Journal of General Virology (2008), 89, 1402–1410
DOI 10.1099/vir.0.2008/000695-0
Slow cell infection, inefficient primary infection and
inability to replicate in the fat body determine the
host range of Thysanoplusia orichalcea
nucleopolyhedrovirus
Lihua Wang,13 Tamer Z. Salem,1 Dwight E. Lynn24
and Xiao-Wen Cheng1
Correspondence
1
Xiao-Wen Cheng
2
Chengx@muohio.edu
Received 22 January 2008
Accepted 25 February 2008
Department of Microbiology, Miami University, Oxford, OH 45056, USA
USDA/ARS, Insect Biocontrol Laboratory, Beltsville, MD 20705, USA
Thysanoplusia orichacea multicapsid nucleopolyhedrovirus (ThorMNPV) carrying an enhanced
green fluorescent protein (EGFP) gene expression cassette (vThGFP) was used to study
host-range mechanisms. Infection kinetics showed that vThGFP replication in Sf21 cells was too
slow to suppress cell growth. Wide-host-range Autographa californica MNPV (AcMNPV) could
speed up vThGFP infection and enhance the vThGFP infection rate in Sf21 cells. The
enhancement was not due to recombination, as no recombinant virus was isolated from coinfection by plaque assay. No improvement of vThGFP infection in Sf21 was found by AcMNPV
cosmid transactivation assay. However, culture medium from Sf21 cells infected with AcMNPV
did enhance vThGFP replication in Sf21. Third-instar larvae of Spodoptera frugiperda, S. exigua
and Helicoverpa zea were not killed by feeding with vThGFP polyhedra but were killed by
intrahaemocoelic injection using budded viruses (BVs). This suggested that insufficient BVs were
generated during the primary infection in the midgut. vThGFP infected haemocytes, tracheae
and Malpighian tubules but not fat bodies of larvae of S. frugiperda, S. exigua and H. zea.
Third-instar S. frugiperda larvae co-infected by injection with vThGFP and vAcDsRed2, an
AcMNPV expressing a red fluorescent protein gene, showed EGFP expression in the fat body.
This result suggests that vAcDsRed2 could help vThGFP to replicate in the fat body or
trans-activate EGFP expression in the fat body. All these results suggested that slow cell
infection, insufficient primary infection and inability to replicate in the fat body control the host
range of ThorMNPV.
INTRODUCTION
The insect-specific baculoviruses, particularly nucleopolyhedroviruses (NPVs), are widely used for basic genetic
studies, protein expression and biological control of insect
pests in agriculture and forestry (Moscardi, 1999). Natural
infection of baculoviruses starts when the insect larvae
ingest vegetation contaminated with virion-containing
polyhedra from a previous infection cycle. The polyhedra
3Present address: Entomology Department, 420 Bio. Science Building,
University of Georgia, Athens, GA 30602, USA.
4Present address: Insell Consulting, 247 Lynch Road, Newcastle, ME
04553, USA.
The GenBank/EMBL/DDBJ accession number of the sequence from
Thysanoplusia orichalcea NPV reported in this paper is EU153368.
Details of cell lines used in this study are available as supplementary
material with the online version of this paper.
1402
dissolve in the alkaline environment (pH 9–11) of the
insect midgut, freeing the virions, which subsequently enter
the midgut cells. In most cases, the virus replicates in these
cells and progeny viruses bud through the basal lamina to
form budded viruses (BV) in the haemocoel. The BVs then
infect tracheal, fat body and other tissue cells or are
transmitted through the tracheal matrix to other parts of
the body (Federici, 1997). Fat body tissue of larvae is one of
the most favourable tissues for NPV replication in natural
infections of susceptible insects (Federici, 1997). Any step
of the infection cycle can be interrupted by the host insect
at the tissue or cellular level, and certain natural
environmental factors can induce the host immune system
against viral infection. However, factors that determine the
host range of NPVs are not well understood.
When the BVs from the midgut invade the haemocoel, they
enter and replicate in susceptible cells of specific tissues and
2008/000695
Printed in Great Britain
Host-range determination mechanisms of ThorMNPV
produce progeny BVs to infect other cells. NPV BVs
apparently do not require particular receptors to enter
cells (Miller & Lu, 1997). BVs can enter many cell types in
the insect host as well as in mammalian cells, but only
certain tissues or cell types support replication of the
invading NPVs. Most investigations of viral entry into
different cell lines have been conducted with Autographa
californica multiple nucleopolyhedrovirus (AcMNPV)
although, recently, Bombyx mori NPV (BmNPV) virions
were shown to be unable to reach the nucleus of Sf21 and
Hi5 cells, resulting in an abortive infection in these cells
(Katou et al., 2006). Studies have also shown that different
NPVs can target different tissues. For example, hymenopteran and dipteran NPVs infect only the midgut of
susceptible hosts. In contrast, in lepidopteran NPVs,
replication of NPVs in the midgut is transient, and the
most productive infection is in tissues like the fat body,
trachea and epidermis of susceptible host larvae (Federici,
1997).
