J. Microbiol. Biotechnol.
(2007), 17(4), 632–637
A Cytoplasmic Polyhedrosis Virus Isolated from the Pine Processionary
Caterpillar, Thaumetopoea pityocampa
INCE, IKBAL AGAH, ISMAIL DEMIR, ZIHNI DEMIRBAG, AND REMZIYE NALCACIOGLU*
Department of Biology, Faculty of Arts and Sciences, Karadeniz Technical University, 61080, Trabzon, Turkey
Received: October 10, 2006
Accepted: November 28, 2006
Abstract A cytoplasmic polyhedrosis virus (CPV) was
isolated from the larvae of Thaumetopoea pityocampa and
shown to cause an infection of midgut cells. This viral infection
revealed several important diagnostic symptoms, including
discoloration of the posterior midgut, reduced feeding, and
extended development time of the larvae. The virus infection
is lethal to Thaumetopoea pityocampa, and with the increasing
doses kills the larvae within 4-5 days post infection. Electron
microscopy studies showed typical cytoplasmic polyhedral
inclusion bodies that are icosahedral, and ranged from 2.4 to
5.3 µm in diameter. Electrophoretic analysis of the RNA
genome showed that the virus has a genome composed of 10
equimolar RNA segments with the sizes of 3,907, 3,716,
3,628, 3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp,
respectively. Based on morphology and nucleic acid analysis,
this virus was named Thaumetopoea pityocampa cytoplasmic
polyhedrosis virus (TpCPV), and belongs to the genus
Cypovirus, family Reoviridae.
Keywords: Thaumetopoea pityocampa, cytoplasmic
polyhedrosis virus, Cypovirus, anaphylactic shock, biological
control
Thaumetopoea pityocampa (the pine processionary moth)
is one of the most important pine pests in the forests of
Mediterranean countries, Central Europe, the Middle East,
and North Africa [31]. The caterpillars cause severe damage
to pine plantations, especially in warm districts and low
altitudes. Young pine plantations are the most susceptible,
and may be completely destroyed if the attack is severe
enough. Less severe larval feeding damage can pave the
way for harmful secondary pests and pathogens. Mature
trees may suffer reductions in growth but are rarely killed
outright by the pest. The caterpillars are highly social. At
*Corresponding author
Phone: 90-0-462-377-3554; Fax: 90-0-462-325-3195;
E-mail: remziye@ktu.edu.tr
first, they are nomadic, spinning, and abandoning a series
of flimsy shelters constructed by enveloping a few needles
in silk, but in the third instar, they initiate the construction of
a permanent nest and settle down to become central place
foragers. By that way, they form the processionaries on
pine tree. Controlling this type of moth is an important part
of an integrated pest management system. So far, efforts to
control T. pityocampa have mainly involved the use of
chemical insecticides, particularly insect growth inhibitors.
However, these agents can have undesirable side-effects
on humans, plants and other animal species, particularly
predators and parasites of T. pityocampa. Therefore, it is
necessary to find alternative and environmentally friendly
control methods, such as the utilization of viruses.
Caterpillars of T. pityocampa not only cause significant
damage to forest trees but are also responsible for dermatitis,
ocular lesions, and, more rarely, respiratory signs and
anaphylactic reactions in humans and animals [5, 32].
Airborne urticating hairs from these caterpillars can be
detected using techniques designed for airborne microorganism
and pollen collection [33]. An IgE-mediated mechanism of
hypersensitivity to this caterpillar has been demonstrated
in a study [34], and anaphylactic shock in a pine-forest
worker was described by Vega et al. [32].
Cytoplasmic polyhedrosis viruses (CPVs) belong to the
genus Cypovirus in the family Reoviridae [18, 22]. CPV
virions are occluded by the viral polyhedrin protein,
forming inclusion bodies (polyhedra) within the host cell
cytoplasm [26]. These icosahedral, cubic, or sometimes
irregular polyhedra dissolve in the insect gut, infecting the
midgut cells of a wide range of insect species and have
been considered as potential biological control agents
for harmful insects. CPVs are commonly isolated from
insects belong to Lepidoptera and occasionally Diptera or
Hymenoptera but only rarely Coleoptera or Neuroptera
[2, 3, 6, 13, 20].
