Journal of General Virology (2007), 88, 2329–2336
DOI 10.1099/vir.0.83101-0
Vertical-transmission routes for deformed wing
virus of honeybees (Apis mellifera)
Constanze Yue, Marion Schröder, Sebastian Gisder and Elke Genersch
Correspondence
Institute for Bee Research, Friedrich-Engels-Str. 32, D-16540 Hohen Neuendorf, Germany
Elke Genersch
[email protected]
Received 18 April 2007
Accepted 23 April 2007
Deformed wing virus (DWV) is a viral pathogen of the European honeybee (Apis mellifera),
associated with clinical symptoms and colony collapse when transmitted by the ectoparasitic
mite Varroa destructor. In the absence of V. destructor, DWV infection does not result in visible
symptoms, suggesting that mite-independent transmission results in covert infections. True covert
infections are a known infection strategy for insect viruses, resulting in long-term persistence
of the virus in the population. They are characterized by the absence of disease symptoms in
the presence of the virus and by vertical transmission of the virus. To demonstrate vertical
transmission and, hence, true covert infections for DWV, a detailed study was performed on the
vertical-transmission routes of DWV. In total, 192 unfertilized eggs originating from eight virgin
queens, and the same number of fertilized eggs from the same queens after artificial insemination
with DWV-negative (three queens) or DWV-positive (five queens) semen, were analysed
individually. The F0 queens and drones and F1 drones and workers were also analysed for viral
RNA. By in situ hybridization, viral sequences were detected in the ovary of an F0 queen that
had laid DWV-positive unfertilized eggs and was inseminated with DWV-positive semen. In
conclusion, vertical transmission of DWV from queens and drones to drone and worker offspring
through unfertilized and fertilized eggs, respectively, was demonstrated. Viral sequences in
fertilized eggs can originate from the queen, as well as from drones via DWV-positive semen.
INTRODUCTION
In insect virology, the descriptive terms overt and covert
are used widely to categorize the infections caused by insect
viruses. Typically, overt infections are those in which the
virus-infected host develops obvious disease symptoms,
and covert infections are defined as conditions in which the
virus is present within the host in the absence of clear
disease symptoms. Additional characteristics of covert
infections, deduced from work on baculoviruses, are (i)
that the viruses remain fully competent and can re-emerge
to cause overt infections (Burden et al., 2003) and (ii) that
the viruses persist beyond the current life stage and can be
transmitted vertically to a subsequent generation (Burden
et al., 2002; Kukan, 1999). This definition of a covert infection gives a clear distinction from an inapparent infection,
which is also characterized by the absence of disease
symptoms. True inapparent viral infections are defined as
short-term infections with high levels of virus production,
albeit without visible symptoms, and are transmitted solely
horizontally (Dimmock & Primrose, 1987).
Deformed wing virus (DWV) is a positive-stranded RNA
virus (Lanzi et al., 2006) that is pathogenic for both
honeybees and bumblebees (Genersch et al., 2006). DWV
A supplementary table showing primers used for the detection of DWV
sequences via RT-PCR is available with the online version of this paper.
0008-3101 G 2007 SGM
can be detected in all life stages of the honeybee in the
absence of visible disease symptoms (Chen et al., 2005a;
Tentcheva et al., 2006; Yue & Genersch, 2005). Transmission of DWV by Varroa destructor is often associated
with clinical symptoms (crippled wings, bloated and
shortened abdomen and discoloration) (Ball & Allen, 1988;
Bowen-Walker et al., 1999; Martin, 2001; Martin et al., 1998;
Tentcheva et al., 2006; Yue & Genersch, 2005), suggesting
that, even in the absence of disease symptoms, infecting
viruses remain fully competent and can result in clinically
apparent infections when vectored by V. destructor.
Whilst presence of the virus in the absence of disease
symptoms and occasional overt outbreaks fit with the
definition of a covert infection outlined above, the possibility that the asymptomatic DWV infection is an inapparent one, rather than a genuine covert infection, cannot
be ruled out. To distinguish between a covert and an
inapparent infection, the mode of transmission is crucial
(Dimmock & Primrose, 1987). Inapparent infections are
transmitted horizontally, whereas covert infections can also
be transmitted vertically from parent to offspring. It has
been suggested that differences in the mode of transmission
influence the virulence of a pathogen (Fries & Camazine,
2001; Lipsitch et al., 1996). Horizontal transmission allows
the development of more virulent forms of the pathogen
with high negative impact on the fitness of the host.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 08:58:32
Printed in Great Britain
2329
C. Yue and others
Table 1. Detection of DWV sequences in unfertilized and fertilized eggs, in sperm used for insemination, in offspring and in larval
food
Abbreviations: h/t/a, head/thorax/abdomen; t/sv/vg, testis/seminal vesicles/vesicular glands;
weakly positive.
Minicolony
No. unfertilized
eggs (n524)
DWVnegative
66
48
67
65
68
69
49
28
24
24
21
24
24
24
0
0
DWVpositive
0
0
3
0
0
0
24
24
No. fertilized eggs
(n524)
DWVnegative
24
21
0
0
0
0
0
0
DWVpositive
0
3
24
24
24
24
24
24
ND,
not determined. 2, Negative; +, positive; (+),
DWV detection in:
F1 drones (n52)
Sperm used
for AI
h/t/a
t/sv/vg
2/+/+
2/+/+
2/+/+
2/+/+
+/+/+
+/+/+
+/+/+
+/+/+
ND
ND
ND
ND
2/+/+
2/+/+
+/+/+
+/+/+
Vertical transmission, however, relies on host survival and
reproduction and, therefore, less virulent forms will be
favoured, allowing for long-term persistence of the virus
within the host population (Burden et al., 2003; Oldstone,
2006).