An NPV was isolated from Thysanoplusia orichacea
(ThorMNPV) and several genes were sequenced for
phylogenetic analysis (Cheng et al., 2005). Phylogenetic
analysis placed ThorMNPV within the group 1 cluster,
consisting of AcMNPV, Rachiplusia ou NPV and
BmMNPV. In the initial host-range test of ThorMNPV
against seven lepidopteran insects, only two species,
Pseudoplusia includens and Trichoplusia ni, were found to
be susceptible via infection per os with occlusion bodies
(OBs). No mortality was observed in other tested insects
such as Spodoptera frugiperda, S. exigua and Helicoverpa
zea. The LD50 of ThorMNPV to T. ni is 17 OBs per larva,
which is lower than the 31 OBs per larva of the wide-hostrange AcMNPV to the same third-instar T. ni larvae
(Cheng et al., 2005). Therefore, ThorMNPV is a promising
candidate for the development of viral insecticides for
control of T. ni and P. includens in agriculture.
In this report, we report that ThorMNPV could not kill
resistant insects because it replicates slowly in semipermissive cells. Moreover, it was restricted in the primary
infection cycle and was not able to infect the fat body.
Furthermore, we also provide evidence that AcMNPV can
assist ThorMNPV by increasing replication in Sf21 cells
and can activate reporter gene expression in the fat body of
resistant S. frugiperda larvae.
METHODS
AcMNPV with an EGFP expression cassette at the gp37 locus
(AcGFP) (Cheng et al., 2001) were also included for viral infection
enhancement and OB formation and were propagated in Sf21 cells.
A recombinant AcMNPV containing the red fluorescent protein
(RFP) gene (DsRed2; Clontech) was generated using the bacmid
system (Invitrogen) for tissue tropism study (vAcDsRed2). All
AcMNPV-based viruses were amplified in Sf21 cells. Virus
concentration was estimated by the end-point dilution method
(O’Reilly et al., 1992). Insects used in this test included P. includens,
S. frugiperda, S. exigua and H. zea. Insects were reared according to
Cheng & Carner (2000).
Cell infection. Cell infection studies of vThGFP were carried out in
96-well tissue-culture plates. About 1000 cells of each cell line
(Supplementary Table S1) were seeded in each of the wells. BVs
(56107 p.f.u. ml21) were serially diluted 10-fold and added to the
wells containing cells. Negative controls contained cells without viral
infection. At day 7 post-infection (p.i.), the plates were scanned with a
Typhoon 9200 variable mode imager (Molecular Dynamics) and the
data were processed following Ogay et al. (2006).
To examine whether the wider-host-range AcMNPV could enhance
infection of vThGFP in semi-permissive Sf21 cells, 36106 cells per
25 cm2 T-flask were infected at an m.o.i. of 10 p.f.u. per cell with
either AcBacmid or vThGFP or co-infected with AcBacmid/vThGFP
at an m.o.i. of 10 p.f.u. each per cell. The same number of cells was
mock-infected by addition of similar volumes of GM (O’Reilly et al.,
1992). Infection was examined daily and EGFP expression was
documented by fluorescence microscopy. Infection rates were
calculated based on the numbers of infected cells showing EGFP
expression over the total number of cells counted. This experiment
was run in triplicate. Regression analyses were performed to compare
the difference in infection kinetics between vThGFP and vThGFP/
AcBacmid in Sf21 using Microsoft Excel.
We also examined polyhedron production of vThGFP in Sf21 cells.
Sf21 cells were infected by either vThGFP or vThGFP/AcBacmid or
AcGFP as described above and incubated for 5 days. Polyhedron
formation was analysed by phase-contrast and GFP fluorescence
microscopy.
DNA replication assay. To correlate EGFP expression with vThGFP
replication in Sf21, real-time quantitative PCR (qPCR) was used to
estimate copy numbers of vThGFP replicating in Sf21. Sf21 cells were
infected as described above with either vThGFP or vThGFP/
AcBacmid. Cells were harvested every 24 h until 120 h p.i. and used
for DNA extraction (O’Reilly et al., 1992); purified DNA was
dissolved in equal amounts of water. Equal volumes of DNA from
each time point were used as templates in real-time quantifications of
vThGFP genome copies with a pair of primers specific for vThGFP
but not for AcBacmid and an iQ SYBR Green Supermix kit following
conditions recommended by Bio-Rad. The primers were Thc4-F (59ACGGAAACGGGCAGAAAT-39) and Thc4-R (59-TTAGCGGTGCAAACAGAA-39), giving an amplicon of 87 bp. AcBacmid (10 ng
DNA) was used as a control. vThGFP DNA of known concentration
was serially diluted to construct a standard curve for copy number
estimates. The qPCR was run in triplicate.
Cells, viruses and insects. Insect cells used in the project included
IPLB-SF21AE (Sf21) from S. frugiperda and BTI-TN-5B1-4 (Hi5)
from T. ni. Cells were cultured in Grace’s medium supplemented with
10 % fetal bovine serum (GM). Other cells used in the in vitro
infection were from the USDA/ARS Insect Biocontrol Laboratory
(Beltsville, MD, USA) (Supplementary Table S1). Viruses used in this
project included ThorMNPV containing an enhanced green fluorescent protein (EGFP) gene expression cassette at the gp37 locus
(vThGFP), constructed according to Cheng et al. (2001). vThGFP was
propagated in Hi5 cells. An AcMNPV bacmid (AcBacmid,
bMON14271; Invitrogen) containing no polyhedrin gene and an
http://vir.sgmjournals.org
Plaque assay. Co-infection of vThGFP/AcBacmid might produce
recombinant viruses, and these were analysed by plaque assay in Sf21
cells (O’Reilly et al., 1992). Viral plaques were amplified in Hi5 cells
for DNA extraction to verify their authenticity by PCR and restriction
endonuclease (REN) analyses (O’Reilly et al., 1992). Plaque-purified
viruses (vThGFP1) and the parental vThGFP were compared for
infectivity in Sf21 as described above.