The CPV genomes usually consist of 10 double-stranded
RNA (dsRNA) segments (Seg-1 to Seg-10), although for

some viruses, a small eleventh segment (Seg-11) has been
reported [7, 9]. Each dsRNA segment is composed of a
plus-strand mRNA and its complementary minus strand, in
an end-to-end base-paired configuration, except for a
protruding 5' cap on the plus strand. On the basis of
electrophoretic migration patterns of the dsRNA segments
in agarose or acrylamide gels, CPVs have been classified into
16 different species [9, 16-18, 21-23] by the International
Committee for the Taxonomy of Viruses (ICTV) with five
further proposed types (see http://www.iah.bbsrc.ac.uk/
dsRNA_virus_proteins/CPV-RNA-Termin.htm).
The occurrence of polyhedral inclusion bodies from
Thaumetopoea pityocampa have been reported [4, 10, 11,
24, 25, 27-29]. A cytoplasmic polyhedrosis virus was
the first agent to be mass produced and used to control
Thaumetopoea pityocampa in field conditions in 1959 at
France [15]. However, no further detailed studies of these
viruses were subsequently published.
Here, we report the isolation and characterization of a
CPV isolated recently from Thaumetopoea pityocampa larvae
in Turkey. The structure of this virus, which we have called
TpCPV, was analyzed by light and electron microscopies.
The RNA genome was analyzed electrophoreticaly and has
a migration pattern by agarose gel electrophoresis (AGE)
that shows some similarity to that of CPV-5 [21]. Bioassays
were performed to study the pathogenicity of this virus and
evaluate its potential as a biocontrol agent.
MATERIALS AND METHODS
Field Collections
Dead Thaumetopoea pityocampa larvae were collected
from pine trees during 2004-2005 field studies in Samsun,
located in the middle of the Blacksea Region of Turkey.
Fifty processionaries were checked for infected virus. All
dead larvae in these processionaries were stored individually
and screened for virus infection by light microscopy.
The ones that have polyhedral inclusions were kept at
-20oC.
Virus Production and Purification of Polyhedral Bodies
Third instar T. pityocampa larvae were used for virus
production. They were put in cups without food overnight,
and then exposed to polyhedral inclusion bodies (PIB)-
positive, homogenized T. pityocampa cadavers in phosphate-
buffered saline (PBS) with fresh pine leaves as food, at
10oC. Larvae were incubated for 7 days and than examined
under a light microscope for patent infection. The ones that
have PIB were stored at -20oC. CPV polyhedra were
purified from the infected T. pityocampa midgets. Infected
midguts were first dissected from the insect body, homogenized
in PBS, and purified first by filtration through cheesecloth
to remove the debris and then centrifuged at 3,500 ×g for
A CPV FROM T. PITYOCAMPA 633
10 min. The pellets containing polyhedra were further purified
by Percoll density gradient centrifugation at 12,000 ×g for
20 min. A nine-to-one ratio of Percoll to PBS was employed
for this purpose. The resulting band containing purified
polyhedra was washed three times with PBS and finally
suspended in TE [12].
Light and Electron Microscopies
Different organs of T. pityocampa were dissected and
investigated under bright field microscopy. Differantial
Giemsa staining method was used to stain polyhedral
inclusion bodies in squash preparations [35]. A suspension
of purified polyhedra was air-dried on coverglass, coated
with gold, and examined in a JSM 6400 scanning electron
microscope operated at 15 kV, and photographed. For
electron microscopy, sections of midgut were fixed in
2.5% glutaraldehyde and 0.14 M NaCl in 0.2 M phosphate
buffer (pH 7.4) followed by a secondary fixation for 1 h at
room temperature, with 2% OsO4 and 1.25% (w/v) sodium
bicarbonate at pH 7.4. The primary fixation solution was
changed several times. Between the primary and secondary
fixations, the specimens were washed several times with
0.2 M phosphate buffer containing 0.3 M NaCl. Subsequently,
they were rinsed in phosphate buffer and dehydrated in a
graded series of ethanol-acetone, infiltrated, and embedded
in Epon resin. Thin sections were stained with 5% acidic
uranyl acetate and Reynold’s lead citrate and examined
under a JEOL 100 CX transmission electron microscope at
50 kV.
Isolation of Total Genomic RNA
After dissolving the purified polyhedral in alkali with trizol
reagent (Gibco, BRL) according to the manufacturer’s
instructions. Genomic RNA was extracted from purified
polyhedra by a standard guanidium isothiocyanate method
[10]. RNA was then separated in 1% agarose gel in Tris-
phosphate buffer. RNA segments were visualized on the
ethidium bromide stained (final concentration of 0.5 mg/ml)
gel. Size markers (Bio Basic Inc. Canada) of 1,000 bp
were used to determine segment sizes.
Pathogenicity Test
Third instar T. pityocampa larvae were used for the
bioassay. Experiments were performed with 20 larvae per
dose and were replicated three times with the control group.