Recent studies on the localization of DWV in honeybee
queens and drones revealed that the reproductive organs of
both were strongly positive for viral sequences (Chen et al.,
2006b; Fievet et al., 2006), suggesting the possibility of
vertical-transmission routes. In addition, DWV sequences
were recently demonstrated in sperm (Yue et al., 2006),
further supporting the hypothesis that DWV may be transmitted vertically through drones. Concerning the detection
of DWV RNA in eggs, conflicting results are reported
(Chen et al., 2005a, b, 2006a; Tentcheva et al., 2006), preventing researchers from finally answering the question of
vertical DWV transmission through the queens’ eggs.
Here, we present a detailed analysis of the presumed
vertical-transmission routes of DWV to answer the question of whether DWV can cause true covert infections. By
using RT-PCR and in situ hybridization, we analysed viral
RNA in reproductive organs of both queens and drones, in
sperm before using it for artificial insemination (AI),
individually in unfertilized and fertilized eggs, in larval
food and in offspring. Our results strongly support the
hypothesis that DWV is transmitted vertically through
queens and drones and, therefore, that DWV can cause
genuine covert infections. The impact of true covert DWV
infections on the maintenance of the virus in the
population will be discussed.
METHODS
Sample collection and mating scheme. Eight healthy-looking,
2
2
+
+
+
+
2
+
F1 workers (n53); h/t/a
w1
w2
w3
2/(+)/2
2/+/+
2/2/2
2/+/+
2/+/+
2/2/2
2/+/+
2/2/2
2/+/2
2/(+)/+
2/2/(+)
2/+/+
2/+/+
2/+/+
(+)/+/+
ND
ND
ND
2/+/+
(+)/+/+
2/+/+
(+)/+/+
2/+/+
(+)/+/+
Larval
food
+
+
+
+
+
+
+
+
into eight queenless experimental mini-colonies free of V. destructor
(Table 1). Only in colony 65 were three DWV-positive mites observed
at the end of the experiments. Unfertilized eggs laid by these virgin
queens were analysed for viral sequences (24 eggs each). Some
unfertilized eggs were allowed to develop into F1 drones, which were
then analysed for viral RNA. Semen for AI of the virgin queens was
collected from sexually mature honeybee drones showing no overt
signs of DWV infection. These F0 drones originated from four different, apparently healthy colonies (Table 2). Drones were stimulated to
ejaculate by pressing on the thorax until the cream-coloured semen
and a white mucus plug were released on the end of the penis. Under
a dissecting microscope (610), the semen was collected without
mucus into a syringe, designed by Schley (1982). As only a small
quantity, ,1 ml, of semen can be collected from each drone, the
semen of five drones per colony was pooled to obtain a volume of
around 4 ml. Half of the volume was used for the detection of viral
RNA; the remaining volume was used for AI. Subsequent to the
collection of the F0 drones’ semen, these drones were analysed for
DWV RNA. The eight virgin queens (after analysing the viral status of
their unfertilized eggs) were inseminated artificially with either DWVnegative or DWV-positive sperm to obtain the following combinations (Table 1): (i) DWV-negative unfertilized eggs/DWV-negative
sperm (n52), (ii) DWV-negative unfertilized eggs/DWV-positive
Table 2. Detection of DWV sequences in testis, seminal
vesicles and vesicular glands of the F0 drones (one per donor
group), shown with respect to the viral status of pooled semen
Abbreviation: t/sv/vg, testis/seminal vesicles/vesicular glands. 2,
Negative; +, positive.
Drone colony
Viral status of:
Sperm (pooled from five drones)
t/sv/vg
2
2
+
+
+/+/+
+/+/+
+/+/+
+/+/+
91
Gc
268
GVB
virgin queens were selected randomly from different colonies and put
2330
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 08:58:32
Journal of General Virology 88
Vertical transmission of DWV
sperm (n53), (iii) DWV-positive unfertilized eggs/DWV-negative
sperm (n51), and (iv) DWV-positive unfertilized eggs/DWV-positive
sperm (n51). One queen that had produced a small proportion of
DWV-positive unfertilized eggs (three of 24) was also inseminated
with DWV-positive sperm. Subsequently, fertilized eggs (24 eggs per
colony), as well as F1 workers, were analysed for viral sequences. At
the end of the experiment, the F0 queens were analysed for DWV
RNA by using RT-PCR and in situ hybridization. Larval food was
collected from uncapped cells of larvae not older than 2 days. Eggs,
bees and larval food were frozen at 270 uC immediately after
sampling and stored at 270 uC until analysis.
Tissue dissection. After sperm collection and at the end of the
experiment, the F0 drones and queens were sacrificed for tissue
dissection and analysis of viral RNA. Some of the F1 drones were also
sacrificed for tissue dissection. The bee heads were cut off and the
eviscerated body was fixed with insect pins on the wax top of a
dissecting dish. The abdomen was opened with scissors; the tissues
were removed carefully from the body with forceps and separated into
testis, vesicular glands and seminal vesicles (drones) or gut, spermatheka and ovaries (queens) under a dissecting microscope (610).
