
which
allowed
the
recovery
of
more
recombinants
as
linearized
vectors
lacked
functional
Orf1629
(Kitts
&
Possee,
1993).
The
orf1629
is
an
important
gene
that
is
involved
in
nuclear
actin
during
baculovirus
infection
(M.
van
Oers,
2011).
In
the
BacPAK6
system,
orf1629
is
restored
after
viral
genome
and
transfer
plasmid
are
recombined,
thus
giving
90%
recombinantion
frequency
(M.
van
Oers,
2011).
The
BacPAK6
system
deletes
polyhedrin
gene
with
the
lacZ
gene,
which
has
restriction
enzymes
Bsu361
on
either
side
of
lacZ.
(Kitts
&
Possee,
1993).
Digestions
31. 19
with
Bsu361
removes
ORF1629
and
lacZ
gene,
causing
linear
virus
DNA
that
is
not
capable
of
replication
with
insect
cells
(Kitts
&
Possee,
1993).
The
co-‐transfection
with
Bsu361,
re-‐circularises
the
virus
DNA
by
replacing
orf1629.
The
restoration
of
orf1629
allows
replication
in
insect
cells
and
constructing
high
frequency
production
of
recombinant
viruses
(Kitts
&
Possee,
1993;
Haines,
Possee,
&
King,
2006).
Eventhough
addition
of
Bsu361
sites
in
BacPAK6
increases
recovery
of
recombinants
the
Bsu361
digestion
is
not
100
efficient
and
will
always
have
mixture
of
parental
and
recombinant
virus
(Possee,
&
King,
2006).
Figure
1.7.
A.
DNA
containing
Escherichia
coli
(E.
coli)
lacZ
inserted
at
polh
locus.
B.
Digestion
of
viral
DNA
removes
lacZ
and
partially
deletes
orf1629-‐coding
region
C.
Cotransfection
and
insertion
of
foreign
gene
into
the
virus
DNA
and
restoration
of
orf1629
and
recircularization
of
DNA
thus
permitting
replication
within
insect
cells.
D.
Plaque
assay
is
used
to
isolate
recombinant
virus
(Possee,
&
King,
2006).
1.13.4
Bac-‐to
Bac®
The
Bac-‐to-‐Bac
Baclovirus
Expression
System
represents
an
efficient
way
to
make
recombinant
baculoviruses.
The
advantage
is
that
recombinant
baculovirus
can
be
obtained
fairly
quickly
in
7-‐19
days
(Anderson,
et
al.,
32. 20
1996).
This
system
does
not
require
separation
and
purification
of
recombinant
viruses
by
using
plaque
–assay
(Anderson,
et
al.,
1996).
The
vector
of
this
system
contains
kanamycin
resistance
gene
and
lacZa
(Anderson,
et
al.,
1996).
The
Bac-‐to-‐Bac
Baculovirus
expression
System
has
site
specified
transposition
properties
of
the
Tn7
transposon.
This
aspect
makes
it
easier
to
generate
recombinant
bacmid
DNA.
The
transposition
connection
does
not
interfere
with
the
functions
of
the
gene
(Anderson,
et
al.,
1996).
This
plasmid
is
designed
to
form
blue
colonies
as
bacmid
is
propagated
in
E.
coli
on
differentiation
medium
with
kanamcycin,
X-‐gal
and
IPTG.
The
recombinant
bacmid-‐containing
colonies
can
be
isolated
by
plating
them
on
a
selective
media
(Anderson,
et
al.,
1996).
The
bacmid
recombinant
DNA
is
isolated
by
alkaline
lysis
procedure
from
the
E.coli
cells.
Thereafter,
the
obtained
DNA
is
then
used
to
transfect
insect
cells
and
this
does
not
require
any
further
selection
before
virus
amplification
(Anderson,
et
al.,
1996).
1.13.5
flashBACTM
The
flashBac
is
a
new
technique
developed
by
Oxford
Brookes
University,
which
is
sold
by
Oxford
Expression
Technologies
(Hitchman
et
al.,
2011).