Transactivation assay. Five cosmid clones covering the entire
genome of AcMNPV kindly provided by Dr G. R. Rohrmann (Oregon
1403
L. Wang and others
State University) were used for the transactivation assay (Li et al.,
1999). Sf21 cells were co-transfected with each individual
cosmid DNA (5 mg each) and sets of all but one cosmid, leaving a
different one out from each co-transfection, with 250 ng vThGFP
viral DNA (O’Reilly et al., 1992; Thiem et al., 1996). The other
control was vThGFP DNA in the transfection. All co-transfections
were performed in triplicate. EGFP expression was observed
daily by fluorescence microscopy and, at day 8 post-transfection,
cells were lysed in 0.1 % SDS for EGFP fluorescence
measurements. Fluorescence intensities from different transfections
were analysed statistically by one-way ANOVA and the Tukey
HSD multiple comparison test using the MiniTab software
package.
Conditioned medium enhancement assay. To explore the
possibility that conditioned medium of Sf21 cells infected by
AcBacmid might enhance vThGFP infection in Sf21 cells, we infected
Sf21 cells with either AcBacmid or AcGFP as described above. At day
4 p.i., medium was removed and centrifuged at 196 408 g for 15 h to
remove BVs. The supernatants were filtered through 0.22 mm filters to
produce enhancing medium (EM). Sf21 cells were infected by
vThGFP at an m.o.i. of 10 p.f.u. per cell in a mixed medium of
equal volumes of EM and fresh GM. The other controls in this
experiment were Sf21 cells infected only with vThGFP in GM
and cells in EM/GM alone without vThGFP. EM from AcGFP
infection was used to inoculate Sf21 cells in order to evaluate the
efficiency of removal of BV by centrifugation and filtration. Viral
infection in cells was observed daily and documented by GFP
fluorescence microscopy.
Per os infection. Per os infection studies of vThGFP on P.
includens, S. frugiperda, S. exigua and H. zea followed Cheng et al.
(2001).
Infectivity through intrahaemocoelic injection. The barriers to
vThGFP infection in larvae of resistant species were examined by
inoculating different larvae with BVs of vThGFP at the third instar by
intrahaemocoelical injection. Inocula (1.256105 p.f.u.) were delivered into the haemocoel of each larva. For each species, 30 larvae were
injected. In the control group, larvae were injected with GM. Injected
larvae were allowed to feed on artificial diet and were incubated until
they either died or pupated.
A more detailed bioassay was performed with S. frugiperda larvae by
intrahaemocoelic injection. A 10-fold serial dilution of the inoculum
was made by adding cell growth medium (1.256105 to 1.256101
p.f.u.). For each dilution, 30 third-instar S. frugiperda larvae were
injected. Control larvae (30) were injected with cell growth medium.
Larvae that survived for the first 24 h were incubated on diet cup.
Mortality was scored and infectivity was calculated by the computer
program POLO-PC (Le Ora Software).
Tissue specificity. Larvae of S. frugiperda, S. exigua and H. zea were
fed on diet plugs containing 2.56104 vThGFP OBs as described in the
in vivo infection studies. For each species, 10 larvae were used.
Control larvae were fed on diet inoculated with water. At day 7 p.i.,
larvae were dissected and different tissues such as fat body, trachea,
Malpighian tube, epidermis and haemocytes were examined by
fluorescence microscopy in the GFP channel and documented. To
further understand the tissue tropism, S. frugiperda larvae were
injected with two viruses (vThGFP and vAcDsRed2). Third-instar S.
frugiperda larvae were first injected with vThGFP (1.256105 p.f.u.),
and larvae surviving at day 2 p.i. were subsequently injected again
with vAcDsRed2 (1.256105 p.f.u.). Control larvae were injected with
GM. Larvae were dissected 2 days after injection with vAcDsRed2 for
tissue examination by fluorescence microscopy in the GFP and RFP
channels.
1404
RESULTS
In vitro cell infectivity of vThGFP
Cell line infection screen. Initial studies showed that
ThorMNPV replicated well in Hi5 cells but not in Sf21
(Cheng et al., 2005). In the current study, we used vThGFP
to test cell infectivity in 28 insect cell lines. Fifteen cell lines
were permissive for vThGFP infection at different rates, as
suggested by green fluorescence indicating viral replication.
The most susceptible cell lines included cells from
Anticarsa gemmatalis (AG286), Ephestia kuenella (Ekx4T,
Ekx4T-lt and Ekx4V-lt), T. ni (TN5B1-4) and Manestra
brassicae (MB0503). Cell lines from Heliothis virescens
(HvE6s and HvT1), Lymantria dispar (LdEp), S. frugiperda
(Sf21), S. exigua (SE-1) and T. ni (TND1 and TN368) were
less susceptible and were only infected at the highest
concentration of vThGFP. EGFP expression was not
detected in the remaining 13 cell lines. Three of the
insects from which the permissive cells were derived, S.
exigua, S. frugiperda and A. gemmatalis, were not
susceptible in previous host-range studies (Supplementary Table S1; Cheng et al., 2005), suggesting that the
blockage of infection in these species does not exist in all
cell/tissue types.
Viral infection enhancement. Since vThGFP showed semipermissive infection in Sf21 cells and Sf21 cells were highly
permissive to AcMNPV infection (Supplementary Table
S1), we hypothesized that AcMNPV might help vThGFP to
infect the semi-permissive Sf21 cells. Separate flasks of Sf21
were inoculated with vThGFP and AcBacmid (10 p.f.u.
each per cell) and a third flask was inoculated with both
vThGFP and AcBacmid. In the flask of Sf21 cells infected
by vThGFP alone, no EGFP was detected until day 3 p.i.