Contaminating haemolymph was washed off by rinsing the tissues
carefully with 16 PBS and nuclease-free water. One ovary was used
for RNA extraction and subsequent RT-PCR analysis; the other ovary
was embedded in a polymerizing resin (Technovit 8100; Heraeus
Kulzer) according to the manufacturer’s protocol and subjected to in
situ hybridization. All other tissues were used for RNA extraction and
RT-PCR analysis only.
RNA extraction. Total RNA extraction from eggs, sperm, adult bees
(body parts and tissue of F0 queens, F0 drones, F1 drones and F1
workers) and larval food was performed as described previously (Yue &
Genersch, 2005; Yue et al., 2006), using standard methods following the
manufacturer’s protocols (RNeasy kit for eggs, sperm and bees; Viral
RNA kit for larval food; both from Qiagen). The air-dried RNA pellets
were resuspended in elution buffer (Qiagen) and stored at 270 uC.
One-step RT-PCR for the detection of DWV RNA. One-step
RT-PCR was performed according to standard protocols (One-Step
RT-PCR kit; Qiagen) and as described previously (Genersch, 2005).
The following temperature scheme was used: 30 min at 50 uC, 15 min
at 95 uC, followed by 35 cycles of 1 min at 94 uC, 1 min at 54.3 uC
and 1 min at 72 uC, including a final elongation step for 10 min at
72 uC. The following primer pairs, covering different regions of the
viral genome, were used (see Supplementary Table S1, available in
JGV Online): F0/B0 (designed for this study), F1/B1, F10/B16 and
F15/B23 (Genersch, 2005). PCR products (8 ml per reaction) were
analysed on a 1.0 % agarose gel. The DNA bands were stained with
ethidium bromide and visualized by UV light. Specificity of the
amplicons was further verified by sequencing (Medigenomix,
Germany) of random samples.
In situ hybridization. 59-Digoxigenin (DIG)-labelled oligonucleo-
tides (TIB MOLBIOL) were used as probes for the detection of viral
positive- and negative-stranded RNA. Six oligonucleotides hybridizing to three different regions of the DWV genome were used (positions according to GenBank accession no. NC_004830): 4780–4810
[DIG-TAACTGAGACACCAGTGAGAAGACATTTGCT (DWV4810minus) and DIG-AGCAAATGTCTTCTCACTGGTGTCTCAGTTA
(DWV4780plus)], 6580–6610 [DIG-TAGAGCCTGTGATGTGAATTCAGTGTCGCCC (DWV6610minus) and DIG-GGGCGACACTGAATTCACATCACAGGCTCTA (DWV6580plus)] and 9240–9270
[DIG-TCCGTTATTGGAGAACCTGATGGAATTCCAC (DWV9270minus) and DIG-GTGGAATTCCATCAGGTTCTCCAATAACGGA
(DWV9240plus)], as well as a nonsense oligonucleotide [DIG-GCGTAGTGCAAGCTGATCCGCTAGTGACTG (nonsense)]. Oligonucleotides DWV4810minus, DWV6610minus and DWV9270minus
http://vir.sgmjournals.org
represent antisense probes; oligonucleotides DWV4780plus, DWV6580plus and DWV9240plus are sense probes. For hybridization,
tissues were fixed in 4 % paraformaldehyde in 16 PBS at 4 uC for
24 h and embedded in a polymerizing resin (Technovit 8100; Heraeus
Kulzer) at 4 uC according to the manufacturer’s protocol. Polymerized resin blocks were stored at 4 uC until the preparation of
semi-thin sections (4 mm), which were mounted on poly-L-lysinecoated slides and stored at 4 uC until use. The in situ hybridization
protocol was adapted from Heiles et al. (1988) and Mitta et al. (2000).
Briefly, semi-thin sections were incubated for 20 min at room
temperature in 0.2 M HCl, washed twice for 5 min each at 37 uC in
26 SSC (standard saline citrate) and then incubated for 15 min at
37 uC in 1 mg proteinase K ml21. Subsequently, the sections were
washed three times for 5 min each at 37 uC in 16 PBS, incubated
for 10 min at room temperature in 0.1 M glycine, 0.2 M Tris/HCl
(pH 7.5), and washed twice for 5 min each at 37 uC in 26 SSC,
5 mM EDTA. A post-fixation step was performed for 20 min at room
temperature in 4 % paraformaldehyde in 16 PBS, followed by a final
washing step for 5 min at room temperature with 16 PBS. DIGlabelled oligonucleotides (mixture of DWV4780plus, DWV6580plus
and DWV9240plus, 20 ng each, or DWV4810minus, DWV6610minus and DWV9270minus, 20 ng each, or 20 ng nonsense oligonucleotide, DWV4810minus, DWV6610minus or DWV9270minus
alone) were diluted in 50 ml hybridization buffer containing 66 SSC,
45 % formamide, 10 % dextran sulfate, 0.5 % SDS, 56 Denhardt’s
solution, 0.2 mg salmon sperm DNA ml21. Hybridization was
performed overnight at 37 uC in a humid chamber. The sections
were then washed twice in 66 SSC, 45 % formamide for 15 min each
at 42 uC, once in 66 SSC for 5 min at 42 uC, twice in 26 SSC for
10 min each at 50 uC and twice in 0.26 SSC for 15 min each at
50 uC. Detection of DIG-labelled, hybridized oligonucleotides was
performed by using alkaline phosphatase-conjugated anti-DIG antibodies according to the manufacturer’s protocol (Roche). Bound
antibodies were visualized by incubation (overnight, darkness, room
temperature) in 0.1 M Tris/HCl (pH 9.5), 50 mM MgCl2, 0.1 M
NaCl, 375 mg nitro blue tetrazolium ml21, 188 mg 5-bromo-4-chloro3-indolyl phosphate ml21. The chromagen reaction was stopped by
rinsing the slides in 0.1 M Tris/HCl (pH 7.5), 0.1 M NaCl, and then
coverslips were mounted on the slides with AquaPolyMount
(Polysciences Europe).