Essentially,
flashBAC
comprises
a
copy
of
the
AcMNPV
genome
with
a
partial
deletion
of
ORF1629,
which
is
amplified
in
bacterial
cells.
This
DNA
cannot
replicate
and
produce
infectious
virus
in
insect
cells
unless
it
is
cotransfected
with
a
plasmid
transfer
vector
containing
the
complete
ORF1629
gene.
The
flashBAC
technology
is
one
of
the
best
platforms
to
be
used.
It
does
not
require
selection
pressure
to
separate
recombinant
virus
from
the
non-‐recombinant
parental
virus
(Hitchman
et
al.,
2011).
This
system
is
dependent
on
homologous
recombinant
of
insect
cells
and
transfer
plasmids.
The
flashBAC
provides
many
advantages
to
the
existing
baculovirus
expression
vector
system
(Hitchman
et
al.,
2011).
This
technology
allows
the
user
to
skip
the
necessity
which
required
carrying
out
plaque-‐purification
(Hitchman
et
al.,
2011).
The
gene
deletion
restrains
virus
replication
in
the
insect
cells,
but
BAC
permits
the
viral
DNA
for
the
33. 21
propagation
of
bacterial
genome
(Hitchman
et
al.,
2011).
In
addition,
the
bacterial
circular
DNA
is
isolated
by
lysis
and
purification
by
making
use
of
flashBACTM
Kit
and
the
DNA
is
ready
for
use
in
co-‐transfection
(Hitchman
et
al.,
2011).
Genes
under
the
control
of
any
promoter
can
be
expressed
provided
that
the
ORF1629
deletion
is
rescued(Fig.
3).
The
recombinant
parent
virus
is
not
required
as
the
parental
viruses
propagate
in
the
insect
cell
lines.
The
flashBAC
DNA
system
is
as
simple
as
BacPAK6
system,
when
making
recombinant
viruses
in
insect
cells
and
removal
of
the
step
of
plaque
purification
is
one
of
the
concerns.
The
flashBAC
also
enhances
the
protein
secretion
and
membrane
proteins
(Hitchman
et
al.,
2011).
The
removal
of
chitinase
from
flashBAC
has
improved
the
secretory
pathway
and
produces
great
yield
of
recombinant
proteins
(up
to
60
folds)
which
is
secreted
(Hitchman
et
al.,
2011).
Figure
1.8.
The
flashBAC
baculovirus
expression
system
used
for
production
of
recombinant
baculoviruses.
A.)
Shows
the
deleted
genes.
B.)
Containment
of
gene,
insert,
lef2,
and
orf1629
gene
in
transfer
vector.
C.)
Recombinant
virus
with
repaired
orf1629
gene
(Rohrmann,
2013).
1.14
Multiplicity
of
Infection
The
virus
multiplicity
of
infection
(MOI)
describes
the
infectious
virus
particle
ratio
to
the
quantity
of
cells
in
the
culture
(Kool
et
al.,
1991).
It
can
be
used
to
estimate
the
amount
of
cells
infected
by
the
viruses
(Kool
et
al.,
34. 22
1991).
A
high
MOI
is
needed
to
achieve
synchronous
infection
of
the
cells,
which
usually
range
between
5-‐10
pfu/cell,
but
when
the
MOI
is
low
only
a
part
of
the
cell
population
is
infected
(Kool
et
al.,
1991).
The
use
of
high
MOIs
is
used
for
quick
determination
of
the
protein
production
in
a
small
scale
(Kool
et
al.,
1991).
On
the
other
hand,
it
becomes
difficult
on
a
larger
scale
as
producing
high
multiplicities
of
infection
tends
to
be
a
problem.
It
becomes
a
problem
as
a
larger
quantity
of
virus
is
needed
and
it
becomes
unrealistic.