Larvae were exposed to four different concentrations of
virus (2×104, 2×105, 1×106, 2×106 PIBs). They were starved
overnight and then fed with pine leaves contaminated with
one of the four virus concentrations and reared separately
for 10 days. After they consumed the entire contaminated
leaves, they were given fresh leaves. The larvae were
incubated at 10oC. The mortalities of larvae were recorded
every 24 h, with all dead larvae removed from the containers.
Data were evaluated using Abbott’s formula [1].
634 INCE et al.

RESULTS
Field Collection
The first TpCPV-infected T. pityocampa larvae were found
in March 2004 in a field-collected processionary. Dead or
diseased larvae were frequently found in the field, suggesting
an epizootic was effecting the pest populations. We observed
40% infection among 50 examined processionaries and
90 percent infection of T. pityocampa larvae in one
processionary. A similar result was also observed in 2005.
The infected larvae were smaller, not feeding and not
moving, and became moribund. Many of the diseased T.
pityocampa larvae were observed to be hanging down by
their prolegs immediately prior to death. Since this behaviour
has previously been observed in insect larvae infected with
nuclear polyhedrosis viruses [8], these T. pityocampa
larvae were checked for the presence of such viruses.
However, the analysis failed to detect any NPVs.
Light and Electron Microscopy Studies
Polyhedra were observed by light microscopy in midgut
cells from tissue smear samples of field-collected dead
pine processionary larvae. Differential Giemsa staining
showed a typical cytoplasmic polyhedrosis virus infection.
Whereas the first and the second zones stained colourless
and purple, respectively, the third zone was black, correlating Fig. 1. Electron micrographs of virus samples from Thaumetopoea
with the CPV staining in the literature (data not shown). pityocampa.
A. Scanning electron micrograph showing inclusion bodies (×5,000). B.
Scanning electron microscopy (Fig. 1A) demonstrated that Transmission electron micrograph of virions exhibiting surface spikes
the occlusion bodies were of irregular shape and ranged (×15,000).
from 2.4 to 5.3 µm in diameter. This was confirmed by
transmission electron microscopy studies, which showed
typical cytoplasmic polyhedral inclusion bodies (Fig. 1B). segments. Approximate sizes of segments were estimated
with size markers as follows: Seg-1, 3,907 bp; Seg-2,
Electrophoretic Analysis of dsRNA 3,716 bp; Seg-3, 3,628 bp; Seg-4, 3,249 bp; Seg- 5, 2,726 bp;
An initial analysis of the TpCPV genome by 1% agarose Seg-6, 1,914 bp; Seg-7, 1,815 bp; Seg-8, 1,256 bp; Seg-9,
gel using a 14-cm gel at 75 V for 4 h generated seven RNA 1,058 bp; Seg-10, 899 bp.
bands, some of which stained more intensely and appeared
to contain more than one genome segment each (the first Pathogenicity Test
intense band contained segments 1, 2, and 3 and the fourth In comparison to an unifected control group, TpCPV
band contained segments 6 and 7). Analysis using a longer infection affected the development, feeding, and behaviour
agarose gel with a lower voltage resolved the first band of the larvae, correlating with the virus dosage. The
into three bands and the fourth band into two single posterior midgut also appeared white (due to the presence
bands, confirming that the genome contains a total of 10 of large numbers of polyhedra) in infected caterpillars.

Table 1. Mortality rate of TpCPV on Thaumetopoea pityocampa exposed as third instar larvae and examined 5 days and 15 days post
inoculation.
Dosage After 5 days After 15 days
(PIBs/larva) % feeding larvae % cessation of feeding % larval death % feeding larvae % cessation of feeding % larval death
Control 100.0 00.0 00.0 95 00.0 05.0
2×104 070.0 30.0 00.0 00 40.0 60.0
2×105 057.2 33.3 09.5 00 33.3 66.6
1×106 040.0 50.0 10.0 00 15.0 85.0
2×106 010.0 60.0 30.0 00 10.0 90.0

Electrophoretic separation of TpCPV:1 total genome in
Fig. 2.
1% agarose gel.
Lane 1: DNA molecular weight marker (1 kb); lane 2: TpCPV genome
segments. The arrows indicated that viral segments are 3,907, 3,716, 3,628,
3,249, 2,726, 1,914, 1,815, 1,256, 1,058, and 899 bp. in size, respectively.