(Fig. 1b); more cells become infected by vThGFP at day 8
p.i., as indicated by strong EGFP expression, although the
cells became overgrown (Fig. 1c). When AcBacmid was
mixed with vThGFP to infect Sf21 cells, some (about
2.5 %) cells were detected showing EGFP expression at day
1 p.i. and, by day 5 p.i., over 40 % of the Sf21 cells showed
EGFP expression in the co-infection flask (Fig. 1d)
compared with ,3 % of cells in the vThGFP-inoculated
flask at this time (Fig. 1d). When the infection kinetics
were calculated, AcBacmid increased vThGFP infection
rates significantly in Sf21 cells by 25-fold (P50.0000298,
Fig. 1e). In contrast, more than 98 % of cells infected by
AcBacmid alone showed cytopathic effects due to viral
infection. When the numbers of cells expressing EGFP were
compared, there was a 117-fold increase in cells infected by
vThGFP in the co-infection compared with the vThGFP
infection alone at 72 h p.i. (Fig. 1e).
By real-time qPCR, similar to the changes in cellular EGFP
expression, we found increases in vThGFP DNA replication
rates in Sf21 cells co-infected with vThGFP/AcBacmid. We
found no DNA amplification when AcBacmid DNA was
used as a template. This suggested that the increases in
qPCR amplification using DNA from vThGFP/AcBacmid
Journal of General Virology 89
Host-range determination mechanisms of ThorMNPV
UV/visible
(a)
UV
(c)
Infection rate (%)
50
AcBacmid not only enhanced vThGFP infection rates in
Sf21 cells, but also helped OB formation of vThGFP in
Sf21. Positive-control AcGFP showed OB formation at
48 h p.i. with strong EGFP expression in infected Sf21 cells
(Fig. 2a, b). No OBs were formed in cells showing EGFP
expression with vThGFP alone at day 8 p.i., but EGFP
expression in these cells was high (Fig. 2c, d). OBs were
visible in cells showing EGFP expression in the vThGFP/
AcBacmid co-infection at day 3 p.i. (Fig. 2e, f).
(b)
UV
60
infection of vThGFP, vThGFP genome replication in the
co-infection also showed a significant increase compared
with replication of vThGFP alone in Sf21 (P50.015417,
Fig. 1f). This suggested that AcBacmid helped vThGFP
replication.
UV/visible
(d)
(e)
vThGFP/
AcBacmid
y=0.4032x–3.1921
r2=0.97
40
Bright field
30
20
P=0.0000298
y=0.0166x–0.4492
r2=0.6928 vThGFP
10
24
48
72
96
120
AcGFP
144
Time (h p.i.)
vThGFP genome copies (×1011)
UV/GFP filter
70
60
(a)
(b)
(c)
(d)
(e)
(f)
(f)
y=0.6188x–19.917
r2= 0.9479
50
vThGFP/
AcBacmid
vThGFP
40
P=0.015417
30
y=0.1376x–5.311
r2=0.8614 vThGFP
20
10
24
48
72
96
120
144
Time (h p.i.)
vThGFP/
AcBacmid
Fig. 1. Cell infection by vThGFP in Sf21 and enhancement of
vThGFP infection and DNA replication by AcBacmid. (a–d) vThGFP
infection and enhancement by AcBacmid. Sf21 cells were either
infected by vThGFP or co-infected by vThGFP/AcBacmid and
examined by fluorescence microscopy. (a) Mock infection of Sf21 at
72 h p.i.; (b) vThGFP infection in Sf21 at 72 h p.i.; (c) vThGFP
infection in Sf21 at 192 h p.i.; (d) co-infection of Sf21 by vThGFP/
AcBacmid at 72 h p.i. Bars, 150 mm. (e) Infection kinetics by vThGFP
and vThGFP/AcBacmid (n53). Sf21 cells were either infected by
vThGFP alone or co-infected by vThGFP/AcBacmid and infection
rates were compared by regression analysis (n53). (f) Comparison of
vThGFP DNA replication between vThGFP infection and vThGFP/
AcBacmid co-infection in Sf21 by real-time qPCR (n53).
co-infection were due to increases in replication of
vThGFP. Similar to the increase of Sf21 cells showing
EGFP expression in co-infection compared with the single
http://vir.sgmjournals.org
Fig. 2. Comparison of OB formation in Sf21 cells by vThGFP
infection and vThGFP/AcBacmid co-infection. (a) Sf21 cells
infected by AcGFP at 48 h p.i. showing OBs in the nuclei under
phase-contrast. (b) Cells shown in (a) viewed under UV light. (c)
Sf21 cells infected by vThGFP at 120 h p.i. under phase-contrast,
showing no formation of OBs (arrow). (d) Cells shown in (c)
viewed under UV light, showing strong EGFP expression in one
cell (arrow). (e) Sf21 cells co-infected by vThGFP/AcBacmid at
60 h p.i. under phase-contrast, showing OBs (arrow). (f) Cells
shown in (e) viewed under UV light, showing strong EGFP
expression (arrow). Bars, 10 mm.