In
the
past
studies,
it
has
been
noted
that
defective
particles
form
during
the
passage
of
baculovirus,
as
a
result
to
loss
of
partial
genome
(Kool
et
al.,
1991).
This
then
grows
in
cell
culture
and
causes
decrease
in
protein
production.
Rather
this
can
be
avoided
with
the
production
of
baculoviruses
infected
with
low
multiplicities
(Zwart
et
al.,
2008).
Those
cells
that
are
infected
with
low
multiplicities
of
infection
tend
to
only
affect
small
proportion
of
cells.
Later,
they
spread
the
virus
to
other
cells
in
the
culture
(Radford
et
al.,
1997).
In
addition
the
non-‐infected
cells
continue
to
divide,
thus
low
MOI
system
cannot
be
predicted
easily
(Wong
et
al.,
1996).
Another
tactics
that
can
play
a
role
towards
predictability
of
low
MOI
infection
is
the
sensitivity
to
conditions
such
as
cell
density
and
virus
titers.
It
can
produce
improper
quantification
for
baculovirus
titration
(Radford
et
al.,
1997).
In
low
MOI
if
time
is
not
reduced
while
attempting
to
increase
protein
production
then
the
proteolytic
degradation
of
the
proteins
take
place
which
can
be
problemetic
in
low
MOI
infection
(Radford
et
al.,
1997;
Wong
et
al.,
1996).
The
low
MOI
infection
also
carry
the
risk
of
having
high
cell
density.
This
leads
to
nutrient
depletion
and
causes
decrease
in
production
(Radford
et
al.,
1997).
If
cell
density
and
time
of
infection
is
taken
care
of
then
the
use
of
low
MOI
becomes
possible.
It
also
requires
appropriate
baculovirus
titers
as
inaccurate
MOI
can
cause
cells
to
die
immediately
(Radford
et
al.,
1997;
Wong
et
al.,
1996).
Other
factors
that
lead
to
low
production
can
be
due
to
inaccurate
cell
density,
or
inaccurate
cell
number
to
be
infected.
This
aspect
can
either
lead
to
high
cell
density
and
low
protein
production
as
well
(Licari
&
Bailey,
1991).
In
previous
studies,
35. 23
it
has
been
noted
that
if
cells
are
infected
in
early
exponential
phase,
there
is
no
significant
correlation
to
MOI.
But
if
cells
are
infected
in
the
last
exponential
phase,
then
there
must
have
been
significant
correlation
to
MOI,
resulting
in
high
yield
(Wong
et
al.,
1996).
If
the
cell
density,
time
of
infection,
and
MOI
are
taken
in
careful
consideration,
then
the
low
MOI
can
also
produce
proteins
equal
to
those
with
high
MOI
(Radford
et
al.,
1997;
Wong
et
al.,
1996).
The
lower
MOIs
tend
to
infect
only
the
portion
of
the
cell
while
the
remainder
grow
uninfected.
The
cells
may
have
a
high
yield
instead
of
the
cell
density,
when
the
uninfected
cells
and
their
progeny
get
infected
in
secondary
infection.
Further,
the
secondary
infection
will
lead
to
release
of
progeny
virus.
Thus,
to
obtain
high
production,
cells
should
be
infected
at
low
cell
density
lower
than
cell
yield.
1.15
Serial
Passaging
Baculovirus
go
through
genetic
variation
during
passaging
in
a
bioreactor
to
produce
few
polyhedra
and
defective
interfering
particle
(DIP)
(Giri
et
al.,
2012).
Due
to
the
cumulation
of
the
mutants
it
results
in
a
decrease
in
production
of
polyhedra
thus
reduces
the
virulence
leading
to
being
inefficient
at
being
biopesticide
(Giri
et
al.,
2012).
In
previous
studies
passaging
effect
has
been
found
to
be
a
problem
in
development
of
large-‐
scale
batch
of
recombinant
proteins/vaccines
with
the
use
of
baculovirus
expression
vector
system
(BEVS),
as
mutants
tend
to
pile
up
after
a
few
passages
to
produce
necessary
baculovirus
stock
(Giri
et
al.,
2012).