RESULTS
Detection of DWV sequences in unfertilized eggs
and sperm
To evaluate the incidence of maternally originating DWV
sequences in eggs, unfertilized eggs laid by eight asymptomatic, virgin queens selected randomly from different,
healthy-appearing colonies were analysed for the presence
of DWV RNA (Table 1; Fig. 1a). Five of eight queens
produced only DWV-negative eggs (24 of 24), one queen
laid DWV-negative (21 of 24) as well as DWV-positive
(three of 24) eggs, and two queens laid 100 % DWVpositive eggs (24 of 24). Analysis of sperm for DWV
sequences revealed that two of the four randomly selected
colonies produced drones with DWV-positive sperm and
the other two produced drones with DWV-negative sperm
(Table 2), supporting earlier results (Yue et al., 2006).
Further analysis of the virus-positive samples with a panel
of PCR primers covering four evenly spaced segments of
the viral genome (Supplementary Table S1, available in
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 08:58:32
2331
C. Yue and others
Fig. 1. Detection of DWV sequences in
unfertilized and fertilized eggs and sperm. (a)
Unfertilized and fertilized eggs were analysed
for DWV sequences by using RT-PCR and the
primer pair F15/B23 as described in Methods.
Representative results are shown for two
queens laying DWV-negative eggs and subsequently inseminated with DWV-negative
sperm (colonies 66 and 48), one queen laying
DWV-negative eggs and subsequently inseminated with DWV-positive sperm (colony 65)
and one queen laying DWV-positive eggs and
subsequently inseminated with DWV-positive
sperm (colony 49). The results for 12 of the
analysed 24 eggs per colony are shown. (b)
Sperm and unfertilized and fertilized eggs were
analysed for DWV sequences by using RTPCR and a panel of primer pairs covering
several evenly spaced segments of the viral
genome. Representative results are shown
for sperm collected from drones originating
from colony GVB, for two unfertilized eggs
from colony 49 and for two fertilized eggs from
colony 65.
JGV Online) was successful, thus excluding the possibility
that the positive signals in sperm and unfertilized eggs
represent defective interfering (DI)-like RNAs (Fig. 1b).
Detection of DWV sequences in fertilized eggs
To evaluate the impact of virus transmission via semen,
fertilized eggs laid after AI with DWV-negative or DWVpositive semen were analysed for the presence of DWV
sequences (Table 1; Fig. 1a). AI with DWV-positive sperm
resulted in 100 % DWV-positive fertilized eggs. One
control queen with DWV-negative unfertilized eggs and
inseminated artificially with DWV-negative sperm laid
100 % DWV-negative eggs. The other control queen
showed a small proportion of DWV-positive eggs (three
of 24), suggesting a low background of DWV-positive
unfertilized eggs not detected in the first batch of analysed
eggs. Again, analysis of DWV-positive fertilized eggs with a
panel of PCR primers (Supplementary Table S1, available
in JGV Online) revealed the presence of the entire viral
genome rather than the presence of DI-like RNAs (Fig. 1b).
Detection of DWV sequences in the F1 generation
None of the drones or workers of the F1 generation showed
any visible symptoms of disease or DWV infection (crippled
wings, discoloration and bloated abdomen). Detailed
analysis of two drones of the F1 generation from each
colony revealed the presence of DWV sequences in thorax
and/or abdomen or, when analysed in detail, in testis,
seminal vesicles and vesicular glands (Table 1). This was
true even for drones originating from queens with 100 %
2332
DWV-negative unfertilized eggs. No DWV sequences could
be detected in total RNA isolated from head, supporting
earlier reports that asymptomatic bees rarely test positive
for DWV sequences in head (Yue & Genersch, 2005).
Detailed analysis of three F1 workers from each colony
showed no strong correlation between the detection of
DWV sequences in fertilized eggs and in workers. Although
no DWV sequences could be detected in fertilized eggs in
mini-colony 66, only one F1 worker was DWV-negative; in
two F1 workers, weak signals for DWV could be detected
in RNA isolated from thorax (w1) or abdomen (w3). In
contrast, in mini-colony 67, all eggs tested positive for
DWV but, in two of three workers, no DWV sequences
could be detected. All other workers analysed were DWVpositive. Surprisingly, we detected a weak signal for DWV
in total RNA isolated from head in four F1 workers. Three
belonged to mini-colony 28 (Fig. 2a) and originated from
insemination of a queen laying DWV-positive unfertilized
eggs with DWV-positive sperm (Table 1). The presence of
the entire viral genome in DWV-positive F1 bees could be
substantiated by further analysing DWV-positive bees with
a panel of PCR primers representing different regions of
the viral genome (Fig. 2b). It is noteworthy that, in all
colonies, larval food tested positive for DWV sequences
(Table 1).