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L. Wang and others
Mechanisms of AcBacmid enhancement of vThGFP
infection in Sf21 cells. We considered two hypotheses to
explain how AcBacmid enhanced vThGFP replication in
Sf21 cells. These involved recombination between vThGFP
and AcBacmid or transactivation of vThGFP genes by
AcBacmid products in Sf21 cells. We used medium from
Sf21 cells infected by either vThGFP alone or vThGFP/
AcBacmid as well as vThGFP from infection of Hi5 cells to
infect Sf21 and performed plaque assays. We could not find
Sf21 cells showing EGFP expression by using medium from
Sf21 cells infected by vThGFP alone. This might suggest
that vThGFP had difficulty in budding from Sf21 cells.
vThGFP BVs from Hi5 cells were able to infect Sf21 cells,
but could not form plaques. Only individual cells showed
EGFP expression, suggesting vThGFP infection, at day 5
p.i. (Fig. 3a, b). However, typical viral plaques with EGFP
expression were identified in the plaque assay using
medium from Sf21 cells co-infected by vThGFP/
AcBacmid (Fig. 3c, d). This suggested that AcBacmid
helped vThGFP to form plaques in Sf21 cells. When four of
these plaques (vThGFP1) were purified and used to infect
Sf21 cells, poor infection similar to that shown in Fig. 1 was
observed. PCR and REN analyses confirmed that these
plaques were vThGFP.
We tested the second hypothesis of transactivation of
vThGFP genes by AcBacmid gene products. We used a
cosmid library of AcMNPV and performed transactivation
assays on vThGFP in Sf21; we did not find any genes of
AcMNPV in the cosmids that transactivated vThGFP in
Sf21 (not shown).
Bright field
Since the reported mechanisms of one NPV helping
another NPV to improve infection in non-permissive cells
were not able to explain how AvBacmid helped vThGFP to
infect Sf21, we sought to use EM from Sf21 cells infected by
AcBacmid to enhance vThGFP infection in Sf21 cells. To
our surprise, the EM enhanced vThGFP infection in Sf21
substantially. About 2 % of cells in EM/GM with vThGFP
showed EGFP expression at 24 h p.i., but no EGFP was
detected in Sf21 cells infected by vThGFP alone in GM. At
day 5 p.i., about 15 times more cells showed EGFP
expression by vThGFP in the EM/GM compared with
vThGFP in the GM (Fig. 4). However, trace amounts of
AcGFP were present in the EM, since we detected a few
Sf21 cells (,0.1 %) infected by AcGFP by fluorescence
microscopy at day 5 p.i. (data not shown). Even though
this test was not conclusive, it suggested that there might be
secreted products from Sf21 cells infected by AcBacmid
that helped vThGFP infection in Sf21. This also suggested
that, under the centrifugation conditions we used, there
were still trace amounts of AcBacmid BV that escaped
centrifugation and filtration.
In vivo tests of vThGFP and vAcRed
Per os and intrahaemocoelic infection. We examined
whether the midgut of resistant species such as S.
frugiperda, S. exigua and H. zea is a major barrier for
vThGFP-induced mortality in these insects. When OBs
(2.56104) were used to infect these resistant insects, with
the susceptible host P. includens as a positive control, no
mortality was observed for S. frugiperda, S. exigua or H. zea
larvae. Larvae showed no noticeable symptoms of viral
infection such as sluggishness or epidermal colour changes.
UV/GFP filter
Bright field
UV/GFP filter
vThGFP
(a)
(b)
vThGFP
vThGFP1
(c)
(a)
(b)
(c)
(d)
(d)
vThGFP/
EM
Fig. 3. Analysis of plaque formation by vThGFP from Hi5 cells and
from vThGFP from vThGFP/AcBacmid co-infection in Sf21 cells.
Sf21 cells were infected either with vThGFP from Hi5 or vThGFP
from vThGFP/AcBacmid co-infection in Sf21 cells and overlaid
with agarose. At day 5 p.i., GFP plaques were screened and
documented by fluorescence microscopy. (a) Sf21 cells infected
by vThGFP under bright field. (b) Sf21 cells infected by vThGFP
under UV light in the GFP channel. (c) Sf21 cells infected by
vThGFP/AcBacmid under bright field. (d) Sf21 cells infected by
vThGFP/AcBacmid under UV light in the GFP channel. Bars,
10 mm.
1406
Fig. 4. Enhancement of vThGFP infection in Sf21 by conditioned
medium from Sf21 cells infected by AcBacmid. Conditioned
medium of Sf21 cells infected by AcBacmid at 72 h p.i. was
centrifuged to produce EM. Sf21 cells were infected by vThGFP
either in GM (a, b) or in EM/GM (c, d) and photographed in the
bright field channel (a, c) or GFP channel (b, d) at 72 h p.i. Bars,
100 mm.
Journal of General Virology 89
Host-range determination mechanisms of ThorMNPV
However, mortality could be readily observed in positive
control larvae of P. includens.
When intrahaemocoelic injection of BVs (1.256105) of
vThGFP was performed with these non-susceptible insects,
all larvae were killed prior to pupation or in the pupal
stage, but larvae did not liquefy. No mortality was found in
the control injected with cell-growth medium. Before the
larvae died, they were checked for EGFP expression under a
portable UV lamp at 365 nm. Each larva of the three
species sacrificed showed obvious green fluorescence,
especially on the ventral side of the body, where the colour
is lighter than on the dorsal side. No fluorescence could be
detected in control larvae without viral inoculation (not
shown). This suggested that, at high inoculation doses by
intrahaemocoelic injection, vThGFP could kill insects that
were resistant to infection per os by OBs. Therefore, the
numbers of BVs produced from the primary infection in
the midgut might be a factor in determining the fate of
these insects following infection.