Patent infections developed by 4-5 days post inoculation
(p.i.). Infected larvae stopped feeding and some deaths
were observed from 5 days p.i. onwards, with an increasing
percentage of infected individuals observed with increasing
doses of virus (Table 1). At the end of the 15th day, the
percentages of the mortality at higher and lower doses used
were 90% and 60%, respectively. Percent mortality of the
T. pityocampa increased with increasing dose of the virus.
At lower doses, the development of the larvae was very
slow. The mortality of larvae in the control group was 5%.
These larvae died at the end of the 15th day.
DISCUSSION
An occluded virus with a 10-segmented dsRNA genome
was isolated from Thaumetopoea pityocampa larvae collected
from pine trees in Samsun, Turkey. The characteristics of this
A CPV FROM T. PITYOCAMPA 635
virus, determined by light microscopy, electron microscopy,
and electrophoresis identified it as a CPV, a member of the
genus Cypovirus of the family Reoviridae. CPVs usually
possess a 10-segmented dsRNA (Seg-1 to Seg-10) genome,
although for some viruses, a small eleventh segment (Seg-11)
has been reported [9, 22]. The pattern of size distribution
of the RNA genome fragments after electrophoresis in 1%
agarose or 3-5% polyacrylamide gel provided an original
basis for classification of CPV isolates into different
electropherotypes [9, 16-18, 21-23]. Different CPV types
can also be distinguished by sequence analysis and
comparison of the viral genome segments (for example, by
comparisons of Seg-10, the polyhedrin gene) and the
different electropherotypes are now recognized as distinct
Cypovirus species. Sequencing studies have also demonstrated
that in many cases the CPV genome segments are considerably
larger than initially estimated from their electrophoretic mobility
[36]. Sixteen Cypovirus species have now been formally
recognized by the International Committee for the Taxonomy
of Viruses [18] with a further five proposed species to date
(see http://www.iah.bbsrc.ac.uk/dsRNA_virus_proteins/
CPV-RNA-Termin.htm). TpCPV shows some similarity in
its electropherotype to CPV- 5 [21], although because it is
not feasible to make a direct electrophoretic comparison with
the initial isolate of CPV-5 on the same gel, a conclusive
identification of the species to which TpCPV belongs will be
made after sequence analysis of the genome in further studies.
A cytoplasmic polyhedrosis virus from Thaumetopoea
pityocampa was reported earlier in 1958 [29]; however, it
has not been characterized at the structural and molecular
levels.
Until now, cypoviruses have only been isolated from
arthropods [18]. Experiments conducted to infect vertebrates
or vertebrate cell lines have been unsuccessful, and thus
CPVs appear as safe biocontrol agents. Although more
than 230 cypoviruses have been reported, mainly from
Lepidoptera and some also from Diptera and Hymenoptera
[18], members of the genus are not frequently regarded as
potential biocontrol agents, as they generally produce only
chronic diseases, generally without extensive larval mortality.
However, different virus strains can vary significantly in
virulence [30, 36], and TpCPV exhibits a high level of
virulence, particularly with increasing virus concentrations.
However, even at the low concentration used in the studies
described here, rapid and extensive larval mortality was
observed (Table 1).
Another feature of TpCPV that has also been reported
for other cypoviruses is the rapid cessation of larval
feeding activity after infection. This behavioral change
could significantly reduce the damage to trees before the
insect is killed by the virus. Even if some of the infected
individuals survive to adulthood (as observed with other
cypoviruses), their reproductive potential may be significantly
reduced.
636 INCE et al.
The pine processionary moth caterpillar causes heavy
defoliations in various stands of pines. In Turkey, T.
pityocampa occurs over a forest area of 1,500,000 ha [15].
Pine processionary moth caterpillar also causes health
problems because of their urticating hairs [31]. Contact
with caterpillars induces dermatitis and ocular lesions by a
mechanic and toxic mechanism. Additionally, IgE-mediated
hypersensitivity to this caterpillar has been demonstrated
in two recent studies [32, 34]. Controlling this type of
moth is an important key for an integrated pest management
system.
The results in this article will offer useful information for
future studies on TpCPV molecular biology or implementation
as an insecticide. We are currently intending to determine
the genome sequence and physical map of TpCPV.
Acknowledgments
The authors thank Dr. Peter Mertens (Pirbright Laboratory,
Pirbright, U.K.) and Monique van Oers (Virology Laboratory,
Wageningen University, The Netherlands) for their critical
reading and comments on an earlier draft of the manuscript.
They also acknowledge the Medical Faculty of Karadeniz
Technical University for providing the electron microscopy
service. The project was financially supported by the
Turkish State Planning Organisation and The Scientific
and Technological Research Council of Turkey.
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