Detection of DWV sequences in the F0 generation
Although none of the queens or drones of the F0 generation showed any pathological signs of DWV infection,
they were all DWV-positive, as revealed by qualitative RTPCR analysis of different body parts and tissues. F0 drones
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 08:58:32
Journal of General Virology 88
Vertical transmission of DWV
Fig. 2. Detection of DWV sequences in F1
worker bees. (a) Total RNA extracted from
head, thorax and abdomen of F1 worker bees
was analysed for DWV sequences by using
RT-PCR and the primer pair F15/B23 as
described in Methods. Shown are the results
obtained for three worker bees of colony 28
where, in addition to strong signals in RNA
isolated from thorax (t) and abdomen (a), faint
signals in head RNA (h) could be detected. (b)
Total RNA samples that gave positive signals
obtained with primer pair F15/B23 were analysed further by using a panel of primer pairs
covering several evenly spaced segments of
the viral genome. Representative results
obtained with total RNA extracted from the
thorax of one worker (w1) of colony 28 are
shown.
were positive for DWV RNA in testis, seminal vesicles and
vesicular glands, regardless of whether they had produced
DWV-negative or -positive sperm (Table 2). All queens
tested positive for the entire viral genome in gut, spermatheka and ovaries (Table 3; Fig. 3a, b). Surprisingly, the
ovaries of the queen heading mini-colony 66 were DWVpositive, although this queen laid 100 % DWV-negative
fertilized eggs and produced DWV-negative F1 workers
(Table 1). Similarly, the spermatheka was DWV-positive
even for the queens of mini-colonies 66 and 48, which had
laid DWV-negative unfertilized eggs and were inseminated
with DWV-negative sperm, resulting in 100 and 88 %
DWV-negative fertilized eggs, respectively (Table 1). No
DWV RNA could be detected in head (Table 3).
In situ hybridization analysis of DWV RNA in
ovaries
Table 3. Detection of DWV sequences in head, gut, spermatheka and ovaries of the F0 queens, shown with respect to the
viral status of unfertilized eggs, sperm used for AI and fertilized
eggs
The puzzling results concerning DWV detection in ovaries
and eggs prompted us to analyse the ovaries of the queens
heading mini-colonies 66 and 49 via in situ hybridization.
Strong, DWV-specific signals could be observed in the
ovary of the queen heading mini-colony 49 (Fig. 4), which
had produced 100 % DWV-positive unfertilized eggs and
had been inseminated with DWV-positive sperm (Table 1;
Fig. 1). Positive-stranded viral RNA was detected in the
fully developed oocytes, as well as in the surrounding tissue
of the terminal part of the ovary. No viral negativestranded RNA could be detected, although it was present
according to strand-specific RT-PCR (data not shown).
Localization of viral sequences in the ovary of the queen
heading mini-colony 66 (DWV-negative unfertilized eggs,
inseminated with DWV-negative sperm, DWV-negative
fertilized eggs; Table 1) was unsuccessful, despite the
presence of DWV RNA in the ovary of this queen when
analysed by RT-PCR (Table 3; Fig. 3).
Abbreviation: h/g/sp/o, head/gut/spermatheka/ovaries. 2, Negative;
+, positive; ±, some eggs were DWV-positive and others were
DWV-negative. See Fig. 1 for details.
DISCUSSION
Mini-colony
66
48
65
68
69
67
49
28
Viral status of:
DWV sequences
in h/g/sp/o
Unfertilized
eggs/sperm
Fertilized
eggs
2/2
2/2
2/+
2/+
2/+
±/+
+/2
+/+
2
±
+
+
+
+
+
+
http://vir.sgmjournals.org
2/+/+/+
2/+/+/+
2/+/+/+
2/+/+/+
2/+/+/+
2/+/+/+
2/+/+/+
2/+/+/+
DWV is widespread all over the world (Berenyi et al., 2006;
Chantawannakul et al., 2006; Chen et al., 2004; Martin
et al., 1998; Tentcheva et al., 2004) and outbreaks of clinical
DWV infection have been reported to be associated with
the collapse of honeybee colonies due to infestation with
the ectoparasitic mite V. destructor (Bailey & Ball, 1991;
Ball & Allen, 1988; Martin, 2001; Martin et al., 1998;
Nordström et al., 1999). Vectorial transmission of DWV
through V. destructor is well documented (Bailey & Ball,
1991; Bowen-Walker et al., 1999; Chen et al., 2005a; Shen
et al., 2005) and recent studies suggested that V. destructor
might serve as a biological rather than a mechanical vector
(Yue & Genersch, 2005). As DWV was also demonstrated
in bees that had never had contact with V. destructor (Yue
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 08:58:32
2333
C. Yue and others
exclusively (n55) or predominantly (n51) DWV-negative
unfertilized eggs. However, the unfertilized eggs of two of
eight analysed queens contained DWV RNA, indicating the
possibility of a transovarial route for the vertical transmission of DWV. Analysis of the F1 drones revealed that all
drones, including those hatching from DWV-negative eggs,
were positive for DWV, emphasizing the impact of the
horizontal route through feeding of DWV-containing
larval food on the viral status of the F1 generation.