We then estimated how many BVs of vThGFP from the
primary infection in the midgut were required to produce
mortality in S. frugiperda by performing a bioassay study.
We found that about 47 BVs injected into third-instar
larvae of S. frugiperda were needed to produce mortality.
The calculated LD50 was 240 BVs per larva (CI 140–407
BVs per larva) (Fig. 5). This suggested that, during primary
infection of vThGFP in the midgut of S. frugiperda, fewer
than 47 BVs were budded out to the haemocoel, suggesting
that the primary infection in the midgut was limited.
Tissue tropism. When different tissues from larvae
infected by inoculation of vThGFP per os were examined
by fluorescence microscopy, only haemocytes, tracheae and
Malpighian tubules showed EGFP expression, indicating
100
Mortality (%)
80
60
40
20
0
12.5
125
1250 12500 125000
Dose (p.f.u. per larva)
Fig. 5. Bioassay of susceptibility of third-instar larvae of S.
frugiperda to vThGFP by intrahaemocoelic injection with BVs of
vThGFP. Larvae were infected with different doses of vThGFP by
injection. Mortality was used to establish the dose–mortality
response (n53).
http://vir.sgmjournals.org
infection of vThGFP in these tissues of the three resistant
insects. Only tissues from S. frugiperda are presented
(Fig. 6a). Tissues from the other two insects were similar.
In the control larval tissues, no fluorescence signals were
detected (Fig. 6b, c). This suggested that the EGFP signals
detected by fluorescence microscopy were due to vThGFP
infection in these tissues. No OBs were observed in these
tissues with EGFP and no EGFP was seen in the fat body or
epidermis in these insects. However, EGFP expression was
detected in fat body, haemocytes, tracheae and Malpighian
tubules of the susceptible P. includens larvae when infected
per os with vThGFP and OBs were observed in cells where
EGFP expression was detected (not shown).
Since vThGFP alone could not infect the fat body of S.
frugiperda larvae, we performed a co-injection of vThGFP/
vAcDsRed2 into the haemocoel of S. frugiperda larvae. In
the control larvae injected by vThGFP, no signal of EGFP
could be detected (Fig. 6d, e). After injection of vAcDsRed2
into larvae previously injected with vThGFP, EGFP could
be detected in the fat body tissue (Fig. 6f, g). At the same
time, RFP from vAcDsRed2 could be detected in the fat
body, indicating infection of vAcDsRed2 in the fat body of
S. frugiperda (Fig. 6h). When the colour images were
merged, a yellow colour was observed, suggesting that the
two viruses (vThGFP and vAcDsRed2) have replicated in
the same cells of the fat body (Fig. 6i). This also suggested
that vAcDsRed2 assisted the replication of vThGFP or
trans-activated the EGFP gene from vThGFP.
DISCUSSION
Our findings that AcMNPV (AcBacmid) can enhance
vThGFP infection in Sf21 cells in co-infection experiments
suggest that AcBacmid infection in Sf21 may stimulate
production of secreted products that help vThGFP
infection in Sf21 cells, since recombinant viruses were
not isolated and the transactivation assay did not show
enhancement. This is the first time, to our knowledge, that
a secretion from cells infected by a virus has been shown to
enhance infection by another virus in less-permissive
cells. Even though we could not remove all the virus in
the EM by centrifugation, the residual AcBacmid in the EM
should be very minimal under these centrifugation
conditions. Both transactivation and recombination
require the physical presence of the AcBacmid genome in
the Sf21 cells with vThGFP. Plaque assays are based on the
fact that virus can not move freely in the agarose overlay
(O’Reilly et al., 1992). Both AcBacmid and vThGFP
attacking the same cell is unlikely to occur at high
frequencies, as only minimal residual AcBacmid was
present in the EM. However, secreted products such as
proteins can readily diffuse through the agarose, reach cells
attacked by vThGFP and make the cells more susceptible to
vThGFP infection (Pluen et al., 1999). Furthermore,
vThGFP shares only 70–80 % DNA sequence identity with
AcMNPV. This may be too low for homologous recombination to occur.
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L. Wang and others
Haemolymph
Malpighian tubules
Trachea
UV
(a)
(d)
(e)
(b)
(f)
(g)
(c)
(h)
(i)
UV
Bright
field
Fig. 6. Tissue tropism of vThGFP in S. frugiperda larvae by GFP fluorescence microscopy. (a–c) Infection of vThGFP in
different tissues of S. frugiperda. Comparable tissues are shown from vThGFP-infected larvae (a), control larvae, not showing
fluorescence (b), and control larvae under phase-contrast (c). (d–i) Fat body infectivity of S. frugiperda by vThGFP. (d–e) Fat
body of S. frugiperda infected by vThGFP under phase-contrast with visible illumination (d) and under UV light (e). (f–i) Fat body
of S. frugiperda larvae infected by vThGFP/vAcDs Red2 days after vAcDs Red2 infection under phase-contrast with visible
illumination (f), in the UV GFP channel (g) and under UV in the RFP channel (h); (i) merged image of (g) and (h). Bars, 100 mm.
The inability of NPVs to infect different cell lines has been
investigated in various systems. For example, AcMNPV is
not able to replicate in CF-203, a midgut cell line from
Choristoneura fumiferana, as a result of induced apoptosis.