Mutation
can
occur
in
passaged
viruses
for
many
reasons,
such
as:
deletion
of
the
gene
fragment,
point
mutations,
frame
shift
mutations,
insertions
of
transposonmediated
host
cell
and
viral
genome
sequencing
(Giri
et
al.,
2012).
The
virus
variants
can
also
cause
virus
titers
to
drop
drastically,
in
addition,
virus
variants
with
a
large
number
of
deletions
have
been
found
among
many
virus
families
(Zwart
et
al.,
2013).
The
DIP
mutants
are
deletion
mutant
viruses
that
take
place
during
passaging
and
usually
compete
for
growth
with
normal
wild-‐type
virus.
A
decrease
in
recombinant
36. 24
protein
production
is
usually
due
to
DIP
mutants
as
they
lack
foreign
gene
of
interest
or
genes
that
are
involved
in
very
late
gene
expression
(Giri
et
al.,
2012).
In
particular,
AcMNPV
DIPs
arise
very
quick
in
cell
culture,
usually
two
passages,
and
they
tend
to
become
diverse
in
the
late
passages
(Zwart
et
al.,
2013).
It
is
suggested
from
observations
of
previous
studies,
a
possible
reason
for
genome
deletion
can
be
due
to
recombination
between
any
two
of
the
eight
homologous
repeat
regions
in
the
AcMNPV
genome
(Giri
et
al.,
2012).
Defective
interfering
(DI)
viruses
replicate
at
much
faster
pace
due
to
small
genome
size
than
viruses
that
have
the
full-‐length
genome
(Zwart
et
al.,
2013).
They
can
also
develop
a
better
way
to
compete
with
helper
viruses,
for
example,
accumulation
of
origins
of
DNA
with
a
single
genome.
However,
DI
viruses
are
unable
to
replicate
as
they
do
not
carry
the
essential
genes
to
do
so,
therefore
DI
viruses
must
co-‐infect
a
cell
with
helper
virus
for
it
to
replicate
(Zwart
et
al.,
2013).
Upon
passaging
cell
culture,
few-‐
polyhedra
(FP)
mutants
are
found
to
have
decrease
infectivity
with
reduced
yield
of
occluded
virus
(polyhedra).
FP
mutations
usually
occur
due
to
transposon
insertion
in
fp25k
gene,
which
leads
to
decrease
FP25K
protein
synthesis
(Giri
et
al.,
2010).
Due
to
repeat
passaging
baculoviruses
can
have
mutant
accumation
in
cell
cultures.
Also,
polyhedra
yield
and
virus
number
also
decreased
due
to
few-‐polyhedra
(FP)
and
defective
interfering
particle
(DIP)
(Giri
et
al.,
2010).
For
large-‐scale
production
it
is
necessary
to
come
over
mutations,
thus
will
enable
them
to
be
cost
effective.
FP
mutants
occur
due
to
low
amount
of
cells
containing
polyhedron.
Most
of
the
FP
can
disrupt
the
FP25K
protein
sysnthesis
by
placing
host
cell
DNA
into
a
various
region
of
the
baculovirus
genome
(Giri
et
al.,
2010).
In
previous
studies
it
has
been
found,
that
deletion
or
insertion
of
fp25k
gene
can
lead
to
increase
of
BV
production,
increase
in
BV
structural
proteins
(GP64,
BV
E-‐26
and
VP9),
decrease
in
occluded
virus
proteins,
decrease
in
liquefacation
of
the
larval
host,
decrease
in
E66
protein,
and
production
of
deviant
virions
morphology
(Giri
et
al.,
2010).
Thus,
fp25k
gene
mutation
can
take
place
during
passaging
of
the
viruses
and
can
decrease
the
production
of
polyhedra
and
ODV
in
continuous
cells