Fig. 3. Detection of DWV sequences in F0 queens. (a) Total RNA
extracted from head, gut, spermatheka (sp.theka) and ovaries of F0
queens heading colonies 66 and 49 were analysed for DWV
sequences by using RT-PCR and the primer pair F15/B23 as
described in Methods. (b) Total RNA samples that gave positive
signals obtained with primer pair F15/B23 were analysed further
by using a panel of primer pairs covering several evenly spaced
segments of the viral genome. Representative results obtained
with total RNA from the ovaries of the F0 queens heading colonies
66 and 49 are shown.
& Genersch, 2005), Varroa-independent transmission
routes must exist. Horizontal transmission within an
infected colony through feeding has been suggested by
the recent detection of DWV RNA in larval food (Yue &
Genersch, 2005). Vertical transmission of DWV from
queens to their larval offspring has been proposed recently,
due to the detection of DWV sequences in sperm (Yue
et al., 2006). However, although queens and their reproductive organs were shown to be positive for DWV
RNA, studies on the presence of DWV sequences in eggs
have so far yielded contradictory results (Chen et al., 2005a,
b, 2006a; Fievet et al., 2006; Tentcheva et al., 2006).
Here, we provide evidence that DWV can be transmitted
vertically through the maternal as well as through the
paternal route. Although the reproductive organs of all
F0 queens tested positive for DWV RNA by RT-PCR,
the majority (six of eight) of the analysed queens laid
2334
Recently, we demonstrated the presence of DWV RNA in
semen collected from healthy drones (Yue et al., 2006).
Here, we present further evidence for the vertical transmission of DWV through semen. AI of queens laying
100 % DWV-negative unfertilized eggs with DWV-positive
semen resulted in 100 % DWV-positive fertilized eggs,
demonstrating venereal transmission of an insect virus for
the first time and indicating that DWV transmission via
semen is a very efficient route. All F1 workers originating
from these eggs were DWV-positive. As the F1 workers that
developed from DWV-negative eggs were also DWVpositive, the influence of the horizontal-transmission route
through feeding of DWV-containing larval food is demonstrated further. Unfortunately, our experimental design did
not allow us to exclude the influence of larval food, which
was positive for DWV RNA in all colonies. Further studies
involving laboratory-reared F1 drones and workers are
necessary to analyse separately the true impact of the
vertical routes on the viral status of the offspring.
Recently, the existence of DI-like RNAs was demonstrated
for Israeli acute paralysis virus (IAPV), a honeybeepathogenic dicistrovirus (Maori et al., 2007). As our RTPCR primer pairs and in situ hybridization probes cover
the entire DWV genome from the 59 end, coding for the
structural proteins, to the 39 end, coding for the nonstructural proteins (Lanzi et al., 2006), we can rule out the
possibility that our results originate solely from such DIlike RNAs. Likewise, we can rule out the possibility that
they are due to the detection of transcribed viral sequences
integrated into the host genome, as only a small, 428 bp
fragment of the structural-protein cistron of IAPV was
found to be integrated into the host genome (Maori et al.,
2007), whereas we can demonstrate the presence of the
entire DWV genome, as well as viral replication.
Bergem et al. (2006) recently reported the long-term maintenance of honeybee embryonic cells. As our findings show
that DWV can infect embryonic cells, these cells should
provide a good cell-culture model to study cellular and
molecular aspects of the pathogenesis of DWV.
RT-PCR analysis revealed that the reproductive organs of
all queens contained DWV RNA, irrespective of the viral
status of their unfertilized eggs. In situ hybridization of the
reproductive organs of the queens laying DWV-positive
unfertilized eggs revealed positive signals for DWV in the
terminal part of the ovaries. This is in contrast to a recent
report by Fievet et al. (2006), who failed to detect DWV in
ovaries via in situ hybridization, although the reproductive
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 08:58:32
Journal of General Virology 88
Vertical transmission of DWV
(a)
(b)
(c)
(d)
(e)
(f)
organs tested positive for DWV RNA in RT-PCR. We
believe that our positive in situ hybridization result is due
to the fact that the analysed queen laid DWV-positive eggs,
as we also failed to detect DWV sequences in the ovaries of
the analysed queen laying DWV-negative eggs, although
these ovaries were positive for DWV according to RT-PCR
analysis. Therefore, the detection of DWV in ovaries via
in situ hybridization might correlate with the viral status
of the unfertilized eggs. In situ hybridization analysis of
ovaries for DWV will probably give negative results for
queens laying DWV-negative eggs and positive results for
queens laying DWV-positive eggs, because only then is
enough viral RNA present to be detected by in situ
hybridization. Further studies to elucidate this transovarial-transmission route for DWV are necessary.
In contrast to an earlier report, where we did not find
DWV sequences in total RNA extracted from the heads of
healthy workers (Yue & Genersch, 2005), in the current
study, four bees showed weak signals for DWV sequences
in head RNA. Attempts to localize DWV via in situ hybridization of head sections of healthy bees failed. Therefore, we
can only speculate that, for example, hypopharyngeal
glands might be infected. Persistent infection of the hypopharyngeal glands of some otherwise-healthy worker bees
would be a plausible explanation for the DWV-positive
larval food and, thus, could be the source of horizontal
transmission of DWV within an infected colony.
All F0 bees and almost all (30 of 33) F1 bees were DWVpositive, but none showed any visible symptoms of disease,
suggesting that, in the absence of V. destructor, even
the combination of horizontal- and vertical-transmission
routes within the colony does not have a high negative
impact on the fitness and fecundity of the infected bees.