However, if CF-203 cells are infected by C. fumiferana
MNPV (CfMNPV) prior to infection by AcMNPV,
AcMNPV can establish a full infection in CF-203. The
explanation for this observation is that CfMNPV provides
a trans-acting factor(s) which inhibits AcMNPV-induced
apoptosis of CF-203, allowing AcMNPV to replicate (Palli
et al., 1996). We did not observe obvious cytopathic effects
seen in apoptotic cells, such as blebbing, during the
vThGFP infection in Sf21 cells, so we do not believe that
apoptosis is active in this system. In fact, Sf21 cells without
EGFP expression looked no different from healthy Sf21
cells (Fig. 1a, b). The only abnormality we observed was the
low speed of infection or DNA replication by vThGFP in
Sf21 cells (Fig. 1e, f).
We confirmed that the slow infection of vThGFP in Sf21
cells is due to slow DNA replication. It is unlikely that the
increase in Sf21 cells expressing EGFP is due to the transfer
of the egfp gene from vThGFP to AcBacmid. If so, the
number of vThGFP genome copies should not increase in
line with the increase of Sf21 cells with EGFP (Fig. 1e, f). It
is also unlikely that vThGFP received genes from
AcBacmid. If so, vThGFP1 should infect Sf21 better. In
an earlier report, AcMNPV helped SeMNPV to replicate
better in Sf21, which was poorly permissive for SeMNPV
infection in a co-infection experiment (Yanase et al., 1998).
The enhancement was suggested to be via transactivation
by AcMNPV, but this was not proved as recombinants
were not isolated (Yanase et al., 1998).
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DNA helicase plays an important role in DNA replication
by unwinding double-stranded DNA before DNA polymerase synthesizes the daughter DNA strand (LeBowitz &
McMacken, 1986). The p143 gene of AcMNPV has helicase
activity (McDougal & Guarino, 2001) and has been
implicated as a host-range factor (Croizier et al., 1994;
Kamita & Maeda, 1997). For example, although AcMNPV
and BmNPV share high DNA sequence similarity,
AcMNPV could not replicate in B. mori cells and
BmNPV could not replicate in Sf21 cells. The inability of
AcMNPV to replicate in B. mori cells is due to the p143
gene of AcMNPV (Croizier et al., 1994; Kamita & Maeda,
1997).
High-dose feeding of vThGFP OBs to S. frugiperda, S.
exigua and H. zea did not produce mortality, but
intrahaemocoelic injection of low doses of vThGFP BV
could kill these insects. This suggested that primary
infection of vThGFP in the midgut of these insects did
not produce enough BVs to initiate secondary infection in
the haemocoel. We further traced the tissue infection in
these insects by EGFP expression in these insects. The
infection was apparently restricted to haemocytes, tracheae
and Malpighian tubules, and did not kill these insects
presumably because the number of BVs from the primary
midgut infection was insufficient and replication of
vThGFP was slow (Figs 1 and 6a). In natural hosts, NPVs
in lepidopteran insects always infect the fat body (Federici,
1997). We could not detect EGFP in the fat body of these
insects infected either per os or by intrahaemocoelic
injection. However, we detected EGFP expression in the
fat body of S. frugiperda when the insects were co-infected
by haemocoelic injection with vAcDsRed2. Both EGFP and
Journal of General Virology 89
Host-range determination mechanisms of ThorMNPV
RFP were detected in the fat body of S. frugiperda coinfected by vThGFP and vAcDsRed2, suggesting that both
viruses were present in the fat body cells (Fig. 6d–i). How
vThGFP and vAcDsRed2 interacted, however, is unclear.
vThGFP may enter the fat body cells of S. frugiperda but may
not be able to infect the cells either through not being able to
reach the nucleus (Katou et al., 2006) or, having reached the
nucleus, by being unable to replicate or because the late
genes are not trans-activated. When vAcDsRed2 entered
cells in which vThGFP was already present, it started early
and late gene transcription. Replication of vAcDsRed2
eventually assisted vThGFP replication in the fat body
through an undefined mechanism. It is also possible that the
egfp gene in vThGFP was trans-activated to produce the
EGFP protein detected by fluorescence microscopy. The
inability of vThGFP to infect the fat body of resistant insects
may explain why infection per os could not kill S. frugiperda,
S. exigua and H. zea.
In terms of mortality, some NPVs have a wide host range, in
that they kill many insect species. This is typified by the type
species of Baculoviridae, AcMNPV, which can kill at least 33
species in 10 families (Groner, 1986). It is not known what
gives AcMNPV its wider host range. ThorMNPV has a
narrow host range, in that it can kill T. ni and P. includens at
low doses but can not kill S. frugiperda, S. exigua or H. zea per
os. Certain NPVs can kill only one host species. This is
exemplified by SeMNPV, which can kill S. exigua larvae only,
as proved by mortality produced by infection of OBs per os to
specific insect larvae (Hara et al., 1995). When SeMNPV was
used to infect other Spodoptera species either per os with OBs
or by intrahaemocoelic injection of occlusion-derived virus,
all classes of SeMNPV gene transcripts were detected in the
midgut columnar epithelial cells and haemocoelic tissues,
without any mortality. Despite SeMNPV replicating in the
midgut and haemocoelic tissues, no fatal infection could be
detected in S. frugiperda or S. littoralis. Thus, the narrow host
range of SeMNPV was believed to be controlled at the level of
the primary infection cycle in the midgut, and secondary
infection of the haemocytes in heterologous insects was not
the only or major factor restricting host range (Simon et al.,
2004). Infectivity of vThGFP in S. frugiperda was different,
with high mortality when the BVs were introduced into the
haemocoel by injection.
In conclusion, we provide evidence that a combination of
factors governs the infectivity of vThGFP in resistant
insects at both the cellular and species levels. These factors
include slow infection rates, low BV production during the
primary infection cycle in the midgut and inability to infect
the fat body. Although the detailed mechanism underlying
the poor replication in Sf21 cells is unclear, ThorMNPV
offers another system for further investigation of hostrange determination in NPVs.
ACKNOWLEDGEMENTS
We especially thank Dr Basil Arif for his help during the early stage of
this project. This research is partially supported by an Ohio Plant
http://vir.sgmjournals.org
Biotechnology Consortium grant (401120) awarded to X.-W. C. and a
start-up fund to X.-W. C. from the College of Art and Science, Miami
University.
REFERENCES
Cheng, X. W. & Carner, G. R. (2000). Characterization of a single-
nucleocapsid nucleopolyhedrovirus of Thysanoplusia orichalcea L.
(Lepidoptera: Noctuidae) from Indonesia. J Invertebr Pathol 75,
279–287.
Cheng, X., Krell, P. & Arif, B. (2001). P34.8 (GP37) is not essential for
baculovirus replication. J Gen Virol 82, 299–305.
Cheng, X. W., Carner, G. R., Lange, M., Jehle, J. A. & Arif, B. M. (2005).
Biological and molecular characterization of a multicapsid nucleopolyhedrovirus from Thysanoplusia orichalcea (L.) (Lepidoptera:
Noctuidae). J Invertebr Pathol 88, 126–135.
Croizier, G., Croizier, L., Argaud, O. & Poudevigne, D. (1994).
Extension of Autographa californica nuclear polyhedrosis virus host
range by interspecific replacement of a short DNA sequence in the
p143 helicase gene. Proc Natl Acad Sci U S A 91, 48–52.
Federici, B. A. (1997). Baculovirus pathogenesis. In The Baculoviruses,
pp. 33–59. Edited by L. K. Miller. New York: Plenum Press.
Groner, A. (1986). Specificity and safety of baculoviruses. In The
Biology of Baculoviruses, vol. 1, pp. 177–202. Edited by R. R. Granados
& B. A. Federici. Boca Raton, FL: CRC Press.
Hara, K., Funakoshi, M. & Kawarabata, T. (1995). A cloned cell line of
Spodoptera exigua has a highly increased susceptibility to the
Spodoptera exigua nuclear polyhedrosis virus. Can J Microbiol 41,
1111–1116.
Kamita, S. G. & Maeda, S. (1997). Sequencing of the putative DNA
helicase-encoding gene of the Bombyx mori nuclear polyhedrosis virus
and fine-mapping of a region involved in host range expansion. Gene
190, 173–179.
Katou, Y., Ikeda, M. & Kobayashi, M. (2006). Abortive replication of
Bombyx mori nucleopolyhedrovirus in Sf9 and High Five cells:
defective nuclear transport of the virions. Virology 347, 455–465.
LeBowitz, J. H. & McMacken, R. (1986). The Escherichia coli dnaB
replication protein is a DNA helicase. J Biol Chem 261, 4738–4748.
Li, L., Harwood, S. H. & Rohrmann, G. F. (1999). Identification of
additional genes that influence baculovirus late gene expression.
Virology 255, 9–19.
McDougal, V. V. & Guarino, L. A. (2001). DNA and ATP binding
activities of the baculovirus DNA helicase P143. J Virol 75, 7206–7209.
Miller, L. K. & Lu, A. (1997). The molecular basis of baculovirus host
range. In The Baculoviruses, pp. 217–235. Edited by L. K. Miller. New
York: Plenum Press.
Moscardi, F. (1999). Assessment of the application of baculoviruses
for control of Lepidoptera. Annu Rev Entomol 44, 257–289.
Ogay, I. D., Lihoradova, O. A., Azimova, S. S., Abdukarimov, A. A.,
Slack, J. & Lynn, D. (2006). Transfection of insect cell lines using
polyethylenimine. Cytotechnology 51, 89–98.
O’Reilly, D. R., Miller, L. K. & Luckow, V. A. (1992). Baculovirus Expression
Vectors: a Laboratory Manual. New York: W. H. Freeman & Co.
Palli, S. R., Caputo, G. F., Sohi, S. S., Brownwright, A. J., Ladd, T. R.,
Cook, B. J., Primavera, M., Arif, B. M. & Retnakaran, A. (1996).
CfMNPV blocks AcMNPV-induced apoptosis in a continuous midgut
cell line. Virology 222, 201–213.
Pluen, A., Netti, P. A., Jain, R. K. & Berk, D. A. (1999). Diffusion of
macromolecules in agarose gels: comparison of linear and globular
configurations. Biophys J 77, 542–552.
1409
L. Wang and others
Simon, O., Williams, T., Lopez-Ferber, M. & Caballero, P. (2004).
Virus entry or the primary infection cycle are not the principal
determinants of host specificity of Spodoptera spp. nucleopolyhedroviruses. J Gen Virol 85, 2845–2855.
Thiem, S. M., Du, X., Quentin, M. E. & Berner, M. M. (1996).
Identification of baculovirus gene that promotes Autographa
1410
californica nuclear polyhedrosis virus replication in a nonpermissive
insect cell line. J Virol 70, 2221–2229.
Yanase, T., Yasunaga, C., Hara, T. & Kawarabata, T. (1998).
Coinfection of Spodoptera exigua and Spodoptera frugiperda cell lines
with the nuclear polyhedrosis viruses of Autographa californica and
Spodoptera exigua. Intervirology 41, 244–252.
Journal of General Virology 89