We therefore conclude that, in the absence of V. destructor,
http://vir.sgmjournals.org
Fig. 4. Detection of DWV by in situ hybridization in one ovary of the queen heading colony
49. Consecutive ovary sections were challenged with DIG-labelled antisense (a, d, e, f),
sense (b) and nonsense (c) probes. Signals
were visualized after chromagen reaction by
light microscopy. The terminal part of the
ovaries, containing nearly fully developed
oocytes, is shown. Positive signals were
obtained with a mixture of the antisense
probes DWV4810minus, DWV6610minus
and DWV9270minus (a), as well as with
DWV4810minus (d), DWV6610minus (e) or
DWV9270minus (f) alone.
DWV causes true covert infections, with DWV being
present in the absence of disease symptoms and being
transmitted vertically from parent (queen, drones) to
offspring (workers, drones, queens). Vertical transmission
of pathogens selects for the development of less virulent
forms, as the pathogen needs a relatively fit host that is able
to reproduce at least once to be passed on the next host
generation (Burden et al., 2003; Fries & Camazine, 2001;
Oldstone, 2006). Hence, our findings that DWV transmitted vertically within the colony in the absence of V.
destructor had little, if any, impact on the fitness of the
individual infected honeybee are not surprising, as this is a
prerequisite for the vertical transmission of DWV between
colonies. An infected colony, even when harbouring predominantly such covertly infected animals, will develop
normally and eventually swarm, transmitting the virus
vertically to the next colony generation, allowing long-term
persistence of DWV in the honeybee population. So far,
clinical outbreaks of DWV infections followed by colony
collapse have been associated with infestation of V. destructor (Ball & Allen, 1988; Bowen-Walker et al., 1999;
Martin, 2001; Martin et al., 1998; Tentcheva et al., 2006;
Yue & Genersch, 2005), suggesting that DWV has
developed a very well-balanced co-existence with honeybees and, therefore, needs a strong trigger, such as
immunosuppression by V. destructor (Yang & Cox-Foster,
2005) or V. destructor as biological vector (Yue & Genersch,
2005), to re-emerge as an overt infection lethal to colonies.
This principal ability to re-emerge as an overt infection
demonstrates the full competence of the infecting virus and
is the third hallmark of genuine covert infections. We
therefore propose to classify DWV as a covert-infecting
virus. Further studies will identify the target tissues and
cells that allow the virus to persist, and unravel the
mechanisms of viral persistence.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 08:58:32
2335
C. Yue and others
ACKNOWLEDGEMENTS
Genersch, E., Yue, C., Fries, I. & de Miranda, J. R. (2006). Detection
This work was supported by the EU (according to regulation 797/
2004) and by grants from the Ministries of Agriculture of Brandenburg, Sachsen, and Thüringen, Germany, and the Senate of Berlin,
Germany.
of Deformed wing virus, a honey bee viral pathogen, in bumble bees
(Bombus terrestris and Bombus pascuorum) with wing deformities.
J Invertebr Pathol 91, 61–63.
Heiles, H. B. J., Genersch, E., Kessler, C., Neumann, R. & Eggers,
H. J. (1988). In situ hybridization with digoxigenin-labeled DNA of
human papillomaviruses (HPV 16/18) in HeLa and SiHa cells.
Biotechniques 6, 978–981.
REFERENCES
Kukan, B. (1999). Vertical transmission of nucleopolyhedrosisvirus in
Bailey, L. & Ball, B. V. (1991). Honey Bee Pathology, 2nd edn. London:
Academic Press.
Ball, B. V. & Allen, M. F. (1988). The prevalence of pathogens in honey
bee (Apis mellifera) colonies infested with the parasitic mite Varroa
jacobsoni. Ann Appl Biol 113, 237–244.
Berenyi, O., Bakonyi, T., Derakhshifar, I., Köglberger, H. & Nowotny,
N. (2006). Occurence of six honeybee viruses in diseased Austrian
apiaries. Appl Environ Microbiol 72, 2414–2420.
Bergem, M., Norberg, K. & Aamodt, R. M. (2006). Long-term
insects. J Invertebr Pathol 74, 103–111.
Lanzi, G., De Miranda, J. R., Boniotti, M. B., Cameron, C. E., Lavazza,
A., Capucci, L., Camazine, S. M. & Rossi, C. (2006). Molecular and
biological characterization of deformed wing virus of honeybees (Apis
mellifera L.). J Virol 80, 4998–5009.
Lipsitch, M., Siller, S. & Nowak, M. A. (1996). The evolution of
virulence in pathogens with vertical and horizontal transmission.
Evolution 50, 1729–1741.
Maori, E., Tanne, E. & Sela, I. (2007). Reciprocal sequence exchange
maintenance of in vitro cultured honeybee (Apis mellifera) embryonic
cells. BMC Dev Biol 6, 17.
between non-retro viruses and hosts leading to the appearance of new
host phenotypes. Virology 362, 342–349.
Bowen-Walker, P. L., Martin, S. J. & Gunn, A. (1999). The transmission
Martin, S. J. (2001). The role of Varroa and viral pathogens in the
of deformed wing virus between honeybees (Apis mellifera L.) by the
ectoparasitic mite Varroa jacobsoni Oud. J Invertebr Pathol 73, 101–106.
collapse of honeybee colonies: a modelling approach. J Appl Ecol 38,
1082–1093.
Burden, J. P., Griffiths, C. M., Cory, J. S., Smith, P. & Sait, S. M.
(2002). Vertical transmission of sublethal granulovirus infection in
Martin, S., Hogarth, A., Van Breda, J. & Perret, J. (1998). A scientific
the Indian meal moth, Plodia interpunctuella. Mol Ecol 11, 547–555.
note on Varroa jacobsoni Oudemans and the collapse of Apis mellifera
L. colonies in the United Kingdom. Apidologie 29, 369–370.
Burden, J. P., Nixon, C. P., Hodgkinson, A. E., Possee, R. D., Sait, S. M.,
King, L. A. & Hails, R. S. (2003). Covert infections as a mechanism for
Mitta, G., Vandenbulcke, F., Noel, T., Romestand, B., Beauvillain,
J. C., Salzet, M. & Roch, P. (2000). Differential distribution and
long-term persistence of baculoviruses. Ecol Lett 6, 524–531.
Chantawannakul, P., Ward, L., Boonham, N. & Brown, M. (2006). A
defence involvement of antimicrobial peptides in mussel. J Cell Sci
113, 2759–2769.
scientific note on the detection of honeybee viruses using real-time
PCR (TaqMan) in Varroa mites collected from a Tai honeybee (Apis
mellifera) apiary. J Invertebr Pathol 91, 69–73.
Nordström, S., Fries, I., Aarhus, A., Hansen, H. & Korpela, S. (1999).
Chen, Y. P., Pettis, J. S., Feldlaufer, M. F., Collins, A. M. & Smith, I. B.
(2004). Detection of deformed wing virus infection in honey bees,
Oldstone, M. B. A. (2006). Viral persistence: parameters, mechanisms
and future predictions. Virology 344, 111–118.
Apis mellifera L., in the United States. Am Bee J 144, 557–559.
Schley, P. (1982). Neue Kolbenbesamungsspritze mit Sichtkontrolle
Chen, Y. P., Higgins, J. A. & Feldlaufer, M. F. (2005a). Quantitative
und auswechselbaren Einsätzen. Allg Dtsch Imkerzeit 4, 107–108 (in
German).
real-time reverse transcription-PCR analysis of deformed wing virus
infection in the honeybee (Apis mellifera L.). Appl Environ Microbiol
71, 436–441.
Virus infections in Nordic honey bee colonies with no, low or severe
Varroa jacobsoni infestations. Apidologie 30, 475–484.
Chen, Y., Pettis, J. S. & Feldlaufer, M. F. (2005b). Detection of
Shen, M., Yang, X., Cox-Foster, D. & Cui, L. (2005). The role of varroa
mites in infections of Kashmir bee virus (KBV) and deformed wing
virus (DWV) in honey bees. Virology 342, 141–149.
multiple viruses in queens of the honey bee Apis mellifera L. J Invertebr
Pathol 90, 118–121.
Tentcheva, D., Gauthier, L., Zappulla, N., Dainat, B., Cousserans, F.,
Colin, M. E. & Bergoin, M. (2004). Prevalence and seasonal variations
Chen, Y., Evans, J. & Feldlaufer, M. (2006a). Horizontal and vertical
of six bee viruses in Apis mellifera L. and Varroa destructor mite
populations in France. Appl Environ Microbiol 70, 7185–7191.
transmission of viruses in the honey bee, Apis mellifera. J Invertebr
Pathol 92, 152–159.
Chen, Y. P., Pettis, J. S., Collins, A. & Feldlaufer, M. F. (2006b).
Tentcheva, D., Gauthier, L., Bagny, L., Fievet, J., Dainat, B.,
Cousserans, F., Colin, M. E. & Bergoin, M. (2006). Comparative
Prevalence and transmission of honeybee viruses. Appl Environ
Microbiol 72, 606–611.
analysis of deformed wing virus (DWV) RNA in Apis mellifera and
Varroa destructor. Apidologie 37, 41–50.
Dimmock, N. J. & Primrose, S. B. (1987). Introduction to Modern
Yang, X. & Cox-Foster, D. L. (2005). Impact of an ectoparasite on the
Virology. Oxford: Blackwell Scientific.
Fievet, J., Tentcheva, D., Gauthier, L., De Miranda, J. R., Cousserans,
F., Colin, M. E. & Bergoin, M. (2006). Localization of deformed wing
immunity and pathology of an invertebrate: evidence for host
immunosuppression and viral amplification. Proc Natl Acad Sci
U S A 102, 7470–7475.
virus infection in queen and drone Apis mellifera L. Virol J 3, 16.
Yue, C. & Genersch, E. (2005). RT-PCR analysis of Deformed wing
Fries, I. & Camazine, S. (2001). Implications of horizontal and
virus in honey bees (Apis mellifera) and mites (Varroa destructor).
J Gen Virol 86, 3419–3424.
vertical pathogen transmission for honey bee epidemiology.
Apidologie 32, 199–214.
Genersch, E. (2005). Development of a rapid and sensitive RT-PCR
method for the detection of deformed wing virus, a pathogen of the
honeybee (Apis mellifera). Vet J 169, 121–123.
2336
Yue, C., Schröder, M., Bienefeld, K. & Genersch, E. (2006). Detection
of viral sequences in semen of honeybees (Apis mellifera): evidence for
vertical transmission of viruses through drones. J Invertebr Pathol 92,
105–108.
Downloaded from www.microbiologyresearch.org by
IP: 88.99.165.207
On: Sun, 18 Jun 2017 08:58:32
Journal of General Virology 88